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• W1986353200 abstract "Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B., and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254–32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VLκ-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB. Activating transcription factor 1 (ATF1) and cAMP-responsive element (CRE)-binding protein (CREB) activate transcription through CREs located in the promoters of cellular and viral genes. We previously described a monoclonal antibody (mAb41.4) that prevents ATF1 binding to DNA and reduces CRE-driven promoter activity in vitro (Orten, D. J., Strawhecker, J. M., Sanderson, S. D., Huang, D., Prytowsky, M. B., and Hinrichs, S. H. (1994) J. Biol. Chem. 269, 32254–32263). A single chain Fv (scFv) fragment from the mAb41.4-expressing hybridoma was generated to provide a means to investigate transcription factor function via intracellular expression of the scFv fragment. The affinity of scFv4 (subgroup: VLκ-III, VH miscellaneous) for ATF1 was similar to that of the parental mAb and the Fab fragment, but it demonstrated greater inhibitory activity and reacted with CREB. scFv4 disrupted the binding of both ATF1 and CREB in electrophoretic mobility shift assays and reduced expression of CRE-driven expression in vitro. Transient expression of scFv had no effect on the non-CRE-containing adenovirus major late promoter. The proliferating cell nuclear antigen promoter, containing two CREs, was significantly more sensitive to inhibition by scFv than the cytomegalovirus immediate-early promoter, containing five CREs. Cotransfection of either ATF1 or CREB in the presence of scFv restored basal levels of expression. The intracellular expression of scFv provides a unique means to investigate the roles of the transcription factors ATF1 and CREB. Activating transcription factor 1 (ATF1) 1The abbreviations used are: ATF1, activating transcription factor 1; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element; CREM, cAMP-responsive element modulator; mAb, monoclonal antibody; Ab, antibody; scFv, single chain Fv; VH, variable heavy; VL, variable light; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobilityshift assay; PCNA, proliferating cell nuclear antigen; AdML, adenovirus major late; CMV-IE, cytomegalovirus immediate-early; bZIP, basic leucine zipper. 1The abbreviations used are: ATF1, activating transcription factor 1; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element; CREM, cAMP-responsive element modulator; mAb, monoclonal antibody; Ab, antibody; scFv, single chain Fv; VH, variable heavy; VL, variable light; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobilityshift assay; PCNA, proliferating cell nuclear antigen; AdML, adenovirus major late; CMV-IE, cytomegalovirus immediate-early; bZIP, basic leucine zipper. and cAMP-responsive element-binding protein (CREB) are members of the large bZIP superfamily of transcription factors. Members of the CREB/ATF family bind to cAMP-responsive elements (CREs) within the promoter and enhancer sequences of many genes. ATF1, CREB, and the cAMP-responsive element modulator protein (CREM) constitute the CREB/ATF subfamily within the bZIP superfamily, which is defined by their ability to heterodimerize with each other but not with members of other subfamilies (1Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). ATF1, CREB, and CREM have similar structure and are highly homologous at the amino acid sequence level, especially within the bZIP region. Despite these similarities, members of the CREB multigene subfamily have distinct biological activity.ATF1, CREB, and CREM may act as either positive or negative regulators of transcription. Through alternative mRNA splicing, numerous isoforms of ATF1, CREB, and CREM can be expressed that possess differing transcriptional regulatory activities (2Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar, 3Lemaigre F.P. Ace C.I. Green M.R. Nucleic Acids Res. 1993; 21: 2907-2911Crossref PubMed Scopus (34) Google Scholar). Each protein or isoform also possesses differing patterns of phosphorylation, and the specific patterns contribute to their activity as regulators of transcription (1Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). CREB and CREM have been shown to play pivotal roles in basal and hormone-regulated transcription and differentiation; the role of ATF1 is less well defined. ATF1 homodimers appear to be weaker transcriptional activators than either CREB or certain forms of CREM since ATF1-mediated activation is enhanced by heterodimerization with either CREB or CREM (2Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar). The overexpression of ATF1 has recently been correlated with the neoplastic phenotype of lymphoma (4Hsueh Y.-P. Lai M.-Z. J. Immunol. 1995; 154: 5675-5683PubMed Google Scholar) and the continuous proliferation of lymphocytes. Cell cycle progression in T cells stimulated by interleukin-2 has been shown to be regulated by ATF1 and CREB, and transgenic mice expressing a dominant-negative form of CREB are defective in thymocyte proliferation and interleukin-2 production (5Feuerstein N. Huang D. Hinrichs S.H. Orten D.J. Aiyar N. Prystowsky M.B. J. Immunol. 1995; 154: 68-79PubMed Google Scholar, 6Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar).To investigate the functional role of ATF1, we previously generated a panel of anti-ATF1 monoclonal antibodies (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar). One antibody (mAb41.4; hereafter mAb4) was found to inhibit the binding of DNA by ATF1 and to inhibit ATF1-induced transcriptional activation (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar). Fab4, the Fab fragment of mAb4, retained the inhibitory properties of the parental antibody (Ab) (8Gilchrist C.A. Orten D.J. Bosilevac J.M. Sanderson D.S. Hinrichs S.H. Antibodies Immunoconj. Radiopharmacol. 1995; 8: 281-298Google Scholar). To investigate the cellular role of ATF1, we utilized current advances in the engineering of antibodies that have made possible the cloning of small single chain Fv (scFv) fragments. scFv fragments contain the antigen-binding variable domains of the light and heavy chains connected by a peptide spacer (9Raag R. Whitlow M. FASEB J. 1995; 9: 73-80Crossref PubMed Scopus (132) Google Scholar, 10Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (134) Google Scholar, 11Winter G. Milstein C. Nature. 1991; 349: 293-299Crossref PubMed Scopus (839) Google Scholar). When constructed in this manner, a single RNA transcript can be expressed and translated into an active protein that has the potential to interfere with the activity of targeted intracellular proteins. Intracellular scFv fragments have been successfully employed to decrease the expression of ErbB-2 (12Grim J. Deshane J. Feng M. Lieber A. Kay M. Curiel D.T. Am. J. Respir. Cell Mol. Biol. 1996; 15: 348-354Crossref PubMed Scopus (22) Google Scholar, 13Graus-Porta D. Beerli R.R. Hynes N.E. Mol. Cell. Biol. 1995; 15: 1182-1191Crossref PubMed Google Scholar); the α subunit of the high affinity human interleukin-2 receptor (14Richardson J.H. Sodroski J.G. Waldmann T.A. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3137-3141Crossref PubMed Scopus (95) Google Scholar); and proteins of human immunodeficiency virus type 1, including Rev, reverse transcriptase, and envelope protein gp120 (15Maciejewski J.P. Weichold F.F. Young N.S. Cara A. Zella D. Reitz M.S. Gallo R.C. Nat. Med. 1995; 1: 667-673Crossref PubMed Scopus (83) Google Scholar, 16Marasco W.A. Haseltine W.A. Chen S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7889-7893Crossref PubMed Scopus (234) Google Scholar, 17Levy-Mintz P. Duan L. Zhang H. Hu B. Dornadula G. Zhu M. Kulkosky J. Bizub-Bender D. Skalka A.M. Pomerantz R.J. J. Virol. 1996; 70: 8821-8832Crossref PubMed Google Scholar, 18Duan L. Zhu M. Bagasra O. Pomerantz R.J. Hum. Gene Ther. 1995; 6: 1561-1573Crossref PubMed Scopus (48) Google Scholar). We therefore cloned scFv4 of mAb4 and present here its characterization and its functional effect on intracellular gene expression.DISCUSSIONAlthough studied extensively, the cellular roles of ATF1 and CREB have not been fully determined. CREB is an important mediator of cAMP-activated transcription, whereas ATF1 activates transcription in some cell lines (41Rehfuss R.P. Walton K.M. Loriaux M.M. Goodman R.H. J. Biol. Chem. 1991; 266: 18431-18434Abstract Full Text PDF PubMed Google Scholar) and represses transcription in others (3Lemaigre F.P. Ace C.I. Green M.R. Nucleic Acids Res. 1993; 21: 2907-2911Crossref PubMed Scopus (34) Google Scholar). To elucidate the roles of transcription factors, investigators have attempted to knock out the activity of the transcription factors. In studies with ATF1 and CREB, activity has been reduced in vitro with an ATF1-sequestering CRE oligonucleotide (42Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar) andin vivo through the generation and study of transgenic and mutant mice (6Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar, 43Hummler E. Cole T.J. Blendy J. Ganss R. Aguzzi A. Schmid W. Beermann F. Shcutz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5647-5651Crossref PubMed Scopus (334) Google Scholar, 44Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1566) Google Scholar). Other investigators have used intracellularly expressed scFv fragments as modified gene knockout systems (12Grim J. Deshane J. Feng M. Lieber A. Kay M. Curiel D.T. Am. J. Respir. Cell Mol. Biol. 1996; 15: 348-354Crossref PubMed Scopus (22) Google Scholar, 13Graus-Porta D. Beerli R.R. Hynes N.E. Mol. Cell. Biol. 1995; 15: 1182-1191Crossref PubMed Google Scholar). The recent success in the application of these modified knockout systems led us to evaluate the inhibitory activity of the scFv fragment derived from mAb4.The ATF1 binding activity of mAb4 was maintained by scFv in ELISA (displaying a similar affinity for ATF1). In EMSA, scFv inhibited the formation of ATF1·CRE complexes, as had occurred with the mAb, and also inhibited the formation of CREB·CRE complexes, similar to the effect of the Fab fragment. Previous studies in our laboratory demonstrated that both mAb4 and Fab4 inhibited transcript production from the PCNA promoter (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar, 8Gilchrist C.A. Orten D.J. Bosilevac J.M. Sanderson D.S. Hinrichs S.H. Antibodies Immunoconj. Radiopharmacol. 1995; 8: 281-298Google Scholar). The inhibition of ATF1·CRE and CREB·CRE complexes in EMSA correlated with reduction of expression from the CRE-containing promoters in in vitro transcription assays. The cell transfection experiments confirmed the inhibitory effect of scFv on CRE-containing promoters. Therefore, the EMSA andin vitro transcription studies were predictive of the effect of scFv in cells, but the specific level of inhibition was different in each assay.The mAb interfered solely with ATF1, whereas the Fab fragment possessed additional reactivity for CREB as observed by EMSA and in vitro transcription from the CMV-IE promoter. The scFv fragment maintained reactivity for CREB, but the affinity was lower for CREB than for ATF1. Although the scFv fragment has an apparent lower affinity for CREB than for ATF1, it has greater inhibitory activity than either the Fab fragment or mAb in vitro and maintains this activity in cells. The apparent difference in the relative reactivities of the scFv construct and its parental mAb for the two transcription factors could derive, in principle, from several factors that are still to be determined. First, structural constraints in the antigen-binding site may be imposed by the covalent linkage of the VL and VH domains via the 15-residue linker (Gly4-Ser)3) or by the order of the two domains (the VL C terminus linked to the VH N terminusversus the VH C terminus linked to the VL N terminus as in this study). Although scFv constructs generally retain the activity of the parental antibodies, the length and composition of the linker (35Pantoliano M.W. Bird R.E. Johnson S. Asel E.D. Dodd S.W. Wood J.F. Hardman K. Biochemistry. 1991; 30: 10117-10125Crossref PubMed Scopus (155) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar), as well as the assembly order of the VL and VH domains, have been reported to influence the antigen binding affinity and avidity in certain cases (36Tsumoto K. Nakaoki Y. Ueda Y. Ogasahara K. Yutani K. Watanabe K. Kumagai I. Biochem. Biophys. Res. Commun. 1994; 201: 546-551Crossref PubMed Scopus (57) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar). Second, an increased ability of scFv to diffuse into small spaces, due to the smaller size of the former molecule (27 kDaversus 150 kDa for the mAb), may increase the accessibility of the antigenic epitope within the transcription factor-DNA complex. The tertiary structure of CREB may be such that certain domains are accessible by the small scFv fragment, but not by the large intact antibody. Third, aggregation reactions are known to occur in certain Fv preparations (46Whitow M. Fipula D. Rollence M.L. Feng S.-L. Wood J.F. Protein Eng. 1994; 7: 1017-1026Crossref PubMed Scopus (112) Google Scholar, 47Desplanque D. King D.J. Lawson A.D. Mountain A. Protein Eng. 1994; 7: 1027-1033Crossref PubMed Scopus (120) Google Scholar), which may alter the avidity of the binding for the two transcription factors. Significant intermolecular noncovalent interactions between the variable domains present in different scFv molecules could potentially occur at the scFv concentrations employed in our experiments (0.3–3 μm), depending on the affinity constant for noncovalent VL·VH complexations. Fourth, the presence of unusual residues in our scFv fragment may indicate that non-somatic mutations are responsible for the additional activity. The scFv fragment is notable for the lack of a CDR3 in its VH domain. The CDR3 deletion in our scFv fragment most likely arose from a somatic event since nucleotide sequencing of multiple isolated clones indicated the occurrence of this deletion, and examination of the sequence data revealed a frameshift occurring at this point in the VH domain. In consideration of the above issues, we propose that scFv binds to ATF1 or CREB and limits transcription factor interaction with the CRE. Interference with the binding of ATF1 or CREB to the CRE is believed to prevent the recruitment of transcriptional machinery and subsequent gene expression. Although the reactivity of scFv for both ATF1 and CREB is a useful property, further affinity maturation toward either transcription factor may allow specific inhibition of biological activity.Multiple approaches were taken to demonstrate that the activity of scFv was specific for the CRE-binding proteins, ATF1 and CREB. The adenovirus major late promoter was chosen because it contains only a TATA box and a pair of MAZ and Sp1 elements (38Song B. Young C.S. Virology. 1997; 235: 109-117Crossref PubMed Scopus (5) Google Scholar, 39Parks C.L. Shenk T. J. Virol. 1997; 71: 9600-9607Crossref PubMed Google Scholar, 40Horwitz M.S. Fields B.N. Knipe D.M. Fields Virology. 2nd Ed. 2. Raven Press, Philadelphia1996: 1679-1721Google Scholar). No CRE or CRE-like sequences are present in the AdML promoter. Additionally, adding back ATF1 or CREB to the intracellular scFv system reversed the effects of scFv upon the PCNA promoter.In our transfection studies, scFv showed a greater inhibitory effect on the PCNA promoter than the CMV-IE promoter, with 56 and 25% decreases in the promoter activities, respectively. The PCNA construct encompasses −182 to +143 of the promoter and contains single Ap2, Oct, and Sp1 elements as well as a tandem CRE. These two CREs have been shown to contribute the major activity of this promoter and are primarily activated by ATF1 (24Huang D. Shipman A.P. Orten D.J. Hinrichs S.H. Prytowski M.B. Mol. Cell. Biol. 1994; 14: 4233-4243Crossref PubMed Scopus (53) Google Scholar). The CMV promoter we used contained region −760 to +75 of the major IE promoter, and five 19-base pair repeat elements with a CRE at each of their cores are located in this region. In addition to CREs, the CMV-IE promoter contains other binding sites for transcription factors, including NF-κB, Ap1, Sp1, C/EBP, and NF1. The presence of these additional binding sites for other transcriptional activators most likely accounts for the decreased effect of scFv upon the CMV-IE promoter as compared with the PCNA promoter. The relative strengths of ATF1 and CREB as activators of the CRE (48Flint K.J. Jones N.C. Oncogene. 1991; 6: 2019-2026PubMed Google Scholar) and the intracellular ratios of ATF1 to CREB may also contribute to the observed effects. Whether the difference in activation will correlate with a physiologic change in cell viability or activity is yet to be determined. However, due to its ability to interfere with the activity of ATF1 and CREB, the scFv fragment is a useful tool for novel studies of CRE-mediated cellular differentiation, tumorigenesis, and viral pathogenesis. Activating transcription factor 1 (ATF1) 1The abbreviations used are: ATF1, activating transcription factor 1; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element; CREM, cAMP-responsive element modulator; mAb, monoclonal antibody; Ab, antibody; scFv, single chain Fv; VH, variable heavy; VL, variable light; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobilityshift assay; PCNA, proliferating cell nuclear antigen; AdML, adenovirus major late; CMV-IE, cytomegalovirus immediate-early; bZIP, basic leucine zipper. 1The abbreviations used are: ATF1, activating transcription factor 1; CREB, cAMP-responsive element-binding protein; CRE, cAMP-responsive element; CREM, cAMP-responsive element modulator; mAb, monoclonal antibody; Ab, antibody; scFv, single chain Fv; VH, variable heavy; VL, variable light; CDR, complementarity-determining region; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobilityshift assay; PCNA, proliferating cell nuclear antigen; AdML, adenovirus major late; CMV-IE, cytomegalovirus immediate-early; bZIP, basic leucine zipper. and cAMP-responsive element-binding protein (CREB) are members of the large bZIP superfamily of transcription factors. Members of the CREB/ATF family bind to cAMP-responsive elements (CREs) within the promoter and enhancer sequences of many genes. ATF1, CREB, and the cAMP-responsive element modulator protein (CREM) constitute the CREB/ATF subfamily within the bZIP superfamily, which is defined by their ability to heterodimerize with each other but not with members of other subfamilies (1Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). ATF1, CREB, and CREM have similar structure and are highly homologous at the amino acid sequence level, especially within the bZIP region. Despite these similarities, members of the CREB multigene subfamily have distinct biological activity. ATF1, CREB, and CREM may act as either positive or negative regulators of transcription. Through alternative mRNA splicing, numerous isoforms of ATF1, CREB, and CREM can be expressed that possess differing transcriptional regulatory activities (2Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar, 3Lemaigre F.P. Ace C.I. Green M.R. Nucleic Acids Res. 1993; 21: 2907-2911Crossref PubMed Scopus (34) Google Scholar). Each protein or isoform also possesses differing patterns of phosphorylation, and the specific patterns contribute to their activity as regulators of transcription (1Meyer T.E. Habener J.F. Endocr. Rev. 1993; 14: 269-290PubMed Google Scholar). CREB and CREM have been shown to play pivotal roles in basal and hormone-regulated transcription and differentiation; the role of ATF1 is less well defined. ATF1 homodimers appear to be weaker transcriptional activators than either CREB or certain forms of CREM since ATF1-mediated activation is enhanced by heterodimerization with either CREB or CREM (2Lee K.A. Masson N. Biochim. Biophys. Acta. 1993; 1174: 221-233Crossref PubMed Scopus (188) Google Scholar). The overexpression of ATF1 has recently been correlated with the neoplastic phenotype of lymphoma (4Hsueh Y.-P. Lai M.-Z. J. Immunol. 1995; 154: 5675-5683PubMed Google Scholar) and the continuous proliferation of lymphocytes. Cell cycle progression in T cells stimulated by interleukin-2 has been shown to be regulated by ATF1 and CREB, and transgenic mice expressing a dominant-negative form of CREB are defective in thymocyte proliferation and interleukin-2 production (5Feuerstein N. Huang D. Hinrichs S.H. Orten D.J. Aiyar N. Prystowsky M.B. J. Immunol. 1995; 154: 68-79PubMed Google Scholar, 6Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar). To investigate the functional role of ATF1, we previously generated a panel of anti-ATF1 monoclonal antibodies (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar). One antibody (mAb41.4; hereafter mAb4) was found to inhibit the binding of DNA by ATF1 and to inhibit ATF1-induced transcriptional activation (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar). Fab4, the Fab fragment of mAb4, retained the inhibitory properties of the parental antibody (Ab) (8Gilchrist C.A. Orten D.J. Bosilevac J.M. Sanderson D.S. Hinrichs S.H. Antibodies Immunoconj. Radiopharmacol. 1995; 8: 281-298Google Scholar). To investigate the cellular role of ATF1, we utilized current advances in the engineering of antibodies that have made possible the cloning of small single chain Fv (scFv) fragments. scFv fragments contain the antigen-binding variable domains of the light and heavy chains connected by a peptide spacer (9Raag R. Whitlow M. FASEB J. 1995; 9: 73-80Crossref PubMed Scopus (132) Google Scholar, 10Davies J. Riechmann L. Bio/Technology. 1995; 13: 475-479Crossref PubMed Scopus (134) Google Scholar, 11Winter G. Milstein C. Nature. 1991; 349: 293-299Crossref PubMed Scopus (839) Google Scholar). When constructed in this manner, a single RNA transcript can be expressed and translated into an active protein that has the potential to interfere with the activity of targeted intracellular proteins. Intracellular scFv fragments have been successfully employed to decrease the expression of ErbB-2 (12Grim J. Deshane J. Feng M. Lieber A. Kay M. Curiel D.T. Am. J. Respir. Cell Mol. Biol. 1996; 15: 348-354Crossref PubMed Scopus (22) Google Scholar, 13Graus-Porta D. Beerli R.R. Hynes N.E. Mol. Cell. Biol. 1995; 15: 1182-1191Crossref PubMed Google Scholar); the α subunit of the high affinity human interleukin-2 receptor (14Richardson J.H. Sodroski J.G. Waldmann T.A. Marasco W.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3137-3141Crossref PubMed Scopus (95) Google Scholar); and proteins of human immunodeficiency virus type 1, including Rev, reverse transcriptase, and envelope protein gp120 (15Maciejewski J.P. Weichold F.F. Young N.S. Cara A. Zella D. Reitz M.S. Gallo R.C. Nat. Med. 1995; 1: 667-673Crossref PubMed Scopus (83) Google Scholar, 16Marasco W.A. Haseltine W.A. Chen S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7889-7893Crossref PubMed Scopus (234) Google Scholar, 17Levy-Mintz P. Duan L. Zhang H. Hu B. Dornadula G. Zhu M. Kulkosky J. Bizub-Bender D. Skalka A.M. Pomerantz R.J. J. Virol. 1996; 70: 8821-8832Crossref PubMed Google Scholar, 18Duan L. Zhu M. Bagasra O. Pomerantz R.J. Hum. Gene Ther. 1995; 6: 1561-1573Crossref PubMed Scopus (48) Google Scholar). We therefore cloned scFv4 of mAb4 and present here its characterization and its functional effect on intracellular gene expression. DISCUSSIONAlthough studied extensively, the cellular roles of ATF1 and CREB have not been fully determined. CREB is an important mediator of cAMP-activated transcription, whereas ATF1 activates transcription in some cell lines (41Rehfuss R.P. Walton K.M. Loriaux M.M. Goodman R.H. J. Biol. Chem. 1991; 266: 18431-18434Abstract Full Text PDF PubMed Google Scholar) and represses transcription in others (3Lemaigre F.P. Ace C.I. Green M.R. Nucleic Acids Res. 1993; 21: 2907-2911Crossref PubMed Scopus (34) Google Scholar). To elucidate the roles of transcription factors, investigators have attempted to knock out the activity of the transcription factors. In studies with ATF1 and CREB, activity has been reduced in vitro with an ATF1-sequestering CRE oligonucleotide (42Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar) andin vivo through the generation and study of transgenic and mutant mice (6Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar, 43Hummler E. Cole T.J. Blendy J. Ganss R. Aguzzi A. Schmid W. Beermann F. Shcutz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5647-5651Crossref PubMed Scopus (334) Google Scholar, 44Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1566) Google Scholar). Other investigators have used intracellularly expressed scFv fragments as modified gene knockout systems (12Grim J. Deshane J. Feng M. Lieber A. Kay M. Curiel D.T. Am. J. Respir. Cell Mol. Biol. 1996; 15: 348-354Crossref PubMed Scopus (22) Google Scholar, 13Graus-Porta D. Beerli R.R. Hynes N.E. Mol. Cell. Biol. 1995; 15: 1182-1191Crossref PubMed Google Scholar). The recent success in the application of these modified knockout systems led us to evaluate the inhibitory activity of the scFv fragment derived from mAb4.The ATF1 binding activity of mAb4 was maintained by scFv in ELISA (displaying a similar affinity for ATF1). In EMSA, scFv inhibited the formation of ATF1·CRE complexes, as had occurred with the mAb, and also inhibited the formation of CREB·CRE complexes, similar to the effect of the Fab fragment. Previous studies in our laboratory demonstrated that both mAb4 and Fab4 inhibited transcript production from the PCNA promoter (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar, 8Gilchrist C.A. Orten D.J. Bosilevac J.M. Sanderson D.S. Hinrichs S.H. Antibodies Immunoconj. Radiopharmacol. 1995; 8: 281-298Google Scholar). The inhibition of ATF1·CRE and CREB·CRE complexes in EMSA correlated with reduction of expression from the CRE-containing promoters in in vitro transcription assays. The cell transfection experiments confirmed the inhibitory effect of scFv on CRE-containing promoters. Therefore, the EMSA andin vitro transcription studies were predictive of the effect of scFv in cells, but the specific level of inhibition was different in each assay.The mAb interfered solely with ATF1, whereas the Fab fragment possessed additional reactivity for CREB as observed by EMSA and in vitro transcription from the CMV-IE promoter. The scFv fragment maintained reactivity for CREB, but the affinity was lower for CREB than for ATF1. Although the scFv fragment has an apparent lower affinity for CREB than for ATF1, it has greater inhibitory activity than either the Fab fragment or mAb in vitro and maintains this activity in cells. The apparent difference in the relative reactivities of the scFv construct and its parental mAb for the two transcription factors could derive, in principle, from several factors that are still to be determined. First, structural constraints in the antigen-binding site may be imposed by the covalent linkage of the VL and VH domains via the 15-residue linker (Gly4-Ser)3) or by the order of the two domains (the VL C terminus linked to the VH N terminusversus the VH C terminus linked to the VL N terminus as in this study). Although scFv constructs generally retain the activity of the parental antibodies, the length and composition of the linker (35Pantoliano M.W. Bird R.E. Johnson S. Asel E.D. Dodd S.W. Wood J.F. Hardman K. Biochemistry. 1991; 30: 10117-10125Crossref PubMed Scopus (155) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar), as well as the assembly order of the VL and VH domains, have been reported to influence the antigen binding affinity and avidity in certain cases (36Tsumoto K. Nakaoki Y. Ueda Y. Ogasahara K. Yutani K. Watanabe K. Kumagai I. Biochem. Biophys. Res. Commun. 1994; 201: 546-551Crossref PubMed Scopus (57) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar). Second, an increased ability of scFv to diffuse into small spaces, due to the smaller size of the former molecule (27 kDaversus 150 kDa for the mAb), may increase the accessibility of the antigenic epitope within the transcription factor-DNA complex. The tertiary structure of CREB may be such that certain domains are accessible by the small scFv fragment, but not by the large intact antibody. Third, aggregation reactions are known to occur in certain Fv preparations (46Whitow M. Fipula D. Rollence M.L. Feng S.-L. Wood J.F. Protein Eng. 1994; 7: 1017-1026Crossref PubMed Scopus (112) Google Scholar, 47Desplanque D. King D.J. Lawson A.D. Mountain A. Protein Eng. 1994; 7: 1027-1033Crossref PubMed Scopus (120) Google Scholar), which may alter the avidity of the binding for the two transcription factors. Significant intermolecular noncovalent interactions between the variable domains present in different scFv molecules could potentially occur at the scFv concentrations employed in our experiments (0.3–3 μm), depending on the affinity constant for noncovalent VL·VH complexations. Fourth, the presence of unusual residues in our scFv fragment may indicate that non-somatic mutations are responsible for the additional activity. The scFv fragment is notable for the lack of a CDR3 in its VH domain. The CDR3 deletion in our scFv fragment most likely arose from a somatic event since nucleotide sequencing of multiple isolated clones indicated the occurrence of this deletion, and examination of the sequence data revealed a frameshift occurring at this point in the VH domain. In consideration of the above issues, we propose that scFv binds to ATF1 or CREB and limits transcription factor interaction with the CRE. Interference with the binding of ATF1 or CREB to the CRE is believed to prevent the recruitment of transcriptional machinery and subsequent gene expression. Although the reactivity of scFv for both ATF1 and CREB is a useful property, further affinity maturation toward either transcription factor may allow specific inhibition of biological activity.Multiple approaches were taken to demonstrate that the activity of scFv was specific for the CRE-binding proteins, ATF1 and CREB. The adenovirus major late promoter was chosen because it contains only a TATA box and a pair of MAZ and Sp1 elements (38Song B. Young C.S. Virology. 1997; 235: 109-117Crossref PubMed Scopus (5) Google Scholar, 39Parks C.L. Shenk T. J. Virol. 1997; 71: 9600-9607Crossref PubMed Google Scholar, 40Horwitz M.S. Fields B.N. Knipe D.M. Fields Virology. 2nd Ed. 2. Raven Press, Philadelphia1996: 1679-1721Google Scholar). No CRE or CRE-like sequences are present in the AdML promoter. Additionally, adding back ATF1 or CREB to the intracellular scFv system reversed the effects of scFv upon the PCNA promoter.In our transfection studies, scFv showed a greater inhibitory effect on the PCNA promoter than the CMV-IE promoter, with 56 and 25% decreases in the promoter activities, respectively. The PCNA construct encompasses −182 to +143 of the promoter and contains single Ap2, Oct, and Sp1 elements as well as a tandem CRE. These two CREs have been shown to contribute the major activity of this promoter and are primarily activated by ATF1 (24Huang D. Shipman A.P. Orten D.J. Hinrichs S.H. Prytowski M.B. Mol. Cell. Biol. 1994; 14: 4233-4243Crossref PubMed Scopus (53) Google Scholar). The CMV promoter we used contained region −760 to +75 of the major IE promoter, and five 19-base pair repeat elements with a CRE at each of their cores are located in this region. In addition to CREs, the CMV-IE promoter contains other binding sites for transcription factors, including NF-κB, Ap1, Sp1, C/EBP, and NF1. The presence of these additional binding sites for other transcriptional activators most likely accounts for the decreased effect of scFv upon the CMV-IE promoter as compared with the PCNA promoter. The relative strengths of ATF1 and CREB as activators of the CRE (48Flint K.J. Jones N.C. Oncogene. 1991; 6: 2019-2026PubMed Google Scholar) and the intracellular ratios of ATF1 to CREB may also contribute to the observed effects. Whether the difference in activation will correlate with a physiologic change in cell viability or activity is yet to be determined. However, due to its ability to interfere with the activity of ATF1 and CREB, the scFv fragment is a useful tool for novel studies of CRE-mediated cellular differentiation, tumorigenesis, and viral pathogenesis. Although studied extensively, the cellular roles of ATF1 and CREB have not been fully determined. CREB is an important mediator of cAMP-activated transcription, whereas ATF1 activates transcription in some cell lines (41Rehfuss R.P. Walton K.M. Loriaux M.M. Goodman R.H. J. Biol. Chem. 1991; 266: 18431-18434Abstract Full Text PDF PubMed Google Scholar) and represses transcription in others (3Lemaigre F.P. Ace C.I. Green M.R. Nucleic Acids Res. 1993; 21: 2907-2911Crossref PubMed Scopus (34) Google Scholar). To elucidate the roles of transcription factors, investigators have attempted to knock out the activity of the transcription factors. In studies with ATF1 and CREB, activity has been reduced in vitro with an ATF1-sequestering CRE oligonucleotide (42Hai T.W. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar) andin vivo through the generation and study of transgenic and mutant mice (6Barton K. Muthusamy N. Chanyangam M. Fischer C. Clendenin C. Leiden J.M. Nature. 1996; 379: 81-85Crossref PubMed Scopus (214) Google Scholar, 43Hummler E. Cole T.J. Blendy J. Ganss R. Aguzzi A. Schmid W. Beermann F. Shcutz G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5647-5651Crossref PubMed Scopus (334) Google Scholar, 44Bourtchuladze R. Frenguelli B. Blendy J. Cioffi D. Schutz G. Silva A.J. Cell. 1994; 79: 59-68Abstract Full Text PDF PubMed Scopus (1566) Google Scholar). Other investigators have used intracellularly expressed scFv fragments as modified gene knockout systems (12Grim J. Deshane J. Feng M. Lieber A. Kay M. Curiel D.T. Am. J. Respir. Cell Mol. Biol. 1996; 15: 348-354Crossref PubMed Scopus (22) Google Scholar, 13Graus-Porta D. Beerli R.R. Hynes N.E. Mol. Cell. Biol. 1995; 15: 1182-1191Crossref PubMed Google Scholar). The recent success in the application of these modified knockout systems led us to evaluate the inhibitory activity of the scFv fragment derived from mAb4. The ATF1 binding activity of mAb4 was maintained by scFv in ELISA (displaying a similar affinity for ATF1). In EMSA, scFv inhibited the formation of ATF1·CRE complexes, as had occurred with the mAb, and also inhibited the formation of CREB·CRE complexes, similar to the effect of the Fab fragment. Previous studies in our laboratory demonstrated that both mAb4 and Fab4 inhibited transcript production from the PCNA promoter (7Orten D.J. Strawhecker J.M. Sanderson S.D. Huang D. Prystowsky M.B. Hinrichs S.H. J. Biol. Chem. 1994; 269: 32254-32263Abstract Full Text PDF PubMed Google Scholar, 8Gilchrist C.A. Orten D.J. Bosilevac J.M. Sanderson D.S. Hinrichs S.H. Antibodies Immunoconj. Radiopharmacol. 1995; 8: 281-298Google Scholar). The inhibition of ATF1·CRE and CREB·CRE complexes in EMSA correlated with reduction of expression from the CRE-containing promoters in in vitro transcription assays. The cell transfection experiments confirmed the inhibitory effect of scFv on CRE-containing promoters. Therefore, the EMSA andin vitro transcription studies were predictive of the effect of scFv in cells, but the specific level of inhibition was different in each assay. The mAb interfered solely with ATF1, whereas the Fab fragment possessed additional reactivity for CREB as observed by EMSA and in vitro transcription from the CMV-IE promoter. The scFv fragment maintained reactivity for CREB, but the affinity was lower for CREB than for ATF1. Although the scFv fragment has an apparent lower affinity for CREB than for ATF1, it has greater inhibitory activity than either the Fab fragment or mAb in vitro and maintains this activity in cells. The apparent difference in the relative reactivities of the scFv construct and its parental mAb for the two transcription factors could derive, in principle, from several factors that are still to be determined. First, structural constraints in the antigen-binding site may be imposed by the covalent linkage of the VL and VH domains via the 15-residue linker (Gly4-Ser)3) or by the order of the two domains (the VL C terminus linked to the VH N terminusversus the VH C terminus linked to the VL N terminus as in this study). Although scFv constructs generally retain the activity of the parental antibodies, the length and composition of the linker (35Pantoliano M.W. Bird R.E. Johnson S. Asel E.D. Dodd S.W. Wood J.F. Hardman K. Biochemistry. 1991; 30: 10117-10125Crossref PubMed Scopus (155) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar), as well as the assembly order of the VL and VH domains, have been reported to influence the antigen binding affinity and avidity in certain cases (36Tsumoto K. Nakaoki Y. Ueda Y. Ogasahara K. Yutani K. Watanabe K. Kumagai I. Biochem. Biophys. Res. Commun. 1994; 201: 546-551Crossref PubMed Scopus (57) Google Scholar, 45Owens R.J. Young R.J. J. Immunol. Methods. 1993; 168: 149-165Crossref Scopus (61) Google Scholar). Second, an increased ability of scFv to diffuse into small spaces, due to the smaller size of the former molecule (27 kDaversus 150 kDa for the mAb), may increase the accessibility of the antigenic epitope within the transcription factor-DNA complex. The tertiary structure of CREB may be such that certain domains are accessible by the small scFv fragment, but not by the large intact antibody. Third, aggregation reactions are known to occur in certain Fv preparations (46Whitow M. Fipula D. Rollence M.L. Feng S.-L. Wood J.F. Protein Eng. 1994; 7: 1017-1026Crossref PubMed Scopus (112) Google Scholar, 47Desplanque D. King D.J. Lawson A.D. Mountain A. Protein Eng. 1994; 7: 1027-1033Crossref PubMed Scopus (120) Google Scholar), which may alter the avidity of the binding for the two transcription factors. Significant intermolecular noncovalent interactions between the variable domains present in different scFv molecules could potentially occur at the scFv concentrations employed in our experiments (0.3–3 μm), depending on the affinity constant for noncovalent VL·VH complexations. Fourth, the presence of unusual residues in our scFv fragment may indicate that non-somatic mutations are responsible for the additional activity. The scFv fragment is notable for the lack of a CDR3 in its VH domain. The CDR3 deletion in our scFv fragment most likely arose from a somatic event since nucleotide sequencing of multiple isolated clones indicated the occurrence of this deletion, and examination of the sequence data revealed a frameshift occurring at this point in the VH domain. In consideration of the above issues, we propose that scFv binds to ATF1 or CREB and limits transcription factor interaction with the CRE. Interference with the binding of ATF1 or CREB to the CRE is believed to prevent the recruitment of transcriptional machinery and subsequent gene expression. Although the reactivity of scFv for both ATF1 and CREB is a useful property, further affinity maturation toward either transcription factor may allow specific inhibition of biological activity. Multiple approaches were taken to demonstrate that the activity of scFv was specific for the CRE-binding proteins, ATF1 and CREB. The adenovirus major late promoter was chosen because it contains only a TATA box and a pair of MAZ and Sp1 elements (38Song B. Young C.S. Virology. 1997; 235: 109-117Crossref PubMed Scopus (5) Google Scholar, 39Parks C.L. Shenk T. J. Virol. 1997; 71: 9600-9607Crossref PubMed Google Scholar, 40Horwitz M.S. Fields B.N. Knipe D.M. Fields Virology. 2nd Ed. 2. Raven Press, Philadelphia1996: 1679-1721Google Scholar). No CRE or CRE-like sequences are present in the AdML promoter. Additionally, adding back ATF1 or CREB to the intracellular scFv system reversed the effects of scFv upon the PCNA promoter. In our transfection studies, scFv showed a greater inhibitory effect on the PCNA promoter than the CMV-IE promoter, with 56 and 25% decreases in the promoter activities, respectively. The PCNA construct encompasses −182 to +143 of the promoter and contains single Ap2, Oct, and Sp1 elements as well as a tandem CRE. These two CREs have been shown to contribute the major activity of this promoter and are primarily activated by ATF1 (24Huang D. Shipman A.P. Orten D.J. Hinrichs S.H. Prytowski M.B. Mol. Cell. Biol. 1994; 14: 4233-4243Crossref PubMed Scopus (53) Google Scholar). The CMV promoter we used contained region −760 to +75 of the major IE promoter, and five 19-base pair repeat elements with a CRE at each of their cores are located in this region. In addition to CREs, the CMV-IE promoter contains other binding sites for transcription factors, including NF-κB, Ap1, Sp1, C/EBP, and NF1. The presence of these additional binding sites for other transcriptional activators most likely accounts for the decreased effect of scFv upon the CMV-IE promoter as compared with the PCNA promoter. The relative strengths of ATF1 and CREB as activators of the CRE (48Flint K.J. Jones N.C. Oncogene. 1991; 6: 2019-2026PubMed Google Scholar) and the intracellular ratios of ATF1 to CREB may also contribute to the observed effects. Whether the difference in activation will correlate with a physiologic change in cell viability or activity is yet to be determined. However, due to its ability to interfere with the activity of ATF1 and CREB, the scFv fragment is a useful tool for novel studies of CRE-mediated cellular differentiation, tumorigenesis, and viral pathogenesis. We are grateful to Andrew H. Henry for sequence analysis of ScFv." @default.
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• W1986353200 title "Inhibition of Activating Transcription Factor 1- and cAMP-responsive Element-binding Protein-activated Transcription by an Intracellular Single Chain Fv Fragment" @default.
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