ImprovedPharmacokineticsofRecombinantBispecific ...costimulatory signal provided by a B7-DbCEA...

Improved Pharmacokinetics of Recombinant BispecificAntibody Molecules by Fusion to Human Serum Albumin*

Received for publication, January 29, 2007, and in revised form, March 2, 2007 Published, JBC Papers in Press, March 8, 2007, DOI 10.1074/jbc.M700820200

Dafne Muller, Anette Karle1, Bettina Meißburger, Ines Hofig2, Roland Stork, and Roland E. Kontermann3

From the Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany

Recombinant bispecific antibodies such as tandem scFv mol-ecules (taFv), diabodies (Db), or single chain diabodies (scDb)have shown to be able to retarget T lymphocytes to tumor cells,leading to their destruction. However, therapeutic efficacy ishampered by a short serum half-life of these small moleculeshaving molecule masses of 50–60 kDa. Thus, improvement ofthe pharmacokinetic properties of small bispecific antibody for-mats is required to enhance efficacy in vivo. In this study, wegenerated several recombinant bispecific antibody-albuminfusion proteins and analyzed these molecules for biologicalactivity and pharmacokinetic properties. Three recombinantantibody formats were produced by fusing two different scFvmolecules, bispecific scDb or taFv molecules, respectively, tohuman serum albumin (HSA). These constructs (scFv2-HSA,scDb-HSA, taFv-HSA), directed against the tumor antigen car-cinoembryonic antigen (CEA) and the T cell receptor complexmolecule CD3, retained full binding capacity to both antigenscompared with unfused scFv, scDb, and taFv molecules. Tumorantigen-specific retargeting and activation of T cells as moni-tored by interleukin-2 release was observed for scDb, scDb-HSA, taFv-HSA, and to a lesser extent for scFv2-HSA. T cellactivation could be further enhanced by a target cell-specificcostimulatory signal provided by a B7-DbCEA fusion protein.Furthermore, we could demonstrate that fusion to serum albu-min strongly increases circulation time of recombinant bispe-cific antibodies. In addition, our comparative study indicatesthat single chain diabody-albumin fusion proteins seem to bethe most promising format for further studying cytotoxic activ-ities in vitro and in vivo.

Bispecific antibodies are designed to target two differentantigens simultaneously (1). In the context of a tumor therapythey can be applied to selectively recruit potent effector cells ofthe immune system such as cytotoxic T lymphocytes to tumorcells (2). This is achieved by binding on the one side to a tumor-associated antigen and on the other side to a trigger moleculeon the effector cell, leading to the activation of the effector cell

and tumor cell destruction. To reduce potential side effectselicited by the Fc part of antibodies (3) small recombinantbispecific antibody formats composed only of the variableregions, which define the binding unit of an antibody, have beendeveloped (1, 4). These formats include bispecific diabodies(Db),4 single chain diabodies (scDb), and tandem scFv (taFv)molecules, which have been applied successfully in vitro andalso in vivo for the retargeting of cytotoxic T lymphocytes (viaT-cell receptor molecule CD3) to tumor cells (e.g. recognizingCEA, EpCAM, or CD19) (5–8).However, these small bispecific antibody molecules with

molecular masses between 50 and 60 kDa are rapidly clearedfrom circulation with an initial half-life of less than 30 min(9, 10). This puts some obstacles on therapeutic applications,e.g. requirement of high doses and repeated injections orinfusions (6). Hence, therapeutic applications should benefitfrom an improvement of serum half-life. To improve phar-macokinetic properties of small molecules most attemptshave been directed so far to increase the apparent molecularsize of the recombinant protein. One approach compriseschemical coupling of polyethylene glycol (PEG) chains to therecombinant antibody molecules (11). This way, longer cir-culation times could be achieved for scFv and F(ab�) frag-ments (12–14). However, PEGylation can lead to reducedbinding and activity of the proteins (14, 15). Other strategiesto improve pharmacokinetic properties of bispecific recom-binant antibodies employed fusion to heavy chain fragments(Fc/CH3) (16, 17) or multimerization strategies (10, 18).However, in these cases molecules contain two binding sitesfor each antigen bearing the risk of target cell-independentactivation of effector cells.New approaches to improve pharmacokinetics of small

proteins are based on binding to or fusion with long-circu-lating serum proteins such as albumin (19–21). Albumin isthe most abundant protein in the blood plasma. It is pro-duced in the liver as a monomeric protein of 67 kDa. Besidesits role in regulating the osmotic pressure of plasma, thephysiologic functions comprise the transport of metaboliteslike long chain fatty acids, bilirubin, steroid hormones, tryp-tophan, and calcium, among others. Albumin also binds with* This work was supported in part by Deutsche Forschungsgemeinschaft

Grant Ko1461/2. The costs of publication of this article were defrayed inpart by the payment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

1 Current address: Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4070Basel, Switzerland.

2 Current address: GSF-National Research Center for Environment and Health,Marchioninistr. 25, 81377 Munchen, Germany.

3 To whom correspondence should be addressed. Tel.: 49-711-685-66989;Fax: 49-711-685-67484; E-mail: [email protected].

4 The abbreviations used are: Db, diabody; AUC, area under the curve; CEA,carcinoembryonic antigen; HSA, human serum albumin; PBMC, peripheralblood mononuclear cell; PEG, polyethylene glycol; RSA, rat serum albumin;scFv, single chain Fv; scDb, single chain diabody; taFv, tandem scFv; IL,interleukin; PBS, phosphate-buffered saline; ELISA, enzyme-linked immu-nosorbent assay; HPLC, high performance liquid chromatography; FPLC,fast protein liquid chromatography; FAP, fibroblast activation protein; VH,heavy chain variable domain; VL, light chain variable domain.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 17, pp. 12650 –12660, April 27, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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high affinity to a broad range of drugs influencing their phar-macokinetic properties (22). Albumin has a simple molecu-lar structure and is highly stable. It is abundantly present invascular and extravascular compartments with a circulationhalf-life of 19 days in humans. Recent studies have shownthat this long serum half-life is due to a recycling processmediated by the neonatal Fc receptor (FcRn), similar to thatobserved for IgG molecules (23, 24).Taking advantage of these properties, human serum albumin

(HSA) has been employed as macromolecular carrier for drugdelivery or diagnostic purpose (19). Moreover, HSA has alsobeen successfully used to generate fusion proteins, e.g.with hor-mones (insulin, human growth hormone) (25, 26) and cyto-kines (interferon-�, interferon-�, IL-2) (27–29), to reduceimmunogenicity and modulate the pharmacokinetic proper-ties, thus improving therapeutic efficacy of these molecules.Improved pharmacokinetic properties have also beendescribed for a scFv-HSA fusion protein aswell as for F(ab�) andF(ab�)2 conjugated to rat serum albumin (RSA) for the targetingof human tumor necrosis factor (20).Here, we have employed the albumin fusion strategy to

recombinant bispecific antibody molecules. Three forms ofrecombinant bispecific antibody HSA fusion proteins based onsingle chain diabody, tandem scFv, or two different single chainFv fragments were generated and produced in a mammalianexpression system. These novel bispecific antibody moleculesshowed specific binding to both antigens (CEA and CD3) andwere able to retarget and activate effector cells in vitro to vari-ous extents. Compared with the parental antibodies, all bispe-cific albumin fusion proteins showed strong increase of theserum half-life in mice.

EXPERIMENTAL PROCEDURES

Materials—Antibodies were purchased from Santa CruzBiotechnology (CA) (HRP-conjugated anti-His tag antibody),Dianova (Hamburg,Germany) (unconjugated anti-His tag anti-body), and Sigma (Taufkirchen, Germany) (anti-mouse IgG-fluorescein isothiocyanate or phycoerythrin-conjugated anti-body). Carcinoembryonic antigen was obtained from EuropaBioproducts (Cambridge, UK). Total RNA from human liverwas purchased fromStratagene (Amsterdam,Netherlands) andthe First Strand cDNA Synthesis kit from Fermentas (St. Leon-Rot, Germany). The human colon adenocarcinoma cell lineLS174T was purchased from ECACC (Wiltshire, UK) and cul-tured in Earle’s minimal essential medium (Invitrogen,Karlsruhe, Germany) supplemented with 2 mM glutamine, 1%non-essential amino acids, and 10% fetal bovine serum.HT1080#13.8 were a kind gift of W. Rettig (BoehringerIngelheimPharma, Vienna, Austria). HT1080#13.8were grownin RPMI 5% fetal bovine serum in the presence of 200 �g/mlG418. Jurkat and HEK293 were cultured in RPMI, 10 and 5%fetal bovine serum, respectively. Buffy coat from a healthyhuman donor was kindly provided by Prof. G. Multhoff(Regensburg, Germany). IL-2 was purchased from Immuno-tools (Friesoythe, Germany) and phytohemagglutinin-L fromRoche Applied Science. CD1 mice were purchased fromElevage Janvier (Le Genest St. Isle, France).

Oligonucleotides—The following oligonucleotideswere used:HSA-XhoI-back, 5�-ACCGTCTCGAGTGGTGGATCAGGC-GGTGATGCACACAAGAGTGAGGTTGC-3�; HSA-Asc-for,5�-GGCCGAGGCGCGCCCACCGCTGCCACCGGCAGCT-TGACTTGCAGCAACAAG-3�; scFVCD3-Sfi-back, 5�-GAC-GCGGCCCAGCCGGCCGATATCCAGATGACCCAGTCC-CCG-3�; scFvCD3-XhoI-for, 5�-ACCACTCGAGACGGTGA-CTAGGGTTCC-3�; scFvCEA-Asc-back, 5�-GGTGGGCGCG-CCTCGGGCGGAGGTGGCTCAGGAGGGCAGGTGAAA-CTGCAGCAGTCTGGG-3�; scFvCEA-Not-for, 5�-GCTCGA-TGCGGCCGC TTAGTGATGGTGATGATGGTGACCTC-CCCGTTTCAGCTCCAGCTTGGTGCC-3�; XhoI-M6-CEA-back, 5�-CCGCTCGAGTAGTACTGATGGTAATACTCAGG-TGAAACTGCAGCAGTCTGG-3�; Not-HSA-back, 5�-ATAA-GAATGCGGCCGCAGGTGGATCAGGCGGTGATGCAC-ACAAGAGTGAGGTTGC-3�; LMB2, 5�-GTAAAACGACG-GCCAGT-3�; HSA-His-stop-EcoRI-for, 5�-CCGGAATTCTT-AGTGATGGTGATGATGGTGGCCACCGGCAGCTTGACT-TGCAGCAACAAG-3�; NcoI-B7.2-back, 5�-CATGCCATGGC-CGCTCCTCTGAAGATTCAAGCT-3�; B7.2-(2–225)-XhoI-for,5�-TACCGCTCGAGCCACCTCCTGAACCGCCTCCAGGA-ATGTGGTCTGGGGGAGGCTG-3�; XhoI-CEA(VH)-back, 5�-AACCGCTCGAGCGGAGGCGGTTCACAGGTGAAACTG-CAGCAGTCT-3�.Cloning of RecombinantAntibody Fusion Proteins—scFvCEA

corresponds to murine scFvMFE-23 (30) and was expressed intheVH-VL orientation using a (G4S)4 linker. scFvCD3 is derivedfromhumanized variant 9 ofUCHT-1 (31). VLCD3 andVHCD3were linked by the sequence GGGGSGGRASGGGGSGGGGS.scDbCEACD3 is organized VHCEA-VLCD3-VHCD3-VL-

CEA. The cloning strategy for this format has been describedelsewhere (32). All constructs were cloned in pAB1 via SfiI/NotI and exhibit a C-terminal c-Myc and a His6 tag. scFvCEA,scFvCD3, and scDbCEACD3 were further used as startingmaterial for the cloning of taFvCD3CEA and the recombinantantibody-HSA fusion proteins. The coding sequence of HSA(amino acids 25–604 of the precursor protein) was amplified byPCR (primer: HSA-XhoI-back and HSA-Asc-for) with cDNAfrom human liver as a template and cloned into pAB1 via XhoI/AscI. scFvCD3 (primers: scFVCD3-Sfi-back and scFvCD3-XhoI-for) and scFvCEA (primers scFvCEA-Asc-back and scFv-CEA-Not-for) were PCR amplified and introduced N-terminal(scFvCD3) and C-terminal (scFvCEA) of the HSA sequence inthe pAB1 vector, generating the bispecific scFvCD3-HSA-scFv-CEA (scFv2-HSA) construct. Through the primers, an-AAAGGSGG- linker was introduced between scFvCD3 andHSA and a -GGGGSGGRASGGGGS- linker between HSA andscFvCEA. taFvCD3CEA-HSA (taFv-HSA)was cloned by ampli-fying scFvCEA (primers XhoI-M6-CEA-back and LMB2) andHSA (primers Not-HSA-back and HSA-His-stop-EcoRI-for),respectively, and introducing first the HSA (NotI/EcoRI)behind the scFvCD3 into pAB1 scFvCD3-HSA-scFvCEA, gen-erating an intermediate scFvCD3-HSA-scFvCEA-HSA prod-uct. In the next step, the HSA-scFvCEA region was replaced byscFvCEA (XhoI/NotI) introducing a -STDGNT- linkerbetween scFvCD3 and scFvCEA and a -GGSGG- linkerbetween scFvCEA and HSA. scDbCEACD3-HSA (scDb-HSA)was generated by replacing the scFvCD3-HSA-scFvCEA of the

Bispecific Antibody-Albumin Fusion Proteins

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pAB1 scFvCD3-HSA-scFvCEA-HSA intermediate constructby scDbCEACD3 (SfiI/NotI). taFvCD3CEA was cloned byamplifying scFvCEA (primers XhoI-M6-CEA-back and scFv-CEA-Not-for) and cloning the fragment (XhoI/NotI) behindscFvCD3 in pAB1 scFvCD3-HSA-scFvCEA, replacing HSA-scFvCEA. Introduced by primer design, all cloned HSA fusionproteins and taFvCD3CEA contain a His6 tag at their C termi-nus. Finally, all HSA fusion protein constructs, as well as scDb-CEACD3 and the taFvCD3CEAwere cloned as SfiI/EcoRI frag-ments into mammalian expression vector pSecTagA(Invitrogen, Karlsruhe, Germany).For cloning of B7-DbCEA, the extracellular region of B7.2

(amino acids 2–225)was amplified by PCR (primersNcoI-B7.2-back and B7.2-(2–225)-XhoI-for) using cDNA provided byProf. Winfried Wels (Frankfurt, Germany). In parallel DbCEAwas amplified (primers XhoI-CEA(VH)-back and LMB2) fromplasmid pAB1-DbCEA (33). PCR fragments of B7.2 andDbCEAwere digestedwithNcoI/XhoI andXhoI/NotI, respectively, andcloned into pAB1 (NcoI/NotI). Finally, the whole B7-DbCEAconstructwas cloned (SfiI/NotI) into amodified pSecTagAvec-tor (pSecTagA-His) devoid of the c-Myc tag sequence.Expression and Purification of Recombinant Antibodies

and Their Respective HSA Fusion Proteins—scFvCEA,scFvCD3, and scDbCEACD3 were expressed in theperiplasm of Escherichia coli strain TG1. Two liters of 2�TY, 100 �g/ml ampicillin, 0.1% glucose were inoculated with20 ml of overnight culture of transformed TG1 and grown toexponential phase (A600 � 0.8) at 37 °C. Protein expressionwas induced by addition of 1 mM isopropyl 1-thio-�-D-galac-topyranoside and bacteria were grown for an additional 3 hat room temperature. Cells were harvested by centrifugationand resuspended in 100 ml of 30 mM Tris-HCl, pH 8.0, 1 mMEDTA, 20% sucrose. After addition of 5 mg of lysozyme, cellswere incubated for 15–30 min on ice. After addition of 10mM Mg2SO4, cells were centrifuged at 10,000 � g for 30 minat 4 °C. Supernatant was dialyzed against PBS and loadedonto a nickel-nitrilotriacetic acid column (Qiagen, Hilden,Germany) equilibrated with 50 mM sodium phosphatebuffer, pH 7.5, 500 mMNaCl, 20 mM imidazole. After a wash-ing step (50 mM sodium phosphate buffer, pH 7.5, 500 mMNaCl, 35 mM imidazole) the His-tagged recombinant anti-body fragments were eluted with 50 mM sodium phosphatebuffer, pH 7.5, 500 mM NaCl, 100 mM imidazole. Proteinfractions were pooled and dialyzed against PBS. Protein con-centration was determined spectrophotometrically and cal-culated using the calculated � value of each protein.Plasmid-DNA (pSecTagA expression vector) encoding

taFvCD3CEA, scFvCD3-HSA-scFvCEA, scDbCEACD3, scDb-CEACD3-HSA, taFvCD3CEA-HSA, and B7-DbCEA weretransfected with LipofectamineTM 2000 (Invitrogen) intoHEK293 cells. Stable transfectants were generated by selectionwith zeocin (300 �g/ml). Cells were expanded and grown inRPMI, 5% fetal calf serum to 90% confluence. For protein pro-duction cells were cultured in Opti-MEM� I (Invitrogen)replacing media every 3 days for 3–4 times. Supernatants werepooled and proteins were concentrated by ammonium sulfateprecipitation (60% saturation), before loading onto a nickel-nitrilotriacetic acid column (Qiagen) (16). Purification by

immobilizedmetal ion affinity chromatography was performedas described above.Flow Cytometry—1 � 106 cells/well were incubated with 10

�g/ml recombinant antibody or recombinant antibody-HSAfusion protein for 2 h at 4 °C. After washing, cells were incu-bated for 1 h at 4 °C with mouse anti-His tag antibody followedby washing and 30 min incubation with fluorescein isothiocya-nate-labeled anti-mouse IgG.Wash cycles and incubation stepswere performed inPBS, 2% fetal calf serum, 0.02%azide. Finally,cells were analyzed by flow cytometry using an EPICS XL-MCL(Beckman Coulter, Krefeld, Germany).ELISA—Binding properties of recombinant antibodies or

antibody-HSA fusion proteins to CEA were analyzed byELISA as following: 96-well plates were coated with carcino-embryonic antigen (300 ng/well) overnight at 4 °C. After 2 hblocking with 2% (w/v) dry milk/PBS, recombinant antibodyfragments or HSA fusion proteins were titrated in duplicateand incubated for 1 h at room temperature. Detection wasperformed with mouse HRP-conjugated anti-His tag anti-body using 3,3�,5,5�-tetramethylbenzidine substrate (1mg/ml 3,3�,5,5�-tetramethylbenzidine, sodium acetate buffer,pH 6.0, 0.006% H2O2). The reaction was stopped with 50 �l of1 M H2SO4. Absorbance was measured at 450 nm in an ELISAreader.Binding properties of B7-DbCEA were analyzed in the fol-

lowing setting: 96-well plates were coated and blocked asdescribed above, followed by incubation with 1 �M B7-DbCEAfusion protein or DbCEA for 1 h at room temperature. Detec-tion was performed via binding to recombinant CTLA-4-Fc (1h at room temperature) followed by anti-mouse Fc-HRP con-jugate (1 h at room temperature). Plates were developed with3,3�,5,5�-tetramethylbenzidine substrate as described above.Concentration of human IL-2 in the supernatant after

T-cell retargeting was determined by an IL-2 sandwichELISA. Anti-human IL-2 antibodies as well as the standardof recombinant human IL-2 was provided by the DuoSet IL-2ELISA Development System kit (R&D Systems, Norden-stadt, Germany) and the assay was performed following themanufacturer’s protocol.Size Exclusion Chromatography—Apparent molecular

weight of recombinant antibody and recombinant antibody-HSA fusion proteins was determined by HPLC on a BioSep-Sec-3000 column or a BioSep-Sec-2000 column (Phenomenex,Torrance, CA) with a flow rate of 0.5 ml/min and PBS asrunning buffer. The following standard proteins were used:thyroglobulin, apoferritin, �-amylase, bovine serum albu-min, carbonic anhydrase, and cytochrome c.Preparation of Peripheral Blood Mononuclear Cells (PBMC)—

Buffy coat (leukapheresis) from a healthy human donor wasdiluted 1:4 in RPMI 1640, layered onto a LSM 1077 Ficoll/Hypaque gradient (PAA, Colbe, Germany), and centrifuged for20min at 670� g at room temperature. ThePBMC fractionwasaspirated and washed once with medium, before resuspendingin 10% Me2SO, 40% RPMI, 50% fetal calf serum and storing at�80 °C. For flow cytometry PBMCs were preactivated by incu-bation with phytohemagglutinin-L (1 �g/ml) and IL-2 (100units/ml) for 3 days.




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Retargeting of T Cells—1 � 105 LS174T or HT1080#13.8cells/100 �l/well were seeded in 96-well plates. The next daysupernatant was removed and 100 �l of recombinant anti-body � costimulus added. After a 1-h preincubation at roomtemperature, 2 � 105 PBMC/100 �l/well were added.PBMCs had been thawed the day before and seeded on aculture dish, to remove monocytes by attachment to theplastic surface. Only cells that remained in suspension wereused for the assay. After addition of PBMCs, the 96-wellplate was incubated for 22–24 h at 37 °C, 5% CO2. Plateswere centrifuged and cell-free supernatant was collected.IL-2 concentration in the supernatant was determined byELISA.In Vitro Stability—Antibody molecules were incubated with

human serum at a concentration of 10 �g/ml for up to 24 daysat 37 °C. Aliquots were taken at various time points and storedat �20 °C. The concentration of active antibody molecules wasthen determined by ELISA as described above including dilu-tions of untreated antibody molecules as reference. Half-liveswere calculated by linear regression.Pharmacokinetics—Animal care and all experiments per-

formed were in accordance with federal guidelines and havebeen approved by university and state authorities. CD1 mice(female, 9–12 weeks, weight between 30 and 40 g, 3 mice/group) received intraveneous injections of 25 �g of recom-binant antibody or antibody-HSA fusion protein in a totalvolume of 100–150 �l. In time intervals of 3, 10, 30, 60, 120,and 360 min (recombinant antibody) or 3, 30, 60, 120, 360min, 24 h, and 6 days (recombinant antibody-HSA fusionprotein) blood samples (100 �l) were taken from the tail andincubated on ice. Clotted blood was centrifuged at 10,000 �g for 10 min at 4 °C and serum samples stored at �20 °C.

Serum concentration of CEA-binding recombinant antibody orrecombinant antibody-HSA fusionproteins was determined by ELISA(as described above), interpolat-ing the corresponding calibrationcurves. For comparison, the firstvalue (3 min) was set to 100%. Phar-macokinetic parameters AUC, t1⁄2�,and t1⁄2� were calculated with Excelusing the first 3 times points to cal-culate t1⁄2� and the last 3 time pointsto calculate t1⁄2�. For statistics, Stu-dent’s t test was applied.

RESULTS

Generation of Bispecific Recom-binant Antibody-HSA Fusion Pro-teins—The structure of HSAshows that the N and C terminusof the protein are located on oppo-site sites and stick out of the mol-ecule, thus providing a good pre-condition for the generation offusion proteins (Fig. 1). By geneti-cally fusing various antibody for-

mats to HSA, the recombinant bispecific antibody-HSA fusionproteins scFv2-HSA, scDb-HSA, and taFv-HSAwere generated(Fig. 1). In the scFv2-HSA variant scFvCD3 was fused to the Nterminus and scFvCEA to the C terminus of HSA by shortglycine/serine-rich linkers of 5 and 15 amino acids, respec-tively. scDb-HSA and taFv-HSA fusion proteins were gener-ated by fusing scDbCEACD3 or taFvCD3CEA, respectively,by a linker of 8 amino acids to the N terminus of HSA. AllHSA fusion proteins were C-terminal endowed with a Histag for detection and purification.scFvCD3 and scFvCEA were purified from the periplasm

of transformed bacteria, whereas the HSA fusion proteinswere purified from the cell culture supernatant of stablytransfected HEK293 cells. scDb and taFv were produced inboth expression systems. In the bacterial expression systemthe yield of scFv production was 0.3–0.35 mg/liter, i.e. threetimes higher than the yield of the scDb and taFv production(0.1 mg/liter). Higher yields of scDb and taFv were obtainedin the mammalian expression system (5–6 mg/liter cell cul-ture supernatant). The yields of purified HSA fusion proteinswere 5 mg/liter for scFv2-HSA, 9 mg/liter for taFv-HSA, and13 mg/liter for scDb-HSA.In SDS-PAGE analyses all proteins were found to be highly

pure and migrated according to their predicted molecularmasses: scFvCD3 and scFvCEA, 28–30 kDa; scDb and taFv,56 kDa; and scFv2-HSA, scDb-HSA, and taFv-HSA, 121 kDa(Fig. 2). Identities of the recombinant proteins were con-firmed by Western blot analysis with anti-His tag antibody(not shown). Constructs were further analyzed by HPLC sizeexclusion chromatography (Fig. 3). scFvCEA eluted as amajor peak (80%) corresponding to monomeric molecules of29 kDa, but also contained a fraction (20%) of dimeric mol-

FIGURE 1. Schematic presentation of recombinant antibodies and antibody fusion proteins withHSA or B7.2 extracellular domain. A, variable regions of VH and VL were joined by linkers of differentlength generating the recombinant antibody formats of scFv, scDb, taFv, and Db. Fusion of the recombi-nant antibody formats to HSA or B7.2 (ECD) led to the generation of bispecific recombinant antibody-HSAconstructs (with specificity for CD3 and CEA) and the B7-Db fusion protein (with specificity for CEA andCD28/CTLA-4). B, structure of HSA with the N and C terminus marked. Structure was visualized with thePyMOL Molecular Graphics System (DeLano Scientific, San Carlos, CA).




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ecules. Similar results were obtained for scFvCD3 (notshown). scDbCEACD3 showed a major peak (96.3%) elutingwith an apparent molecular mass of 48 kDa, with a smallfraction corresponding in size to dimers. taFvCD3CEA alsoshowed a predominant peak (98%) indicating an apparentmolecular mass of �27 kDa, however. This difference

between calculated and apparent molecular mass was lesspronounced applying fast protein liquid chromatographysize exclusion chromatography using a Sephadex 200 col-umn. In this experiment both scDb and taFv eluted at thesame volume indicating an apparent molecular mass of37–40 kDa applying the same standard proteins used forHPLC analysis (not shown). Thus, taFvCD3CEA but alsoscDbCEACD3 migrate in size exclusion chromatographywith a lower apparent molecular mass as calculated from theamino acid sequence. Similar results were also described forother bispecific taFv molecules, e.g. directed against CD3and fluorescein (34). All HSA fusion proteins eluted with apredominant peak (78.1% for scFv2-HSA, 96.3% for scDb-HSA and 95.1% for taFv-HSA) with an apparent molecularmass of 104–110 kDa, with the remaining protein corre-sponding in size mainly to dimeric molecules. Thus, themajority of HSA fusion proteins are present as monomericmolecules in the preparations.Binding Properties—Flow cytometry analysis of the con-

structs on CEA positive LS174T cells and CD3 positive PBMCsrevealed strong binding of all constructs to the respective anti-gen-positive cells according to their specificities (Fig. 4). AllCD3-specific constructs also bound to CD3� Jurkat cells (notshown). No binding was observed to CD3 and CEA negativeHEK293 cells. Furthermore, binding to purified CEA wasshown in ELISA (Fig. 5). Here, binding properties of scFv, scDb,and taFv were similar, whereas signals of the respective HSAfusion proteins tended to be slightly reduced. No binding wasobserved with bovine serum albumin as control protein (notshown). These experiments clearly demonstrate that antigenbinding of the various antibody molecules is not impaired byfusion to HSA.Target Cell-dependent Effector Cell Activation—Bispecific

antibody-mediated activation of T cells was measured by IL-2release. In this assay, LS174T (CEApositive) target cells or CEA

negative HT1080#13.8 control cellswere preincubated with the con-structs, followed by the addition ofunstimulated PBMCs and quantifi-cation of released IL-2 after 24 h. Toprovide a costimulatory signal for Tcell activation, a B7-DbCEA fusionprotein was employed. This con-struct consists of the extracellularregion of B7.2 (CD86) fused to theVH domain of a CEA specific dia-body (Fig. 1). Due to assembly of twodiabody chains the final constructpossesses two B7.2 ligands as well astwo CEA binding sites. The aminoacid sequence of B7 contains 8potential N-glycosylation sites.According to this, SDS-PAGE (Fig.6A) and Western blot analysis (notshown) showed proteins migratingwith an apparent molecular massbetween 75 and 110 kDa, with a pre-dominant band at 110 kDa, which is

FIGURE 2. Analysis of recombinant antibodies and antibody-HSA fusionproteins by SDS-PAGE (10%). 3 �g of protein/lane was loaded under reduc-ing (A) or non-reducing (B) conditions. The gel was stained with CoomassieBlue. Lane 1, marker; 2, scFvCEA; 3, scFvCD3; 4, scFv2-HSA; 5, scDb; 6, scDb-HSA; 7, taFv; 8, taFv-HSA; 9, HSA.

FIGURE 3. HPLC size exclusion chromatography. HPLC analysis of scFvCEA (A), scDb (B), taFv (C), scFv2-HSA(D), scDb-HSA (E), and taFv-HSA (F) using BioSep-Sec-3000 or BioSep-Sec-2000 columns.




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�60 kDa larger than the molecular mass calculated from theamino acid sequence (54 kDa). This increasedmolecularweightis in accordance with data from a similar B7.2-scFv fusion pro-

tein directed against erbB2 and expressed in Pichia pastoris(35). This fusion proteinmigrated in SDS-PAGEwith an appar-ent molecular mass of �105 kDa, which was reduced to �55kDa after deglycosylation with protein N-glycosidase F. HPLCsize exclusion chromatography revealed the presence of a singleprotein fraction with an apparent molecular mass of 490 kDa(Fig. 6B). This is about twice the size calculated for the dimericB7-DbCEA molecule and might be due to an extended struc-ture of this molecule. In comparison, a B7.2-scFvCEA fusionprotein not prone to dimer formation eluted with a major peakcorresponding to a molecular mass of 200 kDa and a minorpeakwith approximately twice themolecularmass, whichmostlikely represent dimeric molecules (not shown). This finding

FIGURE 4. Analysis of binding properties by flow cytometry. LS174T, preacti-vated PBMC, and HEK293 cells were incubated with 10 �g/ml of the recombinantantibody constructs. Detection was performed with anti-His tag antibody and fluo-rescein isothiocyanate-labeled anti-mouse IgG antibody. Filled, detection system;solid line, antibody molecules.

FIGURE 5. Binding of recombinant antibodies and antibody-HSA fusionproteins to CEA in ELISA. Titration of scDb (A), scFvCEA (B), and taFv (C) andthe respective HSA fusion proteins for binding to CEA. Detection was per-formed with an HRP-conjugated anti-His tag antibody. Duplicate sampleswere analyzed.




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further supports the assumption that the B7-DbCEA protein ispresent as a dimericmolecule. Simultaneous binding of B7-Db-CEA to CEA and the B7 receptor CTLA-4 was demonstrated inELISA (Fig. 6C). Costimulatory activity of B7-DbCEA in com-bination with scDbCEACD3 as first stimulus was seen afterbinding to CEA positive cells incubated with 15 nM of bothproteins (Fig. 6D). In this experiment, IL-2 release of PBMCsincubated with scDbCEACD3 and B7-DbCEA was increased2.5-fold compared with cells incubated with scDbCEACD3 inthe absence of B7-DbCEA (DbCEA or scFvCEA were added inthese experiments as a substitute for B7-DbCEA). Only verysmall amounts of IL-2 (similar to medium control) werereleased after incubation of target and effector cells withB7-DbCEA, indicating that B7-DbCEA is not sufficient to stim-ulate effector cells on its own. Incubation of PMBCs with bothantibody molecules in the absence of target cells caused only amarginal release of IL-2 (�10% of IL-2 release induced by cell-bound molecules) (Fig. 6D), clearly demonstrating that targetcell-mediated presentation of the constructs is necessary foreffector cell activation and costimulatory activity.Next, we analyzed all bispecific antibody molecules for acti-

vation of PBMCs in the presence or absence of a costimulatory

signal applying 40 nM bispecificantibody molecules and B7-DbCEA. Using LS174T as CEA-ex-pressing target cells all CEAxCD3bispecific constructs induced IL-2release fromPBMCs,whichwas fur-ther �2-fold increased in the pres-ence of B7-DbCEA (Fig. 7A). Noincreased IL-2 release comparedwith medium control was observedfor HSA or bispecific scDbFAPCD3directed against fibroblast activa-tion protein (FAP) and CD3 andincluded as negative control protein(see also Fig. 4). The scDb-HSAfusion protein induced IL-2 releasesimilar to scDbCEACD3. scFv2-HSA showed a reduced releasecompared with scDb or scDb-HSA.taFvCD3CEA showed the strongestsignals, whereas IL-2 release in-duced by taFv-HSA was betweenthat of scDb-HSA and scFv2-HSA.In a further experiment we ana-lyzed selectivity of activationusing CEA negative but FAP posi-tive HT1080#13.8 cells (Fig. 7B).As expected, scDb, scDb-HSA,and scFv2-HSA did not induceeffector cell activation, in contrastto scDbFAPCD3, which is able tobind to these cells. Surprisingly,taFvCD3CEA also showed stronginduction of IL-2 releasewhen incu-bated with HT1080#13.8 cells simi-lar to that observed with LS174T.

These resultswere confirmedwith 2 further independently pre-pared taFv samples produced in mammalian cells (not shown).Induction of IL-2 releasewas also observedwhen taFvwas incu-bated with effector cells in the absence of target cells, whereasall other constructs did not induce any IL-2 release in theseexperiments (not shown). taFv-HSA showed some increasedIL-2 release when incubated together with B7-DbCEA on CEAnegative HT1080#13.8 cells. In further experiments we alsotested lower concentrations of our bispecific molecules. Strongactivation and costimulation was still seen using antibody con-centrations of 5 nM.At this antibody concentration IL-2 release,especially that induced by scDb, was stronger compared withIL-2 release at 40 nM. This observation is in accordance with amore detailed titration of scDb for activation of PBMCs indi-cating an optimum between 2.5 and 10 nM under the appliedassay conditions, which might be caused by competing effectsof scFvCEA at higher concentrations (not shown). IL-2 releasewas reduced almost to background signals at 0.2 nM, with theexception of taFvCD3CEA, which induced strong IL-2 releasealso at this concentration (Fig. 7, C and D).In Vitro Stability and Pharmacokinetic Properties—In vitro

stability was analyzed by incubation of the constructs in human

FIGURE 6. Characterization of B7-DbCEA. A, Coomassie-stained SDS-PAGE (10%) of purified B7-DbCEA pro-tein under reducing and non-reducing conditions. B, size exclusion chromatography of B7-DbCEA by HPLC.C, simultaneous binding of B7-DbCEA to CEA and recombinant CTLA-4. ELISA plate was coated with CEA (3�g/ml) and incubated with 1 �M B7-DbCEA or DbCEA. Detection was performed by CTLA-4-Fc, followed byanti-mouse Fc-HRP. D, costimulatory property of B7-Db. PBMCs were incubated for 22 h with 15 nM scDb-CEACD3 in the presence or absence of B7-DbCEA in solution or with the constructs presented on LS174T cells.IL-2 release was determined by ELISA from duplicate samples.




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serum at 37 °C for up to 24 days and subsequent measurementof CEA binding activity in ELISA. All constructs were found tobe highly stable under these conditions (t1⁄2 � 4 days) (notshown). The pharmacokinetic properties of the recombinantproteinswere analyzed byELISAof serumsamples after a singledose injection (25 �g intravenously) into CD1 mice (Fig. 8 andTable 1). This allowed for the detection of molecules in theserum that retained binding activity for CEA. All proteinsshowed a biphasic elimination from circulation. scFvCEA wasrapidly eliminated from circulation (t1⁄2� � 5 min). scDb andtaFv showed a slightly increased circulation time (t1⁄2� � 8–9min). Circulation time was drastically increased for all HSAfusion proteins. Thus, t1⁄2� was increased to 37 min for taFv-HSA, 43 min for scDb-HSA, and even 117 min for scFv2-HSA. The improvement of pharmacokinetic properties wasalso demonstrated by comparison of the area under thecurve (AUC). For the bispecific constructs (scDb, taFv) theAUC(0–24 h) increased by a factor of 6–7 after fusion to HSA.The scFv2-HSA showed the highest AUC, which was �1.3times that of scDb-HSA and taFv-HSA.

DISCUSSION

In this study we show that fusion of recombinant antibodymolecules to HSA leads to a drastic improvement of theirpharmacokinetic properties, without impairing their targetbinding capacity. All HSA fusion proteins could be producedat high yields (5–13 mg/liter cell culture supernatant) inmammalian cells. These yields were similar to that observedfor scDb and taFv molecules indicating that fusion to albu-

min does not interfere with pro-tein synthesis and secretion. Com-parable yields (7.5–9 mg/liter)were also described for scFv-HSA fusion proteins expressed ina P. pastoris expression system(20).Our study showed that the bio-

logical activity, i.e. effector cell acti-vation, is influenced by the formatused for the generation of bispecificantibody albumin fusion proteins.Strong and target cell-dependentactivation of effector cells wasobserved with scDb as well as withthe scDb-HSA fusion protein.scFv2-HSA-mediated PBMC acti-vation was significantly reducedcompared with scDb-HSA. Con-sidering the location of the scFvmoieties on opposite sites of theHSA molecule, the HSA moietymight lead to insufficient cellproximity to promote strongreceptor cross-linking. This issupported by the fact that in scDbmolecules the two antigen-bind-ing sites are �6 nm apart, whereasin scFv2-HSA molecules this dis-

tance varies strongly and can be up to 18 nm.Surprisingly, the taFv construct and to some extent also the

taFv-HSA construct induced strong but nonspecific activa-tion of PBMCs. Size exclusion chromatography indicatesthat this is most likely not caused by taFv multimers oraggregates. In addition, because all other antibody con-structs were produced and purified the same way, it isunlikely that copurified molecules account for this stimula-tory activity. Several other bispecific taFv molecules for theretargeting of T cells via CD3 to tumor cells have beendescribed in the literature using different arrangements ofvariable domains and linker compositions (for an overviewsee Ref. 4). Target cell-specific T cell activation and cytotox-icity was described for these molecules. This points to anintrinsic T cell activating property of the taFvmolecules ana-lyzed in our study. The taFv molecule and the scDb moleculeused in this study are composed of the same variabledomains, differing only in the arrangement and the linkersequences used to connect these domains. The taFv format ispredicted to be more flexible than the scDb format. Thismight be reflected in the unexpected behavior of the taFvmolecule observed in the HPLC analysis. Furthermore, itmight be possible that the taFvmolecule binds to CD3 via theCD3-specific scFv moiety and that due to the flexibility ofthe molecule, especially of the middle linker region, otherparts of the molecule interact with cell surface molecules orreceptors of the T cell leading to target cell-independentactivation and IL-2 release. Further studies, e.g. testing other

FIGURE 7. Target cell-mediated activation of PBMCs by bispecific recombinant antibody-HSA fusionproteins. LS174T (A) and HT1080#13.8 (B) cells were preincubated for 1 h with 40 nM bispecific recombi-nant antibody construct in the presence (B7-DbCEA) or absence (scFvCEA) of 40 nM costimulus. PBMCswere added and IL-2 concentration in the cell culture supernatant measured after 22–24 h by ELISA fromduplicate samples. Antibody constructs were further analyzed on LS174T cells applying 5 (C) or 0.2 (D) nM

antibody molecules.




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taFv molecules or taFv molecules with different linkersequences, are necessary to clarify this issue.Our findings indicate that the scDb and scDb-HSA format

is particularly effective in effector cell activation. Impor-tantly, this activation did not occur in the absence of CEA-expressing target cells or with CEA negative target cells.Thus, T cell receptor clustering requires the membrane-bound presentation of the bispecific antibody moleculemediated by binding to the target antigen. Fusion to HSAdoes not impair this process. Target cell-dependent stimu-

lation by scDb and scDb-HSA was further enhanced by theB7-DbCEA fusion protein. This is in accordance with previ-ous data of a fusion protein of the extracellular region ofB7-2 and a scFv specific for ErbB2 (35). In our hands a B7-2-(2–225)-scFvCEA molecule did not reach the costimulatoryeffect induced by the B7-DbCEA form, probably due toinsufficient cross-linking (not shown). Thus, dimerization ofthe B7 component arising from the fusion to the diabodyformat might facilitate efficient clustering of CD28 on the Tcell, gaining stronger costimulatory effects. For a B7-DbCEAconstruct, composed of the extracellular domain of B7.1fused to DbCEA, cis- and trans-costimulation has beenreported (36). In a gene therapy approach, DNA encodingthis costimulatory molecule was applied together with DNAfor the bispecific DbCEACD3 in a xenograft mouse model,leading to growth delay of established tumors (5). B7-recom-binant antibody fusion proteins are thought to open the pos-sibility to direct and localize the costimulatory activity to thetumor site. If this promising concept is also applicable for theB7-DbCEA in vivo remains to be demonstrated in furtherstudies.Prolonged circulation times were found for all albumin

fusion proteins. The increase in circulation half-lives andAUC correlates with data of Smith and co-workers (20), whoreported pharmacokinetic properties of different recombi-nant antibody-albumin conjugates and fusion proteins in rat.In this study, scFv-HSA fusion proteins showed �12-foldlonger half-lives in comparison with a F(ab�)-Cys recombi-nant antibody. In addition, the conjugation of F(ab�) to RSAas well as an RSA binding approach through bispecificF(ab�)2 (anti-RSA � anti-tumor necrosis factor) resulted in asignificant increase (17- and 8-fold) in AUC(0-∞) comparedwith F(ab�)-Cys and a control F(ab�)2 construct (not specificfor RSA). In rats the pharmacokinetics of RSA and HSA dif-fered markedly, i.e. HSA showed a faster elimination thanRSA. Thus, RSA had a t1⁄2� � 4.1 h and a t1⁄2� � 49.1 h,whereas HSA had a t1⁄2� � 0.8 h (48 min) and a t1⁄2� � 14.8 h.Our HSA fusion proteins showed similar or even better val-ues in mice with t1⁄2� between 37 and 117 min and a t1⁄2�between 40 and 47 h indicating that the antibody fusion part(probably through increase in size of the fusion protein) alsocontributes to prolonged circulation time. This finding is inaccordance with results of various other HSA fusion pro-teins, which showed improved pharmacokinetic propertiesin various animals including mice (25, 27–29). In addition,improved therapeutic activities were observed for HSAfusion proteins (25–28). Very little data are available on thepharmacokinetic properties of albumin fusion proteins in

FIGURE 8. Improved pharmacokinetic properties of recombinant anti-bodies after fusion to HSA. Serum concentrations of the various recombi-nant bispecific antibodies and antibody HSA fusion proteins were deter-mined after various time points after intravenous injection (25 �g) into CD1mice (n � 3). Data were normalized considering maximal concentration at thefirst time point (3 min). A, scDb and scDb-HSA; B, taFv and taFv-HSA; C, scFv2-HSA and scFvCEA.

TABLE 1Pharmacokinetic parameters of recombinant antibodies andantibody-albumin fusion proteins

Construct Molecular mass t1⁄2� t1⁄2� AUCkDa min h h * %

scFv CEA 28 5 � 1 4 � 1 18 � 1scDb 56 8 � 1 16 � 4 61 � 4taFv 56 9 � 2 26 � 8 70 � 4scFv2-HSA 121 117 � 59 47 � 4 586 � 83scDb-HSA 120 43 � 11 43 � 0 433 � 163taFv-HSA 121 37 � 16 40 � 0 426 � 140




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humans. A prolonged circulation time of an albumin-inter-feron-� fusion protein was reported in a phase I/II clinicaltrial. In this study an increase of mean elimination half-lifefrom 2–3 to 159 h (6.6 days) was observed, supporting adosing schedule at 2–4-week intervals (37). This half-life issignificantly shorter than that of albumin, which is 19 days inhumans. Currently, we cannot exclude that fusion of an anti-body molecule or other proteins to the N and/or C terminusof albumin has an influence on pharmacokinetic propertiesmediated by the albumin moiety, e.g. whether FcRn-medi-ated recycling is affected.Other approaches to improve pharmacokinetic properties

of recombinant antibodies focus on increasing the apparentmolecular size by PEGylation. Several recombinant antibodyformats have been conjugated in a site-specific manner toPEG chains of different length (5–40 kDa) increasing theserum half-life significantly (11). However, cases have beenreported where PEGylation of monomeric or dimeric scFvmolecules led to a reduced affinity (38). PEGylated F(ab�)2and diabody fragments specific for CEA have shown inxenograft tumor mouse models prolonged plasma half-lifeand increased tumor accumulation (39, 40). The latter isthought to be attributed in part to the enhanced permeabilityand retention effect (EPR) observed in solid tumors, whereenhanced vascular permeability goes along with the reten-tion of macromolecules inside the tumor. This aspect mightbe also conceivably important for HSA-antibody fusionproteins.In summary, we could show that fusion of recombinant

bispecific antibodymolecules to albumin results in a prolongedcirculation. Furthermore, our findings indicate that the scDb-HSA format is particularly effective in effector cell activation ina target cell-dependentmanner. Further studies have to show ifthis improvement of pharmacokinetic properties translatesinto improved anti-tumor activity in vivo.

Acknowledgments—We thank Katja Stolpa for technical assistance.Furthermore, we thank Genentech for making DNA of the human-ized anti-CD3 antibody UCHT1 available. We also thank Prof.Winfried Wels (Georg-Speyer-Haus, Frankfurt) for providing B7.2cDNA, Prof. Gabriele Multhoff for supplying buffy coat from ahealthy human donor, and Sabine Munkel (IZI, Stuttgart) forHPLC analysis.

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E. KontermannDafne Müller, Anette Karle, Bettina Meißburger, Ines Höfig, Roland Stork and Roland

Fusion to Human Serum AlbuminImproved Pharmacokinetics of Recombinant Bispecific Antibody Molecules by

doi: 10.1074/jbc.M700820200 originally published online March 8, 20072007, 282:12650-12660.J. Biol. Chem.

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