Conjugation and evaluation of 7E3 x P4B6, a chemically crosslinked

P4B6, a chemically crosslinked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the p...
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Blocon/ugate Chem. lQQ2,3, 120-131

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Conjugation and Evaluation of 7E3 X P4B6, a Chemically Cross-Linked Bispecific F(ab’)2 Antibody Which Inhibits Platelet Aggregation and Localizes Tissue Plasminogen Activator to the Platelet Surface Donald S. Neblock,’ Chien-Hsing Chang, Mary Ann Mascelli, Melanie Fleek, Lisa Stumpo, Mary Margaret Cullen, and Peter E. Daddona Department of Immunobiology, Centocor Inc., 200 Great Valley Parkway, Malvern Pennsylvania 19355. Received October 7, 1991

A bispecific F(ab’)z monoclonal antibody which recognizes both the platelet GPIIb/IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis. A stable, thioether-cross-linked bispecific F(ab’)z (7E3 X P4B6) combining the GPIIb/IIIaspecific monoclonal antibody 7E3, which inhibits platelet aggregation, and a nonneutralizing anti-tPA monoclonal antibody (P4B6) was produced. This was performed by coupling each of the parental Fab’ moieties with the homobifunctional cross-linker bis(maleimid0 methyl) ether (BMME). 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction (HIC) HPLC. HIC was shown to completely resolve each of the parental F(ab’)z species from the bispecific one. 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in GPIIb/IIIa and tPA binding EIA’s. The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab. Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251chromogenic substrate assay. 7E3 X P4B6 recruited 12-foldmore tPA to the washed platelets than a mixture of the parental F(ab’)z molecules used as controls.

INTRODUCTION

Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA’) has received widespread clinical use in the treatment of acute myocardial infarction, significantly reducing associated mortality and morbidity (Collen, 1990). Despite the successful use of this thrombolytic agent in the clinic, there are still problems associated with its use. Short serum half-life (requiring high doses and prolonged infusions), serious nonspecific hemorrhagic complications, and risk of reocclusion are among these unresolved problems (Verstaete, 1990). These issues have promoted interest in the development of third-generation plasminogen activators, including forms of tPA modified via recombinant DNA technology to have longer circulating half-lives, and the production of novel antibody-plasminogen activator hybrid molecules and immunoconjugates aimed at increasing the thrombus specificityof tPA and other plasminogen activators (Haber et al., 1989;Dewerchin & Coller, 1991;Hayzer et al., 1991). In addition, the combined use of thrombolytics with potent inhibitors of platelet aggregation is seen as an approach to the reduction of the time to reperfusion and frequency of coronary vessel reocclusion currently accompanying thrombolytic therapy (Coller, 1990).

* Author to whom all correspondence should be addressed.

1 Abbreviationsused: 7E3, murine IgGl anti-GPIIb/IIIa monoclonal antibody; P4B6, murine IgGl anti-tPA monoclonal antibody; 7E3 X P4B6, chemically cross-linked bispecific antiGPIIb/IIIa anti-tPA (F(ab’)z;s-2251,H-D-valyl-L-leucyl-L-lysinep-nitroanilide dihydrochloride;BMME, bis(maleimidomethy1)ether; rtPA, recombinant human tissue-type plasminogen activator; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;Tris,tris(hydroxymethyl)aminomethane; TBS, Tris-buffered saline; PBS, phosphate-buffered saline; DTT, dithiothreitol; DTNB, 5,5’-dithio-bis(2-nitrobenzoic acid);EDTA, ethylenediaminetetraaceticacid; HPLC, high-performanceliquid chromatography; EIA, enzyme immunosorbant assay; OPD, ophenylenediamine;NEM, N-ethylmaleimide;PRP, platelet-rich plasma.

Antibody targeting of tPA and other plasminogen activators to thrombi via a high-affinity monoclonal antibody directed against the amino terminus of the pchain of fibrin has been the topic of recent research which demonstrated an increase rate of fibrinolysis in vitro and in vivo for a number of plasminogen activator-antifibrin antibody conjugates (Bode et al., 1985;Runge et al., 1987) and bispecific antibodies (Runge et al., 1990; Bode et al., 1989;Runge et al., 1987;Runge et al., 1988a,b). As a major component of arterial thrombi, platelets provide an additional target for the antibody recruitment of tPA. This may be especially useful in the case of thrombolysisresistant platelet-rich arterial thrombi occurring in acute myocardial infarction or during acute reocclusion subsequent to thrombolytic therapy (Yasuda et al., 1990; Gold & Leinbach, 1987). As one approach, targeting to activation-specific platelet antigens would direct a plateletspecific thrombolytic agent only to platelet-rich thrombi, circumventing binding to circulating platelets. This approach has been described recently for immunoconjugates of recombinant single-chain urokinase-type plasminogen activator (Dewerchin et al., 1991). However, the use of a potent inhibitor of platelet aggregation which does bind to circulating resting platelets as the fibrinolysis targeting antibody, such as the anti-GPIIb/IIIa monoclonal antibody 7E3, might yield additional benefits in terms of preventing reocclusion as well as hastening reperfusion. Experimentally, this concept was demonstrated recently by acceleration of in vitro platelet-rich clot lysis and inhibition of platelet aggregation with a 7E3-urokinase immunoconjugate (Bode et al., 1991). Toward this end, targeting of tPA to platelets via a bispecific antibody comprised of 7E3 and an anti-tPA monoclonal antibody would combine potent inhibition of platelet aggregation in vivo via the 7E3 half of the molecule (Coller et al., 1988; Gold et al., 1987; Gold et al., 1990) with the recruitment of the plasminogen activator to both the platelet-rich thrombus and circulating platelets. The potential advantages of this approach over currently available or next0 1992 American Chemlcal Society

F(ab’)n Antibody Speclflc for GPIIb/IIIa and rtPA

generation thrombolytics may include increased specificity for platelet-rich thrombi, recruitment of endogenous and pharmacological tissue plasminogen activator, increase of avidity by combining platelet and fibrin binding specificities in one molecule, and combination of antiplatelet and thrombolytic functions in one agent. In the present study, a bispecific F(ab’)2,7E3X P4B6, recognizing both human tissue plasminogen activator as well as GPIIb/ IIIa, was prepared by chemical conjugation of the parental Fab’ antibodies via a nonreducible thioether cross-link and was biochemically and functionally characterized. A bispecific antibody with these dual specificitiesmight allow the targeting of both pharmacologicallyadministered rtPA and endogenously released tPA to circulating platelets and the platelet-rich thrombus in vivo. EXPERIMENTAL PROCEDURES

Recombinant tPA (Activase) was obtained from Genentech (San Francisco, CAI. BMME was purchased from Calbiochem. 5-2251, plasminogen, and fibrin fragments were purchased from Helena Laboratories (Beaumont, TX). All other chemicals were purchased from Sigma (St. Louis, MO). Electrophoresis. Phastaystem gels and supplies (Pharmacia, Piscataway, NJ) were used for all SDS-PAGE analyses. Samples of the BMME cross-linking steps were prepared for electrophoresis by incubating 1 volume of sample for 10 min on ice in the presence of 2 volumes of 200 mM N-ethylmaleimide, followed by dilution 1:2 in 2 X sample buffer (4% SDS, 20% glycerol, 0.250 M Tris-HC1 (pH 6.8), and 0.002% bromphenol blue) and heating for 3 min at 100 “C. Other samples were processed for SDSPAGE by dilution into 2X sample buffer and heating for 3 min at 100 “C in the presence or absence of 100 mM DTT. A mixture of prestained molecular weight standards consisting of myosin (200 kDa), phosphorylase B (97.4 ma),bovineserumalbumin (68kDa), ovalbumin (43kDa), carbonic anhydrase (29 kDa), &lactoglobulin (18.4 kDa), and lysozyme (14.3 kDa) was purchased from Bethesda Research Laboratories (Gaithersburg, MD). Monoclonal Antibodies. An IgGl mouse monoclonal antibody (7E3) specific for the glycoprotein GPIIb/IIIa fibrinogen receptor on platelets was produced by methods previously described (Coller, 1985). The P4B6 hybridoma was provided for research purposes by Dr. Desire Collen (Leuven, Belgium). The hybridoma secretes an IgGl which is specific for tPA. Purification and Characterization of Parental F(ab’)2Antibodies. 7E3 F(ab’)2 fragment was obtained from the pepsin digestion of 7E3 IgG as previously described (Yasuda et al., 1988). P4B6 IgG was purified from tissue culture supernatant fluid using protein A chromatography equilibrated in 1.5 M glycine and 3 M NaCl (pH 8.9). The IgG was eluted from the column by using a pH gradient in 100 mM sodium citrate buffer starting at pH 6.5 and ending at pH 3.5. The purified IgG was diafiltered into 100 mM sodium citrate a t pH 3.9 and digested in the presence of 2% (w/w) pepsin for 16 h at 37 “C. The F(ab’)2 fragment of P4B6 was purified by cation-exchange FPLC using a Mono S HR 10/10 column equilibrated in 50 mM sodium acetate a t pH 4.2. The bound F(ab’)a was eluted with a 0-1 M NaCl gradient under conditions which resolved undigested IgG and low molecular weight fragments from the F(ab’)2. Cross-Linking of Bispecific Antibodies. Immediately before cross-linking, 10 mg of each of the parental F(ab’)2 fragments was reduced to Fab’ fragments using a modification of previously described methods (Chang et al., 1986;Glennie et al., 1987). The fragments were buffer exchanged into 50 mM sodium borate, 100mM NaC1, and

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1mM EDTA (pH 8.0) and reduced with 20 mM L-cysteine (free base form) at 37 “Cfor 1h, yielding Fab’ fragments. Both Fab’ fragments were desalted on a separate 1.0 X 18 cm column of P6DG equilibrated with 50 mM ammonium citrate, 100 mM NaC1, and 1 mM EDTA (pH 6.3) (conjugation buffer). The P4B6 Fab’ was maintained as the free sulfhydryl form (Fab’-SH) while the 7E3 Fab’ was derivatized with BMME. The 7E3 Fab’ was added dropwise to an approximately 30-fold molar excess of 50 mM BMME in dimethylformamide with constant mixing and allowed to react at room temperature for 10 min. The 7E3-BMME derivative was desalted on a 1.8 X 10cm P6DG column equilibrated in conjugation buffer. The BMMEderivatized 7E3 Fab’ was reacted with P4B6 Fab’-SH at a 1:l molar ratio for 60 min at room temperature. Each of the reaction steps was analyzedafter reaction with excess NEM by Phast SDS-PAGE under nonreducing conditions to determine the efficiency of reduction and cross-linking. The reaction products were treated with 1mM DTNB to cap remaining sulfhydryl groups and stored at 4 “C until purification. Purification of Bispecific Antibodies. The reaction mixture containing the bispecific F(ab’)zwas concentrated by ultrafiltration using a 30 kDa cutoff membrane in a stirred cell and was applied to a column of Sephacryl S200 (1.6 X 90 cm) equilibrated in 10mM sodium phosphate and 150 mM NaCl (pH 7.2). The F(ab’)~pool of the 5-200 step was applied to a 7.5 X 75 mm Biogel TSK phenyl5-PW HPLC column for hydrophobic interaction chromatography (HIC). The HIC column was equilibrated with 100 mM sodium phosphate at pH 6.3 containing 1M (NH&S04. The bispecific F(ab’)n fragment was eluted with a 1-0 M ammonium sulfate gradient in the same phosphate buffer over a period of 55 min, followed by isocratic elution with 100 mM sodium phosphate for 15 min using a flow rate of 1 mL/min. The purified bispecific F(ab’)2 was biochemically characterized by gel filtration HPLC using a 9.6 X 250 mm Zorbax GF-250 column equilibrated in 200 mM sodium phosphate at pH 6.8, SDSPAGE under reducing and nonreducing conditions using a Phastsystem, and analytical HIC as described above. Native Reduction Analysis. The presence of the nonreducible cross-link between the parental heavy chains in the bispecific F(ab’)2 was demonstrated by reduction of the bispecific or the parental F(ab’)~species followed by gel-filtration HPLC. All samples were reduced at 37 final volume of 1 M “C for 1 h after the addition of Tris-HC1 (pH 8.0) and DTT to 1mM final concentration. After reduction, the samples were reacted with l/10 final volume of 50 mM NEM and analyzed by GF-HPLC for the presence of Fab’ or F(ab’)z as described previously. Immunoassays. The immunoreactivity of the parental antibodies and the 7E3 X P4B6 specific and control antibodies was quantified using EIA’s specific for GPIIb/ IIIa and tPA, respectively. Polystyrene 96-wellplates were coated with 100pL per well containing 5 pglmL of affinitypurified human platelet GPIIb/IIIa (Blum et al., 1989) in 100mMTris-HC1 (pH 9.5),1 mM CaC12,and 0.02 % sodium azide buffer for 1h at room temperature. The plates were washed in TBS containing 1mM CaClz and 0.05 % Tween80 and blocked in the same buffer containing 1% bovine serum albumin (BSA) for 1h a t room temperature. After washing, the plates were incubated in the presence of dilutions of the test antibodies for 1 h at room temperature, washed, and incubated with a 1:5000 dilution of affinity-purified goat anti-murine F(ab’)z conjugated to horseradish peroxidase (HRP). After the final washing, the plates were developed with 50 pL/well of 1.2 mg/mL OPD and 0.16% H202 in 20 mM sodium citrate and 50 mM sodium phosphate buffer at pH 5. The color was

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read at 490 nm. The format for the t P A EIA was identical to that for the GPIIb/IIIa EIA with the exceptionof coating the rtPA in 50 mM in sodium carbonate (pH 8.9) and performing all incubations in PBS containing 1%(w/v) bovine serum albumin diluent instead of the TBS/CaC12 used in the GPIIb/IIIa assay. The tPA was coated at 5 pg/mL. In both EIA’s, the color development in the bispecific F(ab’)2 was compared to the respective parental antibody as the Fab’ fragment and to nonrelevant control antibodies for the determination of a binding constant defined as the inverse of the concentration corresponding to half of the maximum signal. Platelet Aggregation. The inhibition of platelet aggregationby 7E3 and the 7E3 X P4B6 bispecific antibody was assayed using a two-channel aggregometer (Chronolog). Platelet-rich plasma (PRP) anticoagulated with 15% acidified citrate dextrose (ACD) solution was prepared according to standard centrifugal technique (Coller, 1979). Platelet counts were adjusted to 250000300 OOO per microliter with autologous platelet-poor plasma. Platelet aggregation studies were performed at 37 “C in siliconized glass cuvettes with continuousstirring. A stable baseline was established for each sample, followed by stimulation of the PRP with 10 pM ADP to initiate aggregation. The aggregationresponse in the presence or absence of test antibodies was measured as the rate of change in light transmittance in the platelet aggregometer for sample volumes of 0.5 mL. Test antibodies were preincubated with PRP at room temperature for 5 min prior to initiation of aggregation. tPA Activity Assay. The recruitment of rtPA to human platelets in vitro was assayed by incubation of citrated platelet-rich plasma in the presence of test antibodies and controls at 10 pg/mL for 1 h at room temperature. A total of 1 x lo6 platelets per well were then added to 96-well plates. The platelets were washed by centrifugation at 3000g for 10 min at room temperature and washed with TBS/CaC12. A 5 units/mL portion of recombinant human tPA (Activase) in TBS/CaC12 was added to each well and allowed to incubate for 1h at room temperature. The wells were washed with TBS and incubated for up to 2 h at 37 “C in the presence of S-2251, plasminogen, and fibrin fragments. The color resulting from the plasmin-mediated release of p-nitroaniline was quantified every 15 min in a plate reader set to 405-nm absorbance. The activity recruited to the platelets was compared to a standard curveof tPA in solution containing 10-0.125 units/mL incubated simultaneously in wells containing no platelets. RESULTS

Production of the Bispecific F(ab’)2. The bispecific F(ab’)2 cross-linking was monitored by nonreducing SDSPAGE as shown in Figure 1. The selective, complete reduction of the parental F(ab’)2 hinge-region disulfides by L-cysteine was indicated by the presence of the approximately40 kDa Fab’ band for either parent in these nonreducing gels (lanes 2 and 3), with no detectable 100 kDaF(ab’)2bands. The presence of low molecular weight bands in the Fab’s analyzed in overloaded nonreducing gels (not shown) indicated a low level of reduction of the H-L disulfides did occur. NEM capping of the Fab’ fragments prevented any possible reoxidation to larger fragments in these analyses. Spontaneous reoxidation of either parental Fab’ to F(ab’)2 after desalting into conjugation buffer was prevented by the maintenance of low pH and the presence of 1mM EDTA. Lane 4 depicts the BMME-derivatized 7E3 Fab’, demonstrating that the conjugation of BMME to 7E3 Fab’ had no effect on the molecular weight of the Fab’ and that no formation of

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Figure 1. Nonreducing SDS-PAGE of the BMME cross-linking of 7E3 and P4B6. 10-15% gradient Phastsystem SDS-PAGE (Pharmacia) was electrophoresed under nonreducing conditions according to the manufacturer’s instructions and manually stained with Coomasie Brilliant Blue R. Samples were prepared for electrophoresis by incubation on ice for 10 min in the presence of excess N-ethylmaleimide and then heated to 100 “Cfor 3 min in 2X sample buffer without DTT. The prestained molecular weight calibrators are shown in lane 1,with respective molecular weights as indicated by the arrows; lane 2, P4B6 Fab’, 0.09 pg; lane 3’7E3 Fab’, 0.07 pg; lane 4,7E3 Fab’-BMME, 0.04 pg; lane 5, products of cross-linking P4B6 Fab’ and 7E3 Fab’-BMME for 60 min at room temperature; A, F(ab’)a sized band produced by P4B6 Fab’-7E3 Fab’-BMME conjugation.

BMME-mediated 7E3 homodimers or F(ab’)2 reoxidation occurred. No F(ab’)2 formation was evident for either parent until the addition of the P4B6 Fab’ to the BMMEmodified 7E3. After mixing, the F(ab’)2 formed via the coupling of 7E3-BMME to P4B6 at the end of 60 min is shown in lane 5 by the appearance of a 100 kDa band. A significant level of unreacted Fab’ was present in the final mixture, as well as lower levels of non-F(ab’)2 byproducts of molecular weight intermediate between those of the F(ab’)2 and Fab’. These immunoglobulin-derived fragments likely result from the recombination of intact Fab’ with completely reduced light or heavy chains. S-200 Sephacryl was used to separate the bispecific F(ab’)2 from residual Fab’ in the first purification step after capping the mixture with DTNB (not shown). The reaction mixture chromatographed into two partially resolved peaks of approximately 100 and 50 kDa. The fractions enriched in F(ab’)2 were pooled for further purification by HIC HPLC. The preparative and analytical hydrophobic interaction chromatography HPLC is shown in Figure 2. This step was employed to remove residual parental F(ab’)2 species or parental F(ab’)2 which may have formed via sulfhydryl oxidation, since resolution of the parental F(ab’)2 species was obtained under the conditions used in this separation (panels A and B). The technique was also employed to remove low amounts of BMME-coupled 7E3 homodimer which might have formed during the cross-linkingreaction or other non-F(ab’)2 byproducts of the reaction. The preparative HIC of the S-200F(ab’)a-sized pool containing the bispecific (panel C) species yielded a major peak of UV absorbance with a retention time of 45-53 min, as well as two prominent peaks with retention times of 32 min (compare to 30.2 min for P4B6 F(ab’)2 standard in panel A) and 68 min (compare to 66.4 min for the 7E3 F(ab’)2 in panel B). The 45-53-min peak was pooled, concentrated, and rechromatographedunder the same conditions, resulting in a single peak of approximately 47.5 min (panel D). An overall yield of approximately 16% was obtained for the production of the bispecific ii1(ab’)2 through the two chromatographic steps. The purified bispecific antibody was shown to be predominantly F(ab’)2 by nonreducing SDS-PAGE (not shown), containing low levels of low molecular weight, copurifying byproducts. The pu-

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F(ab‘h Antibody Speclfic for GPIIb/IIIa and rtPA I

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Figure 2. Hydrophobic interaction HPLC of 7E3 X P4B6 and parental F(ab’)z fragments. All samples were diluted with an equal volume of 2 M ammonium sulfate and injected onto a 75 X 7.5 mm Biogel TSK phenyl-5-PW HIC column which had been equilibrated in 1 M ammonium sulfate and 100 mM sodium phosphate (pH 6.3) at a 1 mL/min flow rate. Column elution was performed as described in the text. Protein detection was by UV absorbance at 214 nm (panel C) or 280 nm (panels A, B, and D): panel A, 33 pg of P4B6 F(ab’)z; panel B, 33 pg of 7E3 F(ab’)2; panel C, 1 mL of the S-200 F(ab’h pool containing the bispecific F(ab’)z; panel D, 27 pg of purified bispecific F(ab’)z rechromatographed on HIC.

Figure 3, Native reduction analysis of the bispecific F(ab’)z by gel-filtration HPLC. 7E3 X P4B6 (panel A), P4B6 (panel B), and 7E3 (panel C) F(ab’)2 were reduced and capped with NEM as described in the text. The samples (16-20 pg) were analyzed by GF-HPLC (dotted lines) as described in the text and compared to molecular weight standards and to chromatograms of nonreduced samples (solid lines). The reduced bispecific antibody was retained 8.3 min (panel A, dotted line) and the nonreduced sample was retained 8.3 min. The reduced P4B6 (panel B, dotted line) wasretained9.lminwhile thenonreducedformwasretained 8.2 min. The reduced 7E3 (panel C, dotted line) was retained 9.2 min while the nonreduced (panel C, solid line) form was retained 8.3 min.

rified bispecific F(ab’)2 was shown to be approximately 93 %I pure as determined by gel-filtration HPLC (Figure 3, panel A).

Table I. Binding Constants for 7E3 X P4B6 and Parental Antibodies in Antigen Binding EIA’s GPIIb/IIIaa tPAb test antibody binding constant, M-’ binding constant, M-l 7E3 5x108 ND P4B6 ND 2 x 109 7E3 X P4B6 1 x 109 1 x 1010 Relative binding constant for human GPIIb/IIIa determined in purified GPIIb/IIIa EL4 as described in Experimental Procedures. Inverse molar concentrations yielding half of the maximal signal were compared between 7E3 Fab’ fragment and the monovalent 7E3 X P4B6 F(ab’)z.* Relative binding constant for tPA determined in purified tPA EIA as described in Experimental Procedure. Inverse molar concentrations yielding half of the maximal signal were compared between P4B6 Fab’ and 7E3 X P4B6 F(ab’)z.

Biochemical Characterization. Support for the formation of the nonreducible cross-link in the bispecific F(ab’)z was obtained by performing native reduction analysis by HPLC as shown in Figure 3. The bispecific and parental F(ab’)2 species were subjected to native reduction in the presence of 1mM DTT under conditions shown to result in complete conversion of F(ab’)z to Fab’ (Pak et al,, 1991). After reduction, the antibodies were capped with NEM to prevent in situ reoxidation to F(ab’)z and analyzed by gel-filtration HPLC. Panel A shows a retention time of 8.3 min for the reduced and capped bispecific F(ab’)2 (dotted line) and 8.3 min for the nonreduced bispecific F(ab’)2 (solid line), consistent with approximately 100 kDa molecular weight. In contrast, the retention times for reduced and capped P4B6 (panel B, dotted line) increased to 9.1 min from 8.2 min for the nonreducedsample (solid line). Similarlythe reduced and capped 7E3 (panel C, dotted line) shifted to 9.2 min from 8.3 min (solid line). This shift indicates the conversion to Fab’ by the predicted molecular weight of approximately 40-50 kDa as compared to molecular weight calibrators. These data indicate that the bispecific F(ab’)z was not reducible to Fab’, consistent with the presence of the thioether cross-linkingthe heavy chains of the two parental Fab’ fragments.

Immunoreactivity. The relative association constants of 7E3 X P4B6 relative to each parental immunoreactivity were quantified in antigen binding EIA‘sas described in Experimental Procedures. In these EIA’s,the association constant was defined as the inverse of the concentration corresponding to half of the maximum signal.These data are summarized in Table I and show that the bispecific F(ab’)z fully retained both its anti-tPA (relative binding constant approximately 1 X 1010 M-1) and antiGPIIb/IIIa (relative binding constant approximately 1X lo9 M-l) immunoreactivities. Inhibition of Platelet Aggregation. The ability of the 7E3 X P4B6 bispecific F(ab’)z to inhibit platelet aggregation in vitro was compared to that of the Fab fragment of 7E3. Figure 4 shows the percent inhibition

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by washing the platelets to remove free rtPA and then incubating the wells in the presence of S-2251, plasminogen, and fibrin fragments. As indicated in Figure 5, plasmin activity was associated only with the platelets which had been incubated in the presence of the bispecific F(ab’)z. When compared to a standard curve of soluble rtPA incubated simultaneously in the absence of platelets, it was calculated that approximately 1.5 units/ mL of rtPA activity had been recruited to the platelets (1 X 106/well),a difference of approximately 12-foldover the background binding of tPA in the presence of the mixture of the parental F(ab’)z antibodies. DISCUSSION

The anti-GPIIb/IIIa monoclonal antibody 7E3 and the anti-tPA monoclonal P4B6 have been chemically crosslinked as Fab’ fragments to produce a bispecific F(ab’)z molecule containing both parental specificities. 7E3 was ANTIBODY CONCENTRATION (nM) chosen for its ability to target platelets and inhibit Figure 4. Inhibition of platelet aggregation by 7E3 X P4B6. fibrinogen-mediated platelet aggregation, and P4B6 was Human platelet-rich plasma was preincubated in the presence selected for its ability to bind tPA with high affinity and of test samples and then aggregated at 37 “C by stimulation with preservation of plasminogen activation. The combination 10pM ADP. Aggregation was quantified in a two-channel platelet of these two immunoreactivities in one F(ab’)z species in aggregometer as described in the text, and the percent inhibition postulated to have clinical utility via the targeting of enof aggregation in the presence of antibody was calculated: 0 , dogenous or pharmacologically administered tPA to plate7E3 Fab; a, 7E3 X P4B6 F(ab’),; A, P4B6 F(ab’)z. let-rich thrombi. The cross-linking of Fab’ fragments via bis-maleimides has been widely used for its speed, simplicity, yields (Glennie et al., 1987), and predicted resistance of the resulting thioether linkage toreduction in vivo (Stickney et al., 1989). BMME conjugates of this type have been used in human clinical trials successfully in a cancer imaging application (Stickney et al., 1989) and have been used to target cytotoxic T-cells to ovarian carcinoma cells in vitro and in vivo (Mezzanzanicaet al., 1991). In the case of the present report, the BMME-cross-linked bispecific species was produced and purified to an overall yield of approximately 16?6 using a combination of gel filtration and hydrophobic interaction chromatography. The conjugation step was shown to result in the formation of an F(ab’)z containing a nonreducible cross-link. This immunoconjugate was 0 50 100 150 shown to retain both parental immunoreactivities, inhibit the aggregation of platelets in vitro, and recruit 12-fold TIME (minutes) more active tPA to platelets in vitro than mixtures of the Figure 5. Measurement of tPA activity in the S-2251 assay. parental antibodies, resulting in the activation of soluble The amount of tPA activity recruited to 1X lo6platelets per well plasminogen to plasmin in the S-2251assay. While double by 7E3 X P4B6 or controls was quantified by incubation of the immunoaffinity chromatography of bispecific antibodies washed platelets in the presence of the plasmin substrate 5-2251 has been used (Runge et al., 19901,this purification strategy as described in the text. The activation of plasmin from plaswould be difficult to perform in large-scale production. In minogen was detected by the release of p-nitroaniline with absorbance at 405 nm. A, 7E3 X P4B6 plus 5 units/mL rtPA; ., contrast, hydrophobic interaction chromatography is no antibody or rtPA; A, rtPA with no antibody; 0,mixture of scalable and nondenaturing. In this study, HIC was 7E3 F(ab’)n and P4B6 F(ab’)n plus rtPA; overlap of data points capable of resolving F(ab’)z fragments (Figure 2) derived has occluded certain symbols. The experiment shown was from the two parental murine IgGl antibodies based on representative of three separate experiments. their relative hydrophobicities. The retention times observed for the bispecific and parental F(ab’)z species in of ADP-induced platelet aggregation associated with Figure 2 were consistent with the intermediate hydrovarying concentrations of 7E3 X P4B6,7E3 Fab, or P4B6 phobicity expected for the heterodimer containing both F(ab’)a. The inhibition curves for both 7E3 Fab and 7E3 of the parental Fab’ fragments. This chromatographic X P4B6 were comparable, showing equivalent levels of technique is thus capable of extremely powerful resolution inhibition at all concentrations tested. The P4B6 parent based on the hydrophobicity differences attributable to showed no inhibition of platelet aggregation a t comparable the variable region of murine antibodies or primary concentrations. sequence and posttranslational modification differences Plasminogen Activation Assay. The recruitment of in nonvariable regions within an isotype. This separation tPA to the surface of platelets via 7E3 X P4B6 with technique may lend itself to similar applications such as concomitant activation of plasminogen to plasmin was in the resolution of complex product mixtures secreted by demonstrated in the 5-2251 assay as indicated in Figure hybrid-hybridomas (quadromas). Purification of distinct 5. Human platelets which had previously been incubated parental antibodies must nevertheless be evaluated on a in the presence of saturating levels of the bispecific or case-by-case basis for this application. control antibodies (10 kg/mL) were washed and then It is believed that a potential benefit of platelet targeting incubated in the presence of 5 units/mL of rtPA. The specificrecruitment of rtPA to platelets was demonstrated of tPA through this bispecific antibody may reside in com-

F(ab‘)n Antlbody Specific for GPIIb/IIIa and rtPA

bining targeting to lysis-resistant, platelet-rich thrombus and to circulating platelets, potentially hastening the rate of lysis and subsequent reperfusion of the occluded vessel as well as decreasing reocclusion. The recruitment of tPA to circulating platelets via 7E3 X P4B6 may be advantageous in coronary thrombolysis by preventing aggregation through the 7E3 function of the bispecific antibody, as shown in this study, and promoting disaggregation by generation of a fibrinolytic surface at the platelet membrane (Loscalzo and Vaughan, 1987). This second mechanism may be responsible for the observed potentiation of platelet aggregation inhibiting capacity of 7E3-urokinase compared to 7E3 alone (Bode et al., 1991). Future evaluation of the effects of this bispecific antibody on the rates of reperfusion and reocclusion in vivo in animal models of thrombolysis will provide valuable insight into these mechanisms. ACKNOWLEDGMENT

We thank Dr. Desire Collen for the generous gift of the anti-tPA hybridoma P4B6 and other anti-tPA murine hybridoma lines and Ms. Joani Wendel and Ms.Camille Thompson for preparation of the manuscript. LITERATURE CITED Blum, M., Jordan, R., Wagner, C., and Mattis, J. (1989) Immunopurification and Functional Characterization of Platelet GPIIb/IIIa Complexes. Blood 74 (Suppl. I), 398a. Bode, C., Matsueda, G. R., Hui, K. Y., and Haber, E. (1985) Antibody-Directed Urokinase: A SpecificFibrinolytic Agent. Science 229, 765-767. Bode, C., Runge, M. S., Branscomb, E. E., Newell, J. B., Matsueda, G. R., and Haber, E. (1989) Antibody-Directed Fibrinolysis: An Antibody Specificfor Both Fibrin and Tissue Plasminogen Activation. J. Biol. Chem. 264, 944-948. Bode, C., Meinhardt, G., Runge, M. S., Freitag, M., Nordt, T., Arens, M., Newell, J. B., Kubler, W., and Haber, E. (1991) Platelet-Targeted Fibrinolysis Enhances Clot Lysis and Inhibits Platelet Aggregation. Circulation 84, 805-813. Chang, C.-H., Ahlem, C. N., Wolfert, B., Hochswender, S. M., Jue, R., Frincke, J. M., and Carlo, D. J. (1986) Preparation and Characterization of Bifunctional Antibodies with Reactivity to Carcinoembryonic Antigen and Indium Benzyl EDTA. J. Nucl. Med. 27, 1041. Collen, D. (1990) Coronary Thrombolysis: Streptokinase or Recombinant Tissue-Type Plasminogen Activator. Ann. Int. Med. 112,529-538. Coller, B. S. (1979) Platelet Aggregation by ADP, Collagen and Ristocetin: A Critical Review of Methodologyand Analysis in CRC Handbook Series in Clincal Laboratory Science (R. M. Schmidt, Ed.) Vol. 1, p 301, CRC, Boca Raton, FL. Coller,B. S. (1985)A New Murine MonoclonalAntibody Reports an Activation-Dependent Change in the Conformation and/ or Microenvironment of the Platelet Glycoprotein IIb/IIIa Complex. J . Clin. Inuest. 97, 101-108. Coller, B. S. (1990) Platelets and Thrombolytic Therapy. N . Engl. J. Med. 322, 33-42. Coller, B. S., Scudder, L. E., Berger, H. J., and Iuliucci, J. D. (1988) Inhibition of Human Platelet Function In Vivo with a Monoclonal Antibody with Observations on the Newly Dead as Experimental Subjects. Ann. Int. Med. 109,635-638. Dewerchin, M., and Collen, D. (1991)Enhancement of the Thrombolytic Potency of Plasminogen Activators by Conjugation with Clot Specific Antibodies. Bioconjugate Chem. 2, 293300. Dewerchin, M., Lijnen, H. R., Stassen, J. M., DeCook, F., Quertermous, T., Ginsberg,M. H., Plow, E. F., and Collen, D. (1991) Effect of Chemical Conjugation of Recombinant Single-Chain Urokinase-Type Plasminogen Activator with MonoclonalAntiplatelet Antibodies on Platelet Aggregation and Plasma Clot Lysis in Vivo. Blood 78, 1005-1018. Glennie, M. J., McBride, H. M., Worth, A. T., and Stevenson, G. T. (1987) Preparation and Performance of Bispecific F(ab’)z Antibody Containing Thioether-Linked Fab’ Fragments. J. Zmmunol. 139,2367.

Bloconlugete Chem., Vol. 3, No. 2, 1992

131

Gold, H. K., and Leinbach, R. C. (1987) Prevention of Acute Reocclusion after Thrombolysis with Intravenous Recombinant Tissue Plasminogen Activator. In Tissue Plasminogen Activator in Thrombolytic Therapy (B. E. Sobel, D. Collen, and E. B. Grossbard, Eds.) p 115, Marcel Dekker, New York. Gold, H. K., Coller, B. S., Yasuda, T., Saito, R., Fallon, J. T., Guerrero, J. L., Leinbach, R. C., Ziskind, A. A., and Collen, D. (1987) Rapid and Sustained Coronary Artery Recanalization with Combined Bolus Injection of Recombinant Tissue-Type Plasminogen Activator and Monoclonal Antiplatelet GPIIb/ IIIa Antibody in a Canine Preparation. Circulation 77,670. Gold, H. K., Gimple, L. W., Yasuda, T., Leinbach, R. C., Werner, W., Holt, R., Jordan, R., Berger, H., Collen, D., and Coller, B. S. (1990) Pharmacodynamic Study of F(ab’)z Fragments of Murine Monoclonal Antibody 7E3 Directed Against Human Platelet Glycoprotein IIb/IIIa in Patients with Unstable Angina Pectoris. J. Clin. Inuest. 86, 651-659. Haber, E., Quartermous, T., Matsueda, G. R., and Runge, M. S. (1989) Innovative Approaches to Plasminogen Activator Therapy. Science 243, 51-56. Hayzer, D. J., Lubin, I. M., and Runge, M. S. (1991) Conjugation of Plasminogen Activators and Fibrin-Specific Antibodies To Improve Thrombolytic Therapeutic Agents. Bioconjugate Chem. 2,301-308. Loscalzo, J., and Vaughan, D. E. (1987) Tissue Plasminogen Activation Promotes Platelet Disaggregation in Plasma. J. Clin. Znuest. 79, 1749-1755. Mezzanzanica, D., Garrido, M. A., Neblock, D. S., Daddona, P. E., Andrew, S. M., Zurawski, V. R., Jr., Segal, D. M., and Wunderlich, J. R. (1991) Human T-Lymphocytes Targeted Against an Established Human Ovarian Carcinomawith a Bispecific F(ab’)z Antibody Prolong Host Survival in a Murine Xenograft Model. Cancer Res. 51, 5716-5721. Pak, K. Y., Nedelman, M. A., Fogler, W. E., Tam, S. H., Wilson, E., van Haarlem, L. J. M., Colognola, R., Warnaar, S. O., and Daddona, P. E. (1991) Evaluation of the 323/A3 Monoclonal Antibody and the Use of Technetium-99m Labeled 323/A3 Fab’ for the Detection of Pan Adenocarcinoma. Nucl. Med. Biol. 18, 483-497. Runge, M. S., Bode, C., Matsueda, G. R., and Haber, E. (1987) Antibody-enhanced thrombolysis: Capture of Tissue Plasminogen Activator by a Bispecific Antibody and Direct Targeting by an Antifibrin-Tissue Plasminogen Activator Conjugate In Vivo. Trans. Assoc. Am. Phys. 100, 250-255. Runge, M. S., Quartermous, T., Matsueda, G. R., and Haber, E. (1988a) Increasing Selectivity of Plasminogen Activators with Antibodies. Clin. Res. 56, 501-506. Runge, M. S., Bode, C., Matsueda, G. R., and Haber, E. (1988b) Conjugation to an Antifibrin MonoclonalAntibody Enhances the Fibrinolytic Potency of Tissue Plasminogen Activator in Vitro. Biochemistry 27, 1153. Runge, M. S.,Bode, C., Savard, C. E., Matsueda, G.R.,and Haber, E. (1990)Antibody-DirectedFibrinolysis: A Bispecific F(ab’)z that Binds to Fibrin and Tissue Plasminogen Activator. Biconjugate Chem. 1,274-277. Stickney, D. R., Slater, J. B., Kirk, G. A., Ahlem, C., Chang, C.-H. and Frincke, J. M. (1989) Bifunctional Antibody: ZCH/CHA 111Indium BLEDTA-IV Clinical Imaging in Colorectal Carcinoma. Immunoconjugates Radiopharm. 2, 1-13. Verstraete, M. (1990) Thrombolytic Treatment in Acute Myocardial. Circulation 82 (Suppl. 111, 11-96-11-109. Yasuda, T., Gold, H. K., Fallon, J. T., Leinbach, R. C., Guerrero, J. L., Scudder, L. E., Kanke, M., Shealy, D., Ross, M. J., Collen, D., and Collen,B. S. (1988) MonoclonalAntibody Against the Platelet Glycoprotein (GP) IIb/IIIa Receptor Prevents Coronary Artery Reocclusion after Reperfusion with Recombinant Tissue-Type Plasminogen Activation in Dogs. J.Clin. Invest. 81, 1284-1291. Yasuda, T., Gold, H. K., Yaolta, H., Leinbach, R. C., Guerrero, J. L., Jang, I. K., Holt, R., Fallon, J. T., and Collen, D. (1990) Comparative Efforts of Aspirin, A Synthetic Thrombin Inhibitor and a Monoclonal Antiplatelet Glycoprotein IIb/IIIa Antibody on Coronary Artery Reperfusion and Bleeding with Recombinant Tissue-Type Plasminogen Activator in a Canine Preparation. J. Am. Coll. Cardiol. 16, 1728-1735. Registry No. PA, 105913-11-9.