A Single Chain Fv Fragment of P-glycoprotein-specific Monoclonal Antibody C219

Abstract

A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 × 0.2 × 0.2 mm were grown in 100 mmcitrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 Å with an R factor of 20.6, anR free of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule. A construct encoding a single chain variable fragment of the anti-P-glycoprotein monoclonal antibody C219 was made by combining the coding sequences for the heavy and light chain variable domains with a sequence encoding the flexible linker (GGGGS)3, an OmpA signal sequence, a c-myc identification tag, and a five-histidine purification tag. The construct was expressed in Escherichia coli and purified from the periplasmic fraction using a nickel chelate column and ion exchange chromatography. Three-step Western blot analysis showed that the construct retains binding affinity for P-glycoprotein. Crystals of 1.0 × 0.2 × 0.2 mm were grown in 100 mmcitrate, pH 4.5, 21% polyethylene glycol 6000 in the presence of low concentrations of subtilisin, resulting in proteolytic removal of the linker and purification tags. The structure was solved to a resolution of 2.4 Å with an R factor of 20.6, anR free of 28.5, and good stereochemistry. This result could lead to a clinically useful product based on antibody C219 for the diagnosis of P-glycoprotein-mediated multidrug resistance. The molecule will also be useful in biophysical studies of functional domains of P-glycoprotein, as well as studies of the intact molecule. The Fv portion of an antibody comprises the variable domains of a heavy and a light chain, and it is the smallest fragment that maintains the binding specificity and affinity of the entire antibody (1Bedzyk W.D. Weidner K.M. Denzin L.K. Johnson L.S. Hardman K.D. Pantoliano M.W. Asel E.D. Voss Jr., E.W. J. Biol. Chem. 1990; 265: 18615-18620Abstract Full Text PDF PubMed Google Scholar). This antibody fragment is produced most easily using recombinant DNA technology because, unlike Fab fragments (antigen-binding fragments), it cannot be readily produced proteolytically (2Huston J.S. Levinson D. Mudgett-Hunter M. Tai M.S. Novotna J. Margolies M.N. Ridge R.J. Bruecolen R.E. Haber E. Crea R. Oppermann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5879-5883Crossref PubMed Scopus (1399) Google Scholar). Usually a single chain construct (scFv) is made by linking the heavy and light chains with a flexible linker (3Glockshuber R. Malia M. Pfitzinger I. Plückthun A. Biochemistry. 1990; 29: 1362-1367Crossref PubMed Scopus (450) Google Scholar, 4Johnson S. Bird R.E Methods Enzymol. 1991; 203: 88-98Crossref PubMed Scopus (47) Google Scholar). Several linkers have been reported in the past, but the linker (GGGGS)3 has become most popular (2Huston J.S. Levinson D. Mudgett-Hunter M. Tai M.S. Novotna J. Margolies M.N. Ridge R.J. Bruecolen R.E. Haber E. Crea R. Oppermann H. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5879-5883Crossref PubMed Scopus (1399) Google Scholar, 3Glockshuber R. Malia M. Pfitzinger I. Plückthun A. Biochemistry. 1990; 29: 1362-1367Crossref PubMed Scopus (450) Google Scholar, 5Tai M.S. Mudgett-Hunter M. Levinson D. Wu G.M. Haber E. Oppermann H. Huston J.S. Biochemistry. 1990; 29: 8024-8030Crossref PubMed Scopus (89) Google Scholar, 6Anand N.N. Mandal S. MacKenzie C.R. Sadowska J. Sigurskjold B. Young N.M. Bundle D.R. Narang S.A. J. Biol. Chem. 1991; 266: 21874-21879Abstract Full Text PDF PubMed Google Scholar). This linker spans the 35-Å distance between the carboxyl terminus of one variable domain and the amino terminus of the other without distorting the conformation of the antigen binding site (3Glockshuber R. Malia M. Pfitzinger I. Plückthun A. Biochemistry. 1990; 29: 1362-1367Crossref PubMed Scopus (450) Google Scholar). Because of their small size, scFv fragments could be useful in tumor imaging and therapeutic strategies (7Chaudhury V.K. Queen C. Junghans R.P. Waldmann T.A. FitzGerald D.J. Pastan I. Nature. 1989; 339: 394-397Crossref PubMed Scopus (393) Google Scholar, 8Yokota T. Milenic D.E. Whitlow M. Schlom J. Cancer Res. 1992; 52: 3402-3408PubMed Google Scholar, 9Reiter Y. Brinkmann U. Lee B. Pastan I. Nat. Biotechnol. 1996; 14: 1239-1245Crossref PubMed Scopus (143) Google Scholar). The murine monoclonal antibody C219 was raised against plasma membranes of multidrug-resistant Chinese hamster ovary cells (10Kartner N. Evernden-Porelle D. Bradley G. Ling V. Nature. 1985; 316: 820-823Crossref PubMed Scopus (832) Google Scholar), and is directed against Chinese hamster P-glycoprotein (Pgp), 1The abbreviations used are: Pgp, P-glycoprotein; scFv, single chain variable fragment; ABC, ATP binding cassette; NBD, nucleotide binding domain; V L, light chain variable domain; V H, heavy chain variable domain; RMS, root mean square; CDR, complementarity-determining region;R, residual; Fo, observed structure factor amplitudes; Fc, calculated structure factor amplitudes. 1The abbreviations used are: Pgp, P-glycoprotein; scFv, single chain variable fragment; ABC, ATP binding cassette; NBD, nucleotide binding domain; V L, light chain variable domain; V H, heavy chain variable domain; RMS, root mean square; CDR, complementarity-determining region;R, residual; Fo, observed structure factor amplitudes; Fc, calculated structure factor amplitudes. an ATP-dependent transporter. Pgp is a member of a large family of ATP-driven transmembrane transporters called ABC transporters, present in both eukaryotes and prokaryotes (11Higgins C.F. Annu. Rev. Cell Biol. 1988; 8: 67-113Crossref Scopus (3350) Google Scholar). Overexpression of Pgp has been associated with multidrug resistance in some cancer cells undergoing chemotherapy (12Kartner N. Riordan J.R. Ling V. Science. 1983; 221: 1285-1288Crossref PubMed Scopus (879) Google Scholar). Based on amino acid sequence analysis, Pgp is thought to consist of two halves, each with six transmembrane helices and a cytoplasmic nucleotide binding domain (NBD). C219 recognizes a continuous peptide epitope present in both NBDs of Pgp (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar). The sequence of the epitope has been determined by Georges et al. (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar) to be VQ(E/A)ALD in Chinese hamster Pgp. C219 has been shown to react with all P-glycoproteins studied so far but not with other ABC transporters (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar, 14Barrand M.A. Twentyman P.R. Br. J. Cancer. 1992; 65: 239-245Crossref PubMed Scopus (33) Google Scholar, 15Lee J.J. Hughes C.S. Fine R.L. Page R.L. Cancer. 1996; 77: 1892-1898Crossref PubMed Scopus (102) Google Scholar). The antibody therefore has been used widely for Pgp immunohistochemistry (14Barrand M.A. Twentyman P.R. Br. J. Cancer. 1992; 65: 239-245Crossref PubMed Scopus (33) Google Scholar, 15Lee J.J. Hughes C.S. Fine R.L. Page R.L. Cancer. 1996; 77: 1892-1898Crossref PubMed Scopus (102) Google Scholar, 16Krishan A. Sauerteig A. Stein J.H. Cytometry. 1991; 12: 731-742Crossref PubMed Scopus (44) Google Scholar), and has also been used to characterize novel Pgp cDNAs (17Norris M.D. Gilbert J. Madafiglio J. Haber M. Gene. 1995; 1565: 313-314Crossref Scopus (5) Google Scholar). A potential disadvantage of using the whole C219 antibody in immunohistochemical studies or in immunotherapy might be that its epitope lies in the cytoplasmic portion of Pgp and is therefore relatively inaccessible. Furthermore, it has been shown that some commercial lots of the C219 monoclonal antibody contain antibodies to blood group A carbohydrate determinants (18Finstad C.L. Yin B.W.T. Gordon C.M. Federici M.G. Welt S. Lloyd K.L. J. Histochem. Cytochem. 1991; 39: 1603-1610Crossref PubMed Scopus (40) Google Scholar) and that commercially available C219 may cross-react with unrelated proteins (19Beaulieu E. Demeule M. Pouliot J.F. Averill-Bates D.A. Murphy G.F. Beliveau R. Biochim. Biophys. Acta. 1995; 1233: 27-32Crossref PubMed Scopus (27) Google Scholar). The use of a recombinant version of C219 may improve the uptake into cells and at the same time rule out contamination with other antibody specificities. Furthermore, it would facilitate mutagenic studies aimed at improving the binding and selectivity of the molecule and could form the basis for developing immunotoxins against cells overexpressing Pgp. Additionally, the scFv can be a useful tool in the process of crystallization of Pgp or of its separate NBDs. An atomic structure of P-glycoprotein would be of great interest in understanding the process of multidrug resistance, in particular, the atomic basis of substrate recognition and the mechanism by which ATP drives the transport process. Such a structure would also contribute valuable information on other members of the ABC superfamily. At present, structural information on ABC transporters is limited to a low resolution structure of Pgp determined by electron microscopy (20Rosenberg M.F. Callahan R. Ford R.C. Higgins C.F. J. Biol. Chem. 1997; 272: 10685-10694Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar) and two recent theoretical models for NBDs (21Annerau J.-P. Wulbrand U. Vankeerberghen A. Cuppens H. Bontems F. Tümmler B. Cassiman J.-J. Stoven V. FEBS Lett. 1997; 407: 303-308Crossref PubMed Scopus (33) Google Scholar, 22Hoedemaeker P.J. Davidson A.R. Rose D.R. Proteins Struct. Funct. Genet. 1997; (in press)Google Scholar). We have designed, cloned, and expressed a gene encoding a scFv variant of C219. The protein product was purified and crystallized, and the three-dimensional structure was determined to a resolution of 2.4 Å. A cDNA clone encoding the antibody was obtained and sequenced previously. 2S. Sharma, unpublished results. The determination of its peptide epitope sequence has been described (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar). The expression vector pSJF2 was designed and donated by Dr. S. Foote (National Research Council of Canada, Ottawa, Ontario, Canada). The vector is based on pUC8 (23Vieira J. Messing J. Gene. 1982; 19: 259-268Crossref PubMed Scopus (3779) Google Scholar) and contains a multiple cloning site flanked by an amino-terminal OmpA leader sequence and a carboxyl-terminal c-myc epitope tag followed by a five-histidine tag for nickel chelate purification. A single chain Fv version of the monoclonal antibody C219 was obtained by polymerase chain reaction. The light chain sequence was amplified with the sense primer 5′-GGG-GCG-TACGAG-ACA-TTG-TGA-TGA-CAC-AG-3′ and the reverse primer 5′-AAA-ATC-CGG-AGG-CGG-CGG-CGA-GGT-TCA-GCT-GCA-G-3′. The heavy chain sequence was amplified with the sense primer 5′-TTT-TGGATC-CTG-AGG-AGA-CGG-TGA-CCG-TGG-TCC-C-3′ and the reverse primer 5′-TTT-TTC-CGG-AGC-CGC-CGC-CGC-CCG-AGC-CGC-CGC-CGC-CCG-AGC-TCC-AGC-TTG-GTC-CCA-GCA-3′. The light chain polymerase chain reaction fragment was digested with BsiWI and BspEI, and the heavy chain fragment was digested with BspEI and BamHI. Both fragments were simultaneously cloned into pSJF2 as aBsiWI/BamHI insert, thereby combining the light chain sequence, the linker, and the heavy chain sequence. The construct was expressed in Escherichia coli TG1 cells (F′ traD36 lacI q D(lacZ)M15 pro A + B + /supE D(hsdM-mcrB)5(r k −mk − McrB − ) thyD(lac-proAB)). TG1 cells containing the expression vector were grown in M9 medium supplemented with 2% casamino acids, vitamin B1, and 100 μg·ml−1 ampicillin for 31 h at 24 °C. The culture was then induced with 10% (v/v) 10 × Terrific Broth (12% tryptone, 24% yeast extract, and 5% glycerol) and isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm, and the culture was grown for an additional 64 h at 24 °C. Cells were harvested by centrifugation at 5000 ×g and washed once in 10 mm Tris-HCl, pH 8.0, 0.9% NaCl and once in 10 mm Tris-HCl, pH 8.0, 1 mm EDTA, 25% sucrose. Cells were osmotically shocked by rapid dilution in 10 mm Tris-HCl, pH 8.0, 5 mmMgCl2, and the periplasmic fraction was collected by centrifugation at 5000 × g. Osmoshock supernatant was concentrated using ultrafiltration in a stir cell with a 10-kDa cutoff (Amicon Inc., Beverly, MA) and dialyzed against 50 mmsodium phosphate buffer, pH 8.0. The protein was loaded onto a Ni2+-nitrilotriacetic acid agarose column (Qiagen, Chatsworth, CA); washed with 50 mm sodium phosphate buffer, pH 6.0, 300 mm NaCl, and 10% glycerol; and eluted with 50 mm sodium acetate buffer, pH 4.0, 300 mm NaCl, and 10% glycerol. The protein was further purified to homogeneity using anion exchange chromatography on a 1-ml Hi-TRAP Q-column (Pharmacia, Uppsala, Sweden). SDS-polyacrylamide gel electrophoresis gels loaded with whole cell extracts of E. coli cells overexpressing a fusion of the carboxyl-terminal NBD of Pgp to maltose-binding protein (24Sharma S. Rose D.R. J. Biol. Chem. 1995; 270: 14085-14093Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) was electroblotted onto nitrocellulose. Blots were treated with scFv C219 as a primary antibody, mouse anti-c-myc as a secondary antibody, and peroxidase-conjugated goat anti-mouse as a tertiary antibody. Blots were stained with diaminobenzidine and compared with conventional Western blots using intact C219 antibody. The protein was concentrated to 7 mg·ml−1 in 10 mm Tris-HCl, pH 9.0, and crystallized using standard vapor diffusion in hanging drops over reservoirs containing 100 mm sodium citrate, pH 4.5, and 21% (w/v) polyethylene glycol 6000 (Fluka), in the presence of subtilisin Carlsberg (Sigma) in a molar ratios of 1:100. Polyethylene glycol was deionized before use with a mixed were collected from a single with 0.2 × 0.2 × 1.0 mm at with a on a at and using the by al. C. Methods Enzymol. 1985; PubMed Scopus Google Scholar). The were for using the S. An was obtained by with J. Scopus Google Scholar), using the of in the R.L. J. Biol. 1992; PubMed Scopus Google Scholar, M.D. J.R. T. M. J. Biol. PubMed Scopus Google Scholar). The structure was with Scholar), with of the to anR free by and were using a resolution of with and an of the final a low resolution cutoff of Å was and were out with T.A. M. 1991; Scopus Google Scholar). as well as for which all were in of were The of the was the with M.W. J. Google Scholar). The final has been in the and has been M.D. J.R. T. M. J. Biol. PubMed Scopus Google Scholar). A single chain variable domain construct of monoclonal antibody C219 was expressed in E. and purified from the periplasmic the was of protein of cell The protein was and as from Western blot analysis crystallization were by a standard of and to a of × 0.1 × 0.1 SDS-polyacrylamide gel electrophoresis analysis of that the the linker c-myc and tag not of scFv to by in crystallization subtilisin was to the hanging Crystals then and to a of 1.0 × 0.2 × 0.2 A of showed to a resolution of Å and the of the cell to the group with Å. Based on a calculated of two scFv in the resulting in an cell to of J. Biol. PubMed Scopus Google Scholar) and a of in the The in were collected to with an R of on two to the two − over all is the of a observed R − over all is the of a observed in a in The final protein and with a factor of and an R free of mean from were Å for for for and for The showed of the in the most were in V. Chem. PubMed Scopus Google Scholar). other by for a structure M.W. J. Google − B was The final and R − in a B was The final and scFv of two as is in the variable of one each from the light and heavy The two to form an with the antigen binding site at one two scFv in the The two by a the between the a were used The variable domains of the light and heavy chains and V were for the light and heavy chains were and but in one of the the domain was by an to the to V H. This to a of Å the Fv fragments were a between the heavy and light chains by two and the molecule in the the distance between two was by 1 Å of the but the was for the of two between the by with the of a between the chain of in molecule two and the chain of in molecule of the and form some between the two both binding to the DNA a of with the C219 sequences chain, light chain, and could be to or one of the binding Because sequence is in in light chain sequences as by a not to an in the sequence. The final electron is with DNA and the sequence from the of the of the six antigen binding C. J. Biol. PubMed Scopus Google Scholar) showed the S. and D. R. in and from for which have been to not have novel chain 5 A the antigen binding site in to in the some of the and in to be for the epitope peptide to without the which would to the conformation of the peptide in the antigen binding B the of the antigen binding for potential on the and on the The antigen binding of the two in the not the small not The antigen binding a well by and as well as an by and single chain antibody fragments have been made in the past, have been crystallized to M.D. J.R. T. M. J. Biol. PubMed Scopus Google Scholar). the crystallization of scFv once the were to contain with a linker, a small of subtilisin was used as an to the crystallization in a reported scFv structure electron was for the linker the resolution of the A. Y. Bundle D.R. MacKenzie C.R. Narang S.A. Young N.M. M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of subtilisin to the drops may be to other single chain or to proteins in The electron for the light chain the in the primary sequence results. continuous electron with the for two chains and as to a single was at This is well with B well with on the antigen binding and may well contribute or to antigen The of the two scFv in the final the in This of has been observed in antibodies and antibody fragments and can be that observed U. Science. 1992; PubMed Scopus Google Scholar, S. Rose D.R. S. and Inc., Scholar). most of have been reported binding of the small is an to the it is an of the and of antibody and may be an factor in antigen The determination of the three-dimensional structure of the single chain variable fragment of C219 is a information on the atomic structure of Pgp and its functional The epitope peptide of Pgp has been (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar) and a in as determined by A of the sequence an in which the in C219 binding (13Georges E. Bradley G. Gariepy J. Ling V. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 152-156Crossref PubMed Scopus (377) Google Scholar) on one of the This is in with of ABC NBDs P.J. Davidson A.R. Rose D.R. Proteins Struct. Funct. Genet. 1997; (in press)Google Scholar). A of the of epitope binding by based on the structure of an antibody is not based on the in B and the in a can be The in the as the most of would be on the of Pgp and with in a in the antigen binding site of the antibody, between and The and in the epitope could then to a on the antibody, by in and in Crystals of scFv in the presence of its peptide epitope have been obtained but not of and to structure of the scFv with peptide epitope would a of the of the epitope as observed in and a of the binding to Pgp or its separate domains have not been so binding of the scFv to expressed and purified nucleotide binding domains of P-glycoprotein or to the intact molecule will be a valuable in to the of their has been shown that the crystallization of proteins can be by the use of Fv domains to form that the available for C. S. B. H. Nat. Struct. Biol. 1995; PubMed Scopus Google C. M.G. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). in the the antibody C219 has been used in the of the and of P-glycoprotein, as well as in the of P-glycoprotein-mediated This may be clinically in the of the and of of cancer G. G. Ling V. 1995; Full Text PDF PubMed Google Scholar, G. G. Ling V. 1995; Full Text PDF PubMed Google Scholar, C.L. C. J.F. M. M. D. P.J. M. E. R. Cancer Res. 1996; Google Scholar). The scFv described may also be a useful in and is a for produced by that have for particular, the and of immunotoxins based on construct could have a M. R.E. R.J. J. Biol. Chem. 1997; 270: Full Text Full Text PDF Scopus Google Scholar, Jr., F. J. A.R. G. S. D. A. A. J. J. 1997; PubMed Scopus Google Scholar). C219 is directed a cytoplasmic a of to to the by using peptide sequences D. J. Gariepy J. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google or by cell the D.J. Chem. 1996; PubMed Scopus Google An to gene is to M. J. J. M. 1997; PubMed Scopus Google Scholar, 1997; 8: PubMed Scopus Google Scholar). it would be of interest to with affinity and The of will be by the atomic structural analysis of the scFv as and Georges cDNA to We Dr. Ling for and and for and Dr. for with the of