Proc. Nati. Acad. Sci. USAVol. 84, pp. 6904-6908, October 1987Medical Sciences

Construction and expression of a recombinant antibody-targetedplasminogen activator

(anti-fibrin antibody/fusion protein/immunoglobulin gene/thrombolysis)

JANET M. SCHNEE*, MARSCHALL S. RUNGE*, GARY R. MATSUEDA*, NORMAN W. HUDSONt,JONATHAN G. SEIDMANt, EDGAR HABER*, AND THOMAS QUERTERMOUS*§*Cardiac Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114; tDepartment of Medicine, Indiana University, Indianapolis, IN46223; and tDepartment of Genetics, Harvard Medical School, 25 Shattuck Street, Boston, MA 02115

Communicated by Michael Sela, June 8, 1987 (received for review May 6, 1987)

ABSTRACT Covalent linkage of tissue-type plasminogenactivator (t-PA) to a monoclonal antibody specific for the fibrinB chain (anti-fibrin 59D8) results in a thrombolytic agent thatis more specific and more potent than t-PA alone. To providea ready source of this hybrid molecule and to allow tailoring ofthe active moieties for optimal activity, we have engineered arecombinant version of the 59D8-t-PA conjugate. The rear-ranged 59D8 heavy chain gene was cloned and combined in theexpression vector pSV2gpt with sequence coding for a portionof the y2b constant region and the catalytic .3 chain of t-PA.This construct was transfected into heavy chain loss variantcells derived from the 59D8 hybridoma. Recombinant proteinwas purified by affinity chromatography and analyzed withelectrophoretic transfer blots. These revealed a 65-kDa heavychain-t-PA fusion protein that is secreted in association with the59D8 light chain in the form of a 170-kDa disulfide-linkeddimer. Chromogenic substrate assays showed the fusion pro-tein to have 70% of the peptidolytic activity of native t-PA andto activate plasminogen as efficiently as t-PA. In a competitivebinding assay, reconstituted antibody was shown to have abinding profile similar to that of native 59D8. Thus, byrecombinant techniques, we have produced a hybrid proteincapable of high-affinity fibrin binding and plasminogen acti-vation.

The remarkable ability of antibodies to selectively identifyand adhere to an unlimited number of antigenic determinantsmakes them an attractive vehicle for delivering effectorfunctions to cells, tissues, or even soluble molecules. Thehomogeneous specificity ofmonoclonal antibodies makes theprospect ofantibody targeting particularly attractive. The useof antibodies for nuclear imaging and delivery of toxins tomalignant cells shows promise and is already close at hand (1,2). Stimulated by the need for a more thrombus-specificplasminogen activator for the dissolution of intravascularclots, this laboratory has been investigating the use offibrin-specific antibodies for the delivery of various plasmin-ogen activators (3-5). It is apparent that, by coupling aplasminogen activator to a fibrin-specific antibody, the con-version of plasminogen can be concentrated at the clotsurface, resulting in increased clot dissolution and lesssystemic fibrinogenolysis. However, the chemical couplingprocedures required to link the enzymatic molecule to theantibody are laborious and result in compounds that areunstable on storage. Further, because cross-linking agentsare not site specific, the conjugates are a heterogeneouspopulation. For these reasons evaluation of activity amongvarious constructs, and ultimately in vivo testing, thusbecome difficult.

Recombinant techniques provide the opportunity of pro-ducing hybrid molecules that contain only the protein do-mains of interest, attached in a single fashion, and shouldallow for production in significant quantity. Pioneering workby Neuberger et al. indicates the feasibility of using recom-binant techniques to attach peptides to the hinge region oftheimmunoglobulin heavy chain (6, 7). This is accomplished byjoining the gene coding for the immunoglobulin heavy chainwith the gene coding for an effector peptide and transfectingthis construct into a readily available myeloma cell capable ofproducing only immunoglobulin light chain. Their workdemonstrates that folding of the effector peptide at thecarboxyl end ofa hybrid molecule is sufficient to give 10-30%oof the peptide's normal level of activity. This is true evenwhen the carboxypeptide has intrachain disulfide bonds. Inthese earlier studies, the antigen-combining site of the hybridmolecule was derived from a model myeloma protein and wasnot an integral feature of the molecular design. The workreported here builds on these fundamental studies by showingthat antibody specificity and effector function can be selec-tively combined to produce an antibody-targeted molecule.Transfection of the gene construct into a specifically select-ed, hybridoma-derived heavy chain loss variant appears to bea fast and easy means with which to reform antigen-bindingfunction. Also, we show that the activity of the effectorpeptide attached to the carboxyl end of the immunoglobulinheavy chain can approach normal levels, even when theeffector activity depends on complex, multiple, intrachaindisulfide bonds.

MATERIALS AND METHODS

Cloning of 59D8 Heavy Chain Gene. High molecular weightgenomic DNA was made from the 59D8 hybridoma cells asdescribed (8). To identify rearranged heavy chain immuno-globulin genes specific for the 59D8 hybridoma line, Southernblot analysis was performed as described with EcoRI-digested genomic DNA and a 1.7-kilobase (kb) EcoRI/Pst Igenomic joining region probe (9, 10). Two rearrangementswere identified that were not found in either of the cellsoriginally fused to produce the 59D8 hybridoma (SP2/0 andBALB/c). Subsequently, 1 mg ofgenomicDNA was digestedwith EcoRI and size-fractionated on a preparative agarose gel(11). Fractions containing each of the two rearranged frag-ments were identified by hybridization to the joining regionprobe. These fractions were concenttated and ligated intoXgtlO. The two subgenomic libraries thus constructed werescreened with the joining region probe and several potentialclones were isolated from each library (12). Selection of theclone containing the rearranged fragment coding for the 59D8

Abbreviations: CH2, heavy chain constant region gene second exon;t-PA, tissue-type plasminogen activator.§To whom reprint requests should be addressed.

6904

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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antigen-combining site was accomplished by hybridization toa 20-base-pair oligonucleotide that had been constructed onthe basis of the sequence of the 59D8 heavy chain mRNA.RNA isolation and sequencing, 32p labeling of the oligonu-cleotide with T4 polynucleotide kinase, and hybridizationwere carried out according to described techniques (12-14).

Expression Vector Construction. The tissue-type plasmin-ogen activator (t-PA) sequence was derived from a cDNAclone (pPA34'F) that had been constructed from HeLa cellmRNA (15). DNA encoding the 13 chain was isolated in twoparts. First, a 3' fragment extending from the f3 chain Sac Isite to the EcoRI site of pBR322 was isolated and ligated intopGEM3. Next, a contiguous 5' fragment was isolated bydigestion with SfaNI and Sac I. A synthetic oligonucleotide,containing a BamHI end, an Xho I site, and two basesreconstituting a codon for glycine, was added to this secondfragment's 5' end. The modified fragment was then ligatedinto a plasmid already containing the 3' fragment, thusreconstituting the B-chain sequence. The .8 chain was excisedwith Xho I and Sca I-the Sca I site being contributed by thepBR322 sequence.The final construct was assembled in the pSV2gpt vector

that had been modified by the insertion of a polylinkercontaining a 6-kb Xba I restriction fragment encoding themurine 'y2b heavy chain constant region (16, 17). The pro-ductive 59D8 heavy chain rearranged gene that had beencloned on a 2.6-kb EcoRI fragment was inserted in the correctorientation into an EcoRI site in the polylinker 5' of the y2bconstant region. The constant region sequence between theunique Xho I site in the heavy chain constant region genesecond exon (CH2) and a Sal I site in the polylinker wasexcised, the Sal I site was blunted, and the t-PA ,B chain wasligated into place. Nucleotide sequence analysis confirmedthat the junction between the heavy chain and t-PA segmentswas in-frame (18).Monoclonal Antibodies and Selection of Loss Variants.

Fibrin-specific monoclonal antibody 59D8 was raised byimmunization with a synthetic heptapeptide based on theamino-terminal sequence of the fibrin 13 chain, as described(19). Hybridoma cells and loss variants were maintained incomplete medium: Dulbecco's modified Eagle's mediumwith 4.5 mg of glucose per ml, 12% fetal calf serum, 50 ,ug ofgentamicin sulfate per ml, and 0.6 mg of L-glutamine per ml.For selection of heavy chain loss variants, cells were grownin soft agarose. Five milliliters of complete medium plus 0.2%agarose and an additional 8% fetal calf serum was added totissue culture dishes (60 mm) and allowed to solidify at roomtemperature for 3-5 min. Cells (1-2 x 103) to be selected forchain loss were layered over the agarose bed in a 0.5-mlsolution containing complete medium and 0.2% agarose. Theplates were incubated at 37°C in 6% CO2 until clusters ofcellswere formed (2-4 days). To detect heavy chain loss variants,cell clusters were overlaid with an antiserum solution (1.0 ml)containing complete medium with 0.2% agarose and 5-10%orabbit or goat anti-mouse heavy chain. Cell clusters secretingheavy chain developed a precipitin halo. Clusters that did nothave a precipitin halo (-1 in 1000) were picked from softagarose by capillary pipet and subsequently delivered into96-well plates containing complete medium with 8% addi-tional fetal calf serum. Individual subclones were assayed byenzyme-linked immunoabsorbent assay (ELISA) or by elec-trophoretic transfer blotting for the presence of heavy andlight chain.

Transfection and Selection. The construct pD8SVtf3 wastransfected into loss variant cells by electroporation (electricfield-mediated DNA transfer), using an Isco power supply asdescribed elsewhere (20). Optimal transfection conditionswere a 2000-V discharge into 0.8 ml of phosphate-bufferedsaline. Transformants were selected by growth in mycophe-nolic acid, xanthine, and hypoxanthine. Confirmation of

transfection and expression was obtained by RNA transferblot analysis using a 2-kb cDNA probe coding for the 3'portion of the human t-PA 3 chain (12). Transfected cell lineswere subcloned according to standard techniques.

Protein Purification. Protein was purified from cell super-natants and from ascites by sequential double affinity chro-matography on two columns (5). One column was construct-ed by linking the synthetic peptide used for the generation of59D8 to Sepharose. The other consisted of an anti-humant-PA monoclonal antibody linked to Sepharose. We had useda third column, composed of benzamidine linked to Sepha-rose, in our initial purification attempts. However, eventhough benzamidine binds well to the active site of t-PA andbenzamidine-Sepharose can be used to purify the intactmolecule, the column did not retain the recombinant protein.

Purification of the recombinant protein was monitored bytwo solid-phase immunoassays. To detect anti-fibrin anti-body activity, 96-well microtiter plates were coated withfibrin monomer and blocked with 10% horse serum. Theywere then incubated with samples, washed, and probed with1251-labeled goat anti-mouse Fab. The second assay wasdesigned to detect t-PA antigen associated with anti-fibrinantibody activity. In this assay, the fibrin-monomer-coatedplates were incubated with culture supernatant or ascites andprobed with 1251-labeled anti-human t-PA. Because the f3chain of t-PA possesses no fibrin-binding activity, onlyrecombinant protein containing both functional domains isdetected.

Electrophoretic Transfer Blot Analysis. Electrophoretictransfer blots were made from reduced and nonreducedsamples separated on NaDodSO4/polyacrylamide gels usingestablished techniques (21). Either goat anti-mouse Fab or amonoclonal anti-human t-PA antibody labeled with 1251 wasused as a probe.

Antigen-Binding Assay. The original antibody (59D8) andthe recombinant molecule were first assayed for the presenceoffibrin-binding Fab antigen. This was accomplished with thesolid-phase immunoassay described above using 125I-labeledgoat anti-mouse Fab as a probe. Titration curves weregenerated for 59D8 and the recombinant protein by varyingtheir concentrations in the assay. That concentration thatwould yield the same amount of bound 125I-labeled antibodywas then selected from the linear part of each curve. At thisconcentration of either 59D8 or fusion protein, a competitionassay was performed in wells that had been coated with fibrinand filled with various amounts of soluble fibrin. Protein thatbound to the soluble rather than insoluble fibrin was removedby washing before application of the labeled antibody.

Assays of Enzymatic Function. To compare the enzymaticfunction of the recombinant protein with that of native t-PA,its peptidolytic properties were first examined in an assaythat measures cleavage of the nonselective substrate S-2288(Helena Laboratories, Beaumont, TX). The assay was car-ried out in a 50-Al volume ofbuffer (0.15M Tris/0.15 M NaCl)with a 1 mM final concentration of chromogenic substrate.Various concentrations of recombinant protein or t-PA pu-rified from the Bowes melanoma cell line (Bio Response,Hayward, CA) were added and the absorbance at 405 nm wasmeasured at a series of time points.To determine whether the recombinant protein was capa-

ble of activating plasminogen, a second assay was performedutilizing the chromogenic substrate S-2251 (Helena Labora-tories). The activities of melanoma t-PA, the recombinantprotein, and bovine trypsin were first determined in theS-2288 assay and the concentrations were adjusted such thateach enzyme was present at 100 units/100 Al. One hundredmicroliters of melanoma t-PA, recombinant protein, or bo-vine trypsin was then added in serial dilution to 100/l ofhuman plasminogen (0.15 mg/ml) and 800 Aul of S-2251

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substrate. The samples were incubated for 60 min at 370C.The reaction was terminated by the addition of 1 ml of 50%acetic acid and absorbance at 405 nm was determined.

GAM-Fabat-PA Ab

RESULTS

GAM-Fabat-PA Ab

-2000.- 97.4

- 68 0

Electroporation of the construct pSVD8T,8 (Fig. 1) into the59D8 heavy chain loss variants provided numerous trans-fected clones. When -1 x 108 hybridoma cells were mixedwith 50 ,ug of circular plasmid DNA in 0.8 ml of phosphate-buffered saline and subjected to a discharge of 2000 V, -15of the wells on a 96-well plate contained drug-resistantclones. Approximately 75% of these clones were shown tosecrete the recombinant protein. Five clones were chosen forfurther analysis on the basis of their growth rate and expres-sion of mRNA coding for the fusion protein.

Electrophoretic transfer blot analysis of the affinity-puri-fied recombinant protein is shown in Fig. 2. Blots of reducedgels probed with an iodinated anti-human t-PA monoclonalantibody revealed labeling of a 65-kDa peptide. This is theexpected size for a heavy chain-t-PA fusion protein. The 83chain of t-PA is -33 kDa and the truncated heavy chainshould contribute 30 kDa. Several lines of evidence indicatethat the 65-kDa peptide is not a t-PA-like molecule contrib-uted by fetal calf serum. The 65-kDa band is observed whenthe transfected cell lines are grown in serum-free medium orin the intraperitoneal space of mice. Also, when we purifiedbovine t-PA from fetal calf serum by benzamidine affinitychromatography, even though it was labeled by the antibodyon electrophoretic transfer blots, the size of the molecule was75 kDa (data not shown).

Electrophoretic transfer blots of reduced samples probedwith a goat anti-mouse Fab derived from polyclonal serarevealed labeling of a 25-kDa protein, which is the expectedsize of the 59D8 K light chain. Although on such blots thisreagent usually labels the mouse immunoglobulin heavychains also, the absence oflabeling ofthe fusion peptide is notsurprising since most of the heavy chain constant region hasbeen removed. Blots produced with unreduced samplesshowed labeling of a single band at a molecular mass of170-180 kDa by both of the iodinated antibodies (Fig. 2). Thisprovides strong evidence that the hybridoma cells are pro-ducing a molecule containing immunoglobulin and t-PA

3 -UT

t-PA-B 0 a

XholIpj>(SfaNl ) g\

y2b-CH pSVD8t TXbolRi

gba1~~RRIA-mpRVDJ

FIG. 1. Structure of expression plasmid pSVD8tB, which codesfor the heavy chain-t-PA fusion protein. Coding sequences areindicated by labels outside the circle; restriction sites used inconstruction are indicated inside the circle. See text for details ofplasmid construction. VDJ, productive 59D8 heavy chain rearrange-ment; y2b-CH, genomic sequence of the murine y2b heavy chainconstant region; t-PA-,8, cDNA sequence coding for the human t-PAf chain; 3'-UT, 3' untranslated sequence of human t-PA cDNA;AmpR, pBR322 ampicillin-resistance gene; gpt, Escherichia coliguanine phosphoribosyltransferase gene driven by simian virus 40promoter; R1, EcoRI.

-430

-25.7

25- "

reduced nonreduced

FIG. 2. Electrophoretic transfer blots of recombinant proteinseparated by reducing and nonreducing NaDodSO4/PAGE andprobed with "25I-labeled goat anti-mouse Fab (GAM-Fab) or mono-clonal anti-human t-PA antibodies (at-PA Ab). The molecular massmarker on the left (given in kDa) applies to the first lane only;markers on the right apply to the other lanes. Size markers (approx-imate) are given in kDa.

peptides. The 170- to 180-kDa value suggests that the inter-heavy-chain disulfide bonds have formed to give a F(ab')2-like molecule that contains two antigen-combining sites andtwo t-PA moieties.The peptidolytic activity of the t-PA portion of the mole-

cule was initially assessed by measuring the cleavage of thenonspecific substrate S-2288. Cleavage of this tripeptide canbe accurately monitored by following the production ofparanitroaniline, which absorbs light at a wavelength of 405nm. Fig. 3A shows a typical assay, which employs differingconcentrations of the recombinant protein and comparesdirectly the activity of differing concentrations of puremelanoma t-PA. Activity in this assay is defined as the rateof change in absorbance. When a comparison is made on amolar basis between the recombinant protein and nativet-PA, the recombinant protein possesses 70% of the activityof native t-PA.To determine whether the catalytic f subunit maintained

activity against plasminogen (its physiologic substrate), anS-2251 assay was performed. Here the plasminogen activatoris required to convert plasminogen to plasmin and theplasmin subsequently liberates paranitroaniline from a syn-thetic tripeptide. Neither plasminogen activator nor trypsincan directly convert the S-2251 substrate. The amidolyticactivities of the recombinant protein, melanoma t-PA, andtrypsin were first determined in the S-2288 assay, and thenthe ability of comparable amounts of each to convertplasminogen was determined. Fig. 3B reveals that the abilityof the recombinant protein to act upon the physiologicsubstrate is very similar to that of native t-PA. Although anonspecific serine protease such as trypsin is able to convertplasminogen to plasmin, it does so much less efficiently thandoes either the native or recombinant plasminogen activator.The purification scheme and the assays used to follow

purification required an intact and functional antigen-com-bining site. To more quantitatively compare the recombinantmolecule with antibody 59D8, we employed a simple com-petition assay. This assay measured the ability of solublefibrin monomer to compete for antibody-binding sites againstfibrin bound to the bottom of a 96-well plate. Although theassay indicates that the native antibody binds fibrin monomerbetter than does the recombinant protein, the difference intheir binding activities is <10-fold (Fig. 4). It is evident thatantibody binding is not significantly impaired in the fusionprotein.

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100r

80

-o b.0,, 40

.o0 .5 1.0 1.5 2.0 2.5 3.0 3.5Time, hr 20~

0

\"'A

.1 1 10 100Soluble fibrin, /g

FIG. 4. Comparison of the binding behavior of 59D8 anti-fibrinantibody and recombinant protein. Curves represent the inhibition ofantibody binding to solid-phase fibrin monomer by competition withvarious concentrations of soluble fibrin monomer. Recombinantantibody (dashed lines) requires a slightly higher concentration ofsoluble fibrin for 50o inhibition than does the antibody (solid lines)and thus binds fibrin somewhat less avidly. This difference is<10-fold.

I 1 10

Activator, units100

FIG. 3. Chromogenic substrate assay comparing the catalyticactivity of the recombinant protein with that of melanoma t-PA. (A)As shown by the dashed lines, the S-2288 assay was performed with105 ng (0) or 70 ng (o) of recombinant protein. Solid lines representthe catalytic activity of 50 ng (e), 40 ng (n), or 30 ng (A) of melanomat-PA used as standards. Relative molar activity of the recombinantprotein was determined by comparison with t-PA standards withsimilar rates of catalysis. (B) The S-2251 assay was conducted withvarying activities of melanoma t-PA standard (o), recombinantprotein (o), and bovine trypsin (A). Units of activity of each proteinwere determined in the S-2288 assay.

DISCUSSIONExtensive analysis of the secreted protein indicates that a59D8 heavy chain-t-PA fusion protein is being expressed andsecreted in association with light chain in the manner pre-dicted. The amount of recombinant protein present in cellculture supernatants, however, appears to be only 10% ofthat expected for monoclonal antibodies. By affinity purifi-cation, we routinely obtained only 0.1 pug of purified proteinper ml of cell culture supernatant or 10 ,ug/ml in ascites. Wemonitored the purification with solid-phase immunoassays asdescribed above, and our recoveries from the affinity col-umns were within the expected range. There are a number ofpossible reasons for the limited production of recombinantprotein. One is that the recombinant protein is being degrad-ed during cell growth or protein purification. In an attempt tolimit proteolytic degradation, we have added protease inhib-itors to the cell cultures. Although no improvement in yieldwas observed, proteolytic degradation remains a concern.Other more fundamental problems could be the cause of

the low yields of protein. Although mRNA of the appropriatesize can be seen on RNA transfer blot, transcription of theconstruct may occur at a low level. Transcription is driven bythe natural heavy chain promoter and enhancer, but 3'sequences, which have been shown to be important inregulation of immunoglobulin expression, have been exclud-

ed from this construct (22, 23). In addition, the 3' untrans-lated region of the chimeric gene is from t-PA, a protein thatis produced at a low level under normal conditions, and issubsequently stored in the cells where it is produced. It ispossible that the 3' untranslated region of the t-PA gene leadsto low levels of transcription or translation or interferes withsecretion of the recombinant protein from the cell. Experi-ments aimed at quantitation of mRNA synthesis, proteinsynthesis, and stability of the recombinant peptide shouldallow resolution of this problem.Heavy chain loss variants provide a convenient tool for the

reconstitution of the antibody-combining site. Their avail-ability makes it unnecessary to clone and transfect theproductive light chain rearrangement. This approach, ofcourse, depends on being able to transfect these variant celllines. The two lines used in these experiments were easilytransfected using standard techniques, but it is not yet clearwhether other SP2/0-derived lines will behave similarly. Theamount of light chain that heavy chain loss variants secretevaries. However, some loss variants that secrete smallquantities of light chain may be capable of secreting normalamounts of this same light chain when heavy chain synthesisis resumed (24). Little is known about the biological basis forloss of immunoglobulin chain production in these cells and itis possible that the ability of some loss variants to producelight chain as well as heavy chain may be impaired. Ourrecombinant protein's low level of production could be theresult of depressed light chain expression.The recombinant t-PA chain has a high level of catalytic

activity, and it retains the specific ability to convert plas-minogen to plasmin. Earlier studies, which linked staphylo-coccal nuclease and Escherichia coli DNA polymerase func-tions to immunoglobulin heavy chain, yielded considerablyless effector function activity than the 70% measured in theS-2288 assay (6, 7). This retention of enzymatic activity andsubstrate specificity indicate that even complex moleculesrequiring strict folding and formation of multiple intrachaindisulfide bonds can be used to form hybrid recombinantproteins. Others have shown that the chain of t-PA iscapable of folding correctly and maintaining activity in theabsence of the a chain (25, 26). Our results confirm theactivity of the catalytic chain alone and indicate that the chaincan fold correctly in the context ofa different amino-terminal

A.20 H

.15 F

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sequence. Together, these observations provide evidence forthe independent folding of different protein domains.

This molecule represents the first attempt to combine achosen antibody specificity and effector function so as toproduce a recombinant antibody-targeted hybrid molecule.In this initial construct, we wanted to limit the size of eachcomponent to its functional minimum to determine thestructures necessary for activity. Although both functionalends of this molecule are active, it is not yet known whetherthey can act in concert and whether they will recreate theenhanced fibrinolysis provided by coupling plasminogenactivators to fibrin-specific antibodies. There are a number ofwell-defined in vitro and in vivo assays for evaluatingthrombolysis. Such assays make this antibody-targeted mol-ecule particularly useful because it can be applied in inves-tigations of the rules that govern the construction of hybridmolecules. Even if this recombinant protein is not effectivein vivo, it should still point to modifications that will allowconstruction of an effective molecule.

In summary, we have cloned the heavy chain gene codingfor the antigen-combining site of an anti-fibrin antibody andproduced a construct that codes for a truncated heavychain-t-PA ,6-subunit fusion peptide. The construct wassubsequently transfected into heavy chain loss variants of theanti-fibrin hybridoma. Electrophoretic transfer blot analysisindicates that the fusion protein is secreted in associationwith light chain. The recombinant protein has anti-fibrinantibody activity and retains a level of plasminogen-activat-ing activity high enough to be considered similar to that ofnative t-PA.

We thank Dr. R. I. Near for providing the pSV2gpt expressionvector containing the y2b constant region sequence. Dr. D. J. Pankais gratefully acknowledged for his contribution of the 59D8 mRNAsequence. We also extend our appreciation to Dr. S. J. F. Degen,without whose generous gift of the t-PA cDNA this project wouldhave been considerably more difficult. J.M.S. was supported by afellowship from the Stanley Sarnoff Endowment for the Cardiovas-cular Sciences. M.S.R. is a Merck Fellow of the American Collegeof Cardiology. J.G.S. is supported by grants from the NationalInstitutes of Health (AI-19938 and AI-18436) and by a grant from theMallinckrodt Foundation. Other support is provided by grants fromthe National Institutes of Health (HL-19259 and HL-28015) and bya grant from the Schering Corporation.

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