HETEROBIFUHC IOK-L ANTIBODIES AND METHOD OF USE

TECHNICAL FIELD

This invention relates to heterobifunctional anti¬ bodies having dual specificities, one specificity directed against a thrombus and the other specificity directed against a thrombolytic agent. This invention further relates to a method of using these heterobi¬ functional antibodies in imrnunodiagnostic and im uno- therapeutic processes.

BACKGROUND ART

Most myocardial infarctions are caused by coronary thrombosis (DeWood et al., N. Eng. J. Med. , 303: 897 (1983). The coronary thrombosis that causes the myo¬ cardial infarction can be lysed by thrombolytic agents. These thrombolytic agents are plasminogen activators that activate the conversion of plasmino¬ gen to the fibrinolytic enzyme plasmin. Plasmin will then lyse the fibrin present in the thrombus. This treatment with plasminogen activators is not without side effects. Plasmin acts non-selectively and there¬ fore, not only lyses the fibrin in the thrombus, but also attacks fibrinogen and clotting factors, often resulting in severe bleeding diathesis.

Streptokinase, urokinase and tissue-type plasmino¬ gen activator (TPA) are three known plasminogen acti¬ vators for lysing thrombi. These activators are indi¬ cated for the treatment for acute cardiovascular dis¬ ease such as infarct, stroke, pulmonary embolism, deep

vein thrombosis, peripheral arterial occlusion, and other venous thrombosis. Both streptokinase and uro- kinase, however, have severe limitations. Due to a low affinity for fibrin, both activators will activate circulating and fibrin-bound plasminogen indiscrimi¬ nately. The plasmin formed in circulating blood is neutralized before it can be used in thrombolysis. Residual plasmin will degrade several clotting factor proteins, for example, fibrinogen, factor V, and fac¬ tor VIII, causing he orrhagic potential. Further, streptokinase is strongly antigenic and patients with high antibody titers respond inefficiently to treat¬ ment and cannot remain on continuous treatment.

Human tissue-type plasminogen activator can bind to fibrin and therefore favors the activation of plas¬ minogen in close proximity to the thrombus, potential¬ ly sparing fibrinogen elsewhere in the circulation. However, at doses required for prompt lysis of coron¬ ary thrombi, the use of tissue-type plasminogen act¬ ivator can also result in hemorrhage.

In order to increase the specificity of the throm¬ bolytic agents to the thrombus, it has been shown that covalent linkage of urokinase to a fibrin-specific antibody results in marked enhancement of fibrinolytic potency and specificity Bode et al. , Science, 22£:765-767 ⁽ 1985 ⁾ .

One function characteristic of every antibody molecule is specific binding to an antigenic determi¬ nant. Antibodies in vivo are bivalent and monospeci- fic, containing two identical antigen binding sites. The specific binding of antigen by an antibody mole¬ cule is determined by the antibody's structure of the variable regions (F , ) of both heavy and light chains.

Antibodies having dual specificities have been pre¬ pared by subjecting antibodies of different specifici¬ ties to a selective cleavage of the disulfide bridges that link the two heavy chains together. Antibody half-molecules are then reassociated under nuetral pH to produce the hybrid antibodies having dual specifi¬ cities.

Nisonhoff et al.. Nature (London), 194; 355 (1962) describes the in vitro production of a bispecific antibody molecule from a polyclonal rabbit antibody, anti-ovalbumin, and an anti-bgg antibody. The mono- specific antibodies were treated with pepsin to remove the F portion of the antibody, leaving the two anti¬ gen-binding sites (F , ) covalently linked by a single disulfide bond. This bond was then split under reduc¬ ing conditions and the two antibody molecules reasso¬ ciated under oxidizing conditions to produce a bispe¬ cific antibody.

In Brennan et al.. Science, 229: 31 (1985) a chem¬ ical procedure is described for preparing bispecific antibody fragments from monoclonal antibodies. In this procedure, a modification of the Nisonoff tech¬ nique was used in cleaving the F . fragments, followed by reconstituting the half-fragments to form the bi¬ specific antibody molecule. The F . fragments were reduced in the presence of sodium arsenite to stabi¬ lize vicinal dithiols and impede intramolecular disul¬ fide formation. The other modification involved ac¬ tivating the thiols of one of the half-F . fragments as a thionitrobenzoate derivative. By this process, a bispecific antibody was produced from anti-avidin F , and anti- luciferase F . was produced.

Liu et al., Proc. Nat'l. Acad. Sci. USA, 82: 8648 (1985) discloses a chemical procedure for forming a bispecific antibody in which anti-T3 antibody was covalently linked to a second monoclonal antibody, anti-Igld specific for the idiotype of the surface immunoglobulin of a human B lymphoma. The anti-T3 and anti-Igld antibodies were first reacted with N-suc- cinimidyl-3-(2-pyridyldithio) propionate (SPDP). Thiol groups were attached to the cleaved anti-T3 an¬ tibody using 2-iminothiolane. Then the two modified half- antibodies, anti-T3 and anti-Igld, were mixed to covalently link the two antibodies. The result showed that the T8 cytotoxic T lymphocytes lysed the human B-lymphoma cells, but no lyses was observed when T4 cytotoxic T lymphocyte cells were used.

Bispecific antibodies have also been produced from hybridomas. The preparation of bispecific monoclonal antibodies by fusion of antibody-producing hybridoma cells is described in Milstein and Cuello, Nature (London), 305: 537 (1983). This reference describes the fusion of two hybridomas, or the fusion of one hybridoma with spleen cells from an immunized rat, to produce hybrid hybridomas. These hybrid hybridomas secrete predefined bispecific monoclonal antibodies as well as monospecific antibodies. Anti-somatostatin/ anti-pluroxidase and anti-substance P/ anti-peroxidase bispecific monoclonal antibodies were prepared in this manner. The bispecific monoclonal antibodies produced by hybrid hybridomas were complete molecules, contain¬ ing the F • region as well as the antigen-combining sites.

PCT application, WO83/03679, describes the produc¬ tion of a bispecific antibody having dual specifici¬ ties obtained by fusion two hybridomas. This applica¬ tion describes procedures for producing and selecting hybrid hybridomas. The bispecific antibodies therein are described as having many potential uses, ranging from immunodiagnostic procedures to targeted delivery of drugs.

It would be desirable to have a bispecific anti¬ body having dual specificity such that one specificity would be directed against a thrombus and the other specificity would be directed against a thrombolytic agent. With this bispecific antibody, a thrombus would be detected. This thrombus then could be lysed by the action of a thrombolytic agent that becomes or is attached to the anti-thrombolytic antibody. The lysis of thrombi is complicated; it was not known whether a bispecific antibody would block or inhibit the thrombolytic activity by the thrombolytic agent.

DESCRIPTION OF THE INVENTION The present invention provides for a heterobifunc¬ tional antibody having dual specificity, with one specificity directed against a thrombus and the other specificity directed against a thrombolytic agent. The heterobifunctional antibody according to this in¬ vention can be used for immunodiagnosis and immuno- therapy. Accordingly, the present invention also pro¬ vides processes for immunodiagnosis and immunotherapy employing the heterobifunctional antibodies.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the release of labeled fibrin from fibrin-Sepharose by the following compounds: (1) a heterobifunctional antibody specific for both fibrin and tissue plasminogen activator (TPA) complexed to TPA (BI-AB), then TPA); (2) a heterobifunctional anti¬ body specific for both fibrin and TPA, first added to fibrin-Sepharose followed by the addition of TPA after washing (BI-AB + TPA); (3) a urokinase-antifibrin antibody covalent complex (UK-AB); (4) TPA alone; and (5) urokinase alone (UK). Lysis was expressed as the quotient of released radioactivity and total radio¬ activity. Each point represents the mean of three separate experiments with a mean standard deviation of 1.23.

Figure 2 shows the release of labeled peptides from fibrin-Sepharose (1) by TPA alone and (2) by TPA captured by a heterobifunctional antibody specific for both fibrin and TPA. For clarity, data at 0.1 and 0.34 ng/ml are not shown in this figure but are incorporated into Figure 3. Each point represents the mean of three experiments with a mean standard devia¬ tion of 0.74.

Figure 3 shows the enhancement of fibrinolysis computed as the maximal quotient of percent lysis in the presence and absence of the heterobifunctional antibody. The data are taken from those shown in Figure 2, with the addition of experiments at 0.1 and 0.3 ng/ml, not shown in Figure 2. Error bars repre¬ sent standard deviations of the mean of the quotients. This figure shows that the relative potency of TPA in the antibody-pretreated samples increases as TPA concentration decreases.

Figure 4 shows the demonstration of antigen bind¬ ing by heterobifunctional antibodies F32.1 and F36.23 produced by somatic fusion.

Figure 5 shows the ability of the heterobifunc¬ tional antibodies F32.1 and F36.23 to enhance fibrino- lysis.

BEST MODE OF CARRYING OUT THE INVENTION

This invention is directed to heterobifunctional antibodies having dual specificity with one specifi¬ city directed against a thrombus and the other speci¬ ficity directed against a thrombolytic agent. The individual specificities are to (a) antigenic deteimi- nants on a thrombus and (b) antigenic determinants on a thrombolytic agent.

Throughout this specification, the term "heterobi¬ functional antibody" is used to designate a single antibody molecule having two specificities or two molecules linked to each other each having different specificities. Other terms have been used to describe heterobifunctional antibodies including heteroanti- body, bispecific antibody, hybrid antibodies, hetero- ligating antibody, antibody duplex, hetereodimer, among others. It will be understood by one of skill in the art that these are all equivalent terms.

The antibodies usable in preparing the heterobi¬ functional antibodies of the present invention may be either polyclonal or monoclonal antibodies. In the preferred embodiments of this invention, monoclonal antibodies are used in preparing heterobifunctional antibodies.

The anti-thrombus specificity as used herein re¬ fers to antibodies raised against fibrin or fibrino-

gen. Blood clots when thrombin cleaves two pairs of small peptides from fibrinogen to yield fibrin mono¬ mers (Blomback et al.. Ark. Kemi., 12; 173 (1958) and Doolittle, R.F. , Adv. Protein Che ., 27: 1 (1973)). Fibrin monomers spontaneously aggregate to form an insoluble gel that is covalently stabilized by Factor Xllla. Fibrin retains 98% of the original covalent structure of fibrinogen. Thus, in the preferred em¬ bodiment of this invention the antibodies that are used to form one-half of the heterobifunctional mole¬ cule are any antibodies which are fibrin-specific and

_* are substantially devoid of fibrinogen cross-reac¬ tivity.

For example, antibodies with this specificity have been described in Hui et al. , Science, 222; 1129 (1983). Further description of the same type of anti¬ bodies can be found in commonly assigned co-pending U.S. Patent Application Serial No. 824,228, filed Jan¬ uary 30, 1986, for "Fibrin-Specific Monoclonal Anti¬ bodies Lacking Fibrinogen Cross-Reactivity." Fibrin- specific monoclonal antibodies with substantially no fibrinogen cross-reactivity are also described in com¬ monly assigned co-pending United States Patent Appli¬ cation Serial No. 851,514, filed concurrently here¬ with. Other examples of antibodies with a specificity against a thrombus include Kudryk et al, Mol. Imm. , 21; 89 (1984). All of the above references are herein incorporated by reference.

Antibodies specific against thrombolytic agents may also be polyclonal or monoclonal antibodies, pre¬ ferably monoclonal antibodies. When plasminogen is converted by an activator to plasmin, the active fibrinolytic enzyme of plasma, it develops a marked

affinity for its substrate, fibrin. Three plasminogen activators are currently available for converting plasminogen to plasmin: streptokinase, urokinase, and human tissue plasminogen activator (TPA). The term "thrombolytic agent" as used in this specification is therefore meant to include broadly any agent utilized for inducing or initiating the lysis of a thrombus. Other terms are known in the art for the lysis of a thrombus, including fibrinolysis. Although the most common thrombolytic agents are streptokinase, urokin¬ ase, and tissue-type plasminogen activator, any other thrombolytic agent can be utilized as defining the specificity of the relevant portion of the heterobi¬ functional antibodies of the invention.

Antibodies specific against thrombolytic agents may be raised according to means known in the art. In order to have enhanced specificity, it is preferred that monoclonal antibodies be raised against the thrombolytic agents. (Kohler and Milstein, Nature, 5 :495 (1975).

The process for obtaining a heterobifunctional antibody according to the present invention requires one antibody specific against a thrombus and the other antibody specific against a thrombolytic agent. These two antibodies may be modified by chemical procedures for preparing antibody fragments therefrom that can then be recombined to produce the heterobifunctional antibody. Alternatively, the heterobifunctional anti¬ body can be prepared from the fusion of two hybridomas producing a hybrid hybridoma which secretes predefined heterobifunctional antibodies.

One procedure in which the anti-thrombus antibody and the anti-thrombolytic agent antibody may be chemi-

cally modified to produce the heterobifunctional anti¬ body is described in Liu et al, Proc. Nat'l. Acad. Sci. (U.S.A.), 82; 8648 (1985), incorporated herein by reference. In this procedure, each antibody is treat¬ ed separately with a molar excess of N-succinimidyl- 3(2-pridyldithio) propionate (SPDP) dissolved in absolute ethanol. Thiol groups are then added to one of the modified antibodies by reacting a molar excess of 2-iminothiolanine dissolved in sodium borate. Then equal molar amounts of the two modified antibodies are mixed and after sufficient reaction time, the reaction is stopped by adding an excess of iodoacetamide. The reaction mixture is then passed over an affinity col¬ umn to separate the bispecific antibody from uncoupled antibodies. In this p ^e rocedure, the Fc/Fa.b one-half of the antibody molecule is kept intact.

In another chemical procedure for producing het¬ erobifunctional antibodies, the F fraction of the antibody is cleaved from the F , fraction. This pro¬ cedure is described in Brennan et al., Science, 229; 31 (1985), incorporated herein by reference. In this procedure, the F portion of the antibody is cleaved by pepsin hydrolysis to yield the F , portion. The F , portion is then reduced by mercaptoethylamine in the presence of sodium arsenite to cleave the disulfide bonds. The bonds are stabilized with Ellman's rea¬ gent, 5,5'-dithiobis(2-nitrobenzoic) acid. One of the stable F . half-molecules is then treated with mer¬ captoethylamine and is mixed with an equimolar amount of the other untreated stable F . half-molecule to form the heterobifunctional antibody.

As usual herein, the term "modification" refers to onospecific antibodies that are chemically altered.

such as in the above two described procedures, to dis¬ associate the antibodies and then reassociate them, producing the heterobifunctional antibody having dual specificity.

Alternatively, the heterobifunctional antibody may be produced by the fusion of hybridomas to produce a hybrid hybridoma that secretes predefined bispecific heterobifunctional antibodies. This procedure is de¬ scribed in Milstein and Cuello, Nature (London), 305; 537 (1983), incorporated herein by reference. Methods for producing a hybrid hybridoma which secretes a hy¬ brid monoclonal antibody having dual specificity against two different antigenic determinants is also described in PCT application, WO 83/03679, also incor¬ porated herein by reference. In the PCT application a procedure is described for using hybridomas with se¬ lectable markers, such that the monospecific hybridoma cannot survive in a medium in which the hybrid hybrid¬ oma is cultured. Thus, by the fusion of the two hy¬ bridomas, each conferring to the other the ability to grow in selected medium, the hybrid hybridoma can be easily selected. Examples of such selectivity include the inability to produce the enzyme HPRT, HAT-ouaban selection, HAT sensitivity, and antibiotic resistance.

The present invention also provides methods for immunotherapy and immunodiagnosis using heterobifunc¬ tional antibodies having a dual specificity, wherein one of the dual specificities is against a thrombus and the other specificity is against a thrombolytic agent. In the immunotherapeutic and immunodiagnostic applications, heterobifunctional antibodies produced from either chemical means or hybrid hybridomas can be used.

The heterobifunctional antibody of dual specifi¬ city can be used in immunotherapy by constructing it to have a specificity against a thrombus and a speci¬ ficity against a thrombolytic agent. In the preferred embodiment of this invention, the anti-thrombus anti¬ body is a monoclonal antibody against fibrin with sub¬ stantially no cross-reactivity to fibrinogen and the thrombolytic agent is TPA. In this application, after the heterobifunctional antibody is administered to the patient, the heterobifunctional antibody then becomes localized at the site of the thrombus. During this time, the endogenous TPA will become attached to the heterobifunctional antibody.

Surprisingly, it has been found by the inventors that endogenous TPA is captured by the heterobifunc¬ tional antibody, enhancing the potency of endogenous TPA and fibrinolysis with increasing efficacy at de¬ creasing concentrations of TPA (See example below) .

This method may also be used by first administer¬ ing to the patient a low dosage of a thrombolytic agent. The heterobifunctional antibody is then admin¬ istered to the patient, and it localizes at the site of the thrombus. The heterobifunctional antibody will capture the administered thrombolytic agent, directly it to the thrombus site. In results shown in in vitro testing, the hetereobifunctional antibody may be first administered, followed by administration of a low dos¬ age of the thrombolytic agent. As will be appreciated by one of skill in the art, the low dosage of the thrombolytic agent will reduce the risk of serious side effects, such as hemorrhage.

In another embodiment of this invention, the thrombolytic agent, for example streptokinase, uroki-

nase, or TPA, is attached to the heterobifunctional antibody prior to administration to the patient. In this drug-targeted system, the specificity of the anti-thrombus portion of the heterobifunctional anti¬ body permits selectivity of the antithrombolytic agent to lyse the thrombus.

The heterobifunctional antibody may also be used in immunodiagnostic applications. In vivo immunodi¬ agnosis can be performed using the heterobifunctional antibody of this invention. The heterobifunctional antibody, having one specificity against a thrombus and the second specificity against a thrombolytic agent, is first administered to the patient. After sufficient time has passed for the antibody to local¬ ize at the thrombus and unbound antibody has been per¬ mitted to clear from healthy tissue in the patient, the thrombolytic agent bearing a radionuclide is ad¬ ministered. The radionuclide must be of the type of decay which is detectable for a given type of instru¬ ment. 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 radionu- clides to the protein agents is known in the art and is often accomplished either directly or indirectly using an intermediary functional group. Examples of radioisotopes that can be used for in vivo diagnosis are ⁹⁹ Tc, ¹²³ I, ¹³¹ I, ^nl ln, ⁹⁷ Ru, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, and ²⁰¹ T1.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be used according to the methods of this invention. Examples of elements that are par-

ticularly useful for use in Magnetic Resonance Energy techniques include ¹⁵⁷ Gd, ⁵⁵ Mn, ¹⁶² Dy, ⁵² Cr, and ⁵⁶ Fe.

The heterobifunctional antibody having dual speci¬ ficity can further comprise a pharmaceutical composi¬ tion, with a pharmaceutically acceptable carrier. These carriers are well known in the art and can in¬ clude aqueous or solvent emulsions or suspensions, including saline and buffered media. The pharmaceuti¬ cal compositions may be prepared by any of the methods that are well-known in the pharmaceutical art, for example, as described in Remington's Pharmaceutical Sciences (16th Edition, 1980).

The dose ranges for administration of the hetero¬ bifunctional antibody are those that are large enough to detect the presence of thrombi. The dosage should not be so large as to cause adverse side effects, such as unwanted cross rashes, and anaphylactic rashes and the like. Generally, the dosage will vary with the age, condition, sex, and extent of disease in the patient. Counter indications can include immune tolerance and other variables and can be adjusted by the individual physician. Dosage can range from 0.01 mg/kg to 500 mg/kg of body weight, preferably 0.01 mg/kg to 200 mg/kg. The heterobifunctional antibodies can be administered parentally by injection or by gradual perfusion over time. They can also be admin¬ istered intravenously, intraperitoneally, intramuscu¬ larly, or subcutaneously.

Having now generally described this invention, the same will become more readily understood by reference to specific examples included herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.

EXAMPLES

Example 1

PREPARATION OF MONOCLONAL ANTIBODIES SPECIFIC FOR HUMAN TISSUE TYPE PLASMINOGEN ACTIVATOR (ANTI-tPA)

IgGl mouse monoclonal antibodies specific for human tissue-type plasminogen activator (anti-TPA) were raised from A/J mice that had been immunized with human recombinant two-chain tissue-type plasminogen activator (TPA) (Genentech Inc). To select the appropriate clones, spleen cells from the immunized mice were fused with SP2/0 myeloma cells and the mixture was then distributed into ten 96-well icro- titer plates containing macrophage feeder layers. Hybridoma colonies were observed in over 95% of the wells. Supernatants from 12 wells showed evidence of anti-TPA binding in a solid phase immunoassay. The cloning and subcloning of these colonies by the limiting dilution method resulted in 33 stable lines, each producing anti-TPA antibody. Of these stable lines, 27 were IgG, Kappa and 6 were IgG~, Kappa as isotyped by the Boehringer kit. Two IgG, lines were selected for expansion in ascites and the antibodies subsequently purified by ammonium sulfate precipita¬ tion (45%) and DEAE-cellulose chromatography. The resulting antibody preparations were homogeneous by SDS-PAGE electrophoresis and demonstrated binding to the heavy chain of TPA by Western blotting. The apparent dissociation constants of the two anti-TPA antibodies was estimated by determining the half maxi- mal binding concentration of 125I-TPA. One antibody was determined to have a K, of 4.5 x 10 and the

other 6.5 x 10~ . Neither antibody inhibited the activity of TPA as measured either against an esterase substrate, S2444, or in a fibrinolysis assay against fibrin monomer (described in Example 3, below). The anti-TPA hybridoma cell line TCL8 was placed on deposit at the American Type Culture Collection (ATCC), Rockville, Maryland and designated ATCC No. HB9090. Hybridoma line TCL8 produces a monoclonal antibody specific for the catalytically active chain of TPA.

Example 2

PREPARATION OF HETEROBIFUNCTIONAL ANTIBODY HAVING DUAL SPECIFICITY FROM FIBRIN SPECIFIC MONOCLONAL ANTIBODY AND ANTI-TPA MONOCLONAL ANTIBODY

An IgGl mouse monoclonal antibody specific for fibrin and not cross-reacting with fibrinogen (59D8) has been previously described in Hui , et al. , Science, _22_2:1129-1131 (1983). Anti-TPA monoclonal antibody described in Example 1 was covalently coupled to anti- fibrin monoclonal antibody 59D8 utilizing the cross- linking reagent (N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP). Lui _______. , Proc Nat'l. Acad. Sci.

USA, 8_2_:8648-8652 (1985). In a typical experiment, 8.4 mg anti-fibrin antibody at a concentration of 2.4 mg/ml in 0.0IM phosphate, 0.15M NaCl, 0.02% NaN , pH 7.4, (PBSA) was reacted with 50 ul of 20mM SPDP in absolute ethanol. After 30 minutes at room tempera¬ ture, the reagents were removed by gel filtration on a Sephadex G-25 column (2.5 x 30 cm) equilibrated with 0.14M NaCl, lmM KC1, 3.7mM sodium phosphate, pH 7.4 (NaPi). A modification of from two to four 2-pyridyl-

disulfide groups per more of anti-fibrin antibody was obtained. Grassetti et al. , Arch. Biochem. Biophys.,

119;41-49 (1967); Stuchbury et al. , Biochem. J. ,

151;417-432 (1975).

Thiol groups were attached to the anti-TPA anti¬ bodies by reacting 8.4 mg of either of the two anti- TPA monoclonal antibodies at a concentration of 2.4 ng/ml in NaPi with a 200-fold molar excess of 2-imino- thiolane in 25mM sodium borate, pH 9.1. After 45 minutes at room temperature, the mixture was frac¬ tionated on a Sephadex G-25 column (2.5 x 30 cm) equilibrated with O.IM NaCl, O.IM sodium phosphate, pH 6.6. This protocol introduces 1 to 2 thiol groups per antibody molecule. Lui et al., supra.

Equimolar amounts of the anti-fibrin and anti-TPA modified antibodies were mixed and stirred at room temperature for 3.5 hours. The reaction was then stopped by addition of 0.5 ml IM iodoacetamide in IM sodium phosphate, pH 8.0. At this point the mixture was no longer reactive with Ellman's reagent.

The sample was next concentrated to a volume of 9 ml in a 10 ml Amicon ultrafiltration cell using a YM 30 membrane and then applied to a calibrated Sephacryl S-300 column (2.5 x 85 cm) equilibrated with 0.1M phosphate, 0.1 M NaCl, 1.0M urea, pH 6.6. Two peaks were clearly resolved. The first peak, eluting at approximately 300 Kd, was consistent with heterobi¬ functional antibodies (heterodimers). The second peak, eluting at 150 Kd, was presumed to contain unreacted antibody monomers. Small amounts of higher molecular weight materials were also observed, which were presumed to be higher polymers. The material from the 300 Kd peak was pooled and dialyzed against the buffer to be used in subsequent assays.

To bind TPA to the heterobifunctional antibody, 3.5 mg of TPA (0.5 mg/ml) was mixed with 5 mg of heterobifunctional antibody (0.5 mg/ml) for 2 hours at room temperature. The solution was concentrated to a volume of 9 ml in a 10 ml Amicon ultrafiltration cell on a YM 30 membrane and applied to a Sephacryl S-300 column. Two peaks were resolved. One peak eluted with a slightly smaller volume than that of the heterobifunctional antibody peak, and with a molecular weight of approximately 400 Kd. Since the apparent molecular weight of TPA was 70 Kd on this column, the first peak was presumed to be an ?>ntibody-TPA complex and the second peak unbound TPA. On the basis of enzymatic activity (described in Example 3), it was estimated that about 1.5 moles of TPA were bound to each mole of heterobifunctional antibody.

EXAMPLE 3

PEPTIDASE ENZYMATIC ACTIVITY ASSAY

The plasminogen-activating potency of TPA, the heterobifunctional fibrin-TPA antibody, urokinase, and a covalent complex of urokinase and a fibrin-specific antibody (urokinase-antibody conjugate) (Bode et al. , Science, 229;765-767 (1985)), were compared at equiva¬ lent peptidase activities. Peptidase activity inde¬ pendent of fibrin binding was measured with the chromogenic substrate, S-2288 (Helena Labs). Uroki¬ nase (Abbokinase, Abbot Lot #82-087-AF)) was the reference standard. The peptidase activity of 1 unit of urokinase was equal to that of 7 ng of TPA.

EXAMPLE 4

PLASMINOGEN ACTIVATOR ASSAY

The lysis of 125I-fibrin monomer covalently linked to Sepharose 4B-C1 (fibrin-Sepharose) provided the end-point for the plasminogen-activator assay. Hui et al. , supra. In this assay, the test substance was incubated with a 300 ul of a suspension of fibrin- Sepharose in a buffer containing 10 raM sodium phos¬ phate, 0.1% BSA, 0.01% Tween-80, pH 7.4. The fibrin-Sepharose was washed with the same buffer and then incubated with 1 ml of plasminogen solution (0.15 mg/ml). Release of labeled peptides was measured at varying intervals by counting aliquots of the super¬ natant after centrifugation of the fibrin-Sepharose and was expressed as a percentage of the total initial radioactivity.

The test substances assayed included: i) the heterobifunctional antibody described in Example 2; ii ) the heterobifunctional antibody pretreated with TPA; iii) TPA iv) urokinase (UK), and v) urokinase-anti-fibrin conjugate. An additional sample, consisting of the heterobifunc¬ tional antibody that had been incubated with fibrin- Sepharose, washed, mixed with TPA, and washed again, was incubated with plasminogen as described above.

Figure 1 shows that the concentration of TPA re¬ quired to release labeled peptides from fibrin-Sepha¬ rose is about 1/10 that of urokinase and that the TPA-

heterobifunctional antibody is 10-fold more potent than TPA alone. There was no significant difference in fibrinolytic effectiveness between a heterobifunc¬ tional antibody that had been treated first with TPA and then added to the fibrin-Sepharose, and that of heterobifunctional antibody mixed first with fibrin- Sepharose and then treated with TPA. The TPA-hetero- bifunctional antibody is equipotent to the urokinase- anti-fibrin complex described in Bode et al., supra.

The relative efficacy of TPA relative to urokinase has been attributed to the capacity of TPA to selec¬ tively bind to fibrin. Collen et al.. Circulation, 2_:1012-1017 (1984); Bergmann et al.. Science,

120:1181-1183 (1983); Hoylaerts et al., J^ Biol.

Chem., 252:2912-2919 (1982). Urokinase does not have a fibrin binding site. The even greater fibrinolytic potency observed with the TPA-heterobifunctional anti¬ body, and with the urokinase-anti-fibrin complex, may be explained by the considerable difference in the relative affinity for fibrin that exists between TPA and fibrin-specific antibody 59D8. The K, of TPA for fibrin is 0.1 mM, whereas that of antibody 59D8 is approximately 0.1 microM. In the heterobifunctional antibody, the affinity of anti-TPA for TPA is O.lnM, not a limiting factor.

EXAMPLE 5

TPA CAPTURE AT LOW CONCENTRATIONS

The plasma concentration of TPA is reported to be in the range of 5ng/ml. Hamster et al., N. Enql. J. Med., 313;1557-63 (1985). To determine whether the

fibrinolytic efficacy of TPA would be enhanced at these and lower TPA concentrations, 1 ml aliquots of fibrin-Sepharose were incubated both with or without 330 ug/ml heterobif nctional antibody in a volume of 1.0 ml for 4 hours at 20°C. A volume of 2.7 1 of 100 mM phosphate, 0.1% BSA, 0.01% Tween-80 containing 0 (control), 0.1, 0.3, 1.0 or 5.0 ng/ml TPA was then run through a column at a flow rate of 180 ml/hr. After the fluid had passed through the column, the fibrin- Sepharose was removed and placed in a test tube at 25 C. One ml of plasminogen (0.15 lmg/ml) was then added. The Sepharose was allowed to settle at the bottom of the test tube. At 20, 45, 60, 90, 120, 150 and 180 min, 0.6 ml of the supernatant solution was removed, counted in a gamma scintillation counter and then returned to the fibrin-Sepharose. Percent lysis was computed as the fraction of total counts released into the supernatant.

Figure 2 compares the fibrinolytic rates by TPA, both with and without pretreatment of fibrin-Sepharose with the heterobifunctional antibody. At each TPA concentration, the data indicate substantially enhanced fibrinolysis in the samples heated with heterobifunctional antibody.

In Figure 3, enhancement of lysis is defined as the ratio maximal of fibrinolysis in the presence and absence of heterobifunctional antibody. It is apparent that the relative potency of TPA-heterobi- functional antibody complex increases as TPA concen¬ tration decreases. This observation may be considered in light of the relative affinities of TPA and anti¬ body for fibrin as discussed above. Anti-fibrin

antibody has a higher affinity for fibrin than does TPA. The heterobifunctional antibody is consequently capable of binding TPA to fibrin with an affinity greater than that which could be effected by TPA alone.

Thus a heterobifunctional antibody composed of a fibrin-specific antibody and an anti-TPA antibody bound to fibrin enhances the fibrinolytic potency of TPA, with increasing efficacy at decreasing TPA. This phenomenon is readily demonstrable in vitro at or below TPA plasma concentrations. Extending these observations in vivo, the treatment of thrombosis may be without the administration of exogenous plasminogen activators. Since the affinity of fibrin-specific antibodies for fibrin is greater than that of TPA, the risk of fibrinogenolysis or of the destruction of other clotting proteins is minimized and thereby the risk of bleeding is likely to be diminished.

EXAMPLE 6

The following example shows the production of two different heterobifunctional antibodies from somatic cell fusion of two hybridoma lines. The resulting cell lines secrete asymetic antibodies (heterobifunc¬ tional antibodies) capable of binding both fibrin and TPA and sharing with the chemically produced product the ability to enhance fibrinolysis. Both heterobi¬ functional antibodies contained a TPA binding site and an additional binding site: one antibody possessed an antibody binding site specific for the amino terminus of fibrin's beta chain (F36.23); the other antibody possessed an antibody binding site specific for the amino terminus of fibrin's alpha chain (F32.1).

ANTI-TPA, ANTI-BETA CHAIN HETEROBIFUNCTIONAL ANTIBODY

Hybridoma line TCL8 produces a monoclonal antibody specific for the catalytically active B chain of TPA. TCL8 cells were treated with 6-thioguanine to select for hypoxanthine phosphoribosyl transferase deficient (HPRT-MINUS) variants, subcloned and then tested for viability in HAT (hypoxanthine, aminopterin, thymi- dine) medium. Hybridoma line 59D8, which produces a monoclonal antibody specific for the beta chain of human fibrin, was grown in bromodeoxy uridine in order to select thymidine kinase deficient subclones that were not viable in HAT medium. The HPRT-MINUS and the thymidine kinase deficient subclones were fused in polyethylene glycol and desired clones selected that were viable in HAT medium. Further subclones were selected by screening with solid phase radioimmuno- assay using a synthetic fibrin-like peptide and TPA as antigens. One cell line called F36.23 possessed both anti-human fibrin and anti-human TPA immunoreactivi- ties. The products of this cell line were then puri¬ fied to remove unproductive chain recombinants by serial affinity chromatography. Antibodies were first fractionated with beta chain peptide-Sepharose column and sequentially absorbed to and eluted from TPA-Seph- arose. The purified products were then characterized by two additional assays that demonstrated their bi- functionality.

A solid-phase immunoradiometric assay was con¬ structed in which fibrin monomer was adsorbed to a plastic surface, the test antibody solution added, followed by 1251 labeled tPA (with intervening washing steps). Figure 4, column A shows evidence of binding

to both fibrin and TPA. In Figure 4, column B, a sim¬ ilar assay was constructed in which TPA was adsorbed to the plate and 1251 labeled D2E (a fragment of fibrin containing that amino terminus of the beta and alpha chains) was the probe. Here too the binding by the test antibody compared favorably to the control. These assays indicate that the purified product of clone F36.23 is a heterobifunctional antibody capable of binding both fibrin and TPA.

ANTI-TPA, ANTI-ALPHA CHAIN HETEROBIFUNCTIONAL ANTIBODY

A second method was used to yield heterobifunc¬ tional antibody, F32.1. The F32.1 cell line was selected after fusing TCL8 (HPRT-MINUS) cells with spleen cells from a mouse immunized with a fibrin-like peptide corresponding to the amino terminus of fibrin alpha-chains. Fusion products were similarly screened using TPA and an alpha-chain peptide as antigens to yield the desired hybridoma which produced both acti¬ vities. The isotype of this monoclonal antibody was Gammal, Kappa.

After affinity chromatography similar to that described for antibody F36.23, except that an amino terminal alpha chain peptide was used instead of the beta chain peptide, a similar immunoradiometric assay was performed. Figure 4, column C, shows the binding of F32.1 when fibrin monomer is bound to the solid phase and 1251 tPA is the probe whereas Figure 4, column D, shows the binding of the same antibody when TPA is bound to the solid phase and 1251 D2E is the probe. These assays also indicate that the purified product of clone F32.1 is also a heterobifunctional antibody capable of binding both fibrin and TPA.

Both antibodies were tested for their ability to enhance fibrinolysis in an assay previously described above and in Bode et al. , Science, 229;765-767 (1985). As is apparent from Figure 5, the potency of TPA in fibrinolysis is enhanced five- to ten-fold by the presence of either F36.23 or F32.1.

It is understood that these descriptions, examples and embodiments are for illustrative purposes only, and that various modifications would be suggested within the spirit and purview of this application and the scope of the appended claims.

PCT Appl icant's Guide - Volume I - Annex M3

ANNEX H3

Intemttionel Application No: PCT/