Proc. Nati. Acad. Sci. USAVol. 88, pp. 10337-10341, November 1991Medical Sciences

A recombinant chimeric plasminogen activator with high affinity forfibrin has increased thrombolytic potency in vitro and in vivo

(urokinase/tssue plminogen activator/fibrinolysis/antibody targeting)

MARSCHALL S. RUNGE*t, THOMAS QUERTERMOUSt§, PAUL J. ZAVODNY¶, TED W. LOVE0§,CHRISTOPH BODE", MATHIAS FREITAGII, SHYH-YU SHAW**, PAUL L. HUANGU§, CHUAN-CHU CHOU¶,DEBORA MULLINs¶, JANET M. SCHNEEt, CHRISTOPHER E. SAVARDt, MARK E. ROTHENBERG*,JOHN B. NEWELLt, GARY R. MATSUEDA**tt, AND EDGAR HABERt§*Cardiology Division, Emory University, Atlanta, GA 30322; tCardiac Unit of the General Medical Services, Massachusetts General Hospital and §HarvardMedical School, Boston, MA 02114; ISchering-Plough Research, Bloomfield, NJ 07003; IlMedizinische Klinik III (Kardiologie) der Universitat Heidelberg,6900 Heidelberg, Federal Republic of Germany; **Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, NJ 08543; and ttPrincetonUniversity, Princeton, NJ 08544

Communicated by Eugene Braunwald, July 22, 1991 (received for review October 30, 1990)

ABSTRACT A recombinant plasminogen activator withhigh fibrin affinity and specificity was expressed by transfect-ing hybridoma cells with a plasmid that combines sequencecoding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)J and anti-fibrinmonoclonal antibody 59D8. The expression of the recombinantmolecule [r-scuPA(32kDa)-59D8] was optimized by replacingthe 3' untranslated region (initially that of high molecular massscuPA) in the plasmid with the 3' untranslated region of either13-globin or mouse immunoglobulin. This modification resultedin a >100-fold improvement in the level of protein expression.The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalyticactivity indistinguishable from that of high molecular massscuPA and fibrin binding comparable to that of native antibody59D8. r-scuPA(32kDa)-59D8 was 6 times more potent thanhigh molecular mass scuPA in lysing a human plasma clot invitro and was 20 times more potent than high molecular massscuPA in the rabbit jugular vein model of thrombolysis.Molecules of this type may serve as prototypes for highlyspecific, antibody-targeted enzymes suitable for human use.

Acute thrombotic occlusion of a major epicardial coronaryartery results in myocardial infarction, the single most com-mon cause of death in industrialized societies. The use ofthrombolytic therapy in patients with acute myocardial in-farction has resulted in a significant reduction in mortality(1-3). At its present stage of development, however, throm-bolytic therapy is limited by (i) significant bleeding at highdoses (intracranial hemorrhage causes stroke or death in0.1-0.5% of patients who receive plasminogen activators)(1-4), (ii) the failure to restore blood flow in 20%o ofpatientsor the fact that thrombotic reocclusion after cessation oftherapy occurs in 15-25% of patients (5), and (iii) the lagbetween initiation of therapy and clot lysis, which averagesabout 60 min (5).These limitations have prompted generation (by recombi-

nant DNA technology) of hundreds of plasminogen activatormutants, which have produced, at best, only modest im-provements in thrombolytic efficacy. Most investigatorshave either rearranged (or duplicated or deleted) variousplasminogen activator functional domains or altered theirposttranslational modification (6-10). We and others (11-16)have pursued an alternative strategy, the generation ofchem-ical conjugates or recombinant molecules with domains forboth plasminogen activator activity and high-affinity fibrinbinding [conferred by a monoclonal antibody (17) that binds

to fibrin, the principal component of a thrombus, but notfibrinogen, its circulating precursor]. Here we report thegeneration and characterization (in vitro and in vivo) of arecombinant molecule that is fibrin selective by two differentmechanisms. It combines a high-affinity anti-fibrin antibody,59D8, with a low molecular mass (32 kDa) single-chainurokinase-type plasminogen activator [scuPA(32kDa)], a fi-brin-selective plasminogen activator that does not bind di-rectly to fibrin but does bind to fibrin-bound plasminogen(18). In vitro and in vivo, the recombinant (r) molecule[r-scuPA(32kDa)-59D8] lysed human thrombi with a cata-lytic efficiency far greater than that of high molecular mass(54 kDa) scuPA (hereafter referred to as scuPA).

MATERIALS AND METHODSCloning and Expression of Recombinant Proteins. We pre-

viously cloned into expression plasmid pSV2gpt genomicsequence coding for a portion of the mouse immunoglobulinIgG2b constant region, genomic DNA for the rearrangedheavy-chain gene of antibody 59D8, and a cDNA coding forthe catalytic light chain ofhuman tissue plasminogen activator(tPA) (15, 16). The resulting expression plasmid, pSVD8T(Q),was used as the starting point for the constructs describedhere.scuPA genomic DNA encoding amino acids 144-411 was

inserted into pSVD8T(,8) in place of the tPA light-chain gene.(A plasmid containing genomic sequence encoding the entirescuPA molecule was a gift from F. Blasi, InternationalInstitute of Genetics & Biophysics, CNR, Naples, Italy.) Anadditional portion of the mouse immunoglobulin IgG2b con-stant region, containing sequence coding for the entire sec-ond constant domain and a portion of the third constantdomain of the heavy chain, was also ligated into pSVD8T(,3).The resulting expression plasmid, pSVUKG(UK), was trans-fected into 59D8 heavy-chain loss variant hybridoma cells asdescribed (15, 16).

Production of Large Quantities of r-scuPA(32kDa)-59D8.Milligram quantities of r-scuPA(32kDa)-59D8 protein wereproduced by growing cells to high density (total mass of -4x 101° cells) in Dulbecco's modified Eagle's medium with10% fetal calfserum in the extrafiber space ofa CellMax (typeB) bioreactor (Cellco, Kensington, MD) containing celluloseacetate hollow fibers with a sieving coefficient of -4 kDa.r-scuPA(32kDa)-59D8 was purified from the medium (the

Abbreviations: scuPA(32kDa), low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator; tPA, tissue plasmino-gen activator; UT, untranslated; r, recombinant.tTo whom reprint requests should be addressed.

10337

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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initial concentration of recombinant protein varied between0.05 and 0.3 mg/ml) by affinity chromatography on a resincontaining Sepharose linked to the heptapeptide epitope forantibody 59D8 (19). The eluate contained a mixture ofr-scuPA(32kDa)-59D8 (15-35%) and enzymatically cleaved(by either thrombin or plasmin) r-scuPA(32kDa)-59D8 (85-65%). Benzamidine-Sepharose chromatography was per-formed as described (11) to remove two-chain r-scuPA(32-kDa)-59D8 from the mixture of single- and two-chain forms.Purified r-scuPA(32kDa-59D8 (0.05-0.5 mg/ml in Tris/glycine buffer, pH 7.4) was used immediately in assays orfrozen at -70°C, at which it remained stable for up to 6weeks.

Protein Determination. The concentrations in solution ofscuPA, r-scuPA(32kDa)-59D8, and 59D8 were determined asdescribed by Bradford (20).SDS/PAGE and Western Blotting. SDS/PAGE was per-

formed according to the method ofLaemmli (21) as described(19). Proteins were either visualized with Coomassie brilliantblue R or transferred by electrophoresis to a nitrocellulosefilter for Western blotting (12).

Kinetic Assays. The amidolytic activity of scuPA, two-chain urokinase, and r-scuPA(32kDa)-59D8 was measured inthe S-2444 assay as described (11, 14) in the presence andabsence of plasmin. In individual experiments, duplicatedeterminations were performed. In general, correlations be-tween S-2444 assay results and protein determinations by theBradford assay were excellent. Where there was a significantdifference, we chose to use the S-2444 assay results in thekinetic or thrombolytic experiments that followed.

A SUCH CH2H

E coRlIHEcoR1CH

sacI ~~~~CHI So

pSVUKG(UK) pS\

59OD8 Eco I59D 8

Eco RI

ORI amp

Measurement of Fibrin Binding. ELISA plates (96-well;Falcon) were coated with fibrin monomer (5 ug/ml). Samplesof either antibody 59D8 (serially diluted over 7 orders ofmagnitude from an initial concentration of 1.3 mg/ml) orr-scuPA(32kDa)-59D8 (serially diluted over 7 orders of mag-nitude from an initial concentration of 1.7 mg/ml) wereincubated on the plates for 1 hr, washed extensively withTris-buffered saline (pH 8, with 0.05% Tween-20), blockedagain with bovine serum albumin, and then probed with aF(ab')2 preparation of polyclonal rabbit anti-mouse IgG an-tibodies that had been labeled with biotin (Bethesda ResearchLaboratories).Thrombolysis Assays. Thrombolytic potency was measured

in vitro and in vivo. The in vitro plasma clot assay wasperformed as described (19) based on the method of Lijnen etal. (22). The rabbitjugular vein model of Collen et al. (23) wasmodified as described (11, 13). Plasminogen activators (orsaline) were administered by infusion ofa bolus (consisting of20% the total dose) over 1 min, along with a heparin bolus(300 units/kg) over 1 min, followed by continuous infusionover the next 60 min of the remainder of the plasminogenactivator dose and continuous infusion of heparin over thenext 180 min (60 units-kg-1 hr-1). Thrombolysis was mea-sured by y counting of the remaining vein segment. Fibrin-ogen was measured by the method of Clauss (24), anda2-anti-plasmin was measured by the method of Edy et al.(25).Measurement of Plasma Clearance. To determine their

plasma clearance rates in the rabbit, we labeled 54-kDascuPA and r-scuPA(32kDa)-59D8 with 1251 by the chloram-

EmoROI

O ORIamp SVORORI

B

8000

co 6000 4--

2-4000 -l

-71200 -

FIG. 1. Expression plasmids for r-scuPA(32kDa)-59D8. (A) pSVUKG(UK) contains a genomic heavy-chain variable region fromfibrin-specific monoclonal antibody 59D8, the cloned genomic constant region of mouse IgG2b [first constant domain of the heavy chain (CH1),hinge (H), and the second constant domain of the heavy chain (CH2)], and the coding region from a genomic clone of scuPA(32kDa) (containingexons VII-XI). In pSVUKG(UK) the 3' UT region is that of scuPA, beginning at Leu'44. Three modified plasmids were made by substitutingeither the 3' UT region of P-globin [pSVUKG((3)i or the 3' UT region of mouse immunoglobulin (from antibody 59D8) [pSVUKG(Ig) andpSVUKc(Ig)]. pSVUKc(Ig) also differed in that the genomic DNA encoding scuPA(32kDa) was replaced by cDNA encoding the same region(exons VII-XI). (B) Protein expression levels. The values for supernatants (in ng/ml) are based on the presence of mouse immunoglobulinmeasured by reference to a standard curve (as described in Fig. 3B). Results represent the means of duplicate determinations. Bar 1, supernatantfrom 59D8 cells; bar 2, supernatant from IgG2b cells (heavy-chain loss variant cells that had been transfected with an expression plasmid encodingonly the heavy chain of antibody 59D8); bar 3, supernatant from pSVUKG(UK); bar 4, supernatant from pSVUKG(Q3); and bar 5, supernatantfrom pSVUKG(Ig). amp, Gene conferring ampicillin resistance; SV 40 ORI, simian virus 40 origin of replication; gpt, guanine phosphoribo-syltransferase gene.

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ine-T method (26). After -500,000-1,000,000 cpm of labeledmaterial had been injected into the marginal ear vein of arabbit, blood samples were taken from the contralateralmarginal ear vein as described (11).

RESULTS AND DISCUSSIONInitial assays of culture supernatants from cells transfectedwith pSVUKG(UK) indicated the presence of scuPA(32kDa)antigen and amidolytic activity as well as 59D8 antigen andfibrin binding activity. However, the protein levels wereextraordinarily low (Fig. 1B), on the order of 30-200 ng/mlof culture supernatant. Northern blot analysis with poly(A)+RNA from P220R-15 cells [a stable, subcloned line expressingpSVUKG(UK)I showed low steady-state mRNA levels. Nu-clear run-off experiments indicated that this low steady-statemRNA level was likely due to mRNA instability rather thanto a reduced transcription rate (data not shown). We thenreplaced the 3' untranslated (UT) domain [that of scuPAin pSVUKG(UK)] with the 3' UT domain of f3-globin[pSVUKG(f3); Fig. 1A] or immunoglobulin [pSVUKG(Ig);Fig. 1A]. This produced a >100-fold improvement in levelsofprotein expression (Fig. 1B), with corresponding increasesin steady-state mRNA levels (data not shown). The proteinexpression level was similarly greater for the plasmidpSVUKc(Ig) (Fig. 1A), in which the genomic DNA encodingamino acids 144-411 of scuPA had been replaced with cDNAencoding the same sequence and the tPA 3' UT domain had

NON-REDUCED

been replaced with the immunoglobulin 3' UT domain (datanot shown). The r-scuPA(32kDa)-59D8 protein tested in thein vitro and in vivo studies that follow came from thetransfection of the pSVUKG(q) plasmid into 59D8 heavy-chain loss variant cells (L2LV).

Preparations of r-scuPA(32kDa)-59D8 were subjected toSDS/PAGE under nonreducing and reducing conditions(Fig. 2). In addition, Western blot analysis was performedusing antibodies specific for either mouse immunoglobulin orurokinase. Fig. 2 Left shows nonreduced samples stainedwith Coomassie dye or, after electrophoretic transfer, sub-jected to Western blotting. The only significant species wasa single band at 103 kDa that contained both urokinase andmouse immunoglobulin epitopes. Fig. 2 Right shows reducedsamples treated in the same way. In lane C of Fig. 2 TopRight, the reduced recombinant protein shows four bands byCoomassie blue staining. By densitometry of the Coomassieblue-stained bands, 77.6% of the material is accounted for bythe sum of the predicted 78-kDa fusion peptide containingimmunoglobulin heavy-chain domains and scuPA(32kDa)(48.7%) and the 25-kDa immunoglobulin light chain (28.9%).Although Western analysis showed that both bands con-tained the expected immunoglobulin epitopes, only the 78-kDa band contained the predicted urokinase epitopes. The48-kDa band, which accounts for 12% of the material, cor-responds to the predicted size of the amino-terminal prote-olytic fragment of the 78-kDa fusion peptide containingimmunoglobulin heavy-chain domains and scuPA(32kDa)

REDUCED

kDakDa

200-

116-97-

Coomassie 66- .. --=45-

31-

97- f _66- -

45-

31 -

22-

22-

A B

Goat anti-Mouse IgG

14-

C D E

kDa

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1 6-97-

66-

45-

31-

22-

C D E

kDa

200-

Goat anti HumanUrokinase

115-97- 'I'

66-

A B C D E

kDa

97-

66--

45-

kDa

97-

66-

45-

31-

22-

C D E

FIG. 2. Affinity-purified r-scuPA(32kDa)-59D8 analyzed by SDS/PAGE under nonreducing (Left) and re-ducing (Right) conditions. (Top)Coomassie-stained gels. (Middle) West-ern blots using goat anti-mouse IgG.(Bottom) Western blots using goat anti-human urokinase. Lanes A and B, mo-lecular mass standards. Lanes C, affini-ty-purified r-scuPA(32kDa)-59D8.Lanes D, scuPA(32kDa) (Green Cross,Osaka). Lanes E, affinity-purified anti-

C D E body 59D8.

31-45-

2'2-

1 --

31-

22-

C D E

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A 16-

10-

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-0.6-0.4,

B3 1.21

E0

0_v

sw(.)n

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.0

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0.4

0.0-1 --2 -3 -4 -5 -6 -7

Log dilution

FIG. 3. Kinetic and fibrin binding properties of r-scuPA(32kDa)-59D8. (A) Initial rates of reaction for two-chain urokinase (A) and thetwo-chain form of r-scuPA(32kDa)-59D8 (A). (B) Fibrin binding bynative 59D8 (stippled bars) and r-scuPA(32kDa)-59D8 (hatchedbars). The means of duplicate determinations are shown.

after cleavage by either thrombin (at Arg156-Phe'57) or plas-min (at Lys158-11e159). However, because plasmin-cleavedscuPA binds efficiently to benzamidine-Sepharose butthrombin-cleaved scuPA does not, it is more likely that thetwo-chain material on these gels was thrombin-cleavedr-scuPA(32kDa)-59D8. As expected, this 48-kDa fragmenthad immunoglobulin but not urokinase epitopes. The thirdband in lane C of Fig. 2 Top Right is likely the carboxyl-terminal proteolytic fragment, which accounts for 10.4% ofthe material. As predicted, its molecular size is 32 kDa, thesame as that of low molecular mass scuPA, and it containedonly urokinase epitopes. Because the predicted products ofthrombin and plasmin cleavage would vary in molecular massby only two amino acids and would not be distinguishable byelectrophoresis, we have not ascertained the relative contri-butions of thrombin and plasmin to proteolytic cleavage ofthe fusion peptide containing immunoglobulin heavy-chaindomains and scuPA(32kDa). The increase in amidolytic ac-tivity of the recombinant protein after plasmin treatment was80% that of native scuPA, which is consistent with thedensitometry results from Fig. 2 indicating 77.6% single-chain material.To evaluate the functional properties of r-scuPA(32kDa)-

59D8, we compared the specific amidolytic activity andkinetic parameters for activation of plasminogen by plasmin-cleaved r-scuPA(32kDa)-59D8 with those of low molecularmass two-chain urokinase and also compared the fibrin-binding activity with that of native 59D8. The specific ami-dolytic activity of tissue culture-derived scuPA was 85,000units/mg. This material was >95% uncleaved (i.e., singlechain). Preparations of r-scuPA(32kDa)-59D8 were 77.6%single chain, with a catalytic activity of 26,000 units/mg ofprotein. Given the contribution, on a molar basis, of the32-kDa scuPA portion of the 103-kDa r-scuPA(32kDa)-59D8molecule, the activity of the scuPA portion was 83,900units/mg of scuPA. This is not significantly different from theactivity of native scuPA. Further, the Km for activation of

A

(fi

e>1a5minogen (,uM-0.4[Piasminogen (AM)[-'

1.0

0 1000 2000 3000 4000 5000scuPA, units

B l0-

80-

c 60-

20

1 2scuPA, mg/kg

3 4

FIG. 4. (A) Human plasma clot lysis assay. Clot lysis is shown atdifferent concentrations (in units/ml) of scuPA (--A--) or r-scuPA-(32kDa)-59D8 (-A-). Points represent clot lysis at 2 hr. The foldincrease in potency was calculated by comparison ofthe percent lysiscurves in the plasma clot and rabbit jugular vein assays, which werefit using a two-parameter exponential function (19). (B) Thrombolysisin vivo. Data represent the means of values from between 3 and 8animals at each point. The 20-fold increase in potency forr-scuPA(32kDa)-59D8 was derived as described in A.

plasminogen by the plasmin-cleaved (two-chain) form ofr-scuPA(32kDa)-59D8 (16.6 ,uM) did not differ significantlyfrom that of low molecular mass two-chain urokinase (9.1,uM) (Fig. 3A). The fibrin-binding activity ofr-scuPA(32kDa)-59D8 [native scuPA(32kDa) does not bind fibrin directly] wascompared with that of native 59D8. The data shown in Fig.3 demonstrate that r-scuPA(32kDa)-59D8 bound to fibrin ina manner similar to that of native 59D8. Although there wasa trend toward diminished fibrin binding by r-scuPA(32kDa)-59D8 (in comparison with fibrin binding by native 59D8) atdilutions of 10'-, 10-6, and 10-7, the observed differenceswere not statistically significant.

In a human plasma clot assay, r-scuPA(32kDa)-59D8 was6 times more potent than scuPA (P < 0.0001) (Fig. 4A). Atequivalent thrombolytic doses, r-scuPA(32kDa)-59D8 wasmore fibrin specific; i.e., there was less consumption offibrinogen and a2-anti-plasmin than with scuPA. In a typicalplasma clot assay, at 3 hr with 800 units of scuPA, there was66% ± 5% clot lysis. The fibrinogen level was 56% + 5% thatof the control, and the a2-anti-plasmin level was 35% ± 9%othat of control. With 200 units of r-scuPA(32kDa)-59D8, clotlysis was 53% ± 4%, and fibrinogen and a2-anti-plasminlevels were 95% + 1% and 85% ± 5%, respectively, those ofthe controls. These data are comparable with those of Bodeet al. (12), who compared the fibrin specificity of scuPA withthat of scuPA chemically coupled to antibody 59D8. In vivoresults in the rabbit jugular vein model were even morestriking. Compared with scuPA, r-scuPA(32kDa)-59D8 dis-played a remarkable 20-fold increase in thrombolytic potencyover the entire dose-response range (P < 0.0001) (Fig. 4B).r-scuPA(32kDa)-59D8 did not cause a decrease in fibrinogenconcentration until 83% lysis was reached, at which point the

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E80-

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2 40-

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1 10 160 i000Time, min

FIG. 5. In vivo plasma clearance of 1251-labeled scuPA and1251-labeled r-scuPA(32kDa)-59D8. 125I-labeled scuPA (A) orr-scuPA(32kDa)-59D8 (v) was injected intravenously into rabbits,and the percentage of maximal initial radioactivity remaining wasdetermined. Each point represents the mean of results from twoanimals.

fibrinogen concentration was 79o ± 4% that of the control.This represents a small but probably significant decrease infibrinogen concentration.We used the rabbit jugular vein model in these studies

because it allows quantitative comparisons of thrombolyticpotency. It is not ideal, however, for comparisons of fibrinspecificity. For example, even very high doses of scuPA donot result in >40% thrombolysis in this model, precluding a

comparison of specificity between scuPA and r-scuPA(32-kDa)-59D8 at a near-maximal thrombolytic dose. Within therange where it was possible to compare plasma fibrinogenand a2-anti-plasmin concentrations between scuPA andr-scuPA(32kDa)-59D8, the data resemble those previouslypublished comparing tPA with tPA chemically conjugated to59D8 (11).

Fig. 5 demonstrates that scuPA clears from the plasmamuch more rapidly than r-scuPA(32kDa)-59D8 (tl2 of 5.5 and25.5 min, respectively). This difference is important in judg-ing the relative thrombolytic potency of the two activators.Collen et al. (13) showed that the plasma clearance of scuPAchemically conjugated to an anti-fibrin antibody was similarlyprolonged. In our study and in theirs, the entire increase inthrombolytic potency could not be attributed to the longerhalf-life in plasma. We estimate that, of the 20-fold enhance-ment in thrombolytic potency with r-scuPA(32kDa)-59D8, 4-to 6-fold was due to the targeting of scuPA to fibrin. This isconsistent with the in vitro plasma clot assay results (Fig. 4A)showing a 4- to 6-fold increase in potency: plasma clearancedoes not play a role in the plasma clot assay. Marder andSherry (27) have proposed that one potential clinical advan-tage of streptokinase over tPA is a longer half-life, which mayreduce the tendency for rethrombosis after successful throm-bolysis. The prolonged plasma clearance ofr-scuPA(32kDa)-59D8 may offer a similar advantage. A precise determinationof the thrombolytic potency and fibrin selectivity ofr-scuPA(32kDa)-59D8 will require characterization in a moreclinically relevant model.

In summary, r-scuPA(32kDa)-59D8 demonstrates that it ispossible to design a plasminogen activator chimera in whichthe activities of the components, though comparable to thoseof the native proteins, manifest increased selectivity andpotency when combined in a single molecule.

We are grateful for the technical assistance of James Anagnost,Jacqueline Carter, Herinder Lonial, Mary Petro, and Samuel With-erspoon and the editorial assistance of Tom McVarish and Kate W.Harris. This work was supported in part by National Institutes ofHealth Grants HL-19259 (to E.H.), HL-28015 (to G.R.M.), andHL-41808 and HL-02414 (to M.S.R.) and by a grant from The RobertWood Johnson Foundation (to T.W.L.).

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