A new chimeric plasminogen activator with high fibrin affinity was designed to bind to a fibrin clot and initiate clot destruction in the presence of thrombin, but not plasmin. The chimeric molecule has an antibody variable region having a fibrin-specific antigen binding site and a single chain urokinase region having a thrombin activation site but not a plasmin activation site. The preferred embodiment, 59D8-ScuPA-T, has an N-terminal fragment of an anti-fibrin antibody (59DB) and a C-terminal thrombin-activatable low molecular weight single-chain urokinase plasminogen activator (scuPA-T). The scuPA-T portion was obtained by deletion of two amino acids (Phe157 and Lys 158) that make up the plasmin activation site from low molecular weight single chain urokinase-type plasminogen activator (scuPA).

FIELD OF THE INVENTION 
This invention relates to plasminogen activators and methods of treatment 
of thrombosis. In particular, this invention relates to single chain 
urokinase and to chimeric immunoglobulin molecules incorporating a single 
chain urokinase region. 
BACKGROUND OF THE INVENTION 
Most myocardial infarctions are caused by coronary thrombosis. DeWood at 
al., N. Eng. J. Med., 303, 897 (1983). The coronary thrombi that cause 
myocardial infarction can be lysed by thrombolytic agents, which 
significantly reduce mortality. ISIS-3 Collaborative Group, Lancet, 339, 
753-70 (1992); Haber, et al., Science, 243, 51-6 (1989). However, 
currently available thrombolytic agents may also cause haemorrhagic 
strokes or other bleeding. ISIS, supra: Marder, V. J. and Sherry, S., N. 
Engl. J. Med. 318, 1512-20 (1988); Collen, D., Am J. Cardiol., 69, 71A-81A 
(1992); Smitherman, T. C. Mol. Biol. Med., 8, 207-18 (1991). 
Plasmin and thrombin are both enzymes that affect coronary thrombi. Plasmin 
is a fibrinolytic enzyme, i.e., it lyses the fibrin present in a thrombus. 
Currently available thrombolytic agents activate the conversion of 
plasminogen to the fibrinolytic enzyme plasmin. Plasmin, however, also 
lyses fibrinogen, resulting in a bleeding diathesis. 
Thrombin, on the other hand, initiates thrombus formation by cleavage of 
fibrinogen and activation of platelets. Mann, K. G. and Lundbald, R. L., 
Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 
(Colman, R. W., et al., eds) 2nd ed., 148-161 (1987). It is produced 
locally around the site of injury and is absorbed into the thrombus by 
interacting with fibrin. Jackson, C. M., Hemostasis and Thrombosis: 
Mechanism of Prothrombin Activation, (Colman, R. W., et al., eds) 2nd 
ed.,148-161 (1987); Fenton, J. W., II, Ann. N.Y. Acad. Sci. 322, 468-495 
(1981). Fibrin-bound thrombin is enzymatically active and is slowly 
released from a thrombus. Liu, C. Y. et al., J. Biol. Chem. 258, 10530-5 
(1979). Therefore, the inventors have identified thrombin as a transient 
marker for a thrombus. 
Single chain urokinase-type plasminogen activator (scuPA) is a zymogen. It 
can be activated by plasmin cleavage between Lys 158 and IIe 159, and 
inactivated by thrombin cleavage between Arg 156 and Phe 157. Lijnen, H. 
R. et al., Semin. Throm. Hemostasis 13, 152-9 (1987); Ichinose, A. et al., 
J. Biol. Chem. 261, 3486-9 (1986). Thus, scuPA-initiated plasma clot lysis 
is apt to be regulated by plasmin or thrombin around the thrombus. 
Plasmin- or thrombin-resistant scuPA mutants have been produced to study 
plasmin activation or thrombin inactivation of scuPA. Nelles, L., Lijnen, 
H. R., Collen, D., and Holmes, W. E. J. Biol. Chem., 262, 5682-9 (1987); 
Lijnen, R., et al. Eur. J. Biochem. 177, 575-82 (1988); Lijnen, R., et al. 
Eur. J. Biochem. 172, 185-8 (1988); Miyake, T.et al., J. Biochem. 104, 
643-7 (1988); Eguchi, Y., et al., J Biochem. 108, 72-9 (1990). In order to 
increase scuPA's fibrin selectivity, chimeric plasminogen activators were 
constructed from an anti-fibrin antibody and low molecular weight scuPA. 
Both in vitro and in vivo, these activator constructs had better fibrin 
selectivity and higher potency than scuPA. Bode, C., et al., Science. 229, 
765-7 (1985); Bode, C., et al., J. Biol. Chem. 262, 10819-23 (1987); 
Runge, M. S., et al., Biochemistry, 27, 1153-7 (1988). Collen, D., et al., 
Fibrinolysis. 3, 197-202 (1989); Dewerchin, M., et al., Eur. J. Biochem., 
185, 141-9 (1989); Holvoet, P., et al., J. Biol. Chem. 266, 19717-24 
(1991); Runge, M. S., et al., Proc. Natl. Acad. Sci. USA. 88, 10337-41 
(1991). A chimeric immunoglobulin molecule having an antibody variable 
region with an antigen binding site specific for fibrin and a fibrinolytic 
enzyme activity region has also been prepared by recombinant DNA 
techniques. European Patent Application 478,366, published 1 April 1992. 
The preferred embodiment in that application is r-scuPA(32)-59 D8. 
The foregoing research has focused on ways to increase the fibrin 
selectivity of potential thrombolytic agents such as the scuPA mutants and 
conjugates. Due to the bleeding side-effect, however, the present 
inventors perceive a need for thrombolytic agents that distinguish between 
thrombi, which lead to myocardial infarction, and hemostatic plugs, which 
are mainly involved in prevention of bleeding. The present inventors 
hypothesized that plasminogen activators such as t-PA cannot distinguish 
fibrin epitopes on a pathogenic thrombus from those on a hemostatic plug. 
According to this hypothesis, thrombolytic agents lyse both thrombi and 
hemostatic plugs, causing bleeding during thrombolytic therapy. See 
Sherry, Mod. Conc. Cardiovas. Disease. 60, 25-30 (1991). 
SUMMARY OF THE INVENTION 
In accordance with the present invention, the present inventors designed a 
chimeric immunoglobulin molecule comprising (a) an antibody variable 
region with an antigen binding site specific for fibrin, covalently linked 
to (b) a single chain urokinase region having a thrombin activatable site 
but not a plasmin activatable site. In a preferred embodiment, the 
antibody variable region of the chimeric immunoglobulin molecule is a 
high-affinity anti-fibrin antibody fragment (Fab), with an antibody 
fragment from 59D8 most preferred. 
Also in accordance with the present invention are nucleic acid molecules 
having sequences coding for the aforementioned chimeric immunoglobulin 
molecule, expression vectors having that sequence, and host cells 
transformed by such vectors. This invention also includes a method for 
preparing the chimeric immunoglobulin molecule in which such host cells 
are grown under conditions that allow expression of the nucleic acid 
sequence of the vector, after which the immunoglobulin molecule may be 
purified. 
Further in accordance with the present invention is a pharmaceutical 
composition comprising the aforementioned chimeric immunoglobulin molecule 
and a pharmaceutically acceptable carrier. The present invention further 
includes a method of lysing a thrombus in a mammalian subject, which 
comprises administering to the subject an effective amount of the chimeric 
immunoglobulin molecule. Further still, the present invention includes a 
method of detecting a thrombus in a mammalian subject, which comprises 
administering the chimeric immunoglobulin molecule having a detectable 
marker and then detecting the presence of the marker.

DETAILED DESCRIPTION OF THE INVENTION 
Definition of Terms 
The following definitions apply to the terms as they appear throughout this 
specification, unless otherwise limited in specific instances. 
The term "scuPA" refers to single chain urokinase-type plasminogen 
activator. It exists in two forms--high molecular weight and low molecular 
weight. High molecular weight scuPA has a molecular weight of about 54 kDa 
and comprises a EGF domain, a kringle domain, a thrombin inactivation 
site, a plasmin activation site and a protease domain. Low molecular 
weight scuPA has a molecular weight of about 34 kDa and is a degradation 
product of high molecular weight scuPA that lacks the EGF and kringle 
domain but has the rest of the domains. Such single chain urokinase 
regions are described, for example, in Bachmann, F., Hemostasis and 
Thrombosis: Basic Principles and Clinical Practice (Colman, R. W., et al., 
eds.) 2nd ed., pp. 318-39 (1987); Stump et al,, J. Biol. Chem., 261 17120 
(1986); Nelles et al., J Biol. Chem., 262, 10855 (1987). The domains 
needed to maintain thrombolytic activity may be determined by cloning, 
subcloning, expression and screening procedures well known to those having 
ordinary skill in the art. The term "single chain urokinase region" 
comprises sequences based on both the low and high molecular weight forms 
of scuPA, although the low molecular weight form is preferred. 
The term "scuPA-T" refers to a scuPA mutant made by deleting the two amino 
acids phenylalanine (157)-lysine (158) located between thrombin and 
plasmin cleavage sites, as shown in FIG. 2. 
The term "59D8" refers to the monoclonal antibody fragment prepared by the 
procedures described in Quertermous et al., J. Immunol., 2687-90 (1987). 
The term "fibrin-specific" as used to describe antibodies refers to 
antibodies raised against fibrin. Such antibodies have been described, for 
example, in Hui et al., Science, 222, 1129 (1983); U.S. Pat. No. 
4,927,916, issued May 22, 1990; U.S. Pat. No. 4,916,070, issued Apr. 10, 
1990; Kudryk, et al., Mol. Immunol., 21, 89 (1984); European Patent 
Application 146, 050, published 26 June 1985; and Australian Patent 
Application AV-A-25387/84. 
Process of Preparation 
In order to design a novel plasminogen activator that is able to 
distinguish new from old clots, a surface marker on a new clot is 
required. The inventors identified active fibrin-bound thrombin as such a 
marker based on certain known characteristics of thrombin: (1) thrombin is 
produced locally around the injury site, (2) thrombin is located around a 
thrombus, (3) thrombin tends to leach out of a fibrin clot during 
extensive washing, and (4) prolonged exposure of surface-bound thrombin in 
circulating plasma may result in inactivation by plasma protease 
inhibitors. 
The regions making up the chimeric molecule of the present invention may be 
prepared as follows. 
Single Chain Urokinase Region 
The single chain urokinase region may be prepared by recombinant nucleic 
acid methods. See, for example, the recombinant DNA methods of Nelles et 
al., J. Biol. Chem., 262, 10855 (1987). 
The DNA sequence may be derived from a variety of sources, including 
genomic DNA, subgenomic DNA, cDNA, synthetic DNA, and combinations 
thereof. Genomic and cDNA may be obtained in a number of ways. Cells 
coding for the desired sequence may be isolated, the genomic DNA 
fragmented (e.g., by treatment with one or more restriction 
endonucleases), and the resulting fragments cloned, identified with a 
probe complementary to the desired sequence, and screened for the presence 
of a sequence coding for fibrin recognition or for thrombolytic activity. 
The cDNA may be cloned and the resulting clone screened with a probe for 
the desired region. Upon isolation of the desired clone, the cDNA may be 
manipulated in substantially the same manner as the genomic DNA. 
To provide for thrombin activation only, the plasmin cleavage site from a 
single chain urokinase region is deleted while the thrombin cleavage site 
remains. This deletion may take place at the nucleic acid level, in which 
the nucleotides encoding the plasmin cleavage site are deleted, or at the 
protein level. For both nucleic acids and proteins, the necessary 
procedures are well known to those having ordinary skill in the art. A 
preferred procedure is site-directed mutagenesis, in which a primer 
lacking the sequence coding for the plasmin cleavage site is used in a T7 
DNA polymerase reaction or a polymerase chain reaction (PCR). 
Fibrin-Specific Antibody Region 
In preparing the chimeric immunoglobulin molecule of the present invention, 
the entire fibrin-specific antibody may be cloned. In order to reduce the 
size of the molecule and to reduce antigenicity, it is preferred to use 
only that region of the antibody that will bind to fibrin. 
The variable and constant regions of the fibrin-specific antibody used may 
be derived from a mammalian source, with a human source preferred. The 
variable and constant regions may, however, be derived from separate 
sources; for example, the variable region may be derived from a nonhuman 
mammalian source and the constant regions from a human source to reduce 
antigenicity. Principles and procedures used to isolate such regions are 
well known in the art. See, for example, European Patent Application 478, 
366, published Apr. 1, 1990. 
For the variable region of the fibrin-specific antibody, the rearranged 
heavy chain coding DNA may include V, D, and J regions. The rearranged 
germlin light chain coding DNA may include the V and J regions. Upon 
identification of the cloned fragment containing the sequence for the 
fibrin-specific binding site, that fragment may be further manipulated; 
for example, to remove superfluous DNA (including all or part of any 
introns present) or to modify one or both termini. 
Control Regions 
To express the chimeric immunoglobulin molecule, transcriptional and 
translational signals recognized by an appropriate host are necessary. 
The promoter region from genomic DNA may be obtained in association with 
the DNA sequence for the fibrin-specific antibody region or the single 
chain urokinase region. To the extent that the host cells recognize the 
transcriptional regulatory and translational initiation signals associated 
with the variable region, the 5' region adjacent to the coding sequence 
may be retained and employed for transcriptional and translational 
regulation. This region typically will include those sequences involved 
with initiation of transcription and translation, such as the TATA box, 
capping sequence, CAAT sequence, and the like. Typically, this region will 
be at least about 150 base pairs long, more typically about 200 bp and 
rarely exceeding about 1 to 2 kb. 
The non-coding 3' region may be retained, as well, especially for its 
transcriptional termination regulatory sequences, such as the stop signal 
and polyadenylated region. In addition, the non-coding 3' region may also 
contain an enhancer in immunoglobulin genes. Where the transcriptional 
termination signals are not satisfactorily functional in the host cell, 
then a functional 3' region from a different gene may be substituted. In 
this method, the choice of the substituted 3' region would depend upon the 
cell system chosen for expression. 
A wide variety of transcriptional and translational regulatory sequences 
may be employed, depending upon the nature of the host. The 
transcriptional and translational regulatory sequences may be derived from 
viral sources (e.g., adenovirus, bovine papilloma virus, Simian virus, and 
the like) where the regulatory signals are derived from a gene that has a 
high level of expression in the host. Alternatively, promoters from 
mammalian expression products (e.g., actin, collagen, myosin, and the 
like) may be employed. Transcriptional initiation regulatory signals may 
be selected that allow for repression or activation, so that expression of 
the genes can be modulated. One such controllable modulation technique is 
the use of regulatory signals that are temperature-sensitive, so that 
expression can be repressed or initiated by changing the temperature. 
Another controllable modulation technique is the use of regulatory signals 
that are sensitive to certain chemicals. 
Formation of the Chimeric Construct 
DNA fragments may be ligated in accordance with conventional techniques 
known in the art. Such techniques include use of restriction enzymes to 
digest DNA fragments, DNA polymerases and nucleotides to fill in sticky 
ends to form blunt ends, alkaline phosphatase to avoid undesired 
ligations, and ligases to join fragments. 
The constructs for the fibrin-specific antibody and single chain urokinase 
regions may be joined together to form a single DNA segment or may be 
maintained as separate segments by themselves. The constructs may be 
introduced into a cell by transformation in conjunction with a gene 
allowing for selection where the construct will become integrated into the 
host genome. Usually, the construct will be part of a vector having a 
replication system recognized by the host cell. 
Expression Vectors 
Expression vehicles for production of the molecules of the invention 
include plasmids or other vectors. In general, such vectors contain 
control sequences that allow expression in various types of hosts, 
including but not limited to prokaryotes, yeasts, fungi, plants and higher 
eukaryotes. Suitable expression vectors containing the desired coding and 
control sequences may be constructed using recombinant DNA techniques 
known in the art, many of which are described in Sambrook, et al., 
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor 
Laboratory, Cold Spring Habor, N.Y. (1989). 
An expression vector as contemplated by the present invention is at least 
capable of directing the replication, and preferably the expression, of 
the nucleic acids of the present invention. One class of vectors utilizes 
DNA elements that provide autonomously replicating extrachromosomal 
plasmids derived from animal viruses (e.g., bovine papilloma virus, 
polyomavirus, adenovirus, or SV40 virus). A second class of vectors relies 
upon the integration of the desired gene sequences into the host cell 
chromosome. 
Expression vectors useful in the present invention include sequences that 
control the replication and expression of the subject DNA sequence. 
Typically, the expression vector contains an origin of replication, a 
promoter located 5' to (i.e., upstream of) the DNA sequence to be 
expressed, and a transcription termination sequence. Suitable origins of 
replication include, for example, the Col E1, the SV40 viral and the M13 
orgins of replication. Suitable termination sequences include, for 
example, the bovine growth hormone, SV40, lac Z and the Autographa 
californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral 
polyadenylation signals. Suitable promoters include, for example, the 
immunoglobulin H chain promoter, the cytomegalovirus promoter, the lac Z 
promoter, the gal 10 promoter and the AcMNPV polyhedral promoter. 
The expression vectors may also include other regulatory sequences for 
optimal expression of the desired product. Such sequences include 
stability leader sequences, which provide for stability of the expression 
product; secretory leader sequences, which provide for secretion of the 
expression product; enhancers, which upregulate the expression of the DNA 
sequence; and restriction enzyme recognition sequences, which provide 
sites for cleavage by restriction endonucleases. All of these materials 
are known in the art and are commercially available. See, for example, 
Okayama, Mol. Cell. Biol., 3 280 (1983). 
A suitable expression vector may also include marking sequences, which 
allow phenotypic selection of transformed host cells. Such a marker may 
provide prototrophy to an auxotrophic host, biocide resistance (e.g., 
antibiotic resistance) and the like. The selectable marker gene can either 
be directly linked to the DNA gene sequences to be expressed, or 
introduced into the same cell by co-transfection. Examples of selectable 
markers include neomycin, ampicillin, hygromycin resistance and the like. 
Host Cells 
The present invention additionally concerns host cells containing an 
expression vector or vectors that comprise a DNA sequence coding for the 
chimeric immunoglobulin molecule. In a preferred embodiment, the 
expression vector for the chimeric molecule only has the sequence for the 
heavy chain of the fibrin-specific antibody and is used with host cells 
that provide the light chain. Alternatively, the expression vector for the 
chimeric molecule could be co-transfected with an expression vector for 
the light chain of the fibrin-specific antibody. 
The preferred hosts are mammalian cells, grown in vitro in tissue culture, 
or in animals. Mammalian cells may provide post-translational modification 
to immunoglobulin protein molecules, including correct folding or 
glycosylation at correct sites. Mammalian cells that may be useful as 
hosts include cells of fibroblast origin (e.g., VERO or CHO-K1) or 
lymphoid origin (e.g., SP2/0-AG14, or P3x63Sg8) or derivatives thereof. 
Preferred mammalian host cells include 5P2/0 and J558L. The host cells of 
Schnee, et al., Proc. Natl. Acad. Sci. USA. 84, 6904-8 (1987) are most 
preferred. 
Immortalized cells, particularly myeloma or Iymphoma cells, are also 
suitable host cells. These cells may be grown in an appropriate nutrient 
medium in culture flasks or injected into a synergenic host (e.g., mouse 
or rat) or an immunodeficient host or host site (e.g., nude mouse or 
hamster pouch). In particular, the cells may be introduced into the 
abdominal cavity for production of ascites fluid and harvesting of the 
chimeric molecule. Alternatively, the cells may be injected subcutaneously 
and the antibodies harvested from the blood of the host. The cells may be 
used in the same manner as the hybridoma cells. See Diamond et al., N. 
Eng. J. Med., 344 (1981); Monoclonal Antibodies: Hybridomas-A New 
Dimension in Biologic Analysis (Kennatt, et al., eds.) Plenum (1980). 
When using co-transfection with a light chain expression vector, many 
different prokaryotic and eukaryotic host cells may be employed. Suitable 
prokaryotic host cells include, for example, E. coli strains HB101, DH5a, 
XL1 Blue, Y1090 and JM101. Suitable eukaryotic host cells include, for 
example, Spodoptera frugiperda insect cells, COS-7 cells, human 
fibroblasts, and Saccharomyces cerevisiae cells. 
Expression of the Chimeric Immunoglobulin Molecule 
Expression vectors may be introduced into host cells by various methods 
known in the art. For example, transfection of host cells with expression 
vectors can be carried out by the calcium phosphate precipitation method. 
However, other methods for introducing expression vectors into host cells, 
for example, electroporation, liposomal fusion, nuclear injection, and 
viral or phage infection can also be employed. 
Host cells containing an expression vector that contains a DNA sequence 
coding for the chimeric immunoglobulin molecule may be identified by one 
or more of the following six general approaches: (a) DNA--DNA or RNA-DNA 
hybridization; (b) the presence or absence of marker gene functions; (c) 
assessment of the level of transcription by measuring production of mRNA 
transcripts encoding the chimeric immunoglobulin molecule in the host 
cell; (d) detection of the gene product immunologically; (e) enzyme assay; 
and (f) PCR. 
In the first approach, the presence of a DNA sequence coding for the 
chimeric immunoglobulin molecule can be detected by DNA--DNA or RNA-DNA 
hybridization using probes complementary to the DNA sequence. 
In the second approach, the recombinant expression vector/host system can 
be identified and selected based upon the presence or absence of certain 
marker gene functions (e.g., thymidine kinase activity, resistance to 
antibiotics, etc.). A marker gene can be placed in the same plasmid as the 
DNA sequence coding for the chimeric immunoglobulin molecule under the 
regulation of the same or a different promoter used to regulate the coding 
sequence. Expression of the marker gene indicates expression of the DNA 
sequence coding for the chimeric immunoglobulin molecule. 
In the third approach, the production of mRNA transcripts encoding the 
chimeric immunoglobulin molecule can be assessed by hybridization assays. 
For example, polyadenylated RNA can be isolated and analyzed by Northern 
blotting or a nuclease protection assay using a probe complementary to the 
RNA sequence. Alternatively, the total RNA of the host cell may be 
extracted and assayed for hybridization to such probes. 
In the fourth approach, the expression of the chimeric immunoglobulin 
molecule can be assessed immunologically, for example, by immunoblotting 
with antibody to either the chimeric immunoglobulin molecule, the single 
chain urokinase region, or the antibody variable region (Western 
blotting). Alternatively, this technique could be carried out with the 
known epitope of the antibody variable region. 
In the fifth approach, expression of the chimeric immunoglobulin molecule 
can be measured by assaying for its activity. For example, the clot lysis 
assay described hereinbelow may be employed. 
In the sixth approach, oligonucleotide primers homologous to sequences 
present in the expression system (i.e., expression vector sequences or the 
chimeric immunoglobulin molecule sequences) are used in a PCR to produce a 
DNA fragment of predicted length, indicating incorporation of the 
expression system in the host cell. 
The expression vectors and DNA molecules of the present invention may also 
be sequenced. Various sequencing methods are known in the art. See, for 
example, the dideoxy chain termination method described in Sanger et al., 
Proc. Natl. Acad. Sci. USA 74, 5463-7 (1977), and the Maxam-Gilbert method 
described in Proc. Natl. Acad. Sci. USA 74, 560-4 (1977). 
Once an expression vector has been introduced into an appropriate host 
cell, the host cell may be cultured under conditions permitting expression 
of large amounts of the desired polypeptide, in this case the chimeric 
immunoglobulin molecule. Expression of the gene or genes encoded by the 
vector results in assembly to form the chimeric immunoglobulin molecule. 
The chimeric immunoglobulin molecule may be isolated and purified in 
accordance with conventional conditions, such as extraction, 
precipitation, chromatography, affinity chromatography, electrophoresis, 
and the like. The preferred method is affinity chromatography with either 
the amino terminal heptapeptide of the fibrin .beta. chain, which binds to 
the antifibrin site, or benzamidine, which binds to the plasminogen 
activator catalytic site. 
It should, of course, be understood that not all expression vectors and DNA 
regulatory sequences will function equally well to express the DNA 
sequences of the present invention. Neither will all host cells function 
equally well with the same expression system. However, one of ordinary 
skill in the art may make a selection among expression vectors, DNA 
regulatory sequences, and host cells using the guidance provided herein 
without undue experimentation and without departing from the scope of the 
present invention. 
Use and Utility 
The chimeric immunoglobulin molecule of the present invention is a 
thrombolytic agent and so will be useful in all ways known for 
thrombolytic agents. For example, it is useful in treatment of a patient 
following acute myocardial infarction, stroke, and deep vein thrombosis. 
The chimeric immunoglobulin molecule is also an anti-thrombotic agent 
useful, for example, to prevent rethrombosis from occurring after 
thrombolytic therapy and angioplasty surgery. 
For such therapeutic applications, the chimeric immunoglobulin molecule is 
administered to a patient and becomes localized to the site of a thrombus 
through the fibrin-specific binding site of the chimeric molecule. The 
chimeric molecule is activated by thrombin present at the site of the 
newly forming clot. The thrombus is lysed by the enzymatic activity of the 
activated urokinase region of the chimeric immunoglobulin molecule. 
One advantage of this chimeric immunoglobulin molecule is that it 
preferentially lyses thrombi rather than hemostatic plugs, thus avoiding 
the bleeding side-effect associated with other known thrombolytic agents. 
Another advantage is that, unlike tPA, the subject chimeric immunoglobulin 
molecule should be stable in human plasma. 
The chimeric immunoglobulin molecule of the present invention may also be 
used in immunodiagnostic applications, including immunodiagnosis. In this 
application, the chimeric molecule is detectably labeled, preferably with 
a radionuclide. The radionuclide must be of the type of decay that is 
detectable by a given type of instrument. Further, the radionuclide for in 
vivo diagnosis should have a half-life long enough that it is still 
detectable at the time of maximum uptake, but short enough that after 
diagnosis unwanted radiation does not remain in the patient. Coupling of 
the radionuclides to the chimeric molecule is known in the art and is 
often accomplished either directly or indirectly using an intermediary 
functional group. Examples of radioisotopes useful for diagnosis are 
.sup.99 Tc, .sup.123 I, .sup.131 I, .sup.97 Ru, .sup.67 Cu, .sup.67 Ga, 
.sup.68 Ga, .sup.72 As, .sup.89 Zr, and .sup.201 Ti. Paramagnetic isotopes 
for purposes of diagnosis can also be used according to the methods of 
this invention. Examples of elements that are particularly useful for 
Magnetic Resonance Energy techniques include .sup.157 Gd, .sup.55 Mn, 
.sup.162 Dy, .sup.52 Cr, and .sup.56 Fe. 
The chimeric immunoglobulin molecule can further be part of a 
pharmaceutical composition that also comprises a pharmaceutically 
acceptable carrier. These carriers are well known in the art and can 
include aqueous or solvent emulsions and suspensions, including saline and 
buffered media. Such formulations are well known in the pharmaceutical 
art. See, for example, Remington's Pharmaceutical Sciences (16th Ed. 
1980). 
The dosage ranges for administration of the chimeric immunoglobulin 
molecules are those that are large enough to detect the presence of 
thrombi. The dosage will vary with the age, condition, sex, and extent of 
disease in the patient. Counterindications can include hypersensitivity 
and other variables and can be adjusted by the individual physician. 
Dosage can range from 0.01 to 500 mg/kg of body weight, preferably 0.01 to 
200 mg/kg. The chimeric immunoglobulin molecule can be administered 
parenterally by injection or by gradual perfusion over time. They can also 
be administered intravenously, intraperitoneally, intramuscularly, or 
subcutaneously. 
Preferred Embodiments 
In the preferred embodiment, two amino acids are deleted from scuPA, Phe157 
and Lys158, to a create a mutant with the sequence 
Arg(154)-Pro-Arg-Ile-Ile(158) around the native plasmin/thrombin sites 
(scuPA-T). Because the native plasmin site at Lys 158 has been removed, 
the mutant sequence can only be cleaved by thrombin and the new N-terminal 
amino acid is isoleucine (See FIG. 5 for 59D8-scuPA-T). As expected, the 
protein containing the mutant sequence cleaved by thrombin (59D8-tcuPA-T) 
is capable of activating plasminogen, although it is not as active as the 
wild type scuPA cleaved by plasmin (59D8-tcuPA; see FIG. 6). In vitro 
plasma clot lysis assays showed that 59D8-scuPA-T lysed thrombin induced 
plasma clots and that lysis was quenched by heparin (FIG. 8B), further 
indicating that scuPA-T is a thrombin-activatable plasminogen activator. 
The 59D8-scuPA and wild type scuPA are both activated by plasmin, and 
therefore lysed thrombin-containing and thrombin-depleted clots equally 
well. 
Clot lysis experiments also revealed that 59D8-scuPA-T was much better at 
dissolving a clot than 59D8-scuPA or high molecular weight scuPA when it 
was present within the forming clot. Since there was more active thrombin 
present in the forming clot as compared to the plasma, they could activate 
59D8-scuPA-T and inactivate 59D8-scuPA and high molecular weight scuPA. 
This result suggests that 59D8-scuPA-T can act as an antithrombotic agent 
working at the site of clots that are being initially formed. 
The invention will now be further described by the following working 
examples, which are preferred embodiments of the invention. These examples 
are illustrative rather than limiting. Unless otherwise indicated, all 
temperatures are in degrees Celsius (.degree.C.). 
EXAMPLE 1 
Construction of 59D8-Scupa-T Chimeric Molecule 
Construction of a Thrombin-Activatable Low Molecular Weight scuPA 
A 3.0 kb human genomic DNA fragment encoding the gene for scuPA, including 
exons 7 to 11, was subcloned into the XhoI/XbaI site of a pBluescriptII KS 
vector (Promega). Phagemid mutagenesis was carried out according to the 
manufacturer's protocol (BioRad). The oligomer used for mutagenesis was a 
28-mer having the following sequence (SEQ. ID. NO. 1), listed here 5' to 
3': 
EQU ACT CTG ASS CCC CGC ATT ATT GGG GGA G' 
SEQ. ID. NO. 1 corresponds to the DNA sequence encoding amino acids 152 to 
163 of scuPA except the deletion of 6 nucleotides that encode amino acids 
157 (Phe) and 158 (Lys). The nucleotide sequence of the mutant was 
verified by dideoxy nucleotide sequencing analysis using T7 Sequenase 
according to the manufacture's protocol (United States Biochemical Corp.). 
Construction and Expression of 59D8-scuPA and 59D8-scuPA-T 
The 3.0 kb XhoI/SalI fragment containing human genomic DNA encoding for low 
molecular weight scuPA (scuPA) or for the deletion mutant of scuPA 
(scuPA-T) were cloned into the XhoI/SalI site of the expression vector 
p220RX. The vector p220RX was derived from expression vector pSVUKG(Ig) by 
deleting CH2 domain with XhoI digestion of the vector. Runge, et al., 
Proc. Natl. Acad. Sci. USA. 88, 10337-41 (1991). Constructs having inserts 
in the correct orientation were screened by XhoI restriction mapping. The 
final expression vectors 59D8-scuPA (shown in FIG. 1) and 59D8-scuPA-T 
were linearized by SaI digestion and 40 .mu.g of each was transformed into 
a 59D8 light-chain producing hybridoma cell line by electroporation as 
previously described. Schnee et al., Proc. Natl. Acad. Sci. USA. 84, 
6904-8 (1987). 
The transformed cells were grown in selection media described previously 
Id. Colonies growing in the selection media were screened for the 
production of chimeric plasminogen activator by incubating 50 .mu.L of the 
culture medium in a 96-well microtiter plate coated with .beta.-7 peptide 
(the heptapeptide epitope for antibody 59D8) for two hours. Bound protein 
was then detected by peroxidase conjugated goat anti-mouse IgG or by 
anti-human urokinase and then by peroxidase conjugated rabbit anti-goat 
antibody. Runge, supra. 
Cell lines producing 59D8-scuPA and 59D8-scuPA-T were grown in a Cellquad 
hollow fiber bioreactor (Cellco). Approximately 1.times.10.sup.7 cells 
were inoculated into a polypropylene bioreactor cartridge (Cellco) with a 
capillary pore size of 0.5 microns and an extra-capillary space volume of 
7.0 mL. The extra-capillary space (ECS) medium and the perfusion medium 
consisted of AIM-V serum-free medium (Gibco) supplemented with 300 IU/mL 
Aprotinin (American Diagnostica) and 10 .mu.g/mL Soybean trypsin inhibitor 
(Sigma). Two weeks after inoculation, chimeric proteins were harvested 
every one or two days from the perfusion medium reservoir bottle. 
FIG. 1 shows the expression vector used in transfecting a 59D8 light 
chain-producing mouse hybridoma cell line. 59D8-scuPA-T was constructed 
and expressed in the same manner, except that the two amino acids between 
the plasmin and thrombin cleavage sites (Phe 157 and Lys158) were removed 
by site-directed mutagenesis (FIG. 2). The schematic structure of 
59D8-scuPA and 59D8-scuPAT are shown in FIG. 3. 
Purification of 59D8-scuPA and 59D8-scuPA-T 
Chimeric plasminogen activators were purified from the perfused bioreactor 
medium by affinity chromatography on a .beta.-7/sepharose matrix prepared 
as previously described. Runge, supra. .beta.-7 peptide is the epitope on 
fibrin monomers that the 59D8antibody recognizes. Chimeric proteins bound 
to the column were eluted with 0.2M Glycine pH 3.5 and the eluted proteins 
were immediately neutralized with the addition of 1/5 volume 1M Tris-HCl 
pH 7.8. The protein solutions were then concentrated using Centriprep 30 
concentrators (Amicon). The pooled, purified samples were passed over the 
affinity column a second time for further purification. The concentrations 
in solution of 59D8-scuPA and 59D8-scuPA-T were determined by the DC 
protein assay as described by the manufacturer (BioRad). 
SDS-PAGE and Western Blotting 
The purity and intactness of the affinity-purified chimeric plasminogen 
activators were checked by SOS-PAGE under reducing (using 
.beta.-mercaptoethanol) and non-reducing conditions. The proteins were 
visualized by either staining with Coomassie brilliant blue R or by 
transferring by electrophoresis to a PVDF membrane (Westran) for Western 
blotting. Alkaline phosphatase-conjugated goat anti-mouse IgG (Kirkegaard 
& Perry Laboratories) was used to detect Fab epitopes. Goat anti-human 
urokinase (American Diagnostica) and alkaline phosphatase-conjugated 
rabbit anti-goat IgG (Kirkegaard & Perry Laboratories) was used to detect 
scuPA epitopes. 
The Coomassie blue-stained gel of the proteins show one band at 91 kDa 
under non-reducing conditions and two bands of 27 kDa and 64 kDa under 
reducing conditions (FIG. 3A). The 91 kDa band can be detected by both 
goat anti-mouse IgG and goat anti-human urokinase antibodies in western 
blot analysis, while the 64 kDa band can only be detected by goat 
anti-human urokinase and the 27 kDa band only by goat anti-mouse IgG (FIG. 
3B and 3C). This result shows that the 91 kDa band is the chimeric protein 
consisting of the 59D8Fd and scuPA fusion protein disulfide linked to the 
59D8 L chain (27 kDa). The 64 kDa band is the fusion of 59D8Fd (31 kDa) 
and scuPA(33 kDa), and the 27 kDa band is the light chain of 59D8 (27 
kDa). The 64 kDa band cannot be detected by goat anti-mouse IgG because 
this antibody cannot recognize the reduced form of 59D8Fd (data not 
shown). 
Amino-Terminal Sequence Analysis 
Intact, thrombin-cleaved (Bovine, Armour Pharmaceutical) or plasmin-cleaved 
(American Diagnostica) chimeric protein was subjected to amino-terminal 
sequence analysis using a gas-phase sequencer (ABI). Amino-terminal 
sequencing revealed that the plasmin cleavage occurred at Lys158 of scuPA 
and the thrombin cleavage occurred at Arg156 (data not shown). Only 
thrombin, however, was capable of digesting 59D8-scuPA-T with the cleavage 
occurring between Arg156 and Ile157 (see FIG. 2). 
EXAMPLE 2 
Demonstration of Clot Lysis Activity 
Plasmin and Thrombin Treatment of 59D8-scuPA and 59D8-scuPA-T 
59D8-scuPA or 59D8-scuPA-T (150 nM final concentration) in TNT buffer 
(0.05M Tris-HCI, pH 7.4, 0.038 M NaCl, 0.01% Tween 80) was treated at 
37.degree. C. with plasmin (5 nM final concentration) or thrombin (15 nM 
final concentration). At timed intervals (0-60 minutes), the 
urokinase-like amidolytic activity was measured using the chromogenic 
substrate s-2444 (0.3 mM final concentration; Kabi Pharmacia) after 
stopping the reaction with either aprotinin (5000 KIU/mL final 
concentration) for the plasmin digestions or hirudin (1 U/mL; Calbiochem) 
for the thrombin digestions. Urokinase activity was expressed in 
International units (IU) by comparison with the International Standard 
(87/594; WHO International Laboratory for Biological Standards). 
Stock solutions of 59D8-tcuPA and 59D8-tcuPA-T were obtained by treating 
59D8-scuPA (10 mM final concentration) with plasmin (2 mol/100 mol) and 
59D8-scuPA-T (10 mM final concentration) with thrombin (1 NIHU/2 nmol) for 
30 minutes at 37.degree. C. Plasmin and thrombin were removed by passing 
the samples over the .beta.-7/sepharose column. The conversion of 
urokinase from the one-chain to the two-chain form was monitored by 
SDS-PAGE on 12% gels after reduction with .beta.-mercaptoethanol. 
Both plasmin and thrombin were able to digest 59D8-scuPA, as shown on the 
SDS-PAGE in FIG. 4. The 64 kDa band was cleaved to yield 31 and 33 kDa 
bands. Amino-terminal sequencing revealed that the plasmin cleavage 
occurred at Lys158 of scuPA and the thrombin cleavage occurred at Arg156 
(data not shown). Only thrombin, however, was capable of digesting 
59D8-scuPA-T with the cleavage occurring between Arg156 and lle157 (see 
FIG. 2). 
Plasmin caused a time-dependent conversion of 59D8-scuPA to its two-chain 
derivative, and the result was the same for thrombin acting on 
59D8-scuPA-T (FIG. 5). The urokinase activity for both two-chain 
derivatives reached approximately 100 IU/.mu.g. In the experiment shown in 
FIG. 5, the molar concentration of thrombin (acting on 59D8-scuPA-T) was 
three times higher than that of plasmin (acting on 59D8-scuPA). As 
expected, 59D8-scuPA treated with thrombin and 59D8-scuPA-T treated with 
plasmin resulted in no urokinase activity. 
Activation of Plasminogen 
Activation of plasminogen (10-50 .mu.M final concentration) was measured at 
37.degree. C. in TNT buffer with 59D8-tcuPA, 59D8-tcuPA-T, or commercially 
obtained high molecular weight urokinase (Serono; 5 nM final 
concentration). Generated plasmin at different time intervals (0-5 
minutes) was measured using the chromogenic substrate S-2251 (1.0 mM final 
concentration: Kabi Pharmacia) after 30-fold dilution of the samples. 
Activation of plasminogen by 59D8-tcuPA and 59D8-tcuPA-T (the two-chain 
derivatives of 59D8-scuPA and 59D8-scuPA-T) obeyed Michaelis-Menten 
kinetics as evidenced by linear double-reciprocal plots of activation 
rates versus the plasminogen concentration shown in FIG. 6. Although the 
k.sub.cat for both proteins are similar (2.16 sec.sup.-1 for 59D8-tcuPA 
and 1.75 sec.sup.-1 for 59D8-tcuPA-T), the km for 59D8-tcuPA-T (66.3 
.mu.M) is approximately three-fold higher than for 59D8-tcuPA (22.5 .mu.M) 
In Vitro Clot Lysis Experiments 
Human plasma clot lysis assays were performed as described in Runge, supra. 
Citrated human plasma (pooled from at least 10 healthy donors) was mixed 
with .sup.125 I-labeled fibrinogen (2.5.times.10.sup.6 cpm/mL: prepared 
using the lactose peroxidase-glucose oxidase method with Enzymobeads 
(BioRAD). Each clot was formed using 0.3 mL of the mixture and adding 
CaCl.sub.2 (25 mM final concentration) and human thrombin 
(4.5.times.10.sup.-3 NIHU/mL). For experiments in which the plasminogen 
activator was added outside the clot, this mixture was pipetted into a 0.2 
mL piece of pipet tubing with parafilm on one end, and allowed to clot for 
at least one hour. The clots were then removed from the pipet pieces to a 
4.5-mL polystyrene tube and washed with TNEA buffer (50 mM Tris-HCl, 100 
mM NaCl, 1 mM EDTA, 0.01% sodium azide). 
Autologous plasma (2 mL) and the plasminogen activator were then added. 
Heparin (50 U/mL; Elkins-Sinn) was also added in some cases. For 
experiments in which the plasminogen activator was added to the forming 
clot, the 0.3 mL mixture was pipetted to the bottom of the polystyrene 
tube with the plasminogen activator and allowed to clot 10-20 minutes. 2 
mL autologous plasma was then added. Both types of clots were rotated at 
37.degree. C., and 0.1 mL aliquots were removed at various time points to 
count the radioactivity on a gamma counter (Packard). High molecular 
weight scuPA (American Diagnostica) was used as a control in all 
experiments. 
In the experiment shown in FIG. 7A, the plasminogen activator was added to 
the clot preparation, which was formed using thrombin. 59D8-scuPA-T was 
able to lyse the clot, indicating that the thrombin cleaved scuPA-T to its 
active two-chain derivative. When the plasminogen activator was present in 
the plasma milieu and not in the clot, 59D8-scuPA (and scuPA) was 
effective at lysing the clot (FIG. 7B). Thrombin inhibitors present in the 
plasma probably inhibit scuPA-T from being activated and lysing the clot. 
To compare the thrombolytic potency of 59D8-scuPA and 59D8-scuPA-T in the 
plasma milieu, the concentrations of these proteins were varied in the 
titration curves shown in FIGS. 8A and B. To reach 30% clot lysis, it took 
59D8-scuPA one hour at a concentration of 3 .mu.g/mL, while 59D8-scuPA-T 
needed five hours at a concentration of 20 .mu.g/mL. Heparin was able to 
effectively inhibit the activity of 59D8-scuPA-T, but had no effect on 
59D8-scuPA. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 3 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
ACTCTGAGGCCCCGCATTATTGGGGGAG28 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ProArgPheLysIleIle 
15 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 4 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: peptide 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
ProArgIleIle 
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