Hybrid monoclonal antibodies, their production and use

The present invention provides a hybrid monoclonal antibody having specificities against fibrin and thrombolytic substance, a polydoma which produces said antibody and thrombolytic agent comprising said antibody and thrombolytic substance which is immunologically coupled thereto, and methods of using said antibody in combination with thrombolytic substance for lysis or removal of thrombi.

BACKGROUND OF THE INVENTION 
The present invention relates to a hybrid monoclonal antibody. More 
specifically, the present invention relates to a hybrid monoclonal 
antibody (hereinafter also simply referred to as hybrid MoAb) having 
specificities against fibrin and thrombolytic substance and a polydoma 
which produces said antibody. 
The present invention also relates to a thrombolytic agent comprising the 
above hybrid MoAb and a thrombolytic substance which is immunologically 
coupled thereto. 
Thrombolytic therapy has long been used to treat thrombotic diseases such 
as myocardial infarction, arterial embolism and cerebral infarction; 
initially, streptokinase (hereinafter also abbreviated SK), urokinase 
(hereinafter also abbreviated UK) etc. were clinically applied as 
efficient thrombolytic agents. Particularly, UK has been relatively 
commonly used since its fibrinolytic action is strong; however, it is 
faulty in that its selectivity for fibrin is low and that it acts on 
fibrinogen as well, and thus it causes a tendency toward bleeding in 
subject patients. In view of this drawback, tissue plasminogen activator 
(hereinafter also abbreviated TPA) and prourokinase (hereinafter also 
abbreviated ProUK) became used as second generation thrombolytic agents. 
With higher selectivity for fibrin in comparison with UK, these agents 
were expected to mitigate the adverse effect, i.e., a tendency toward 
bleeding, of UK; many investigations have so far been made. In recent 
years, there have been an increasing number of attempts to apply these 
agents in clinical situations since their massproduction has become 
possible by use of gene recombination technology [European Cooperative 
Study Group for Recombinant Tissue-type Plasminogen Activator: The Lancet, 
842 (1985)]. 
These attempts at clinical application, however, revealed that TPA etc. 
also have some drawbacks; for example, 1 TPA has a very short half life (2 
to 3 minutes), thus requiring large-amount long-term administration for 
thrombolysis, and 2 such high-dose therapy does not always provide a 
mitigating effect on the tendency toward bleeding. 
Under these conditions, more effective thrombolytic agents were 
investigated and developed; modified TPA, UK-TPA or ProUK-TPA hybrid 
protein etc. have been produced. As to modified TPA, a TPA mutein lacking 
part of the sugar chain structure, which is considered a cause of the 
decrease in half life, has been prepared by gene engineering so that the 
capture of TPA by sugar-chain receptors of hepatocytes is avoided to 
improve TPA kinetics in blood. As to UK-TPA hybrid protein, the strong 
thrombolytic action of UK and the fibrin affinity of TPA are utilized in 
combination to reduce the administration amount. Although these 
thrombolytic agents are expected to slightly mitigate the tendency toward 
bleeding in comparison with conventional thrombolytic agents, significant 
improvement remains to be achieved by further research and development. 
Later, protein complexes based on antibody targeting appeared as third 
generation thrombolytic agents. That is, thrombolytic agents which lyse 
fibrin alone without lysing fibrinogen were developed by chemically 
binding an antibody which does not substantially react on fibrinogen and 
which possesses high affinity with fibrin alone to UK [C. Bode et al.:. 
Science, 229, 765 (1985)] or TPA [M. S. Runge et al.: Proceedings of the 
National Academy of Science, USA, 84, 7659 (1987)]. These 
antibody-targeting thrombolytic agents are all reported to have exhibited 
an effect 3 to 100 times stronger than that of simple UK or TPA in 
experiments in vitro or in vivo. These protein complexes, however, all 
have drawbacks due to the chemical binding of an antibody and a 
thrombolytic enzyme; for example, 1 the antibody activity and enzyme 
activity decrease during the chemical binding procedure, 2 it is difficult 
to obtain a protein complex of antibody and enzyme in a ratio of 1 to 1, 
or 3 protein denaturation accelerates the agent's metabolism in the 
subject patient's body.

DETAILED DESCRIPTION 
With the aim of solving the above problems related to in vivo metabolism, 
fibrin affinity, thrombolytic capability etc., and taking note of the 
recently developed new technology for bi-specific hybrid monoclonal 
antibodies, the present inventors prepared an immunocomplex of bi-specific 
hybrid monoclonal antibody and thrombolytic substance in a ratio of 1 to 
1, substantially free of degradation of their antibody activity and 
thrombolytic activity, by immunologically coupling a thrombolytic 
substance to the bi-specific hybrid monoclonal antibody, thus developing a 
fibrin-specific thrombolytic agent. Accordingly, the present invention 
provides a hybrid MoAb whose two specificities are respectively against 
fibrin and thrombolytic substance and a polydoma which produces this 
antibody. Although the present invention is illustrated by use of 
bi-specific antibodies having specifities against fibrin and a 
thrombolytic agent, it is not so limited. For example, tri-specific 
antibodies could be used in the practice of the present invention so long 
as the speciities against fibrin and thrombolytic agent are retained. 
The present invention also provides a thrombolytic agent comprising the 
above-mentioned hybrid monoclonal antibody and a thrombolytic substance 
which is immunologically coupled thereto. 
Any anti-fibrin-antibody-producing hybridoma can be used for the present 
invention, as long as it produces a fibrin-specific MoAb which does not 
substantially bind to fibrinogen. For example, such a fibrin-specific 
antibody is prepared by the use of an .alpha.-chain N-terminal fragment 
peptide or .beta.-chain N-terminal fragment peptide of the fibrin 
resulting from fibrinolysis as immunogen [K. Y. Hui et al.: Science, 222, 
1129 (1983) or Japanese unexamined Patent Publication No. 93800/1988]. 
Although any fibrin is acceptable, as long as it is of animal derivation, 
human fibrin is preferred; it is especially preferable to use a peptide 
corresponding to the .beta.-chain N-terminal portion of human fibrin. The 
fibrin-specific antibody thus obtained is coupled with carrier protein, 
followed by immunization of an animal (e.g. rabbit, rat, mouse, guinea 
pig) to obtain antibody-producing cells. Antibody-producing cells such as 
splenocytes or lymph node cells are then collected from the immunized 
animal and fused with myeloma cells. The hybridoma cells thus obtained are 
subjected to screening for antibody-producing cells which do not 
substantially react on fibrinogen and which specifically bind to fibrin. 
It is preferable that the human fibrin .beta.-chain N-terminal peptide 
have the following amino acid sequence: 
EQU H-Gly-His-Arg-Pro-Leu-Asp-Lys-R-Cys-OH 
wherein R represents a peptide represented by Lys-Arg-Glu-Glu or a fragment 
thereof. The C-terminal Cys is used for the linker portion for chemical 
binding with carrier protein. Accordingly, it is possible to chemically 
bind the peptide to carrier protein via the SH group of the C-terminal Cys 
of the peptide by previously maleimidating the carrier protein with 
N-(.gamma.-maleimidobutyryloxysuccinimide) (hereinafter also abbreviated 
GMBS) or dithiopyridylating the carrier protein with 
N-succinimidyl-3-(2pyridyldithio)propionate (hereinafter also abbreviated 
SPDP). 
Any thrombolytic substance can be used for the present invention, as long 
as it is a protein capable of thrombolysis or a substance which promotes 
thrombolysis. Examples of such substances include protease or precursors 
thereof and thrombolysis promoters (e.g. TPA, UK, ProUK, SK, trypsin, 
plasmin, protein C, protein S). It is preferable to use protease; TPA, UK 
and derivatives thereof are especially preferable. It does not matter 
whether the TPA is single- or two-chain. As for UK, single- or two-chain 
low molecular weight UK, proUK or derivatives of UK [E. Haber et al.: 
Science, 243,51 (1989)] may be used. For preparing antibody-producing 
hybridomas, the method is available in which an animal is immunized with 
the above-mentioned protein in accordance with a standard method and the 
resulting antibody-producing cells are fused with myeloma cells or other 
cells. Especially, as the method of producing antibody 
producing-hybridomas, it is preferable to use antibody producing-cells 
obtained by immunizing animals with low molecular weight UK. The same 
procedure as the method of producing anti-fibrin-antibody-producing 
hybridomas by animal immunization followed by fusion of the resulting 
antibody-producing cells with myeloma cells or other cells may be used in 
obtaining hybridomas which produce an antibody against thrombolytic 
substances. 
Examples of animals used for immunization include rabbits, rats, mice and 
guinea pigs. It is especially preferable to use mice in the case of MoAb 
production. Inoculation can be achieved in accordance with an ordinary 
method; for example, 1 to 100 .mu.g, preferably 10 to 25 .mu.g of 
immunogen protein, in emulsion in equal amounts (0.1 ml) of physiological 
saline and Freund's complete adjuvant, is subcutaneously or 
intraperitoneally inoculated at the back or abdomen 3 to 6 times at 
intervals of 2 to 3 weeks. 
From these immunized animals, e.g. mice, are selected individuals with high 
antibody titer; spleens and/or lymph nodes are collected at 3 to 5 days 
following the final immunization; antibody-producing cells contained in 
them are fused with myeloma cells. This fusion can be achieved in 
accordance with the procedure of a known method. Examples of the fusogen 
include polyethylene glycol (hereinafter also abbreviated PEG) and Sendai 
virus. It is preferable to use PEG. Examples of myeloma cell lines which 
can be used include NS-1, P3U1 and SP2/0; it is preferable to use P3U1. 
The ratio of e.g. splenocytes and myeloma cells is preferably 1 to 10 of 
.the former to 1 of the latter. It is recommended that PEG with a 
molecular weight of 1,000 to 6,000 be added to the mixture to reach a 
concentration of 10 to 80% and incubation be conducted at 20 to 37.degree. 
C., preferably 30 to 37.degree. C. for 3 to 10 minutes. 
Various methods can be used for the screening of 
anti-fibrin-antibody-producing hybridomas. For example, after fibrinogen 
adsorption onto a microplate, the fibrinogen is converted to fibrin by the 
action of thrombin. Then, a hybridoma culture supernatant is added to the 
fibrin-immobilized microplate in the presence of excess fibrinogen, 
followed by determination of antibody titer in the culture supernatant by 
enzyme immunoassay (hereinafter also abbreviated EIA) for plate-bound 
anti-fibrin specific antibody. The hybridomas found positive for antibody 
titer are selected and bred using a medium supplemented with HAT 
(hypoxantine-aminopterin-thymidine) and immediately subjected to cloning, 
which is normally easily achieved by limited dilution analysis. The 
desired monoclonal anti-fibrin-specific-antibody-producing hybridoma can 
be obtained by determining the antibody titer of the cloned hybridoma 
culture supernatant by the above method and selecting a hybridoma which 
stably produces an antibody with high titer. 
Examples of the anti-fibrin-antibody-producing hybridoma prepared in 
accordance with the above production method include FIB 1-11 and FIB 2-11, 
both described in Example 1 given below. 
Screening of hybridomas which produce an antibody against thrombolytic 
substance can easily be achieved by EIA using a microplate onto which the 
thrombolytic substance has been adsorbed. Cloning can be conducted in 
accordance with the standard method described above to obtain the desired 
antibody-producing hybridoma. 
Examples of the anti-TPA-antibody-producing hybridoma prepared in 
accordance with the above production method include TPA 1-41, TPA 1-70, 
and TPA 2-14 described in Example 2 given below. Examples of the 
anti-LTK-antibody-producing hybridoma include UK 1-3, UK 1-87, both 
described in Example 3 given below, and UK 1-6, described in Example 12. 
Some methods are available for preparation of a polydoma which produces the 
hybrid MoAb [e.g. Hiroshi Aramoto et al.: Protein, Nucleic Acid and 
Enzyme, 33,217 (1988)]. Although any one of these methods can be used to 
prepare a polydoma which produces a hybrid MoAb for use in the present 
invention, mention may be made of 1 (the method in which the above 
HAT-resistant hybridoma which produces an antibody against thrombolytic 
substance is acclimatized step by step to a culture medium supplemented 
with 5-bromodeoxyuridine (hereinafter also abbreviated BrdU), followed by 
cloning of thymidine kinase deficient strain to make it sensitive to HAT; 
similarly, an HAT-resistant anti-fibrin-specific-antibody-producing 
hybridoma is made resistant to 8-azaguanine (hereinafter also abbreviated 
AZG), followed by cloning of a hypoxanthine-guanine-phosphoribosyl 
transferase deficient strain to make it sensitive to HAT; the tetraomas 
obtained by fusing these two cloned hybridomas in accordance with methods 
known to those skilled in the art are subjected to screening using an 
HAT-supplemented medium for a tetraoma which secretes a hybrid MoAb 
capable of binding to both fibrin and thrombolytic substance, which is 
then cloned; and 2 the method in which an 
anti-fibrin-specific-antibody-producing hybridoma is labeled with 
fluorescein isothiocyanate (hereinafter abbreviated FITC) and another 
hybridoma which produces an antibody against thrombolytic substance is 
labeled with tetramethyl rhodamine isothiocyanate (hereinafter also 
abbreviated TRITC), followed by fusion of these two hybridomas in 
accordance with methods known to those skilled in the art; the obtained 
cell suspension is applied to a fluorescein activated cell sorter 
(hereinafter also abbreviated FACS) to select and clone the tetraoma 
having both the green fluorescence of FITC and red fluorescence of TRITC. 
It is also possible to use the markers of both parent strains in reverse 
to select and clone the tetraoma. 
In cell fusion using these procedures, a fusogen such as Sendai virus or 
PEG, electric stimulation or other means can be used. It is preferable to 
use PEG. An example is given below, but this is not to be construed as 
limitative. PEG with a degree of polymerization of about 1,000 to 6,000 is 
normally used at a concentration of about 10 to 80% for a treatment time 
of about 0.5 to 30 minutes. Efficient fusion can be achieved under 
preferable conditions, for example, by keeping about 35 to 55% PEG 6,000 
in contact with cells at 37.degree. C. for about 4 to 10 minutes. 
Polydoma selection can be achieved using the above-mentioned 
HAT-supplemented medium. For this purpose, chemical acclimatization is 
conducted using 8-AZG, 6-thioguanine (6-TG) or 5-BrdU to obtain strains 
resistant to respective chemicals. Various selection media can be used by 
introducing a new marker into fused cells. Examples of such selection 
media include neomycin-supplemented medium and hygromycin B-supplemented 
medium [B. Sugden et al.: Molecular and Cellular Biology, 5, 410 (1985)]. 
Another method is also available in which two hybridomas labeled with 
different fluorescent dyes are fused together, followed by sorting a 
double-labeled hybrid hybridoma using FACS [L. Karawajew et al.: Journal 
of Immunological Methods, 96, 265 (1987)]. 
Various methods can be used for screening of hybrid antibody-producing 
polydomas. For example, it is possible to use in appropriate combination 
the following methods or their modifications: 1 combination of the 
above-mentioned EIA procedures respectively for screening of 
anti-fibrin-specific-antibody-producing hybridomas and for screening of 
hybridomas which produce an antibody against thrombolytic substance, 2 EIA 
for detection of bi-specific hybrid antibody by adding the subject culture 
supernatant to a fibrin-coupled microplate and then adding an HRP-labeled 
thrombolytic substance, and, when using an antibody against a thrombolytic 
substance belonging to a subclass different from that of the anti-fibrin 
specific antibody, 3 EIA for detection of a hybrid antibody by adding the 
subject culture supernatant to a fibrin-coupled microplate and then adding 
the HRP-labeled anti-mouse-IgG-subclass specific antibody. 
The polydomas found positive for hybrid antibody activity are immediately 
subjected to cloning, which is typically achieved by limiting dilution 
analysis. The desired monoclonal hybrid antibody-producing polydoma can be 
obtained by determining the antibody titer of the cloned polydoma culture 
supernatant by the above method and selecting a polydoma which stably 
produces an antibody with high titer. 
The above-mentioned polydoma of the present invention can typically be 
cultivated in liquid medium or in the peritoneal cavity of animals (e.g. 
mammals such as mice) by a known method. Known biochemical techniques can 
be used in combination to purify the antibody in the culture broth and 
ascites fluid. For example, the cell culture broth or ascites fluid is 
centrifuged; the resulting supernatant is taken and subjected to 
salting-out (normally using ammonium sulfate or sodium sulfate). The 
resulting protein precipitate is dissolved in an appropriate solution and 
dialyzed, followed by column chromatography (ion exchange column, gel 
filtration column, protein A column, hydroxyapatite column etc.) to 
separate and purify the desired antibody. This separation purification 
procedure gives about 1 to 5 mg of bi-specific hybrid MoAb with a purity 
of over 80% in a ratio by protein weight from 1 l of culture supernatant, 
for instance. The same antibody can be obtained with a yield of 3 to 10 mg 
from 20 ml of ascites fluid. 
The bi-specific hybrid MoAb thus obtained is a homogeneous protein; its 
treatment with protease (e.g. pepsine) etc. gives F(ab')2 or other 
fragment capable of binding to both fibrin and thrombolytic substance. 
Examples of the bi-specific hybrid antibody-producing polydoma prepared in 
accordance with the above production method include the tetraomas 
described in Examples 4,5 and 13 given below. 
In addition to the tetraomas formed between an anti-fibrin-MoAb-producing 
hybridoma and another hybridoma which produces an antibody against 
thrombolytic substance, mentioned above as examples of the polydoma which 
produces the hybrid MoAb of the present invention, there can be used 
triomas formed between a hybridoma which produces one MoAb and a cell 
which produces another MoAb, hybridomas obtained by fusing cells which 
produce respective MoAb after being immortalized with Epstein-Barr virus, 
and other hybridomas, for the same purpose, as long as they produce the 
hybrid MoAb of the present invention. 
When these polydomas produce mouse IgG MoAb, it is possible to prepare a 
mouse-human chimeric antibody by obtaining a DNA which encodes a variable 
or hyper-variable region containing the antigen recognition site for this 
bi-specific hybrid MoAb and then binding a gene which encodes the constant 
region of human IgG to this DNA using a gene manipulation technique [Z. 
Steplewski et al.: Proceedings of the National Academy of Science, USA, 
85, 4852 (1988)]. This chimeric antibody is preferably used because of its 
low antigenicity in administration to humans. 
Some methods are available for thrombolytic therapy using the hybrid MoAb 
of the present invention or a selective thrombolytic protein complex 
prepared from a thrombolytic substance and the hybrid MoAb, including 1 
the method in which the hybrid MoAb of the present invention is previously 
administered to the thrombotic disease patient and a thrombolytic 
substance such as TPA or UK is administered after elapse of sufficient 
time to allow it to bind to the thrombi formed in the patient's body, 2 
the method in which the hybrid MoAb and a thrombolytic substance are 
administered to the thrombotic disease patient at the same time, and 3 the 
method in which the hybrid MoAb and a thrombolytic substance are 
previously reacted and, after separation of the unreacted portion of the 
thrombolytic Substance, the obtained selective thrombolytic protein 
complex is administered to the thrombotic disease patient. 
The thrombolytic agent, hybrid MoAb or thrombolytic substance of the 
present invention can be prepared as injection directly or in mixture with 
appropriate pharmacologically acceptable carrier, excipient or diluent 
after germ removal by filtration through membrane filter as necessary, and 
administered to mammals (e.g. mice, rats, cats, dogs, swine, bovines, 
monkeys, humans) for the purpose of treatment of thrombotic or obstructive 
diseases such as myocardial infarction, peripheral arteriovenous 
obstruction, retinal arteriovenous obstruction, cerebral infarction and 
pulmonary embolism. 
Although depending upon the type of the target disease, its symptom and 
route of administration, the daily dose of the thrombolytic agent of the 
present invention, in intravenous administration to adult myocardial 
infarction patients, for instance, is typically about 0.02 to 1 mg/kg, 
preferably about 0.04 to 0.4 mg/kg as hybrid MoAb, and, for thrombolytic 
substances, about 0.01 to 0.5 mg/kg, preferably about 0.02 to 0.2 mg/kg as 
TPA and about 0.01 to 0.5 mg/kg, preferably about 0.02 to 0.2 mg/kg as UK. 
The hybrid MoAb of the present invention is capable of specifically binding 
to fibrin while it does not substantially react on fibrinogen and 
specifically binding to a thrombolytic substance without spoiling its 
capability of fibrinolysis. It is therefore possible to easily prepare a 
1:1 immunocomplex of the hybrid MoAb and a thrombolytic substance; their 
use in combination permits selective and efficient lysis or removal of 
thrombi. 
The method described above permits selective and efficient lysis and 
removal of thrombi by the use of the hybrid MoAb of the present invention, 
which is capable of specifically binding to the target thrombus site and 
which does not substantially bind with fibrinogen, in combination with a 
thrombolytic substance. 
EXAMPLES 
The present invention is hereinafter described in more detail by means of 
the following reference examples and working examples; these examples are 
not to be construed as limitative on the scope of the present invention. 
The animal cell lines used in Examples have been deposited with the 
Institute of Fermentation, 17-85, Juso-honomachi 2-chome, Yodogawa-ku, 
Osaka 532, Japan and Fermentation Research Institute, Ministry of 
International Trade and Industry, 1-3, Higashi 1-chome, Tukuba-shi, 
Ibaraki-ken 305, Japan, under accession numbers listed in the table below. 
FIE 1-11, FIB 2-11, TPA 1-41, TPA 1-70, UK 1-3 and UK 1-87 were deposited 
on Oct. 4, 1988; FT2-14 was deposited on Nov. 25, 1988; FU1-74 was 
deposited on Mar. 14, 1989; TPA 2-14 was deposited on Jul. 18, 1989; UK 
1-6 and FU 2-16 were deposited on Aug. 11, 1989. 
______________________________________ 
(IFO) (FRI) 
Animal cell line IFO No. FERM No. 
______________________________________ 
Mouse hybridoma FIB 1-11 
50174 BP-2081 
Mouse hybridoma FIB 2-11 
50175 BP-2082 
Mouse hybridoma TPA 1-41 
50178 BP-2085 
Mouse hybridoma TPA 1-70 
50179 BP-2086 
Mouse hybridoma TPA 2-14 
50194 BP-2519 
Mouse hybridoma UK 1-3 
50176 BP-2083 
Mouse hybridoma UK 1-87 
50177 BP-2084 
Mouse hybridoma UK 1-6 
50208 BP-2548 
Mouse hybrid-hybridoma 
50180 BP-2158 
(tetraoma) FT2-14 
Mouse hybrid-hybridoma 
50185 BP-2334 
(tetraoma) FU1-74 
Mouse hybrid-hybridoma 
50207 BP-2547 
(tetraoma) FU 2-16 
______________________________________ 
IFO: Institute for Fermentation, Osaka 
FRI: Fermentation Research Institute, Ministry of International Trade and 
Industry 
In the present specification, amino acids and peptides are represented by 
abbreviations based on the abbreviation system adopted by the IUIUB 
Commission on Biochemical Nomenclature (CBN); example abbreviations are 
given below. Note that when there is a possibility of the existence of an 
optical isomer in particular amino acid etc., it is an L-amino acid unless 
otherwise stated. 
GIn: Glutamine residue 
Asp: Aspartic acid residue 
Pro: Proline residue 
Tyr: Tyrosine residue 
Val: Valine residue 
Lys: Lysine residue 
Glu: Glutamic acid residue 
Ala: Alanine residue 
Ash: Asparagine residue 
Leu: Leucine residue 
Phe: Phenylalanine residue 
Gly: Glycine residue 
His: Histidine residue 
Ser: Serine residue 
Thr: Threonine residue 
Ile: Isoleucine residue 
Trp: Tryptophan residue 
Arg: Arginine residue 
Met: Methionine residue 
Cys: Cysteine residue 
Reference Example 1 
(EIA for anti-fibrin antibody measurement) 
To a 96-well microplate was dispensed a 1 mg/ml human fibrin monomer 
solution in a 20 mM phosphate buffer solution (PBS, pH 7.3) containing 3.3 
M urea and 0.01% EDTA at 50 .mu.l per well. After leaving this microplate 
at 4.degree. C. overnight, 150 .mu.l of PBS containing 2% casein and 0.01% 
thimerosal was added to prepare a sensitized plate. Then, a 10 mg/ml human 
fibrinogen solution in PBS containing 100 unit/ml heparin and 3 mM 
phenylmethylsulfonyl fluoride was mixed with an equal amount of the 
subject hybridoma culture supernatant. After reaction at room temperature 
for 30 minutes, 100 .mu.l of the mixture was added to the above 
fibrin-sensitized plate, followed by reaction at room temperature for 2 
hours. After thoroughly washing the plate with PBS containing 0.05% Tween 
20 (PBS-TW), a horseradish peroxidase (HRP) labeled rabbit anti-mouse-IgG 
antibody was added, followed by reaction at room temperature for 2 hours. 
After plate washing, a 0.1 M citrate buffer solution containing 
orthophenylenediamine and H.sub.2 O.sub.2 as enzyme substrates was added 
to each well, followed by enzyme reaction at room temperature. After 
termination of the reaction by the addition of 1 N sulfuric acid, the 
coloring pigment was quantitated at a wavelength of 492 nm using the 
Multiscan (produced by Flow Co.). 
Reference Example 2 
(EIA for measuring anti-TPA antibody) 
A 5 .mu.g/ml TPA solution was dispensed to a 96-well microplate at 100 
.mu.l per well and left at 4.degree. C. overnight, followed by addition of 
150 .mu.l of PBS containing 2% casein and 0.01% thimerosal to prepare a 
sensitized plate. After removing the above solution and washing the plate 
with PBS-TW, 100 .mu.l of the subject hybridoma culture supernatant was 
added, followed by reaction at room temperature for 2 hours. Thereafter, 
enzyme reaction was carried out by the method of Reference Example 1 and 
antibody titer was determined. 
Reference Example 3 
(EIA for measuring anti-UK antibody) 
After a UK-sensitized plate was prepared in the same manner as in Reference 
Example 2 but UK was used in place of TPA, anti-UK antibody titer was 
determined in the same manner. 
Reference Example 4 
(EIA for measuring anti-fibrin-anti-TPA hybrid antibody 1) To a 
fibrin-sensitized plate as prepared in Reference Example 1 was added the 
subject hybridoma culture supernatant, followed by reaction at room 
temperature for 2 hours. After plate washing with PBS-T, biotin-labeled 
TPA was added, followed by reaction at room temperature for 2 hours. An 
avidin-HRP complex was then added, followed by reaction at room 
temperature for 1 hour; the activity of HRP bound to the solid phase was 
determined by the method of Reference Example 1. 
Reference Example 5 
(EIA for measuring anti-fibrin-anti-UK hybrid antibody 1) 
The procedure of Reference Example 4 was followed but biotin-labeled UK was 
used in place of biotin-labeled TPA. Thereafter, anti-fibrin-anti-UK 
hybrid antibody titer was determined in the same manner. 
Reference Example 6 
(Fibrinolysis neutralization test) 
To a TPA solution (final concentration 20 ng/ml) or UK solution (final 
concentration 25 ng/ml) was added a dilution of the subject hybridoma 
culture supernatant, followed by reaction at 37.degree. C. for 1 hour. The 
reaction mixture was then dispensed to a fibrin-agarose plate at 5 .mu.l 
per well, followed by incubation at 37.degree. C. for 2 to 6 hours. The 
diameter of the resulting fibrinolysis plaque was measured to determine 
the ability of the MoAb in the hybridoma culture supernatant to neutralize 
the enzyme activity of TPA or UK. 
Reference Example 7 
(EIA for measuring anti-fibrin-anti-TPA hybrid antibody2) 
To a TPA sensitized plate as prepared in Reference Example 2 was added the 
subject solution containing the hybrid antibody, followed by reaction at 
room temperature for 2 hours. After plate washing with PBS-TW, an 
HRP-labeled anti-mouse-IgG.sub.1 (.gamma..sub.1 -chain specific) antibody 
(commercially available from Cosmo-Bio K.K.) was added, followed by 
reaction at room temperature for 2 hours. After thoroughly washing the 
plate with PBS-TW, the activity of IIRP bound; to the solid phase was 
determined by the method of Reference Example 1. 
Reference Example 8 
(EIA for measuring anti-fibrin-anti-TPA hybrid antibody 3) 
To a TPA-sensitized plate as prepared in Reference Example 2 was added the 
subject solution containing the hybrid antibody, followed by reaction at 
room temperature for 2 hours. After plate washing with PBS-TW, the human 
fibrin .beta.-chain N-terminal peptide (1-11)-BSA complex described in 
Example 1-1, labeled with biotin, was added, followed by reaction at room 
temperature for 2 hours. An avidin-HRP complex was then added, followed by 
reaction at room temperature for i hour; the activity of HRP bound to the 
solid phase was determined by the method of Reference Example 1. 
Reference Example 9 
(EIA for measuring anti-fibrin-anti-UK hybrid antibody 2) 
To a UK-sensitized plate as prepared in Reference Example 3 was added the 
subject solution containing the hybrid antibody, followed by reaction at 
room temperature for 2 hours. After plate washing with PBS-TW, the human 
fibrin .beta.-chain N-terminal peptide (1-11)-BSA complex described in 
Example 1-1, labeled with biotin, was added, followed by reaction at room 
temperature for 2 hours. An avidin-HRP complex was then added, followed by 
reaction at room temperature for 1 hour; the activity of HRP bound to the 
solid phase was determined by the method of Reference Example 1. 
Reference Example 10 
(EIA for determination of anti-low-molecular-weight,UK antibody titer) 
After preparation of a plate sensitized with low molecular weight UK 
(two-chain low molecular weight UK, available from JCR Co.) in place of 
the TPA described in Reference Example 2, anti-low-molecular-weight-UK 
antibody titer was determined in the same manner. 
Reference Example 11 
(UK enzyme activity neutralization test) 
To a UK solution (final concentration 1.7 .mu.g/ml) was added a subject 
antibody solution, and reaction was carried out at room temperature for 30 
minutes. To the reaction mixture was added synthetic peptide substrate 
S-2444 (1 mM, pyroglutamyl glycyl arginyl paranitroanilide, produced by 
Kabi Co.). After reaction at 37.degree. C. for 15 minutes, liberated 
paranitroanilide (absorbance at 405 nm) was measured. 
Example 1 
(Preparation: of hybridomas which produce mouse anti-human-fibrin 
monoclonal antibody) 
1 Preparation of immunogen 
To an aqueous solution of 12 mg/2ml bovine serum albumin (BSA), previously 
maleimidated with GMBS (maleimido groups were introduced at 13 mols per 
mol BSA), was added 3.3 mg of a human fibrin .beta.-chain N-terminal 
peptide (1-11)-Cys prepared by the known solid phase synthesis method 
using a peptide synthesizer (Model 430A, Applied System Co.), followed by 
reaction at 30.degree. C. for 1 hour to yield a human fibrin 13-chain 
N-terminal peptide (1-11)-BSA complex. After 3 times of dialysis with 
physiological saline solution (3 l.times.3), this complex was stored under 
freezing conditions and then used as immunogen. 
2 Immunization 
To a 1 mg/ml peptide-BSA complex solution in physiological saline solution 
was added an equal amount of Freund's complete adjuvant, followed by 
subcutaneous immunization of mice (.female.,n=10:0.1 mg/0.2 ml/mouse) at 
the back and abdomen. Additional immunization was conducted by inoculating 
the immunogen in combination with an equal mount of Freund's incomplete 
adjuvant 5 times at intervals of 2 to 3 weeks. 
3 Cell fusion 
At 3 days following the final immunization, spleens were excised and a 
splenocyte suspension was prepared by a standard method [S.M. North et 
al.: Immunology, 47,397(1982)] (about 10.sup.8 cells). After addition of 
2.times.10.sup.7 mouse myeloma cells (P3U1), cell fusion was conducted in 
accordance with the method of Kohler and Milsrein [Nature, 256,495 (1975)] 
using PEG 6000. 
After completion of the fusion, the cell mixture was suspended in HAT 
medium, which contains hypoxanthine, aminopterin and thymidine, followed 
by cultivation for 10 days. Immediately after completion of the selection 
of parent cells, the HAT medium was replaced with HT medium, which lacks 
aminopterin, followed by further cultivation. 4 Selection and cloning of 
hybridomas 
The antibody titer of the hybridoma culture supernatant was determined by 
the EIA procedure described in Reference Example 1, which uses a human 
fibrin monomer adsorbed microplate as the solid phase. At 10 to 20 days 
following the fusion, hybridomas appeared and an antibody which 
specifically bound to human fibrin was detected. The .hybridomas found to 
have especially strong avidity were subjected to cloning by limiting 
dilution method. 
The culture supernatant of cloned hybridomas was subjected to screening by 
EIA; hybridomas with strong avidity to human fibrin were selected. 
As a result, mouse hybridomas FIB 1-11 and FIB 2-11 were obtained, both of 
which produce an MoAb which specifically binds to fibrin in the presence 
of high concentrations of fibrinogen. Antibodies FIB 1-11 and FIB 2-11 
respectively produced by these two hybridomas were both identified as 
IgG.sub.1 in immunoglobulin class and subclass by the Ouchterlony method. 
These anti-human-fibrin specific antibodies FIB 1-11 and FIB 2-11 were 
measured in the presence and absence of fibrinogen by the EIA procedure 
described in Reference Example 1. The results are shown in Table 1. Note 
that anti-human-fibrinogen antibody FIB 2-162 obtained by the procedure 
described in Example 1 was used as control. 
TABLE 1 
______________________________________ 
EIA for Measuring Anti-fibrin Antibody.sup.1) 
Optical absorbance (492 nm) 
In the absence of 
In the presence of 
Monoclonal antibody 
fibrinogen fibrinogen.sup.2) 
______________________________________ 
FIB 1-11 0.50 0.20 
FIB 2-11 0.51 0.16 
FIB 2-162 0.32 0.01 
______________________________________ 
.sup.1) EIA described in Reference Example 1. 
.sup.2) Final concentration 5 mg/ml. 
Example 2 
(Preparation of hybridomas which produce mouse anti-TpA monoclonal 
antibody) 
1 Immunization 
To a solution of 200 .mu.g/ml of a commercially available single-chain TPA 
(available from Chuo Kagaku Kogyo K.K.) in physiological saline solution 
was added an equal mount of Freund's adjuvant. This emulsion was 
subcutaneously administered to BALB/c mice (.female., 20 .mu.g/0.2 
ml/mouse) at the back and abdomen, followed by additional immunization at 
intervals of 2 to 3 weeks. A TPA antigen solution (50 .mu.g/0.1 ml 
physiological saline solution/mouse) was intravenously administered to 
animals which had the maximum serum antibody titer at 10 days following 
the 3 times of additional immunization. 
2 Cell fusion 
Cell fusion was conducted in accordance with the method described in 
Example 1-3. 
3 Selection and cloning of hybridomas 
Hybridomas were screened by the EIA procedure described in Reference 
Example 2, which uses a TPA-bound microplate. Thereafter, the procedure of 
Example 1-4 was followed to obtain anti-TPA-MoAb-producing hybridomas. Out 
of these hybridomas were obtained a mouse hybridoma TPA 1-41, which 
produces an anti-TPA MoAb, which specifically binds to TPA without 
spoiling the capability of fibrinolysis, a mouse hybridoma TPA 1-70, 
which produces a TPA neutralizing antibody and a mouse hybridoma TPA 2-14, 
which produces a TPA non-neutralizing antibody which competitively binds 
to TPA with plasminogen activator inhibitor (PAD. Antibodies TPA 1-41, TPA 
1-70 and TPA 2-14 respectively produced by these three hybridomas were 
respectively identified as IgG.sub.2 b, IgG.sub.1 and IgG.sub.1 in 
immunoglobulin class and subclass by the Ouchterlony method. 
FIG. 1 shows the results of measurement of these anti-TPA specific 
antibodies TPA 1-41 (-- --) and TPA 1-70 (-- --) by the EIA procedure 
described in Reference Example 2. 
Example 3 
(Preparation of hybridomas which produce mouse anti-UK monoclonal antibody) 
1 Immunization 
Mouse immunization was conducted in exactly the same manner as in Example 
2-1 but UK (produced by Nihon Seiyaku) was used in place of TPA described 
in Example 2-1. 
2 Cell fusion 
Cell fusion was conducted in accordance with the method described in 
Example 1-3. 
3 Selection and cloning of hybridomas 
Hybridomas were screened by the EIA procedure described in Reference 
Example 3, which uses a UK-bound microplate. Thereafter the procedure of 
Example 1-4 was followed to obtain anti-UK-MoAb-producing hybridomas. Out 
of these hybridomas were obtained mouse hybridomas UK 1-3 and UK 1-87, 
both of which produce an anti-UK MoAb, which specifically binds to UK 
without spoiling its capability of fibrinolysis. Antibodies UK 1-3 and UK 
1-87 respectively produced by these two hybridomas were respectively 
identified as IgG.sub.1 and IgG.sub.2 b in immunoglobulin class and 
subclass by the Ouchterlony method. 
FIG. 2 shows the results of measurement of these anti-UK specific 
antibodies UK 1-3 (-- --) and UK 1-87 (-- --) by the EIA procedure 
described in Reference Example 3. 
Example 4 
(Production of hybrid monoclonal antibody possessing 
anti-TPA-anti-human-fibrin bi-specificity) 
1 Cell fusion 
The hybridoma FIB 1-11 obtained in Example 1, which produces 
anti-human-fibrin antibody, and the hybridoma TPA 1-41 obtained in Example 
2, which produces anti-TPA antibody, were each incubated in an Iscove-Ham 
F-12 medium containing 0.5 .mu.g/ml FITC and 1.5 .mu.g/ml TRrrC at 
37.degree. C. for 30 minutes for fluorescent staining. An LSM solution 
(commercially available from Wako Pure Chemical Industries Ltd.) was then 
added and the dead cells were removed. These two hybridomas were then 
mixed together at a ratio of 1 to 1, followed by cell fusion by the method 
described in Example 1-3 using PEG 6000. 
After incubation at 37.degree. C. for 2 hours, the cell mixture was applied 
to FACS, whereby 25,000 fluorescein-rhodamine double stained cells. These 
double stained cells were seeded and cultivated on a 96-well microplate at 
10 cells per well, the microplate previously seeded with mouse thymocytes 
at 5.times.105 cells/well as feeder cells. 
2 Selection and cloning of hybrid hybridomas 
The culture supernatants from wells in which cell proliferation occurred in 
1 to 2 weeks after fusion were each subjected to the EIA procedures 
described in Reference Examples 1, 2, 7 and 8 to determine antibody 
activity. The antibody activity of culture supernatants of the hybridomas 
FIB 1-11 and TPA 1-41 respectively obtained in Examples 1-4 and 2-3, as 
controls, was determined in the same manner. The results are shown in 
Table 2. 
The wells which exhibited high hybrid antibody titer were subjected to 
cloning by limiting dilution method; the desired 
bi-specific-antibody-producing mouse hybrid hybridoma (tetraoma) FT 2-14 
was obtained. 
TABLE 2 
______________________________________ 
Specificity of Antibodies Produced by Hybrid Hybridomas 
1): Hybrid hybridoma obtained in Example 4-2. 
2): Hybridoma obtained in Example 1-4. 
3): Hybridoma obtained in Example 2-3. 
EIA antibody titer.sup.4) 
Anti-TPA- 
Anti-TPA- 
anti-fibrin 
Hybridoma anti- .beta.-chain 
culture Anti- mouse- N-terminal 
supernatant 
fibrin.sup.5) 
Anti-TPA.sup.6) 
IgG.sub.1.sup.7) 
peptide.sup.8) 
______________________________________ 
Hybrid hybridoma 
0.66 0.99 1.90 2.45 
A.sup.1) 
Hybrid hybridoma 
0.30 0.60 0.51 2.19 
B.sup.1) 
FIB 1-11.sup.2) 
0.29 0.00 0.01 0.00 
TPA 1-41.sup.3) 
0.01 1.01 0.01 0.00 
______________________________________ 
.sup.4) Expressed in optical absorbence at 492 nm (see Reference Example 
1). 
.sup.5) EIA described in Reference Example 1. 
.sup.6) EIA described in Reference Example 2. 
.sup.7) EIA described in Reference Example 7. 
.sup.8) EIA described in Reference Example 8. 
3 Purification of hybrid antibody 
Mouse hybrid hybridoma (tetraoma) FT 2-14 was intraperitoneally inoculated 
at 5.times.106 cells/mouse to six BALB/c mice which previously received 
0.5 ml of mineral oil by intraperitoneal administration. About 10 to 20 
days later, 20 ml of accumulated ascites fluid was collected and subjected 
to salting-out with 50% saturated ammonium sulfate to yield an IgG 
fraction. After dialysis against 20 mM PBS (pH 7.5), the IgG fraction was 
applied to a column of fibrin-coupled Cellulofine and eluted with a 0.2 M 
glycine-HCl buffer solution of pH 2.9. After dialysis against PBS, the 
acid elution fraction was applied to a column of TPA-coupled Sepharose 4B 
and eluted with a 0.2 M glycine-HCl buffer solution of pH 2.3. The eluate 
was dialyzed against PBS to yield 9.4 mg of bi-specific antibody FT2-14. 
The obtained antibody was subjected to the EIA procedure described in 
Reference Example 8; the dilution curve of the bi-specific antibody was 
drawn. The results are shown in FIG. 3. From FIG. 3, it is evident that 
the obtained antibody exhibited strong avidity to both TPA and fibrin. 
Example 5. 
(Production of hybrid monoclonal antibody possessing 
anti-UK-anti-human-fibrin bi-specificity1) 
1 Cell fusion 
In accordance with the method described in Example 4-1, the hybridoma FIB 
1-11 obtained in Example 1, which produces anti-human-fibrin antibody, and 
the hybridoma UK 1-3 obtained in Example 3, which produces anti-UK 
antibody, were each subjected to fluorescent staining with FITC and TRITC, 
followed by cell fusion using PEG 6000. The cell mixture was applied to 
FACS; double-stained cells were selected and cultivated. 
2 Selection and cloning of hybrid hybridomas 
The culture supernatants from wells in which cell proliferation occurred at 
1 to 2 weeks following the fusion were each subjected to the EIA procedure 
described in Reference Examples 1, 3 and 9 to determine their antibody 
activity. The antibody activity of the culture supernatant of the 
hybridomas FIB 1-11 and UK 1-3 respectively obtained in Examples 1-4 and 
3-3, as controls, was determined in the same manner. 
The well which exhibited the maximum hybrid antibody activity was subjected 
to cloning by limiting dilution method to obtain the desired 
bi-specific-antibody-producing mouse hybridoma FU 1-74. 
3 Purification of hybrid antibody 
Ascites fluid was collected in accordance with the method described in 
Example 4-3, followed by salting-out with ammonium sulfate and 
immunoaffinity chromatography using a fibrin-coupled column and a 
UK-coupled column; 14 mg of FU 1-74, the anti-UK-anti-human-fibrin 
bi-specific antibody of the present invention, was obtained from about 20 
m4of ascites fluid. 
The obtained antibody was subjected to the EIA procedure described in 
Reference Example 9; the dilution curve of the bi-specific antibody was 
drawn. The results are shown in FIG. 4. From FIG. 4, it is evident that 
the obtained FU 1-74 antibody has strong avidity to both UK and fibrin. 
Example 6 
(Property1 of anti-TPA-anti-human-fibrin bi-specific antibody FT 2-14) 
A paper disk of 9.5 mm in diameter was immersed in a 2 ml/me human 
fibrinogen solution, followed by addition of human thrombin (50 unit/ml) 
to form a fibrin clot on the paper disk. After thorough washing, this 
fibrin-coupled paper disk was immersed in a solution of the bi-specific 
antibody FT 2-14 obtained in Example 4-3 in the presence of fibrinogen (3 
mg/ml). The amount of antibody bound to the paper disk was measured using 
HRP-labeled anti-mouse-IgG.sub.2 b antibody. The results are shown in FIG. 
5. 
The fibrin avidity obtained in the presence of fibrinogen (3 mg/ml) () was 
nearly equal to that obtained in the absence thereof (.quadrature.); it 
was demonstrated that the FT 2-14 antibody of the present invention is 
specific to fibrin. 
Example 7 
(Property2 of anti-TPA-anti-human-fibrin bi-specific antibody FT 2-14) 
TPA, at various concentrations, was reacted with a solution of the 
bi-specific antibody FT 2-14 obtained in Example 4-3 of a molar 
concentration of 4.3 times that of the TPA at 37.degree. C. for 30 minutes 
to yield an immunocomplex of TPA and FT 2-14 antibody (TPA/FT 2-14 
antibody), followed by addition of 3 mM synthetic peptide substrate S-2288 
(produced by Kabi Co.) and reaction at 37.degree. C. for 2 hours. The 
optical absorbance of the liberated para-nitroaniline was determined at 
405 nm; the enzyme activity of the above TPA-FT 2-14 antibody complex was 
compared with that of simple TPA. The results are shown in FIG. 6. 
In FIG. 6, shows the enzyme activity of TPA/FT 2-14 antibody complex; 
.largecircle. shows the enzyme activity of unconjugated TPA. It was 
demonstrated that TPA does not reduce its enzyme activity at all even when 
bound with the bi-specific antibody FT 2-14 of the present invention. 
Example 8 
(Property3 of anti-TPA-anti-human-fibrin bi-specific antibody FT 2-14) 
Human fibrinogen was insolubilized by binding it to Cellulofine grains 
(commercially available from Seikagaku Kogyo K.K.) in accordance with a 
known method [Etsuko Ebisui et al.: Seikagaku, 54, 732 (1982)], after 
which it was converted to fibrin by the addition of human thrombin. To 0.2 
g of this fibrin-Cellulofine grain complex was added a 10 .mu.g/500 .mu.l 
of FT 2-14 antibody solution, followed by reaction at 37.degree. C. for 90 
minutes. After thorough washing, the reaction mixture was reacted with 100 
ng of TPA at 7.degree. C. for 40 minutes. After washing, the reaction 
mixture was reacted with added plasminogen (3 unit/ml) at 37.degree. C. 
for 1 hour. The peptide (fibrin degradation product: FDP) which was 
liberated into the supernatant was quantitated using an FDP-EIA kit 
(produced by American Diagnostics Co.). 
The results are shown in FIG. 7. 
TPA had a stronger effect on fibrin-Cellulofine grains pretreated with the 
bi-specific antibody FT 2-14 than on untreated fibrin-Cellulofine grains; 
TPA's capability of fibrinolysis was strengthened by the antibody. 
Example 9 
(Property1 (of anti-UK-anti-human-fibrin bi-specific antibody FU 1-74) 
Both 2.5 .mu.l of a UK solution (25 ng/ml) and a solution (40 ng/ml or 160 
ng/ml) of the bi-specific antibody FU 1-74 obtained in Example 5-3 were 
injected into one well of the fibrin-agarose plate described in Reference 
Example 6, followed by reaction at 37.degree. C. for 4 hours. Then, the 
resulting fibrinolysis plaque was measured and compared with the 
fibrinolysis plaque obtained with unconjugated UK. The results are shown 
in FIG. 8. In comparison with unconjugated UK, the coexistence of FU 1-74 
antibody caused an increase of 3 to 5 times in the capability of 
fibrinolysis. 
Example 10 
(Enhancement of in vitro fibrinolytic capability of anti-TPA 
anti-human-fibrin bispecific antibody FT2-14 and anti-UK anti-human-fibrin 
bispecific antibody FU1-74) 
Columns packed with fibrin-Cellulofine grains obtained in Example 8 were 
respectively coupled with FT2-14 (0 to 100 .mu.g/ml) obtained in Example 
4-3 and FU1-74 (0 to 50 .mu.g/ml) obtained in Example 5-3 and then washed 
with PBS containing 0.01% Triton X-100 (PBS-T). A TPA solution was 
injected to the FT2-14-treated columns and a UK solution to the 
FU1-74-treated columns at 200 .mu.g/400 ml a washings with PBS-T, the 
carrier Cellulofine grains were transferred to test tubes and reacted with 
added plasminogen (4.5 units/1.5 ml) at 37.degree. C. for 2 hours. After 
addition of an equal amount of 6 mM para-amidinophenyl 
methanesulfonylfluoride, Fibrin Degradation Products (FDP) liberated from 
the Cellulofine grains were measured using an EIA kit (produced by 
American Diagnostica Co.). The results are shown in FIG. 9. Enhancement of 
fibrinolytic capability by 3.8 times was noted in the 100 .mu.g/ml FT2-14 
treated fibrin-Cellulofine grains (.largecircle.) and enhancement by 8.5 
times in the 50 .mu.g/ml FU1-74-treated fibrin-Cellulofine grains (), in 
comparison with the fibrinolytic capability of TPA (.largecircle.) and UK 
() on fibrin-Cellulofine grains not treated With antibody. 
Example 11 
(Enhancement of in vivo thrombolytic capability of anti-TPA 
anti-human-fibrin bispecific antibody FT2-14) 
1 Preparation of rabbit jugular vein thrombus model 
After anesthesia of male rabbits (2.5 to 3.5 kg) by intraperitoneal 
injection of pentobarbital (90mg/1.5 ml/rabbit) and urethane (1.5 mg/3 
ml/rabbit), a blood sampling cannula was inserted to the right femoral 
vein and a blood injecting cannula and a piece of woolen thread to the 
left jugular vein. The jugular vein was clipped to block blood flow in a 
portion of about 3 cm in width, and this was followed by vessel inside 
washing with sequential addition of physiological saline and then 0.2 ml 
of a 12mM CaC.sub.12 solution containing 10 U/ml thrombin. To the washed 
vessel was injected 0.2 ml of autoblood collected via the femoral vein. 
The cannulae were immediately removed; the resulting openings were 
ligated. The animal was then kept standing for 30 minutes to allow blood 
to coagulate. 
2 Injection of subject drug solution 
Heparin was intraperitoneally (250 U/kg) and subcutaneously (500 U/kg) 
injected to the thrombus model rabbits; the clip was removed from the 
jugular vein. An injection needle was then inserted to the ear vein on the 
opposite side, and this was followed by intravenous injection of a subject 
drug solution in an mount of 10% of the entire dose volume at 2 ml/rabbit 
and then continuous injection of the remaining 90% volume at a flow rate 
of 6 ml/hour over a period of 4 hours. Four hours later, the thrombus 
adhered to the woolen thread was taken out and weighed on a wet basis. The 
subject drug solutions used were a control physiological saline, TPA 
solutions (0.25 mg/kg and 1.0mg/kg), the FT2-14 antibody solution 
(1.2mg/kg) and TPA/FT2-14 antibody complex solution (0.25 mg/kg TPA+1.2 
mg/kg FT2-14) obtained in Example 4-3. The results are shown in FIG. 10. 
When 0.25 mg/kg TPA was continuously injected over a period of 4 hours, the 
wet weight of thrombus decreased from 61.5 .+-.7.2mg (n=4) in the control 
group to 31.7 .+-.10.1 mg (n=6). In the group receiving 0.25 mg/kg TPA and 
1.2 mg/kg FT2-14 in combination in a molar ratio of 1 of TPA to 2 of the 
bispecific antibody (bs AB), the wet weight became 19.6.+-.6.5 mg (n=7), 
i.e., the thrombolytic capability was enhanced to the extent that it was 
almost equal to the wet weight of thrombus in the 1.0mg/kg TPA group, 
15.5.+-.3.5 mg (n=3). In the 1.2mg/kg FT2-14 group, the wet weight of 
thrombus was 41.5 mg (n=1). 
Example 12 
(Preparation of hybridoma which produces mouse anti-low-molecular-weight-UK 
monoclonal antibody) 
1 Immunization 
Mouse immunization was conducted in exactly the same manner as in Example 
2-1 but commercially available two-chain low molecular weight UK 
(available from JCR Co.; Code No. 300110; molecular weight: 33,000) was 
used in place of the TPA described in Example 2-1. 
2 Cell fusion 
Cell fusion was conducted in accordance with the method described in 
Example 1-3. 
3 Hybridoma selection and cloning 
Hybridomas were screened by the EIA procedure described in Reference 
Example 10 using a microplate bound with low molecular weight UK and 
subjected to the same procedure as Example 1-4 to obtain hybridomas which 
produce anti-low-molecular-weight-UK MoAb. Out of these hybridomas, mouse 
hybridoma UK 1-6 was selected, which produces an 
anti-low-molecular-weight-UK MoAb which specifically binds to UK without 
impairing the fibrinolytic capability of UK. The immunoglobulin class and 
subclass of the antibody UK 1-6 produced by the hybridoma thus obtained 
were identified as IgG.sub.1 (k chain) by the Ouchterlony's method. 
Example 13 
(Production 2 of hybrid monoclonal antibody possessing anti-UK 
anti-human-fibrin bispecificity) 
1 Cell fusion 
FIB 1-11, the hybridoma which produces anti-human-fibrin antibody and which 
was obtained in Example 1, and UK 1-6, the hybridoma which produces 
anti-low-molecular-weight-UK antibody and which was obtained in Example 
12, were subjected to fluorescent staining respectively with FITC and 
TRITC in accordance with the method described in Example 4-1, and then 
subjected to cell fusion using PEG 6000. The cells were then applied to 
FACS to select double-stained cells, which were cultivated. 
2 Hybrid hybridoma selection and cloning 
The culture supernatants obtained from the wells in which cell 
proliferation occurred 1 to 2 weeks after cell fusion were each subjected 
to the EIA procedure described in Reference Examples 1, 9 and 10 to 
determine their antibody activity. 
The well which showed maximum hybrid antibody activity was subjected to 
cloning by limiting dilution method, and the desired mouse hybrid 
hybridoma FU 2-16, which produces bispecific antibody, was obtained. 
The culture supernatant of the hybrid hybridoma FU 2-16 was assayed by the 
EIA procedure described in Reference Example 5. The results are shown in 
FIG. 11. 
3 Purification of hybrid antibody 
Ascites fluid was collected by the method described in Example 4-3 and 
subjected to ammonium sulfate salting-out and immunoaffinity 
chromatography using a fibrin-coupled column and a UK-coupled column to 
yield about 23 mg of FU 2-6, the anti-UK anti-human-fibrin b/specific 
anti-body of the present invention, from about 20 ml ascites fluid. 
Example 14 
(Property1 of anti-UK monoclonal antibody) 
UK 1-3, 1-87 and 1-6, the anti-UK monoclonal antibodies obtained in 
Examples 3 and 12, were subjected to the EIA procedures respectively using 
a UK-bound microplate and a low molecular weight UK-bound microplate 
(described in Reference Examples 3 and 10, respectively), and were 
compared as to avidity to UK [FIG. 12 (A)] and to low molecular weight UK 
[FIG. 12 (B)]. The results are shown in FIG. 12. 
UK 1-3 antibody did not bind to low molecular weight UK, while UK 1and UK 
1-6 antibodies both showed equivalent reactivity to UK and low molecular 
weight UK. 
Example 15. 
(Property2 of anti-UK monoclonal antibody) 
Anti-UK monoclonal antibodies UK 1-3, 1-87 and 1-6 obtained in Examples 3 
and 12 were subjected to a UK enzyme activity neutralization test using 
synthetic peptide substrate S-2444 described in Reference Example 11. The 
results are shown in FIG. 13. 
None of these anti-UK monoclonal antibodies inhibited UK enzyme activity. 
The present invention has been described in detail, including the preferred 
embodiments thereof. However, it will be appreciated that those skilled in 
the art, upon consideration of the present disclosure, may make 
modifications and/or improvements on this invention and still be within 
the scope and spirit of this invention as set forth in the following 
claims.