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• W1983700936 abstract "SummaryBackground and objectives: Thrombin activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) play important roles in fibrinolysis. Both reduce plasmin generation, but they exert their antifibrinolytic effects via different mechanisms. This study reports the cloning and characterization of a heterodimer diabody that inhibits TAFI and PAI-1 simultaneously. Methods and results: The diabody was derived from two inhibiting monoclonal antibodies, i.e. MA-33H1F7, an anti-PAI-1 antibody that induces non-inhibitory substrate behavior of PAI-1, and MA-T12D11, an anti-TAFI antibody that inhibits activation of TAFI by the thrombin–thrombomodulin complex. A single-chain variable fragment (scFv) was derived from MA-T12D11 that displayed slightly reduced binding and inhibitory properties as compared to MA-T12D11. Characterization of the diabody revealed a similar affinity for TAFI and PAI-1 as that of the parental antibodies. Furthermore, the inhibitory properties of MA-33H1F7 and MA-T12D11 were fully preserved in the diabody format. In platelet-free plasma (PFP) clots, addition of the diabody had a stronger effect in shortening lysis times than either MA-T12D11 or MA-33H1F7. A similar reduction in clot lysis time was observed in platelet-rich plasma (PRP) clots. The same effect on clot lysis times in PFP and PRP was also achieved by the combined addition of MA-T12D11 and MA-33H1F7. The lysis rate of human model thrombi, made from whole blood, was approximately doubled after addition of the diabody. Moreover, this effect was significantly better than after the combined addition of the individual antibodies. Conclusions: These observations demonstrate that simultaneous inhibition of TAFI and PAI-1 results in faster lysis of the formed thrombus. Background and objectives: Thrombin activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) play important roles in fibrinolysis. Both reduce plasmin generation, but they exert their antifibrinolytic effects via different mechanisms. This study reports the cloning and characterization of a heterodimer diabody that inhibits TAFI and PAI-1 simultaneously. Methods and results: The diabody was derived from two inhibiting monoclonal antibodies, i.e. MA-33H1F7, an anti-PAI-1 antibody that induces non-inhibitory substrate behavior of PAI-1, and MA-T12D11, an anti-TAFI antibody that inhibits activation of TAFI by the thrombin–thrombomodulin complex. A single-chain variable fragment (scFv) was derived from MA-T12D11 that displayed slightly reduced binding and inhibitory properties as compared to MA-T12D11. Characterization of the diabody revealed a similar affinity for TAFI and PAI-1 as that of the parental antibodies. Furthermore, the inhibitory properties of MA-33H1F7 and MA-T12D11 were fully preserved in the diabody format. In platelet-free plasma (PFP) clots, addition of the diabody had a stronger effect in shortening lysis times than either MA-T12D11 or MA-33H1F7. A similar reduction in clot lysis time was observed in platelet-rich plasma (PRP) clots. The same effect on clot lysis times in PFP and PRP was also achieved by the combined addition of MA-T12D11 and MA-33H1F7. The lysis rate of human model thrombi, made from whole blood, was approximately doubled after addition of the diabody. Moreover, this effect was significantly better than after the combined addition of the individual antibodies. Conclusions: These observations demonstrate that simultaneous inhibition of TAFI and PAI-1 results in faster lysis of the formed thrombus. The fibrinolytic system is initiated after the formation of fibrin, when both plasminogen and tissue-type plasminogen activator (t-PA) bind to C-terminal lysine residues on the fibrin surface. Plasminogen activator inhibitor-1 (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI) are (together with α2-antiplasmin) key inhibitors of the fibrinolytic system. PAI-1, a 50-kDa glycoprotein, is a member of the superfamily of serine proteinase inhibitors and is the primary inhibitor of both t-PA and urokinase-type plasminogen activator (u-PA) [1Pannekoek H. Veerman H. Lambers H. Diergaarde P. Verweij C.L. Van Zonneveld A.J. Van Moerik J.A. Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family.EMBO J. 1986; 5: 2539-44Crossref PubMed Scopus (0) Google Scholar, 2Lijnen H.R. Collen D. Mechanisms of physiological fibrinolysis.Baillieres Clin Haematol. 1995; 8: 277-90Abstract Full Text PDF PubMed Scopus (142) Google Scholar]. In its active form, PAI-1 controls t-PA activity through the rapid formation of an inactive, equimolar PAI-1–t-PA complex [3Kruithof E.K. Tran Thang C. Ransijn A. Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma.Blood. 1984; 64: 907-13Crossref PubMed Google Scholar, 4Thorsen S. Philips M. Selmer J. Lecander I. Astedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2.Eur J Biochem. 1988; 175: 33-9Crossref PubMed Google Scholar]. The active form of PAI-1 is unstable (half-life 1–2 h at 37 °C) and converts spontaneously into a non-inhibitory latent form [5Hekman C.M. Loskutoff D.J. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants.J Biol Chem. 1985; 260: 11581-7Abstract Full Text PDF PubMed Google Scholar]. A third conformation, the non-inhibitory substrate form, interacts with t-PA and u-PA, resulting in the cleavage and irreversible inactivation of PAI-1 and the regeneration of the proteinase activity [6Declerck P.J. De Mol M. Vaughan D.E. Collen D. Identification of a conformationally distinct form of plasminogen activator inhibitor-1, acting as a noninhibitory substrate for tissue-type plasminogen activator.J Biol Chem. 1992; 267: 11693-6Abstract Full Text PDF PubMed Google Scholar, 7Urano T. Strandberg L. Johansson L.B. Ny T. A substrate-like form of plasminogen-activator-inhibitor type 1. Conversions between different forms by sodium dodecyl sulphate.Eur J Biochem. 1992; 209: 985-92Crossref PubMed Scopus (0) Google Scholar, 8Munch M. Heegaard C.W. Andreasen P.A. Interconversions between active, inert and substrate forms of denatured/refolded type-1 plasminogen activator inhibitor.Biochim Biophys Acta. 1993; 1202: 29-37Crossref PubMed Scopus (0) Google Scholar]. TAFI is a 55-kDa plasma zymogen that is activated by either plasmin, thrombin or the thrombin–thrombomodulin (T–TM) complex into an active carboxypeptidase, TAFIa [9Bajzar L. Manuel R. Nesheim M.E. Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor.J Biol Chem. 1995; 270: 14477-84Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar]. During the partial degradation of fibrin clots by plasmin, new C-terminal lysine residues are generated that further stimulate plasmin formation [10Fleury V. Angles-Cano E. Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines.Biochemistry. 1991; 30: 7630-8Crossref PubMed Google Scholar]. TAFIa removes these C-terminal lysines from fibrin, resulting in a decreased rate of plasmin generation and thus downregulation of fibrinolysis [11Wang W. Boffa M.B. Bajzar L. Walker J.B. Nesheim M.E. A study of the mechanism of inhibition of fibrinolysis by activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 1998; 273: 27176-81Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar]. This mode of action is in clear contrast to the working mechanism of PAI-1, which inhibits t-PA by direct interaction. Increased levels of PAI-1 activity resulting in a decreased fibrinolytic capacity have been reported in several thrombotic disease states, including venous thromboembolism, coronary artery disease and acute myocardial infarction [12Lijnen H.R. Pleiotropic functions of plasminogen activator inhibitor-1.J Thromb Haemost. 2005; 3: 35-45Crossref PubMed Scopus (263) Google Scholar]. In humans, a positive correlation was found between TAFI levels and the risk for coronary artery disease, venous thrombosis and angina pectoris [13Silveira A. Schatteman K. Goossens F. Moor E. Scharpe S. Stromqvist M. Hamsten A. Plasma procarboxypeptidase U in men with symptomatic coronary artery disease.Thromb Haemost. 2000; 84: 364-8Crossref PubMed Scopus (156) Google Scholar, 14Van-Tilburg N.H. Rosendaal F.R. Bertina R.M. Thrombin activatable fibrinolysis inhibitor and the risk for deep vein thrombosis.Blood. 2000; 95: 2855-9Crossref PubMed Google Scholar]. Several strategies to inhibit PAI-1 and TAFI are currently being explored. PAI-1 inhibitors include inhibiting peptides, low molecular weight compounds and antisense oligonucleotides blocking PAI-1 synthesis [15Gils A. Declerck P.J. The structural basis for the pathophysiological relevance of PAI-I in cardiovascular diseases and the development of potential PAI-I inhibitors.Thromb Haemost. 2004; 91: 425-37Crossref PubMed Scopus (0) Google Scholar]. Activated TAFI is (relatively non-specifically) inhibited by chelating agents, by compounds that interfere with the disulfide bridges, by small synthetic compounds and by naturally occurring metallocarboxypeptidase inhibitors (e.g. potato tuber carboxypeptidase inhibitor) [16Leurs J. Hendriks D. Carboxypeptidase U (TAFIa): a metallocarboxypeptidase with a distinct role in haemostasis and a possible risk factor for thrombotic disease.Thromb Haemost. 2005; 94: 471-87Crossref PubMed Scopus (0) Google Scholar, 17Wang Y.X. Zhao L. Nagashima M. Vincelette J. Sukovich D. Li W. Subramanyam B. Yuan S. Emayan K. Islam I. Hrvatin P. Bryant J. Light D.R. Vergona R. Morser J. Buckman B.O. A novel inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) - part I: pharmacological characterization.Thromb Haemost. 2007; 97: 45-53Crossref PubMed Scopus (0) Google Scholar]. Furthermore, PAI-1 and TAFI can be inhibited by inhibitory monoclonal antibodies (mAbs) [15Gils A. Declerck P.J. The structural basis for the pathophysiological relevance of PAI-I in cardiovascular diseases and the development of potential PAI-I inhibitors.Thromb Haemost. 2004; 91: 425-37Crossref PubMed Scopus (0) Google Scholar, 18Gils A. Ceresa E. Macovei A.M. Marx P.F. Peeters M. Compernolle G. Declerck P.J. Modulation of TAFI function through different pathways-implications for the development of TAFI inhibitors.J Thromb Haemost. 2005; 3: 2745-53Crossref PubMed Scopus (0) Google Scholar, 19Binette T.M. Taylor Jr, F.B. Peer G. Bajzar L. Thrombin–thrombomodulin connects coagulation and fibrinolysis: more than an in vitro phenomenon.Blood. 2007; 110: 3168-75Crossref PubMed Scopus (0) Google Scholar]. To circumvent the problem of immunogenicity of murine antibodies, derivatives of mAbs can be generated lacking the constant domains. One of the most widely used monovalent antibody fragments is the single-chain variable fragment (scFv), which consists of the variable domains of the heavy (VH) and light (VL) chain linked through a flexible peptide linker (usually (Gly4Ser)3). Given their important role in fibrinolysis, simultaneous inhibition of TAFI and PAI-1 could be a new strategy to improve thrombolytic therapy. The aim of this study was to target TAFI and PAI-1 by one single compound with a dual activity, which constitutes a major advantage over the simultaneous administration of two different antibodies, each with a single inhibitory activity. Therefore, we generated a bispecific heterodimer diabody consisting of the scFvs derived from two inhibitory mAbs against PAI-1 and TAFI (Fig. 1). The diabody was characterized, and its in vitro profibrinolytic effect was evaluated in both clot lysis and thrombus lysis. Recombinant TAFI-Ala147Thr325 (TAFI-AT), recombinant PAI-1, MA-T12D11 and MA-T30E5A2 raised against human plasma-derived TAFI, and MA-33H1F7 and MA-31C9 raised against human PAI-1, were generated as described previously [20Gils A. Alessi M.C. Brouwers E. Peeters M. Marx P. Leurs J. Bouma B. Hendriks D. Juhan-Vague I. Declerck P.J. Development of a genotype 325-specific proCPU/TAFI ELISA.Arterioscler Thromb Vasc Biol. 2003; 23: 1122-7Crossref PubMed Scopus (0) Google Scholar, 21Debrock S. Declerck P.J. Neutralization of plasminogen activator inhibitor-1 inhibitory properties: identification of two different mechanisms.Biochim Biophys Acta. 1997; 1337: 257-66Crossref PubMed Scopus (78) Google Scholar]. All experiments in this study were performed with TAFI-AT. Oligonucleotides used for cloning and sequencing were synthesized by Sigma-Aldrich (St Louis, MO, USA), Pfx50 DNA polymerase was purchased from Invitrogen (Merelbeke, Belgium) and restriction enzymes were provided by New England Biolabs (Hertfordshire, UK). Polymerase chain reaction reactions were carried out with the Mastercycler Gradient from Eppendorf (Hamburg, Germany). Plasmid DNA purification was performed with the Nucleobond AX500 kit (Machery-Nagel, Düren, Germany) or the Plasmid mini kit I (Omega Bio-Tek, Doraville, GA, USA). DNA was sequenced with an ABI Prism 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Human thrombin, rabbit thrombomodulin and H-d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone were obtained from Sigma-Aldrich, American Diagnostica (Greenwich, CT, USA) and Biomol Research Labs (Plymouth Meeting, PA, USA), respectively. Hippuryl-l-arginine was obtained from Calbiochem (La Jolla, CA, USA). Tissue-type plasminogen activator was obtained from Genentech (San Francisco, CA, USA). Chromogenic substrate S2403 was purchased from Nodia/Chromogenix (Antwerp, Belgium). Horseradish peroxidase-conjugated goat anti-mouse antibody was obtained from Bio-Rad (Hercules, CA, USA). For all cloning steps, Escherichia coli strain TOP 10F′ (Invitrogen) was used. Expression plasmid pSKID2 was a kind gift from M. Little (Affimed) [22Cochlovius B. Kipriyanov S.M. Stassar M.J. Christ O. Schuhmacher J. Strauss G. Moldenhauer G. Little M. Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells.J Immunol. 2000; 165: 888-95Crossref PubMed Google Scholar]. ScFv-T12D11 was generated in a similar way as described previously [23Verbeke K. Gils A. Declerck P.J. Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs.J Thromb Haemost. 2004; 2: 298-305Crossref PubMed Scopus (0) Google Scholar]. A glutamic acid at position 6 of the VH region (according to Kabat numbering) was mutated to glutamine, and the cDNA encoding scFv-T12D11 was further ligated into a modified pSKID2 vector. Therefore, an XbaI site was introduced at the 5′-end of the VH region and an AflII site at the 3′-end of the VL region, and this was followed by ligation in an XbaI/AflII-linearized plasmid, pSKID2 (Fig. S1). After transformation of scFv-T12D11 and the diabody into E. coli K12 strain RV308 (LGC Promochem, Teddington, UK), scFv and diabody were expressed as described previously [22Cochlovius B. Kipriyanov S.M. Stassar M.J. Christ O. Schuhmacher J. Strauss G. Moldenhauer G. Little M. Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells.J Immunol. 2000; 165: 888-95Crossref PubMed Google Scholar]. The periplasmic extract was passed through a filter of pore size 0.2 μm and dialyzed against 20 mmol L−1 Tris–HCl and 0.5 mol L−1 NaCl (pH 7.9), and purified as described previously [18Gils A. Ceresa E. Macovei A.M. Marx P.F. Peeters M. Compernolle G. Declerck P.J. Modulation of TAFI function through different pathways-implications for the development of TAFI inhibitors.J Thromb Haemost. 2005; 3: 2745-53Crossref PubMed Scopus (0) Google Scholar]. Affinity constants for the binding of the mAbs, scFv-T12D11 and the diabody to PAI-1 and/or TAFI were determined by surface plasmon resonance using a Biacore 3000 analytical system equipped with the CM5 sensor chip (Biacore AB, Uppsala, Sweden) as described previously [18Gils A. Ceresa E. Macovei A.M. Marx P.F. Peeters M. Compernolle G. Declerck P.J. Modulation of TAFI function through different pathways-implications for the development of TAFI inhibitors.J Thromb Haemost. 2005; 3: 2745-53Crossref PubMed Scopus (0) Google Scholar]. The mAbs, scFv or diabody were covalently coupled to the CM5 sensor chip up to 1500 resonance units (using a concentration of 10 μg mL−1 in 10 mmol L−1 acetate buffer, pH 4.5). PAI-1 and TAFI were injected (40 μL) at concentrations between 10 and 100 nmol L−1 at a flow rate of 30 μL min−1. Dissociation was allowed for 6 min. Ten microliters of a 10 mmol L−1 HCl solution were used to regenerate the chip after each cycle. Association and dissociation rate constants were calculated with the software of the Biacore 3000 (Langmuir binding model). The activity of TAFIa was measured as described previously [24Mosnier L.O. Von-dem-Borne P.A. Meijers J.C. Bouma B.N. Plasma TAFI levels influence the clot lysis time in healthy individuals in the presence of an intact intrinsic pathway of coagulation.Thromb Haemost. 1998; 80: 829-35Crossref PubMed Google Scholar]. To determine the overall inhibitory effect of the antibody (derivative) on TAFI activation by T–TM, purified TAFI was incubated with a 0.5–8-fold molar ratio of MA-T12D11, scFv-T12D11 or diabody as described previously [18Gils A. Ceresa E. Macovei A.M. Marx P.F. Peeters M. Compernolle G. Declerck P.J. Modulation of TAFI function through different pathways-implications for the development of TAFI inhibitors.J Thromb Haemost. 2005; 3: 2745-53Crossref PubMed Scopus (0) Google Scholar]. The percentage inhibition of TAFI activation was calculated relative to TAFI activated by T–TM in the absence of antibody (derivative) (= 100% activation). The functional properties of MA-33H1F7 and diabody were determined by assessing their ability to inhibit active PAI-1. PAI-1 activity was determined using a plasminogen-coupled chromogenic method described by Verheijen et al. [25Verheijen J.H. Chang G.T. Kluft C. Evidence for the occurrence of a fast-acting inhibitor for tissue-type plasminogen activator in human plasma.Thromb Haemost. 1984; 51: 392-5Crossref PubMed Scopus (0) Google Scholar]. A range of molar ratio of antibody or diabody over PAI-1 between 1 and 128 was tested, as described previously [23Verbeke K. Gils A. Declerck P.J. Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs.J Thromb Haemost. 2004; 2: 298-305Crossref PubMed Scopus (0) Google Scholar]. Blood and plasma samples were prepared as described previously [26Mutch N.J. Thomas L. Moore N.R. Lisiak K.M. Booth N.A. TAFIa, PAI-1 and alpha-antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots.J Thromb Haemost. 2007; 5: 812-17Crossref PubMed Scopus (91) Google Scholar]. Clot lysis was performed as described previously, with some modifications [26Mutch N.J. Thomas L. Moore N.R. Lisiak K.M. Booth N.A. TAFIa, PAI-1 and alpha-antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots.J Thromb Haemost. 2007; 5: 812-17Crossref PubMed Scopus (91) Google Scholar]. Pooled normal plasma in the presence and absence of antibody or diabody was incubated for 10 min at 37 °C, and t-PA was then added (final volume of 200 μL). Aliquots from each tube (80 μL) were added in duplicate to microtiter wells, each containing 20 μL of CaCl2, to give the following final concentrations per well: 30% plasma; 10.6 mmol L−1 CaCl2; 180 pmol L−1 t-PA; +/− 65 μg mL−1 MA-33H1F7 and/or 65 μg mL−1 MA-T12D11; and +/− 25 μg mL−1 diabody. These final concentrations of the antibodies and diabody resulted in an 8-fold molar excess over TAFI, assuming a concentration of 10 μg mL−1 TAFI in the plasma pool and using molecular masses of antibody and diabody of 150 and 58 kDa, respectively. The plate was incubated at 37 °C and read at 405 nm at 5-min intervals to determine the 50% clot lysis time. Thrombus lysis was performed as described previously [26Mutch N.J. Thomas L. Moore N.R. Lisiak K.M. Booth N.A. TAFIa, PAI-1 and alpha-antiplasmin: complementary roles in regulating lysis of thrombi and plasma clots.J Thromb Haemost. 2007; 5: 812-17Crossref PubMed Scopus (91) Google Scholar]. Antibody (derivatives) were incorporated both into the blood, prior to thrombus formation, and into the bathing plasma, resulting in the following final concentrations: +/− 65 μg mL−1 MA-33H1F7 and/or 65 μg mL−1 MA-T12D11; +/− 65 μg mL−1 MA-31C9; +/− 65 μg mL−1 MA-T30E5A2; and +/− 25 μg mL−1 diabody. Each antibody (derivative) was evaluated three times per blood donor. Quantitative data were summarized by the mean and standard deviation. Statistical analyses were performed with Graph Pad Prism 4.01 with the unpaired t-test (Graph Pad Software, Inc., San Diego, CA, USA). P-values < 0.05 were considered to be statistically significant. Dose–response curves were fitted to one-site binding hyperbolas. The cDNA encoding the VH and VL regions of MA-T12D11 (Fig. S1) was linked by a 15-residue linker [(Gly4Ser)3] and cloned into the pCANTAB5E vector. The expression yield of scFv-T12D11 in this vector was very low. Therefore, glutamic acid at position 6 of the VH region was mutated into glutamine, as this substitution has been shown to give increased yields of scFvs [27Kipriyanov S.M. Moldenhauer G. Martin A.C. Kupriyanova O.A. Little M. Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity.Protein Eng. 1997; 10: 445-53Crossref PubMed Google Scholar]. The expression yield increased (< 0.050 mg L−1 cultured cells, data not shown), but was still low. Therefore, scFv-T12D11 was ligated into the pSKID2 vector (Fig. S1) [22Cochlovius B. Kipriyanov S.M. Stassar M.J. Christ O. Schuhmacher J. Strauss G. Moldenhauer G. Little M. Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells.J Immunol. 2000; 165: 888-95Crossref PubMed Google Scholar]. This vector contains a gene encoding the periplasmic factor Skp/OmpH, which increases the functional yield of antibody fragments in bacteria [28Bothmann H. Pluckthun A. Selection for a periplasmic factor improving phage display and functional periplasmic expression.Nat Biotechnol. 1998; 16: 376-80Crossref PubMed Scopus (0) Google Scholar]. After expression in the modified pSKID2 vector, approximately 1 mg of scFv-T12D11 per liter of bacterial culture was obtained after purification. The affinity constant of scFv-T12D11 was only slightly decreased as compared to the parental MA-T12D11 (2.5-fold reduced affinity, Table 1). Functional analysis of scFv-T12D11 revealed that this antibody fragment was capable of inhibiting activation of TAFI by the T–TM complex in a dose-dependent manner, reaching virtually 100% inhibition at an 8-fold molar excess (Fig. 2). The IC50 value was slightly increased (17.6 ± 3.7 nmol L−1 vs. 3.8 ± 1.4 nmol L−1 for MA-T12D11), which is in line with the decreased affinity of scFv-T12D11 for TAFI.Table 1Binding parameters of scFv-T12D11, MA-T12D11, MA-33H1F7 and diabody ka, kd and KA for binding of the antibody (derivative) to TAFI and/or PAI-1 were determined using surface plasmon resonance (mean ± SD, n = 3).scFv-T12D11MA-T12D11MA-33H1F7DiabodyTAFIPAI-1TAFIPAI-1TAFIPAI-1TAFIPAI-1ka (m−1s−1)3.6 ± 2 × 105NA1.0 ± 0.3 × 106NANA1.6 ± 0.5 × 1065.6 ± 2 × 1051.3 ± 0.4 × 106kd (s−1)2.6 ± 0.8 × 10−4NA2.9 ± 0.2 × 10−4NANA1.6 ± 0.1 × 10−41.8 ± 0.8 × 10−41.7 ± 0.1 × 10−4KA (m−1)1.4 ± 0.5 × 109NA3.5 ± 1 × 109NANA9.9 ± 3 × 1093.1 ± 0.7 × 1097.7 ± 2 × 109scFv, single-chain variable fragment; TAFI, thrombin activatable fibrinolysis inhibitor; PAI-1, plasminogen activator inhibitor-1.NA, not applicable. Open table in a new tab scFv, single-chain variable fragment; TAFI, thrombin activatable fibrinolysis inhibitor; PAI-1, plasminogen activator inhibitor-1. NA, not applicable. On the basis of this information, we decided to use scFv-T12D11 to construct a bispecific heterodimer diabody, together with scFv-33H1F7. The latter has been characterized previously and displayed similar inhibitory properties towards PAI-1 as its parental antibody MA-33H1F7 [23Verbeke K. Gils A. Declerck P.J. Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs.J Thromb Haemost. 2004; 2: 298-305Crossref PubMed Scopus (0) Google Scholar]. The diabody was ligated successfully into the pSKID2 vector (Fig. S1) and was purified from the periplasmic extract with a yield of approximately 2 mg L−1 bacterial culture. The affinity of the diabody for TAFI and PAI-1 was similar to that of MA-T12D11 and MA-33H1F7 (Table 1). Simultaneous binding of the diabody to TAFI and PAI-1 was also evaluated in an enzyme-linked immunosorbent assay setup. The absorbance increased after addition of the diabody in a dose-dependent manner for both TAFI-coated and PAI-1-coated plates (Fig. S2). This proves that the diabody is capable of binding TAFI and PAI-1 simultaneously, although these results cannot be translated into a quantitative interpretation of the binding capacity of the diabody. The diabody could inhibit PAI-1 in a dose-dependent manner, and this effect was indistinguishable from that of the parental antibody (Fig. 3). The diabody and MA-33H1F7 both reached the same plateau of PAI-1 inhibition (33 ± 7% vs. 32 ± 7%, respectively). The diabody inhibited TAFI activation by the T–TM complex in a dose-dependent manner, reaching virtually 100% inhibition at an 8-fold molar excess. The IC50 value was slightly increased as compared to that of MA-T12D11 (10.4 ± 2.0 nmol L−1 vs. 3.8 ± 1.4 nmol L−1, respectively). To test the functional effects of the diabody in clot lysis and compare this effect with that of the antibodies, platelet-free plasma (PFP) as well as platelet-rich plasma (PRP) was preincubated with buffer, antibody or diabody. An 8-fold molar excess of antibody or diabody over TAFI was used, resulting in a final concentration of 65 μg mL−1 (antibodies) or 25 μg mL−1 (diabody). In the absence of any antibody (derivative), the 50% clot lysis time for PFP was 100 ± 11 min. Preincubation with MA-T12D11 or MA-33H1F7 shortened the clot lysis time to 78 ± 6 min and 78 ± 21 min, respectively, but only neutralization of TAFI activation by MA-T12D11 was significant (Table 2). Preincubation with the diabody resulted in an even stronger effect on 50% clot lysis time, comparable to the effect observed upon combined addition of the two antibodies (55 ± 5 min vs. 56 ± 8 min, respectively). The clot lysis time for PRP in absence of any antibody (derivative) was 176 ± 29 min. After addition of the antibodies, the same pattern was observed as for lysis of PFP clots, except that the reduction of 50% clot lysis time with MA-33H1F7 was now also significant (Table 2). Again, the strongest effect was achieved when plasma was preincubated with the diabody or a combination of the two antibodies (55 ± 12 min vs. 56 ± 4 min, respectively).Table 2Lysis of 30% platelet-free plasma (PFP) clots or 30% platelet-rich plasma (PRP) clots by tissue-type plasminogen activator in the absence or presence of antibody or diabodyAntibody (derivative)PFP clot 50% lysis (min)PRP clot 50% lysis (min)No addition100 ± 11176 ± 29MA-T12D1178 ± 6**128 ± 35*MA-33H1F778 ± 21102 ± 15**MA-T12D11 + MA-33H1F756 ± 8***56 ± 4***Diabody55 ± 5***55 ± 12***Data shown are time to 50% lysis. Significance is shown relative to ‘no addition’. (mean ± SD, n = 5).*P≤0.05; **P≤0.01; ***P≤0.005. Open table in a new tab Data shown are time to 50% lysis. Significance is shown relative to ‘no addition’. (mean ± SD, n = 5). *P≤0.05; **P≤0.01; ***P≤0.005. The profibrinolytic effect of the diabody was also tested in human model thrombi in order to allow assessment of cell and platelet contributions [29Robbie L.A. Young S.P. Bennett B. Booth N.A. Thrombi formed in a Chandler loop mimic human arterial thrombi in structure and pai 1 content and distribution.Thromb Haemost. 1997; 77: 510-15Crossref PubMed Scopus (0) Google Scholar]. Antibodies towards TAFI or PAI-1 or the diabody were incorporated in forming thrombi and bathing fluid (i.e. 30% plasma), using the same concentrations as for the clot lysis assay. Two other antibodies towards human TAFI (i.e. MA-T30E5A2) and human PAI-1 (i.e. MA-31C9) were included as a negative control. These antibodies specifically bind their target, but do not display any inhibitory properties. Figure 4 shows the effects of the different antibodies (and derivatives) on the lysis rate of model thrombi, and represents the mean of three different blood donors. Inhibition of TAFI and PAI-1 by the single antibodies resulted in a slightly increased lysis rate as compared to the negative control antibodies, but this effect was not significant (Table 3). Combined addition of these two antibodies resulted in significantly increased lysis. The most pronounced effect was achieved by addition of the diabody, causing a 2-fold increase in lysis rate. Strikingly, the increase in lysis rate caused by the diabody was significantly greater than the increase observed with combined addition of the two monoclonal antibodies (P=0.019). Thus, data from clot lysis and model thrombi show that simultaneous inhibition of TAFI and PAI-1 results in faster lysis of the formed clot.Table 3Lysis of model thrombi bathed in 30% plasma by 15 nm tissue-type plasminogen activator in the absence or presence of antibody or diabodyAntibody (derivative)FU min−1Fold increaseMA-T30E5A2*1.6 ± 0.2NAMA-31C9*1.6 ± 0.3NAMA-T12D112.0 ± 0.2†1.2 ± 0.1MA-33H1F71.9 ± 0.4†1.2 ± 0.2MA-T12D11" @default.
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• W1983700936 title "Bispecific targeting of thrombin activatable fibrinolysis inhibitor and plasminogen activator inhibitor-1 by a heterodimer diabody" @default.
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