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Thrombosis Research 109 (2003) 137–144

Regular Article Generation and characterization of recombinant single chain Fv antibody that recognizes glycoprotein Iba

Kesheng Dai, Huaiping Zhu, Changgeng Ruan*

Thrombosis and Hemostasis Research Unit, Jiangsu Institute of Hematology, The First Affiliated Hospital of Suzhou University, Suzhou 215006, China Received 2 June 2002; received in revised form 7 January 2003; accepted 3 February 2003

Abstract

A recombinant single chain Fv (scFv) fragment with specific activity against platelet glycoprotein (GP) Iba was developed and characterized. The scFv was generated from the SZ-2 hybridoma, which produced an anti-platelet antibody reactive to GPIba. VH and VL gene segments were generated from the SZ-2 hybridoma by reverse transcribed-polymerase chain reaction (RT-PCR). After cloning into pUCm-T vector, the DNA sequences of both VH and VL genes were analyzed from two different clones, respectively, the same results were obtained. Comparison of SZ-2 variable region to the Kabat database showed that VH belonged to the mouse Ig heavy family XV while VL belonged to the mouse Ig kappa family XXVI. For assembly of the SZ-2 scFv, VH and VL fragments were cloned into pSW1-scFv successively. The scFv was arranged in VH–VL orientation, being joined together with a 15-amino-acid (Gly4Ser)3 linker. The scFv encoding sequence was amplified and cloned into pET22b vector in-frame with a pel B leader sequence to direct secretion of the protein. Escherichia coli strain BL-21(DE3)PlysS was transformed with the recombinant plasmid, and expression of the scFv was induced using isopropyl-h-D-thiogalactopyranoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the recombinant antibody revealed a protein with apparent molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS-PAGE, the production yield of SZ-2 scFv was about 25% of the total cellular proteins. The recombinant SZ-2 scFv antibody was successfully purified using Ni-NTA affinity chromatography with a yield of 120 mg/l. The SZ-2 scFv antibody could bind to demonstrated by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Analyzed by Western blot, it could bind to platelet GPIb. It retained the binding capacity of its parental SZ-2 monoclonal antibody (MoAb). In functional studies, SZ-2 scFv inhibited platelet agglutination and aggregation induced by ristocetin and , respectively, but had no effect on ADP- induced platelet aggregation. Therefore, SZ-2 scFv has the potential to be used as an antithrombotic agent. D 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Platelets; Glycoprotein Ib; Single chain Fv; ; Thrombin; Thrombosis; Monoclonal antibody

Thrombosis is an acute form of cardiovascular disease site for the vWF A1 domain [1,2]. GPIb, a major component wherein sudden aggregation of blood-borne platelets of the platelet membrane, consists of disulfide-linked poly- occludes the arterial blood supply, leading to tissue infarc- peptide chains (a and h), associated with GPIX and GPV to tion. Platelet binding to von Willebrand factor (vWF) at site form a non-covalent, oligomeric complex. GPIb has dual of vascular injury provides a mechanism for the arrest of functions: first, GPIb is a receptor of vWF that is essential for bleeding and also contributes to the occlusion of diseased mediating the initial attachment of platelets to the vessel wall vessels in pathologic states. The process is mediated by the at the sites of injury; second, GPIb plays a role in thrombin- glycoprotein (GP) Ib–IX–V complex in which the amino- induced platelet activation, although this role is poorly terminal domain of the GPIba-chain contains the binding understood [3]. The interaction of GPIb with vWF allows the initial tethering and rolling of platelets before their firm Abbreviations: scFv, single chain Fv; ELISA, enzyme-linked immuno- adhesion and activation [4]. If no other bonds are formed, sorbent assay; GPIb-IX, glycoprotein Ib-IX; MoAb, monoclonal antibody; tethered platelets translocate in the direction of flow, albeit at vWF, von Willebrand factor; RT-PCR, reverse transcribed-polymerase a markedly lower velocity than freely flowing blood cells chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel [1]. On reactive substrates, however, binding of vWF to electrophoresis; IPTG, isopropyl-h-D-thiogalactopyranoside. * Corresponding author. Tel.: +86-512-5101708; fax: +86-512- GPIb–IX–V transduces signals across the plasma mem- 5101708. brane, then activates the platelets. During this process, 2+ E-mail address: [email protected] (C. Ruan). agonists such as ADP are secreted, Cytosolic Ca is

0049-3848/03/$ - see front matter D 2003 Elsevier Science Ltd. All rights reserved. 转载 doi:10.1016/S0049-3848(03)00152-X 中国科技论文在线 http://www.paper.edu.cn 138 K. Dai et al. / Thrombosis Research 109 (2003) 137–144

elevated and the Ca2+-dependent integrin GPIIb–IIIa is (RT-PCR). Briefly, total RNA was extracted using TRIzol activated. These reactions mediate platelet aggregation reagent from 1 Â 107 SZ-2 hybridoma cells secreting MoAb through the binding of GPIIb–IIIa to and result against GPIb. Single-strain cDNA was synthesized from 5.0 in subsequently thrombus formation [4,5]. Therefore, an Ag of total RNAwith 100 pmol random primer using 10 units inhibitor of thrombin-platelet and vWF-platelet interaction of M-MLV reverse transcriptase in a final volume of 30 Al. may be useful in the prevention of thrombotic diseases. The reaction was incubated at 42 jC for 1 h. VH and VL A murine monoclonal antibody (MoAb) SZ-2 against were amplified by polymerase chain reaction (PCR) using GPIba was previously produced and characterized in our the following primers (restriction sites underlined) [11]:VH laboratory [6]. The antibody not only inhibited the ristocetin- Back, 5V-AGGTC(G)C(A)A(A)GCTGCAGGAGTCTGG-3V dependent binding of vWF to platelets and ristocetin-induced incorporates a PstI site; VH For: 5V-TGAGGAGACGGT- platelet agglutination but also inhibited platelet aggregation GACCGTGGTCCCTTGGCCCCAG-3V incorporates a induced by thrombin [6,7]. The epitope for SZ-2 lies between BstEII site; VL Back: 5V-GACATTGAGCTCACCCAGTC- residues Tyr-276 and Glu-282 of GPIba [7]. To investigate TCCA-3V incorporates a SacIsite;VLFor:5V-GTTA- the potential of engineering SZ-2 to avoid the immunores- GATCTCGAGCTTGGTCCC-3Vincorporates a XhoIsite. ponse of the human body to mouse IgG in clinical trials, a PCR amplification was performed using a reaction mix recombinant single chain Fv (scFv) antibody was generated containing 1 Al of cDNA reaction mix, 10 pmol each primer, and characterized. In this paper, we report the construction of 200mMdNTPsand2unitsPfuDNApolymerasein SZ-2 scFv by the joining of variable domains of and 10 Â PCR reaction buffer in a final volume of 50 Al. The heavy chain gene segments derived from SZ-2 hybridoma PCR consisted of an initial reaction for 5 min at 95 jC, 30 with a 15-amino-acid (Gly4Ser)3 linker. This scFv was cycles for 30 s at 55 jC, 1 min at 72 jC and 30 s at 95 jC, produced successfully in Escherichia coli. The molecular and final extension for 7 min at 72 jC. The products were nature and binding properties of this scFv were characterized. separated by 1.5% agarose gel and recovered using the Qiaex II gel extraction . The SZ-2 VH and VL fragments were cloned into pUCm-T vector. Sequencing was done using the 1. Materials and methods dideoxy-chain-termination method. Comparison of DNA sequence to Kabat database was performed through the 1.1. Cells, vectors, bacterial strains, oligonucleotide internet at http://immuno.bme.nwu.edu. primers and reagents 1.3. Construction of SZ-2 scFv plasmid The hybridoma secreting the GPIb-specific monoclonal antibody SZ-2 was generated, as described previously [6], The amplified V genes were digested with the restriction and cells were grown on DMEM, 10% fetal calf serum with enzymes PstI and BstEII for the VH gene, SacI and XhoI for 5% CO2 at 37 jC. Plasmid pSW1-scFv were kindly provided the VL gene. Both digested fragments were electrophoresed by Dr. Greg Winter (MRC Lab., University of Cambridge, in a 1.5% agarose gel and purified. The VH gene fragment Cambridge, UK). Plasmid pET22b and E. coli strain BL- was ligated into a PstI and BstEII digested pSW-scFv vector 21(DE3)PlysS were obtained from Novagen (Novagen, (containing the (Gly4Ser)3 linker sequence). Then the VL Darmstadt, Germany). Qiaex II gel extraction kit, Ni-NTA gene fragment was ligated into the recombinant plasmid Resin and Penta-His antibody were purchased from QIA- containing the VH gene at the 3V-end of the (Gly4Ser)3 linker. GEN (Qiagen, Hilden, Germany). Trizol and moloney mur- The resulting pSW1-2 scFv was identified by restriction ine leukemia virus (M-MLV) reverse transcriptase were from fragment and DNA sequence analysis. The recombinant Gibco BRL (Rockville, MD, USA). Restriction or modifying scFv gene (SZ-2 scFv) was reamplified from the construct enzymes, plasmid pUCm-T, PCR kit and Pfu DNA polymer- pSW1-2 scFv using the following primers (restriction sites ase were purchased from Sangon (Unison Biotek, Canada). underlined): P1,5V-CCAGGTCGACCTGCAGGAGTCA- Oligonucleotide primers for PCR amplification were synthe- GG-3V incorporates a SalIsite;P2,5V-TATGCGGCCGC- sized on DNA synthesizer (Applied Bio-Systems). Horse- CTCGAGCTTGGTCC-3V incorporates a NotIsite.PCR radish peroxidase (HRP)-conjugated goat anti-mouse MoAb conditions were as follows: one cycle for 4 min at 95 jC, was obtained from Immunotech (Marseilles, France). Human 30 cycles for 1 min at 58 jC, 1 min at 72 jC and 1 min at a-thrombin was purchased from American Diagnostica 94 jC and final extension for 7 min at 72 jC. The (Greenwich, CT, USA). Reagents were either analytical or amplified product was digested with SalI and NotI, and molecular biology grade as required. ligated into plasmid pET22b (see Fig. 1). The resulting pET22-2 scFv was analyzed by restriction digestion. 1.2. Amplification and nucleotide sequence analysis of V region genes 1.4. Expression and purification of scFv antibody

VH and VL gene segments were generated from SZ-2 Plasmid containing scFv fragment was transfected into E. hybridoma by reverse transcribed-polymerase chain reaction coli strain BL-21(DE3)PlysS. Individual clone was shaken 中国科技论文在线 http://www.paper.edu.cn K. Dai et al. / Thrombosis Research 109 (2003) 137–144 139

Fig. 1. VH and VL gene segments derived from SZ-2 hybridoma were joined together with a (Gly4Ser)3 linker. An expression cassette was generated by cloning SZ-2 scFv encoding sequence into pET22b.

at 37 jC in LB medium with 100 Ag/ml ampicillin and 34 sonicated to reduce viscosity. The cellular debris were Ag/ml chloramphenicol overnight. Harvested by centrifuga- removedbycentrifugationat4jC for 15 min at tion at 3500 Â g for 10 min at room temperature and washed 8000 Â g. The supernatants applied to His-bind resin col- once with LB broth, the bacteria were resuspended in LB umn and bound antibodies recovered according to the broth containing 100 Ag/ml ampicillin and 34 Ag/ml chlor- manufacturer’s instructions. The eluent was dialyzed against amphenicol and grown at 37 jC until the OD600 reached refolding buffer I (2 mM reduced glutathione and 0.2 mM 0.6. Protein expression was induced by addition of iso- oxidized glutathione) at 4 jC for 10 h, then continued to propyl-h-D-thiogalactopyranoside (IPTG, 1 mM) and cul- dialyze against refolding buffer II (50 mM Tris–HCl, 150 tured at 37 jC for 4.5 h with shaking at 300 rpm. To purify mM NaCl, pH 8.5) at 4 jC for 10 h. Purified antibodies and recombinant scFv antibody, bacteria were harvested by bacteria were electrophoresed through a standard denaturing centrifugation at 4000 Â g for 20 min at 4 jC, resuspended 15% sodium dodecyl sulfate-polyacrylamide gel electro- in 8 M urea, 0.1 M Na-phosphate, 10 mM Tris, pH 8.0 and phoresis (SDS-PAGE). The purified scFv was quantitated 中国科技论文在线 http://www.paper.edu.cn 140 K. Dai et al. / Thrombosis Research 109 (2003) 137–144

by Bradford method, and bovine serum albumin (BSA) was with 0.38% sodium citrate pH 5.0) at 180 Â g for 10 min used as standard. and adjusted to concentration of 3 Â 108 platelets/ml with platelet-depleted plasma (PPP). Platelet agglutination and 1.5. Detection of antigen binding by ELISA aggregation were optically recorded in an aggregometer (Chrono-Log, Havertown, PA, USA) and quantified as Costar Brand 96-well EIA/RIA Plates (Cambridge, MA, change of light transmission. SZ-2 scFv at varying concen- USA) were coated with human platelets (105/well) in PBS at trations were added 3 min before the addition of stimuli at 4 jC for overnight and washed three times with PBS the following final concentrations: ristocetin, 1.25 mg/ml; containing 0.05% Tween-20. Wells were blocked with 200 ADP, 2 Amol/l at 37 jC. Washed platelets were used for Al of PBS containing 1% BSA for 2 h at room temperature platelet aggregation induced by thrombin (0.05 U/ml). To and washed three times with 0.05% Tween-20-PBS. Two prepare washed platelets, blood was anticoagulated with hundred microliters of recombinant antibody in 1% BSA- ACD (2.5% trisodium citrate, 2% D-glucose and 1.5% citric PBS were added to each well and plates were incubated for acid) (1/7 volume). PRP was obtained as usual. PRP were 2 h at room temperature. After washing three times with further centrifuged at 1500 Â g for 20 min. Then the PBS-Tween, plates were then incubated with of Penta-His platelets pellet was washed three times with CGS (0.12 M antibody (200 Al/well, 1:1000, 1% BSA–PBS) for 2 h at NaCl, 0.0129 M trisodium citrate, 0.03 M D-glucose, pH 37 jC. After washing three times, horseradish peroxidase- 6.5) containing 0.1% bovine serum albumin. The washed conjugated goat anti-mouse immunoglobulin antibody (200 platelets were resuspended in modified Tyrode’s solution Al/well, 1:2000 in PBS–1% BSA) was added. Plates were (3 Â 108 platelets/ml). finally washed three times and peroxidase activity was detected with 0.01% tetramethylbenzidine and H2O2 (150 Al/well, 0.05 M phosphate-citrate buffer, pH 5.0) for 10 2. Results min. The reaction was stopped with 50 Alof3NH2SO4 and the optical density at 450 nm was measured by an 2.1. Cloning and sequencing of MoAb SZ-2 variable region enzyme-linked immunosorbent assay (ELISA) reader. genes

1.6. Antigen binding test by Western blot analysis The amplified VH and VL fragment were approximately 360 and 320 bp, respectively (Fig. 2). Then VH and VL The Triton-X-100 lysate of platelets was electrophoresed fragments were cloned into the pUCm-T vector and through a standard native 8% SDS-PAGE with markers and sequenced. The DNA sequences of VH or VL gene from transferred to nitrocellulose membrane. The membrane was two different clones were identical. Comparison of SZ-2 cut into strips for reacting with various antibodies and was variable domains to the Kabat database showed that VH of blocked overnight in 1% (w/v) skim milk in PBS. Strips were SZ-2 antibody belonged to the mouse Ig heavy family XV then incubated for 2 h with SZ-2 scFv (100 Ag/ml) in 1% and VL belonged to the mouse Ig kappa family XXVI. The skim milk in PBS, washed three times with PBS-Tween DNA sequences of VH and VL have been submitted to and reacted to Penta-His antibody (1:1000) for 2 h. After GenBank, and the accession numbers are AF468835 and more than three washes, the strips were incubated with AF468836, respectively. HRP-conjugated goat anti-mouse immunoglobulin (1:2000) for 1 h, and visualized by a combined substrate of H2O2 and 4-chloro-1-naphthol.

1.7. Flow cytometry

The binding of SZ-2 scFv to platelets was examined by flowcytometry(EPICS-XL,Coulter,Hialeah,FL).One microgram SZ-2 scFv was added to 106 cells in 50 Al PBS at 4 jC for 30 min. After washing three times, the cells were incubated with Penta-His antibody for 30 min at 4 jC. The cells were then washed and suspended in FITC-conjugated goat anti-mouse IgG. After a final washing procedure, the cells were suspended in PBS and analyzed by flow cytometry.

1.8. In vitro platelet aggregation Fig. 2. Electrophoresis was performed on 1.5% agarose gel, and PCR Human platelet-rich plasma (PRP) was prepared by products were visualized by ethidium bromide. Line 1: 100 ladder DNA centrifuging freshly drawn citrated blood (anticoagulated marker; line 2: VH gene; line 3: VL gene; line 4: scFv gene. 中国科技论文在线 http://www.paper.edu.cn K. Dai et al. / Thrombosis Research 109 (2003) 137–144 141

2.2. Construction, expression and purification of SZ-2 scFv Table 1 ELISA of the platelet-binding capability of the SZ-2 scFv (OD492) For assembly of the SZ-2 scFv, the pSW1-2 scFv vector Concentration 10 1 0.2 0.1 0.02 was constructed (see Materials and methods and Fig. 1). The (Ag/ml) scFv was arranged in VH–VL orientation and was joined SZ-2 scFv 1.266 1.152 0.894 0.834 0.291 together with a 15-amino-acid (Gly Ser) linker. The scFv SZ-2 MoAb 1.153 1.124 1.050 1.014 0.879 4 3 SZ-29 MoAba 0.337 0.345 0.234 0.215 0.204 encoding sequence was amplified and cloned into pET22b a vector in frame with a pel B leader sequence to direct SZ-29 specific against vWF was used as negative control. secretion of the protein. The presence of a poly-histidine tag at the C-terminal of the product ensures simple detection the total cellular proteins. The SZ-2 scFv was successfully and purification. The recombinant plasmid was confirmed purified using Ni-NTA affinity chromatography with a yield by sequencing, EcoRI and SalI/NotI enzyme digestion, and of 120 mg/l. gel electrophoresis (Fig. 3). As shown in Fig. 3, SDS-PAGE analysis of the cell extract revealed a protein with apparent 2.3. Antigen binding characteristic of SZ-2 scFv antibody molecular weight of approximately 31,000. By comparing band intensity on a Coomassie brilliant blue-stained SDS- The antigen binding capability of the SZ-2 scFv antibody PAGE, the production yield of SZ-2 scFv was about 25% of was assessed by ELISA, Western blot and flow cytometry. To compare the binding activity of SZ-2 scFv with the parental MoAb, an ELISA was conducted in which various concentrations of scFv or MoAb were reacted with a fixed concentration of platelets coated on a microplate solid phase. The binding capacity of SZ-2 scFv was reasonably good compared to the bivalent parental SZ-2 MoAb (Table 1). Western blots were used to test the specificity of the SZ-2 scFv. Similar to its parental MoAb, SZ-2 scFv could bind to GPIb and gave a positive band at approximately 170 kDa (Fig. 4). In addition to ELISA and Western blot, the binding of SZ-2 scFv to platelets was also demonstrated by flow cytometry analysis. The results shown in Fig. 5 clearly demonstrate the binding activity of SZ-2 scFv.

2.4. Effect of SZ-2 scFv on platelet function

SZ-2 scFv was a potent inhibitor of platelet agglutination and aggregation in response to ristocetin and thrombin. When platelets were pre-incubated with SZ-2 scFv for 3

Fig. 3. (A) Identification of recombinant pET22-SZ-2 scFv vector. Line 1: EDNA/EcoRI + HindIII marker; line 2: pET22-SZ-2 scFv vector/EcoRI; line 3: pET22b vector/EcoRI; line 4: pET22-SZ-2 scFv vector/SalI+NotI; line 5: SZ-2 scFv PCR productor; line 6: 100 ladder DNA marker. (B) SDS- PAGE analysis of SZ-2 scFv antibody expressed in BL21(DE3)plysS and Fig. 4. Platelet lysate was electrophoresed on 8% SDS-PAGE, transblotted purified by Ni-NTA chromatography. Proteins were resolved on 15% SDS- and reacted to SZ-2 MoAb (lane 2) or SZ-2 scFv (lane 3). Line 1: Standard polyacrylamide gel under reducing conditions and stained with Coomassie protein markers (kDa: 200, 130, 97, 66, 43); line 2: SZ-2 positive control; blue. Line 1: LMW marker of protein; line 2: uninduced total bacteria line 3: SZ-2 scFv bound to the lysate band at approximately 170 kDa on the proteins; line 3: induced total bacteria proteins; line 4: soluble inclusion nitrocellulose membrane became visible after incubating the membrane bodies in 8 M urea; line 5: supernatant of induced total bacteria proteins; with Penta-His antibody, HRP-goat anti-mouse IgG antibody, visualized by

line 6: purified SZ-2 scFv. a combined substrate of H2O2 and 4-chloro-1-naphthol. 中国科技论文在线 http://www.paper.edu.cn 142 K. Dai et al. / Thrombosis Research 109 (2003) 137–144

Fig. 5. Flow cytometry analysis of the binding of platelets by SZ-2 scFv recombinant protein. (a) Negative control; (b) SZ-2 MoAb positive control; (c) SZ-2 scFv was added to platelets incubated at 4 jC for 30 min, after washing, incubated with Penta-His antibody, FITC-conjugated goat-anti-mouse IgG. The platelets were suspended to 106 cells/ml, analyzed by flow cytometry.

min at 37 jC, SZ-2 scFv (10 Ag/ml) almost completely concentrations of 1 Ag/ml or less only partially inhibited inhibited ristocetin or thrombin-induced platelet agglutina- platelet agglutination or aggregation in response to ristocetin tion or aggregation (Fig. 6). In contrast, SZ-2 scFv at or thrombin (data not shown). However, treatment of platelet-rich plasma with various concentrations of SZ-2 scFv for 3 min at 37 jC had no effect on aggregation that was induced by 2 Amol/l ADP. These inhibitory activities were similar to the parental MoAb.

3. Discussion

Engineered monoclonal antibodies have been proven of value in the therapy and diagnosis of a wide range of diseases [9,12,13]. This strategy has been successfully applied to thrombotic diseases. Abciximab, a chimeric Fab fragment, is effective in the prevention of ischemic compli- cations in patients undergoing high-risk coronary angio- plasty or atherectomy [14,15]. Our previous studies showed that SZ-2 is a potent inhibitor of ristocetin-induced, vWF- dependent platelet agglutination [6] and thus has potential in treatment of thrombotic diseases. Administration of mouse MoAb into human patients, however, creates undesirable side effects. First is the anti-mouse Ab immune response in human patients treated with a mouse antibody [16]. Second is complement-mediated lysis of platelets caused by the Fc region of the antibody, which leads to thrombocytopenia [17]. The use of a smaller size scFv has some advantages over MoAb due to the reduced side effects and improved tissue penetration [18]. The variable region (Fv) of an antibody is the smallest antibody fragment containing a complete antigen-binding site and comprises the antibody VH and VL domains [19]. scFv proteins are consisted of the VH and VL domains of an antibody covalently joined by an engineered polypeptide linker [8,9].Comparedwiththe cross-linking monomeric form of the parental antibody, Fig. 6. (A) Effect of SZ-2 scFv on ristocetin (1.25 mg/ml) induced platelet scFv is monovalent. However, it usually has the same agglutination. (a) Platelet-rich plasma pretreated with buffer (0.15 M NaCl, binding conformation, specificity and affinity, so it retains 50 mM Tris–HCl); (b) platelet-rich plasma pretreated with SZ-2 MoAb (10 the same effects of the parental antibody [10,11,19]. scFv Ag/ml); (c) platelet-rich plasma pretreated with SZ-2 scFv (10 Ag/ml). (B) antibody fragments can also be customized to suit down- Effect of SZ-2 scFv on thrombin (0.05 U/ml) induced platelet aggregation. (a) Washed platelets pretreated with buffer (0.15 M NaCl, 50 mM Tris– stream applications in immunotherapy and immunodiagno- HCl); (b) washed platelets pretreated with SZ-2 MoAb(10 Ag/ml); (c) sis through development of bifunctional fusion protein [20]. washed platelets pretreated with SZ-2 scFv (10 Ag/ml). These proteins can be produced in bacteria in large amounts 中国科技论文在线 http://www.paper.edu.cn K. Dai et al. / Thrombosis Research 109 (2003) 137–144 143

in a short time at a low cost and can be highly purified by References affinity chromatography thus providing an ideal situation for drug preparation on a larger scale [21]. [1] Savage B, Saldivar E, Ruggeri ZM. Initiation of platelet adhesion by In the present study, we constructed from SZ-2 hybridoma arrest onto fibrinogen or translocation on von Willebrand factor. Cell 1996;84:289–97. cell lines the SZ-2 scFv recombinant antibody gene, which [2] Marchese P, Saldivar E, Ware J, Ruggeri ZM. Adhesive properties of was successfully engineered and expressed in E. coli. The the isolated amino-terminal domain of platelet glycoprotein Iba in a recombinant antibody was purified to homogeneity and flow field. Proc Natl Acad Sci U S A 1999;96:7837–42. examined for antigen reactivities. Like the native MoAb from [3] Bouton MC, Thurieau C, Guillin MC, Jandrot-Perrus M. Characteristic which it was derived, the recombinant scFv antibody showed of the interaction between thrombin exosite1 and the sequence 269– binding activity and specificity for platelet GPIb antigen. To 297 of platelet glycoprotein Iba. Thromb Haemost 1998;80:310–5. [4] Goto S, Ikeda Y, Salvidar E, Ruggeri ZM. Distinct mechanisms of increase the probability of producing functional scFv anti- platelet aggregation as a consequence of different shearing flow con- body, we have made our scFv in the VH–VL rather than in the ditions. J Clin Invest 1998;101:479–86. VL–VH arrangement. Although reports indicate that scFvs [5] Ruggeri ZM. Mechanisms initiating platelet thrombus formation. with VH-linker-VL orientation could not be expressed Thromb Haemost 1997;78:611–6. [22,23], we obtained a relatively high yield. The recombi- [6] Ruan C, Du X, Xi X, Castaldi PA, Berndt MC. A murine antigly- coprotein Ib complex monoclonal antibody, SZ-2, inhibits platelet nant-engineered molecule was similar to parental MoAb in aggregation induced by both ristocetin and collagen. Blood 1987;69: binding capacity in ELISA. In aggregation studies, SZ-2 scFv 570–7. was potent in inhibiting platelet agglutination and aggrega- [7] Ward CM, Andres RK, Smith AI, Berndt MC. Mocarhagin, a novel tion induced by ristocetin and thrombin, respectively, but had cobra venom metalloproteinase, cleaves the platelet von Willebrand no effect on ADP-induced platelet aggregation. The success- factor receptor glycoprotein Iba. Identification of the sulfated site ful production of SZ-2 scFv against platelet GPIba opens a for von Willebrand factor and a-thrombin. Biochemistry 1996;35: 4929–38. new avenue to explore antithrombotic agents. Many of the [8] Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee currently investigated antithrombotic drugs interfere with the SM, et al. Single chain antigen binding proteins. Science 1988;242: process of platelet aggregation. However, the inhibition of 423–6. platelet adhesion may be an important and efficient process [9] Todorovska A, Roovers RC, Dolezal O, Kortt AA, Hoogenboom HR, for preventing platelet-rich thrombus formation in damaged Hudson PJ. Design and application of diabodies, triabodies and tetra- bodies for cancer targeting. J Immunol Methods 2001;248:47–66. vessels with high shear rate [24]. The human platelet GPIb– [10] Xu B, Kriangkum J, Nagata LP, Fulton RE, Suresh MR. A single IX complex is a key membrane receptor mediating both chain Fv specific against western equine encephalitis virus. Hybrid- platelet function and reactivity [7]. 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