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
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Abstract |
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Keywords: antibody/chimeric receptor/cytokine receptor/mutation/transmembrane domain
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Introduction |
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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., 2004). 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., 2002
, 2003
, 2004
).
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., 1999, 2001
; Kubatzky et al., 2001
). 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., 1999
). A typical leucine zipper motif in EpoR TM would form an
-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., 2001
). 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.
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Materials and methods |
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The construction of ScFv-gp130 (ScFvg) was as described (Ueda et al., 2000; Kawahara et al., 2001a
,b
, 2004
). 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., 2003
) 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., 2004) 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., 1986), 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., 2000
), 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 vectorFugene6 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., 2004) 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 SDSPAGE 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.
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Results and discussion |
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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., 1999). 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., 1996
, 1998
, 1999
; Syed et al., 1998
; Remy et al., 1999
). 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.
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Acknowledgments |
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References |
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Constantinescu,S.N., Keren,T., Socolovsky,M., Nam,H., Henis,Y.I. and Lodish,H.F. (2001) Proc. Natl Acad. Sci. USA, 98, 43794384.
Gurezka,R., Laage,R., Brosig,B. and Langosch,D. (1999) J. Biol. Chem., 274, 92659270.
Kawahara,M., Natsume,A., Terada,S., Kato,K., Tsumoto,K., Kumagai,I., Miki,M., Mahoney,W., Ueda,H. and Nagamune,T. (2001a) Biotechnol. Bioeng., 74, 416423.[CrossRef][ISI][Medline]
Kawahara,M., Ueda,H., Tsumoto,K., Kumagai,I., Mahoney,W. and Nagamune,T. (2001b) J. Biochem., 130, 305312.[Abstract]
Kawahara,M., Ueda,H., Tsumoto,K., Kumagai,I., Mahoney,W. and Nagamune,T. (2002) J. Biosci. Bioeng., 93, 399404.[CrossRef][ISI]
Kawahara,M., Ueda,H., Morita,S., Tsumoto,K., Kumagai,I. and Nagamune,T. (2003) Nucleic Acids Res., 31, e32.
Kawahara,M., Kimura,H., Ueda,H. and Nagamune,T. (2004) Biochem. Biophys Res. Commun., 315, 132138.[CrossRef][ISI][Medline]
Kubatzky,K.F., Ruan,W., Gurezka,R., Cohen,J., Ketteler,R., Watowich,S.S., Neumann,D., Langosch,D. and Klingmuller,U. (2001) Curr. Biol., 11, 110115.[CrossRef][ISI][Medline]
Livnah,O., Stura,E.A., Johnson,D.L., Middleton,S.A., Mulcahy,L.S., Wrighton,N.C., Dower,W.J., Jolliffe,L.K. and Wilson,I.A. (1996) Science, 273, 464471.[Abstract]
Livnah,O. et al. (1998) Nat. Struct. Biol., 5, 9931004.[CrossRef][ISI][Medline]
Livnah,O., Stura,E.A., Middleton,S.A., Johnson,D.L., Jolliffe,L.K. and Wilson,I.A. (1999) Science, 283, 987990.
May,P., Gerhartz,C., Heesel,B., Welte,T., Doppler,W., Graeve,L., Horn,F. and Heinrich,P.C. (1996) FEBS Lett., 394, 221226.[CrossRef][ISI][Medline]
Morita,S., Kojima,T. and Kitamura,T. (2000) Gene Ther., 7, 10631066.[CrossRef][ISI][Medline]
Ohashi,H., Maruyama,K., Liu,Y.C. and Yoshimura,A. (1994) Proc. Natl Acad. Sci. USA, 91, 158162.[Abstract]
Persons,D.A., Allay,J.A., Allay,E.R., Smeyne,R.J., Ashmun,R.A., Sorrentino,B.P. and Nienhuis,A.W. (1997) Blood, 90, 17771786.
Remy,I., Wilson,I.A. and Michnick,S.W. (1999) Science, 283, 990993.
Sakanaka,M., Wen,T.C., Matsuda,S., Masuda,S., Morishita,E., Nagao,M. and Sasaki,R. (1998) Proc. Natl Acad. Sci. USA, 95, 46354640.
Sugimoto,Y., Aksentijevich,I., Murray,G.J., Brady,R.O., Pastan,I. and Gottesman,M.M. (1995) Hum. Gene Ther., 6, 905915.[ISI][Medline]
Syed,R.S. et al. (1998) Nature, 395, 511516.[CrossRef][ISI][Medline]
Takahashi,T., Tanaka,M., Ogasawara,J., Suda,T., Murakami,H. and Nagata,S. (1996) J. Biol. Chem., 271, 1755517560.
Ueda,H. et al. (2000) J. Immunol. Methods, 241, 159170.[CrossRef][ISI][Medline]
Van Snick,J., Cayphas,S., Vink,A., Uyttenhove,C., Coulie,P.G., Rubira,M.R. and Simpson,R.J. (1986) Proc. Natl Acad. Sci. USA, 83, 96799683.[Abstract]
Wojchowski,D.M., Gregory,R.C., Miller,C.P., Pandit,A.K. and Pircher,T.J. (1999) Exp. Cell Res., 253, 143156.[CrossRef][ISI][Medline]
Received September 6, 2004; accepted October 20, 2004.
Edited by Laurent Jespers
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