enhancement of the thrombolytic potency of plasminogen activators by conjugation with clot-specific...

Bioconjugate ChemBtzy SEPTEMBER/OCTOBER 1991 Volume 2, Number 5 @I Copyright 1991 by the American Chemical Society

REVIEWS Enhancement of the Thrombolytic Potency of Plasminogen Activators by Conjugation with Clot-Specific Monoclonal Antibodies M. Dewerchin and D. Collen’

Center for Thrombosis and Vascular Research, University of Leuven, B-3000 Leuven, Belgium. Received April 11, 1991

Thrombolytic therapy has become a routine treatment of acute myocardial infarction in man. Thrombolytic agents are plasminogen activators which activate the fibrinolytic system in blood (Figure 1) by converting the inactive proenzyme plasminogen to the active enzyme plasmin, a serine protease that degrades the fibrin in the blood clot and thereby causes dissolution of the thrombus. Plas- min, however, also degrades fibrinogen, the circulating precursor of fibrin. Extensive fibrinogen degradation may predispose one to bleeding although such correlation has not been unequivocally observed in clinical trials. Strep- tokinase and urokinase (two-chain urokinase-type plasminogen activator, tcu-PA, UK) are plasminogen activators belonging to the group of the “classic” thrombolytic agents. These indiscriminately activate fibrin-bound as well as circulating plasminogen and consequently induce a systemic fibrinolytic state, characterized by extensive depletion of armtiplasmin, the physiologic plasmin inhibitor in blood, and degradation of fibrinogen and other circulating coagulation factors. With the ‘hew generation” thrombolytic agents tissue-type plasminogen activator (t- PA) on one hand and single-chain urokinase-type plasminogen activator (scu-PA) on the other hand, plasminogen activation is assumed to occur preferentially at the fibrin surface. This fibrin-specific plasminogen activation, resulting in localized generation of active plasmin in the vicinity of the clot, would render these fibrin-specific thrombolytic agents superior to the classic agents. How- ever, clinical experience has revealed that a t the high doses of t-PA or scu-PA required for efficient coronary thrombolysis, the fibrin specificity is only relative and systemic activation of the fibrinolytic system with the risk of bleeding complications may still occur ( I ) .

* Address for correspondence: D. Collen, M.D., Center for Thrombosis and Vascular Research, K.U. Leuven, Campus Gast- huisberg, 0 & N, Herestraat 49, B-3000 Leuven, Belgium.

lQ43-I002/9 1 f2902-O293$O2.5Qf 0

PLASMINOGEN

ACTIVATOR

FIBRINOGEN d DEGRADATION PRODUCTS

4

$. + PLASMIN

1 PLASM I NOGEN

F I B R I N - F I B R I N DEGRADATION PRODUCTS

Figure 1. Schematic representation of the fibrinolytic system. Several approaches to develop more potent and more

clot-selective thrombolytic agents are being explored. These include the construction of mutants and variants of the physiologic plasminogen activators t-PA or scu-PA and of recombinant chimeric molecules containing the protease part of urokinase and fibrin-binding structures of t-PA or of plasminogen (for review, see refs 2 and 3).

In this review, we will focus on an alternative approach toward the development of improved thrombolytic agents, consisting of antibody-mediated targeting of plasminogen activators to the thrombus. The concept of using monoclonal antibodies as carrier molecules for therapeutic agents has proven its value in the area of tumor therapy (4). The same strategy was first applied to cardiovascular disease by Bode et al. (51, who showed successful targeting of a plasminogen activator to fibrin, one of the components of a thrombus, using a conjugate of urokinase with a monoclonal antibody directed against fibrin but not cross- reacting with fibrinogen. Apart from fibrin-like material, a thrombus also contains platelet-rich material, representing an alternative target site in the blood clot. Thus, targeting of plasminogen activators might also be achieved by monoclonal antibodies that discriminate between activated platelets in the thrombus and circulating resting platelets.

0 1991 Amerlcan Chemical Society

294 Bloconjugate Chem., Vol. 2, No. 5, 1991

Table I. In Vitro Fibrinolytic Prowrties of Antifibrin Antibody/Plasminogen Activator Conjugates

Dewerchln and Collen

In vitro fibrinolytic potency

fibrin monomer plmma clot lysisb

plasminogen assay“ enhance- activator enhancement ment fibrinogen

moiety antibody moiety designation conjugation epitope factofl factofl breakdownd refs t-PA-59D8 chemical @-chain 10 3.2 reduced 12 rt-PA IgGi

B-chain t-PA F(ab’)z t-PA-59D8 recombinant @-chain B-chain UK IgGl 64C8-UK chemical @-chain 100 5

64C5Fab’-UK chemical @-chain 95 2-4 - 14 15

LMW-UK Fab’ IgGi 64C5-UK chemical @-chain 250

59D8-UK chemical @-chain 100-250 4.5 reduced 12,15 Fab’ 59D8-UK chemical @-chain 250 - - 15 IgGi

scu-PA-59DBFab’ chemical @-chain 230 1.6-1.8 reduced 16 - 19 SCU-PA‘ Fab’

rscu-PA/MA-l5C5 chemical D-dimer - 6.4 increased 6 - 2.2 reduced 6 rscu-PAf IgGi

rtcu-PA/MA-lBC5 chemical D-dimer - >5(l ? 22 rtcu-PAR IgGi

rtcu-PA/Th IgGl rtcu-PA/MA-l5C5/T chemical D-dimer rscu-PA-32ki F(ab’)z rscu-PA-32k/ chemical D-dimer - 5 increased 24

rtcu-PA-32k F(ab’I2 rtcu-PA-32kI chemical D-dimer - 0.6 increased 25

- ineffective - 17,18 - - - -

LMW-scu-PA CH2-truncated IgG scu-PA-59D8 recombinant @-chain 10-50 -

MA-15C5-F(ab’)~

MA-l5C5:F(ab’)z

MA-15C5-F(ab’)2/T rtcu-PA-BSk/T F(ab’)2 rtcu-PA-32kl chemical D-dimer - > 2, ? 25

rtcu-PA-33k j acFv‘ KizGoSsz rtcu-PA-33Kk scFv K12G&S2 recombinant D-dimer

recombinant D-dimer - 13 reduced 26 - 2.5 increased 26

a The fibrin monomer assay is a solid phase assay in which radiolabeled fibrin monomer, covalently linked to Sepharose, is pretreated with the different plasminogen activators after which it is incubated with plasminogen in buffer. The release of radiolabeled degradation fragments into the supernatant reflects lyeie of bound fibrin. b The plasma clot lysis assay is an in vitro system composed of a [125I]fibrin-labeled normal human plasma clot immersed in normal human plasma. The release of radiolabeled degradation productcl into the supernatant following incubation with the different plasminogen activators reflects the fibrinolytic potency of the agents. Ratio of the activity obtained with conjugated plasminogen activator over the activity obtained with the corresponding unconjugated plasminogen activator. d Fibrinogen breakdown observed with conjugated plasminogen activator as compared to that observed with the corresponding unconjugated plasminogen activator at equieffective dose. e scu-PA single-chain urokinase-type plasminogen activator produced by the human kidney cell line TCL-598. f rscu- PA: recombinant scu-PA, obtained by expression of cDNA in Escherichia coli. 8 rtcu-PA and rtcu-PA Conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugates with plasmin. rtcu-PA/T and rtcu-PA/T conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugates with thrombin. i rscu-PA-32k a low molecular weight form of rscu-PA with M, 32 OOO and NH2 terminal amino acid LeulU. j rscu-PA-33k: a low molecular weight form of rscu-PA with M, 33 OOO and NHz terminal amino acid Ala182.

rtcu-PA-33k in K12G&z: two-chain derivative obtained by treatment of the single-chain u-PA form of K12C‘&&2 with plasmin. acFv: single-chain variable domain fragment of an antibody. ANTIBODY-MEDIATED TARGETING TO FIBRIN

Up to now, two different types of anti-human fibrin antibodies have been used for the construction of antifibrin antibody/plasminogen activator conjugates: the murine monoclonal antibodies 59D8 and 64C5, specifically recognizing the amino terminal residues of the /+chain of human fibrin which become exposed after release of fi- brinopeptide B brought about by thrombin (5), and the murine monoclonal antibody MA-15C5, directed against fragment D dimer of cross-linked human fibrin (6). Both types of antibodies specifically bind to fibrin but not to fibrinogen (7-9) and have been used successfully for thrombus imaging in animal models ( 1 0 , I I ) . Functional properties of conjugates of these antibodies with different types of plasminogen activators are summarized in Tables I and 11.

Covalent Conjugates Targeted to the Fibrin B- Chain. Chemical coupling of recombinant t-PA (rt-PA) with the antifibrin 8-chain antibody 59D8 (12) was found to result in a 10-fold enhancement of its potency toward fibrin monomer in a purified system and a 3.2-fold increase of its potency toward a human plasma clot in a plasma milieu in vitro (Table I). Invivo, the t-PA/59D8 conjugate was shown to possess a 2.8-fold higher thrombolytic potency toward a human plasma clot than rt-PA, by using a jugular vein thrombosis model in the rabbit (13) (Table 11). The enhanced in vitro and in vivo thrombolytic potency of conjugated rt-PA was accompanied by a decrease in the consumption of a2-antiplasmin, plasminogen, and fibrinogen, indicating an increased fibrin selectivity (12, 13).

Similar results were obtained with chemical conjugates of single-chain or two-chain urokinase-type plasminogen activator with the antibodies 59D8 or 64C5 or with fragments of the antibodies (5, 14-16). The purified conjugates had 100-250-fold higher fibrinolytic potencies than their respective unconjugated counterparts toward fibrin monomer and 1.6-4.5-fold enhanced potencies toward human plasma clots in vitro (Table I). In the rabbit jugular vein thrombosis model, a conjugate of scu-PA with 59D8-Fab’ had a 29-fold enhanced in vivo thrombolytic potency as compared to unconjugated scu-PA (Table 11).

Schnee et al. have constructed a recombinant version of the t-PA/59D8 conjugate (17). Therefore, the cDNA encoding the variable domain region of the 59D8 heavy- chain gene was cloned and recombined in an expression vector with the CH1 and hinge sequence of mouse immunoglobulin y2b and with the cDNA coding for the catalytic B-chain of t-PA. This construct was transfected into a heavy-chain loss-variant cell line derived from hy- bridoma 59D8. The resulting fusion protein produced by these cells retained antifibrin antibody activity as well as plasminogen activating potential (1 7). However, the recombinant t-PA/59D8 conjugate was ineffective in plasmaclot lysis (18). In contrast, a recombinant conjugate comprising the 59D8 variable region combined with the mouse immunoglobulin y2b CH1, hinge, and CH2 domains and with a low molecular weight form of scu-PA (LMW- scu-PA) was 10-50-fold more efficient than urokinase in the fibrin monomer assay (19) (Table I).

Covalent Conjugates Targeted to Fibrin Fragment D Dimer. Targeting of recombinant scu-PA (rscu-PA)

Review Bioconlugete Chem., Vol. 2, No. 5, lSS1 295

Table 11. Thrombolytic and Pharmacokinetic Properties of Antifibrin Antibody/Plasminogen Activator Conjugates in Animal Modelr in Vivo

~~ ~ ~~

enhancement factorc - specific

plasminogen thrombo- thrombo- a!r clearance activator antibody lytic lytic fibrinogen antiplasmin prolongation

moiety moiety designation conjugation epitope species potency activityb breakdownd depletiond factor refs rt-PA I S 1 tPA-SgD8 SCU-PA Fab scu-PA-

59D8Fab’ ~scu-PA I F 1 ISCU-PA/

I S 1 rscu-PA/

rtcu-PAI I& rtcu-PA/

rtcu-PAITh IgGl rtcu-PA/

MA-15C5

MA-1C8

MA-15C5

MA-l5C5/T

chemical &chain rabbite chemical @chain rabbit

chemical D-dimer rabbit

chemical - rabbit



bahoonf

baboon

2.8 - 29 -

8 3 3 0.1 0.4 0.1 0.9 0.03 4 0.5

1.4 0.3

reduced equal

equal equal equal equal reduced

equal

reduced - 13 16

4-8 20 equal 23

20

qual 15

equal 1.7-3 equal 15 23 reduced 6 20

equal 4 20

- -

Thrombolytic potency, derived from the doseresponse data based on percent lysis obtained versus the doee (in mg of com und per kg of body weight) adminatered, representing potency at com arable dose. b Specific thrombolytic activit , derived from the dose-response gta based on lysis obtained versus steady-state asma anti en /&el (in pg u-PA or t-PA antigen per mL p6ama) representing rtez at comparable s t e a G 2 plasma antigen levels. C Ratio of t i e activity htained with conjugated plasmin0 en activator over the activity o tain with the correspondmg uncon’ugated plasminogen activator. d Fibrinogen breakdown or a*-antiplasmin dep!etion observed with con ated plasminogen activator as com ared to that observed with the correspondingunconj ated plasminogen activator at equieffective dose. e In the ra%t jugular vein model a [1aI]fibrin-la%eled human plasma clot was produced inn se ment %he ju@ar vein. Thrombol sis was quantitated 30 or 60 min after the end of a 4-h intravenous infusion of the different plasminogen activators,Bby determination of the residual radoactivity in the ‘ugular vein segment. f In the baboon femoral vein model, a thrombus was produced in a segment of a femoral vein using fresh baboon blood mixed with a trace amount of laI-lab@ed human fibrin0 en. Thrombolysis was quantitated 30 min after the end of a 2-h intravenous infusion of the differentplasmino en activators by determination of the resifual radioactivity in the femoral vein s e p t . I rtcu-PA and rtcu-PA conjugates: two-chain derivatives o%tained by treatment of rscu-PA or mu-PA conj ates with plasmin. h rtcu-PA/ and rtcu-PA/T conjugates: two-chain derivatives obtained by treatment of rscu-PA or rscu-PA conjugatae with thro%in.

to fibrin fragment D dimer similarly resulted in an enhancement of the fibrinolytic and thrombolytic potency of the plasminogen activator (Tables I and 11). Chemical conjugation of rscu-PA with MA-15C5 yielded a func- tionally intact conjugate, both with respect to the in vitro enzymatic properties of the rscu-PA moiety and the antigen-binding capacity of the antibody moiety (6). The conjugate, rscu-PA/MA-l5C5, had a 6.4-fold higher fibrinolytic potency than rscu-PA in a human plasma clot lysis assay in vitro (6) and an 8-fold enhanced thrombolytic efficiency toward a human plasma clot in the rabbit jugular vein thrombosis model (20). The enhanced invitro and in vivo thrombolytic potency of the conjugate as compared to unconjugated rscu-PA was not observed when the fibrin clots were produced with rabbit plasma or when rscu-PA was conjugated with a control antibody, MA-1C8 (6,20) (Table 11).

Treatment of scu-PA with plasmin results in conversion of the single-chain molecule to tcu-PA (urokinase), a two- chain derivative in which the constituting A- and B-chains are still linked by a disulfide bond. In contrast to SCU-PA, plasmin-derived tcu-PA indiscriminately activates circulating as well as fibrin-bound plasminogen. The plasmin- derived two-chain form of the conjugate rtcu-PA/MA- 15C5 had a 2.2-fold higher fibrinolytic potency in the in vitro plasma clot lysis assay than unconjugated rtcu-PA (6) and a 4-fold enhanced thrombolytic potency in the rabbit jugular vein model (20). Fibrinogen breakdown or az-antiplasmin depletion during in vitro and in vivo thrombolysis was less pronounced with rtcu-PA/MA-l5CB than with rtcu-PA (Tables I and 11), suggesting that the conjugate has an increased fibrin specificity. In contrast, fibrinogen breakdown during in vitro clot lysis was somewhat more pronounced with the single-chain form of the conjugate than with unconjugated rscu-PA (Table I). No increased fibrinogen breakdown or az-antiplasmin depletion was however observed with rscu-PA/MA-l5C5 during in vivo thrombolysis (Table 11).

Treatment of scu-PA with thrombin converts this single- chain molecule to tcu-PAIT, a two-chain derivative in which the A- and B-chains are still disulfide linked, but

which is cleaved at a site different from the one cleaved by plasmin. Thrombin-derived tcu-PA/T is virtually inactive in in vitro assays but can be converted to active tcu-PA with plasmin. This conversion by plasmin occurs however with a 500-fold lower catalytic efficiency as compared to that of scu-PA (21). In contrast to rtcu- PA/T, the thrombin-derived two-chain form of rscu-PA/ MA-15C5, rtcu-PA/MA-lBCB/T, was found to have a significant fibrinolytic activity in in vitro or in vivo plasma clot lysis and this activity occurred in the absence of fibrinogen degradation or az-antiplasmin depletion (20, 22) (Tables I and 11). These results may be explained by targeting of the conjugate to the fibrin clot and reactivation by plasmin at the fibrin surface (22).

In the rabbit jugular vein thrombosis model, interference of endogenous rabbit fibrin or fibrinogen with clot lysis by rscu-PA/MA-l5C5 is avoided since MA-15C5 does not cross-react with rabbit fibrin (8, 11). Although fibrin fragments do not trigger activation of the fibrinolytic system by rscu-PA/MA-l5C5 in plasma in vitro (6), they might well reduce its thrombolytic efficacy by blocking the antigen binding site of the antibody moiety. Therefore, the thrombolytic properties of rscu-PA/MA-l5C5 were investigated in baboons with an autologous femoral vein clot, after demonstration of cross-reactivity of MA-15C5 with cross-linked baboon fibrin (23). In this system, the thrombolytic potency of rscu-PA/MA-l5C5 was still 3-fold higher than that of rscu-PA (Table 11).

Conjugation of rscu-PA or its two-chain derivatives with MA-15C5 significantly decreased their plasma clearance rate (Table 11), resulting in considerably higher steady- state plasma u-PA antigen levels than observed with unconjugated u-PA (20,23). Therefore, specific thrombolytic activities (reflecting potency at comparable steady-state plasma antigen levels) were determined for the different conjugates in both the rabbit and baboon model. Unlike the thrombolytic potencies (potency at comparable dose), the specific thrombolytic activities of the conjugates in most cases were decreased as compared to those of the corresponding unconjugated moieties (Table 11). However, the targeting effect of MA-15C5 in combination with the

298 Bloconlugete Chem., Vol. 2, No. 5, 1991

prolonged half-life appears to compensate for the reduced specific thrombolytic activity, resulting in significantly enhanced thrombolytic potencies. Indeed, with a control conjugate (rscu-PA/MA-lC8) with similarly decreased plasma clearance rates, both the thrombolytic potency and specific thrombolytic activities were markedly reduced (20, 23).

In order to reduce the molecular size of the conjugates, a low molecular weight form of rscu-PA, rscu-PA-32k (comprising amino acids L e ~ l L L e u ~ ~ ~ ) , was chemically coupled to F(ab’)z fragments of MA-15C5 (24). This yielded a conjugate, rscu-PA-32k/MA-15C5-F(ab’)z, with M, 130 W a n d with similar in vitro biochemical properties for its single-chain form and its plasmin- or thrombin- derived two-chain derivatives as compared to intact rscu- PA/MA-l5C5 and its two-chain derivatives (24,25). The in vitro fibrinolytic potency of rscu-PA-32k/MA-l5C5- F(ab’)zor of the thrombin-derived rtcu-PA-32k/MA-l5C5- F(ab’)a/T was respectively increased 5- and 2-fold over that of the corresponding unconjugated counterpart (Table I). With the plasmin-derived two-chain form rtcu-PA- 32k/MA-15C5-F(ab’)z, however, no enhancement of the in vitro fibrinolytic potency over plasmin-derived rtcu- PA-32k was observed (Table I).

Alternatively, a recombinant 57 kDa single-chain chimeric plasminogen activator, K12GoS32, has been constructed (26), consisting of the variable region fragment (Fv) of MA-15C5 and of a 33 kDa low molecular weight form of rscu-PA (rscu-PA-33k, comprising amino acids Ala132-Leu411). The synthetic single-chain Fv fragment was obtained by linking the carboxy terminal end of the variable domain of the 6-chain of MA-15C5 to the amino terminal end of the variable domain of the MA-15C5 y-chain by means of a seven amino acid residue synthetic linker (27). Purified K12GoS32, produced in an insect cell expression system, retained the in vitro enzymatic properties of low molecular weight rscu-PA (rscu-PA-32k) and the fragment D dimer binding capacity of MA-15C5 (26). In the in vitro plasma clot lysis assay, the single-chain u-PA form of K12GoS32 was found to have a 13-fold higher fibrinolytic potency than rscu-PA-32k. The plasmin- derived two-chain form of the chimera had a 2.5-fold higher in vitro fibrinolytic potency than plasmin-derived rtcu- PA-32k (26) (Table I).

Bispecific Antibodies. In another attempt to con- centrate plasminogen activators to the surface of a fibrin clot, bifunctional antibodies have been prepared that are capable of binding both fibrin and t-PA (28-31) or both fibrin and u-PA (31,32). Such bispecific antibodies have been obtained by chemical coupling (29,32) or by somatic cell fusion (28, 30, 31). In the presence of bispecific antibody, u-PA or t-PA indeed were found to have 5-50- fold enhanced potencies for lysing fibrin monomer (28, 29,32) and 2-&fold higher fibrinolytic potencies toward human plasma clots in vitro (29,32). In the rabbit jugular vein thrombosis model, clot lysis with plasminogen activator administered in combination with bispecific antibody was 1.6-4-fold higher than lysis with plasminogen activator alone (28, 30). In addition, after pretreatment of fibrin with bispecific antibody, subsequently administered plasminogen activator was concentrated at the fibrin surface, both in the in vitro fibrin monomer lysis assay (29,32) and in the rabbit model in vivo (29). The use of bispecific antibodies thus offers the possibility of avoiding administration of exogenous plasminogen activator, by inducing thrombolysis via concentration of endogenous plasminogen activator a t the site of the thrombus.

In summary, antibody-mediated targeting of plasmi-

Dewerchin and Collen

nogen activators to fibrin appears to have the potential to increase the concentration of plasminogen activator in the vicinity of a thrombus, resulting in localized generation of active plasmin and enhanced clot lysis. Urokinase-type plasminogen activator displays fibrin specificity only in its single-chain form (33) and its fibrin specificity is not dependent on the presence of the NH2-terminal143 amino acids (34). With t-PA, the functional domains responsible for ita fibrin affinity reside within the NH2-terminal region (A-chain) of the molecule (35). Thus, conjugation of single chain u-PA molecules (scu-PA or low molecular weight forms of scu-PA comprising amino acids Ala13“Leu411 or LeulU-Leu411) or of the intact t-PA molecule with antifibrin antibodies yields plasminogen activators possessing fibrin specificity by two mechanisms. The results obtained with Fab’ or scFv conjugates (Tables I and 11) and with bispecific antibodies indicate that univalent antibody binding is sufficient for effective targeting. The specificity of the targeting effect is,demonstrated (a) by the finding that the enhancement of the fibrinolytic potency in vitro is inhibited by addition of purified antigen (5, 6 ,15,26) , (b) by the lack of targeting to fibrin clots that do not cross- react with the targeting antibody (6 ,20) , and (c) by the absence of targeting when using conjugates with a control monoclonal antibody (5, 6, 12, 13, 20, 23).

ANTIBODY-MEDIATED TARGETING TO PLATELETS

Recently, evidence has accumulated that platelet-rich zones of a thrombus, particularly arterial thrombi, are very resistant to thrombolysis (36). In addition, platelet deposition may contribute to coronary artery reocclusion following successful thrombolytic therapy ( I ) . Thus, monoclonal antibodies specific for activated plateleta might represent an alternative means to target plasminogen activators to a thrombus.

Bode et al. (37) chemically coupled two-chain urokinase to 7E3, a monoclonal antibody selectively binding to platelet membrane glycoprotein IIb/IIIa, which is the platelet receptor for fibrinogen and other adhesive proteins (38). The resulting conjugate had a markedly enhanced potency toward platelet-rich human plasma clots in vitro (Table 111).

We have chemically coupled rscu-PA to different monoclonal antibodies specifically recognizing surface proteins on activated platelets (39) (Tables I11 and IV): MA-TSPI- 1, directed against human thrombospondin, a platelet a-granule glycoprotein expressed on the surface of stimulated platelets but minimally on the surface of resting platelets; and MA-PMI-2, MA-PMI-1, and MA-LIBS-1, recognizing different epitopes on the platelet surface glycoprotein IIb/IIIa that become exposed only when, upon platelet activation, fibrinogen has bound to the receptor. The purified conjugates retained rscu-PA activity, and binding to ADP-stimulated human platelets in a plasma milieu in vitro was significantly higher than binding to resting platelets (39). However, none of the conjugates appeared to have a higher fibrinolytic potency than rscu- PA toward platelet-rich human plasma clots in a plasma milieu in vitro, and lysis was associated with extensive fibrinogenolysis (Table 111). Nevertheless, the conjugates rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 were found to have a 2.3-3-fold increase in vivo thrombolytic potency as compared to rscu-PA and a 5-7-fold higher potency than a control conjugate rscu-PA/MA-lC8, when tested in hamsters with pulmonary embolism consisting of a platelet-rich human plasma clot (TableIV). The enhanced thrombolytic potency of these rscu-PA/antiplatelet an-

Review Bioconjugate Chem., Vol. 2, No. 5, 1991 297

Table 111. I n Vitro Fibrinolytic Properties of Antiplatelet Antibody/Plasminogen Activator Conjugates in vitro fibrinolytic potency

plasminogen toward platelet-rich clots activator antibody enhancement fibrinogen moiety moiety designation conjugation epitope factorb breakdown refs

- urokinase Fab' UK-7E3Fab' chemical GPIIb/IIIa 1.3-10 37 rscu-PA IgG2a rscu-PA/MA-TSPI-1 chemical thrombospondin <1 increased 39

IgG2a rscu-PA/MA-PMI-2 chemical GPIIIa <1 IgG2b rscu-PA/MA-PMI-1 chemical GPIIb <1 IgGl rscu-PA/MA-LIBS-1 chemical GPIIIa <1 increased 39

39 39

increased increased

a The fibrinolytic potency toward platelet-rich human plasma clots was determined by using an in vitro system composed of a [19]fibrin- labeled plasma clot prepared from platelet-rich human plasma (platelet count > 106/wL) and immersed in buffer containing plasminogen or in normal human plasma. The release of radiolabeled fibrin degradation products into the supernatant following incubation with the different Plasminogen activators reflects the fibrinolytic potency of the agents. Ratio of the activity obtained with conjugated plasminogen activator over the activity obtained with the corresponding unconjugated plasminogen activator.

Table IV. Thrombolytic and Pharmacokinetic Properties of Chemical Antiplatelet Antibody/rscu-PA Conjugates in a Hamster Pulmonary Embolism Model.

~

enhancement factold specific clearance

thrombolytic thrombolytic fibrinogen a2-antiplasmin prolongation conjugate epitope potencyb activity breakdowne depletione factor refs

mcu-PA/MA-PMI-l GPIIb 3.1 0.39 increased increased 8 39 rscu-PA/MA-LIBS-1 GPIIIa 2.3 0.16 increased increased 10 39 rscu-PA/MA-lCS 0.4 0.03 increased increased 9 39

0 In the hamster pulmonary embolism model, a [lBI]fibrin-labeled platelet-rich human plasma clot, produced in vitro from freshly prepared platelet-rich human plasma (platelet count 3 X l@/pL) was injected into the jugular vein, after which it usually embolized into the lunga and occasionally into the heart. Thrombolysis was determined 30 min after the end of a 60-min intravenous infusion of the different plasminogen activators, as the difference between the radioactivity initially incorporated in the clot and the residual radioactivity in the lungs and heart. b Thrombolytic potency, derived from the dose-response data of percent lysis obtained versus the dose (in mg of p-PA per kg of body weight) administered, reflecting potency at comparable dose. Specific thrombolytic activity, derived from the dose-response data of percent lysis obtained versus plasma antigen level (in fig of u-PA antigen per mL), reflecting potency at comparable steady state plasma u-PA antigen levels.

Ratio of the activity obtained with the conjugates over the activity obtained with unconjugated rscu-PA. e Fibrinogen breakdown or a2- antiplasmin depletion observed with the conjugates as compared to that observed with unconjugated rscu-PA at equieffective doses. tibody conjugates was not observed in hamsters with pulmonary embolism consisting of a platelet-poor human plasma clot (39). Thrombolysis with the conjugates was associated with fibrinogen degradation and with extensive az-antiplasmin depletion (Table IV). The reason for the extensive fibrinogenolysis during in vivo or in vitro clot lysis is not known. Increased systemic activation of the fibrinolytic system was also observed with the control conjugate rscu-PA/MA-lC€i (6,391 (Table IV) as well as with the conjugate of rscu-PA with the fragment D dimer specific antibody MA-15C5, rscu-PA/MA-lBCB, described above (6) (Table I). Conversion of these conjugates to two-chain u-PA derivatives by plasmin occurred with initial activation rates (39) or catalytic efficiencies (22) comparable to the values found with rscu-PA (21, 39). Thus, the difference in systemic activation between conjugated and unconjugated rscu-PA appeared not to be related to differences in the rate of activation to two-chain moieties by plasmin. Conjugation of rscu-PA to the antiplatelet antibodies or to the control antibody MA-1C8 decreased ita plasma clearance rate about 10-fold (Table IV), resulting in increased plasma u-PA antigen levels with the conjugates as compared to unconjugated rscu-PA at comparable doses (39). Again, specific thrombolytic activities (potency at comparable plasma u-PA antigen level) for rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, and rscu-PA/MA-lC8 were respectively reduced 2.6-, 6-, and 33-fold as compared to that of rscu-PA (Table IV), suggesting interference of the chemical conjugation procedure with the thrombolytic activity of rscu-PA (39). Impairment of the functional properties of rscu-PA conjugation was however only observed when specific thrombolytic activities in vivo were compared. In all experiments assaying in vitro biochemical properties (specific activities toward chromogenic substrate or on fibrin plates, catalytic efficiency for plasminogen activa-

tion, kinetics of the conversion to two-chain u-PA moieties by plasmin), results obtained with conjugated or unconjugated rscu-PA were comparable (6, 22, 24, 39). With rscu-PA/MA-PMI-1 or rscu-PA/MA-LIBS-1, the targeting effect of the antiplatelet antibody moiety in combination with the prolonged half-life apparently compen- sated for the partial loss of thrombolytic activity of conjugated rscu-PA, resulting in a relatively marginal but statistically significant improvement of their in vivo thrombolytic potency as compared to rscu-PA (Table IV). With rscu-PA/MA-lCS, with similarly prolonged half-life, the 33-fold decrease in specific thrombolytic activity was associated with a reduction of its thrombolytic potency of only 2.5-fold (Table IV). Similar differences between the extent of reduction of specific thrombolytic activity and the extent of reduction of the thrombolytic potency were observed with rscu-PA/MA-lCS in the rabbit and the baboon model (Table 11) and might reflect the effect of the prolonged half-life of conjugated rscu-PA. Prolon- gation of the half-life might also explain the results obtained with rscu-PA/MA-lC8, rscu-PA/MA-PMI-1, or rscu-PA/MA-LIBS-1 in hamsters with pulmonary embolism consisting of a platelet-poor human plasma clot: the specific thrombolytic activity of the control conjugate as well as of the two antiplatelet antibody conjugates was 6-10-fold reduced as compared to that of rscu-PA, whereas no reduction of the thrombolytic potency was found (39). Thus, the increased thrombolytic potency toward platelet- rich human plasma clots in vivo of rscu-PA conjugated with antiplatelet antibodies resulted from the combination of a prolonged half-life and a targeting effect by the antibody moiety of the conjugate.

CHEMICAL CONJUGATION PROCEDURES Chemical conjugates of plasminogen activators with an-

tifibrin or antiplatelet antibodies were prepared with the

298 Bhxonlugete Chem., Vol. 2, No. 5, 1991 Dewerchin and Collen

Scheme I.. Schematic Representation of the Steps Involved in the Preparation of Chemical Conjugates between Plasminogen Activators and Antibodies

PLASMINOGEN ACT TVATOR

0 SPDP

ANT 1 BODY

I

IIa

IIb

Z-iminothiolane

. . 0 II

reaction product I t reaction product IIa t PA 0 IIIb

0 II

reaction p r o d u c t I t reaction product IIb t PA N H - 0 0 II

reaction product I t r e a c t i o n product IIc t @ N H - C - ( C H 2 ) 2 - S - S - / / IIIC

0 Reactive disulfide groups are introduced into the plasminogen activator (PA) molecule by treatment with the heterobifunctional cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (reaction I). Thiol groups are introduced into intact antibody (or its F(ab’)p fragment) either by treatment with SPDP, followed by mild reduction (reaction IIa), or by treatment with 2-iminothiolane (reaction IIb). Alternatively, the antibody is digested by pepsin and the inter-heavy-chain disulfide bond is reduced under mild conditions to yield Fab’ fragments containing h free thiol group (reaction IIc). Finally, the SPDP-treated plasminogen activator is coupled with the thiol-containing antibody following a disulfide-exchange reaction as shown in reaction IIIa (6, 24,39), reaction IIIb (12, 15), and reaction IIIc (14-16).

heterobifunctional cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (40) and/or the thi- olating reagent 2-iminothiolane (41) by using a three-step procedure as outlined in Scheme I. Briefly, reactive disulfide bonds were introduced into the plasminogen activator molecule by treatment with SPDP (reaction I) (6,12,14-16,24,39). In a second step, thiol groups were introduced into the antibody either by treatment with SPDP followed by reduction of the introduced disulfide bonds (reaction IIa) (6,24,39) or by treatment with 2-iminothiolane (reaction IIb) (12,15). Alternatively, I?(ab’)z fragments were prepared by digestion of the antibody with pepsin and the inter-heavy-chain disulfide bond was reduced under mild conditions, yielding Fab’ fragments containing a free thiol group (reaction IIc) (14-16). In the third step, the SPDP-treated plasminogen activator reacts with the thiol-containing antibody via a thiol-disulfide exchange to form a disulfide-linked protein-protein conjugate (Scheme I, reactions IIIa-c). Chemically coupled bispecific antibodies (29, 32) were prepared by using a procedure analogous to steps I, IIb, and IIIb of Scheme I.

CONCLUSION Conjugation of plasminogen activators with antifibrin

antibodies as well as with antiplatelet antibodies has resulted in significantly enhanced thrombolysis, both in vitro and in vivo. Antibody-mediated targeting thus appears to hold promise for the development of plasminogen activators with enhanced potency and clot- selectivity. The use of such agents may allow a significant reduction of the total dose of plasminogen activator which in turn might reduce systemic side effects. On the other hand, the use of bispecific antibodies with high affinities

for both fibrin and plasminogen activator might allow concentration of endogenous plasminogen activator a t the site of a thrombus and might eliminate the need for the administration of high doses of exogenous thrombolytic agent.

Chemical conjugation methods, involving the introduc- tion of reactive groups by treatment with cross-linking agents such as N-succinimidyl 3-(2-pyridyldithio)propionate and 2-iminothiolaneI have the disadvantage of low yields and random coupling. In addition, chemical conjugation may affect the enzymatic properties of the plasminogen activator moiety of covalent plasminogen activator/antibody conjugates. Chemically prepared conjugates are therefore unsuitable for large-scale production for in vivo application. The construction of plasminogen activator/antibody conjugates by recombinant DNA technology may avoid most of these problems, and results obtained with such recombinant conjugates are encour- aging. In addition, this approach permits humanization of the murine monoclonal antibody by substituting the murine constant regions with those of a human antibody (42,43) or by engrafting the complementarity-determining regions of the targeting monoclonal antibody into the corresponding positions of a human antibody (44, 45). In this way, one might circumvent immunogenicity problems associated with the use of nonhuman antibodies in man. Alternatively, recombinant DNA techniques allow the use of small-size binding units of an antibody such as single- chain Fv fragments containing the variable domains of the antibody only (46, 47). Results obtained with the chimera KIZGOS~Z indicate that a plasminogen activator can indeed be targeted to a fibrin clot with a single-chain Fv fragment derived from fibrin-specific monoclonal antibody (26). Changing the size of the conjugate molecule

Review Bioconlugte Chem., Vol. 2, No. 5, 1991 299

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