EP0449879A1

EP0449879A1 - Heterobifunctional antibodies having specificity for platelets and thrombolytic agents

Info

Publication number: EP0449879A1
Application number: EP19900900579
Authority: EP
Inventors: Chien-Hsing Chang; Peter E. Daddona; Donald S. Neblock
Original assignee: Centocor Inc
Current assignee: Janssen Biotech Inc
Priority date: 1988-11-25
Filing date: 1989-11-22
Publication date: 1991-10-09
Also published as: WO1990006133A1

Abstract

Des anticorps hétérobifonctionnels se lient à des plaquettes et à un agent thrombolytique. Ces anticorps hétérobifonctionnels sont utiles en thérapie thrombolytique.Heterobifunctional antibodies bind to platelets and a thrombolytic agent. These heterobifunctional antibodies are useful in thrombolytic therapy.

Description

HETEROBIFU CTIONAL ANTIBODIES HAVING SPECIFICITY FOR PLATELETS AND THROMBOLYTIC AGENTS

Background of the Invention

Heterobifunctional (hereinafter "HBF") antibodies are antibodies or aggregates of

-antibodies which are specific for two different antigens. HBF antibodies can comprise a single antibody or fragment having a bispecific antigen binding region (two different variable regions) or aggregates of two or more antibodies of different specificities.

Different methods of preparing hybrid antibodies have been reported. Auditore-Hargreaves teaches processes for preparing hybrid antibodies by generating "half-molecules" from two parent antibodies and subsequently associating different half molecules. See U.S. Patents 4,470,925 (1984) and 4,479,895 (1984). Various hybrid antibodies were prepared with specificities for horseradish peroxidase, glucose oxidase, digoxin or theophylline.

Reading describes the production of antibodies having binding specificites for two desired antigens using a quadroma cell or a trio a cell. See U.S. Patent 4,474,893 (1984). A quadroma cell is the fusion product of two different hybrido a cells each of which produce an antibody with a different specificity. A trioma cell is the fusion product of a hybridoma and a lymphocyte which produces antibodies with two different binding specificities. Segal et a_l. describe target specific cross- linked heteroantibodies which are used as cytotoxic agents in U.S. Patent 4,676,980 (1987). Staerz et al. (1986) PNAS, j3_:1453-57 teach the use of a hybrid antibody that, can focus effector T cell activity and Milstein ^t al. , (1983) Nature, 305;537-539 describe the use of hybrid antibodies in immunohistochemistry.

Platelet aggregation is an essential event in the formation of blood clots. Blood clots serve to prevent the escape of blood cells from the vascular system under normal circumstances. During certain disease states (e.g., myocardial infarction), however, clots can restrict or totally occlude blood flow resulting in cellular necrosis.

Heart attack patients are typically treated with thrombolytic agents such as tissue plasminogen activator (tPA) or εtreptokinase. Thrombolytic agents dissolve the fibrin component of clots. A major complication associated*with fibrinolysis is reoccl sion based on platelet aggregation which can result in further heart damage.

Glycoprotein (gp)IIb/IIIa receptors on the surface of platelets are known to be responsible for platelet aggregation. Reagents which block these receptors such as receptor-specific antibodies are expected to reduce or prevent reocclusion following thrombolytic therapy and to accelerate the rate of thrombolysis. An example is the urine monoclonal antibody 7E3 which inhibits platelet aggregation. See European Patent Application Nos. 205,207 and 206,532. Su marv of the Invention

This invention pertains to a heterobifunctional antibody (HBF antibody) having a first specificity for a platelet and a second specificity for a thrombolytic agent. The HBF antibody is useful in thrombolytic therapy. The HBF antibody binds to platelets and blocks platelet aggregation while simultaneously recruiting endogenous or pharmalogi- cally-administered thrombolytic agent and it may increase the efficiency of thro bolysis by specifically targeting a thrombolytic agent to a thrombus. The HBF antibody of this invention is formed by combining antigen specificities from at least two parental antibodies, one parent having specificity for a platelet component, preferably a component involved in platelet aggregation such as platelet glycoprotein Ilb/IIIa receptor complex, and the other parent having non-neutralizing specificity for a thrombolytic agent such as tissue plasminogen activator (tPA) , streptokinase, or urokinase. The antigen binding regions of the parental antibodies can be joined chemically. Alternativly, the HBF antibodies can be produced using cell fusion techniques or the HBF antibody can be produced using recombinant DNA techniques.

Brief Description of the Figures

Figure 1 is a photograph of SDS-PAGE analysis of a 25G5x7E3 (anti-platelet x anti-tPA) HBF antibody. The parental F(ab')₂ molecules were reduced, derivatized, and crosslinked, resulting in the appearance of a F(ab')₂ sized band which was not present in either of the reactant Fab' molecules. Figure 2 is a photograph of the F(ab')₂ sized band on non-reducing SDS-PAGE after a two-stage purification process of a P4B6x7E3 (anti-platelet x anti-tPA) HBF antibody. Figure 3 is a photograph which shows that a non-disulfide, covalent bond is present in the 25G5x7E3 (anti-platelet x anti-tPA) HBF antibody by the appearance of a Mr 50-60 kD band on SDS-PAGE in the presence of dithiothreitol. This band is absent in the fully reduced parental antibodies.

Figure 4 is a chromatogram of the analysis of a 25G5x7E3 (anti-platelet x anti-tPA) HBF antibody by gel filtration HPLC, showing the HBF antibody as a single major peak of approximately 115 kD when compared to molecular weight calibrators.

Figure 5 is a chromatogram of the 25G5x7E3 (anti-platelet x anti-tPA) HBF reaction mixture on HIC-HPLC which contains four major peaks. The 25G5x7E3 (anti-platelet x anti-tPA) peak has a retention time of about 43 minutes which differs significantly from the parental F(ab')₂ molecules. Figure 6 (a-c) are graphs showing the results of an immunoassay of a 25G5x7E3 (anti-platelet x anti tPA) HBF antibody detecting the immunoreactivity of both the anti-platelet and anti-tPA specificities.

Detailed Description of the Invention

The HBF antibodies of this invention have combined specificities for platelets and for a thrombolytic agent. The preferred platelet speci¬ ficity of the HBF antibody is for the glycoprotein Ilb/IIIa receptor complex (GP Ilb/IIIa) . The GPIIb/IIIa specificity is preferably provided by the monoclonal antibody 7E3. See Coller, B.S., J. Clin. Invest. 96:101 (1985) and European Patent Applications Nos. 205,207 and 206,532. Antibodies for other platelet antigens can also be employed. For example, antibodies which are reactive with the platelet membrane protein GMP-140 such as the S12 antibody (McEver et al. , J. Biol. Chem. 259:9799 (1984) ) can be used. Binding specificity for a thrombolytic agent is derived from an antibody specific for a thrombolytic agent. The antibody should bind the thrombolytic agent at an epitope such that it does not neutralize or significantly abrogate its activity. The pre- ferred binding specificity is for the thrombolytic agent human tPA, but antibodies with a binding specificity for other thrombolytic agents such as streptokinase and urokinase can also be used.

Antibodies specific for the thrombolytic agent (Holovet et al. , Eur. J. Biochem, 158:173 (1986);

Takada et al. , Throm. Res., _42_:63 (1986) or platelet components (Coller et al. , J. Clin. Invest. , 76:101 (1985)) can be selected from those presently available or can be specially prepared. The anti- bodies can be obtained using conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization techniques of Kohler and Mil- stein, Nature 256:495 (1975). The preferred throm¬ bolytic agent specificity is provided by monoclonal antibodies 25G5 or P4B6, Holovet et al. ; Takada et al. , cited supra, both specific towards tPA. Other possible antibodies which are specific towards tPA include 35F11, 2D7, 31F12, 2D6, 11C5, 13D5, and P4C5F12. Examples of platelet specific antibodies include 7Ξ3, 10E5,and S12, the preferred platelet specific antibody being 7E3. See copending appli¬ cation Serial No. 07/195,720, filed May 18, 1988, the contents of which are incorporated by reference. The HBF antibodies are produced by joining together variable regions of two or more parental antibodies using chemical, cell fusion, or genetic engineering techniques. In a preferred embodiment for chemically joining the variable regions of the parental antibodies, the parental antibodies are cleaved, preferably proteolytically, yielding F(ab-),, fragments of the parental antibodies. The fragments are subsequently subjected to conditions under which the disulfide linkages in the hinge region are selectively reduced yielding Fab' molecules from each parental antibody. One set of parental Fab¹ molecules is maintained in the free sulfhydryl form (Fab'-SH) while the second set of parental Fab' molecules is covalently modified with a chemical σrosslinker. Any sulfhydryl-specific crosslinker capable of crosslinking Fab¹ molecules can be used. The preferred crosslinking agents are those having two maleimide groups such as bis-maleimidomethyl ether (BMME) or o-phenylenedimaleimide. Procedures for crosslinking using these agents are known in the art. See Chang et al. , J. Nucl. Med. , 27:1041 (1986) , and Glennie et al. , J. Immunol. , 139: 2367 (1987) . The derivatized Fab' molecules from one parental antibody are mixed with the Fab'-SH molecules from the second parental antibody under conditions which allow c.ovalent coupling of the two molecules to occur. The resulting product is a heterobifunctional F(ab')₂ molecule (HBF antibody) possessing both of the parental specificities.

The parental antibodies can be selected from any isotypic class of antibodies but it is preferred that the parental antibodies be matched isotypi- cally. When available, the parental antibodies used to produce the HBF antibody can be of chimeric human o^'rigin. Normally, however, they will be murine antibodies.

The HBF antibodies can be purified using any purification process capable of separating HBF antibodies from undesired antibodies or antibody fragments. The preferred purification process is a two step purification process. In the first step, the lower molecular weight contaminants are removed using gel filtration chromatography. Examples of gels which are useful in this first step are dextran crosslinked with epichlorohydrin, allyl dextran crosslinked with N,N'- methylene bisacrylamide, or agarose. The preferred gel is allyl dextran crosslinked with N,N' -methylene bisacrylamide. The second purification step is conducted using hydrophobic interaction chromatography (HIC) , preferably using high performance liquid chromatography (HPLC) , on a phenyl-derivatized packing. In this second purification step, gradient conditions are created using two buffer solutions which may vary depending on the given set of isotype-matched parental antibodies. Examples of possible buffer solutions used as the decreasing gradient (Buffer A) are ammonium sulfate, ammonium acetate, and halide salts such as sodium or potassium chloride. The HIC is capable of separating molecules based on their hydrophobicity.

In another embodiment for chemically joining two or more parental antibodies, whole parental antibodies or antibody fragments can be linked together to produce a heteroaggregate, the HBF antibody. A variety of crosslinking agents, such as protein A, carbodiimide, and N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP) , can be used to link the parental antibodies or antibody fragments. See Kranz et al. , Proc. Natl. Acad. Sci. USA, 7_8:5807 (1981); U.S. Patent 4,474,893.

The HBF antibodies can also be produced using cell fusion techniques as described in U.S. Patent 4,474,893, to Reading. In this technique, hybridoma cells which secrete the parental antibodies are fused together to form quadroma or trioma cells. These quadroma and trioma cells secrete bispecific antibodies possessing the antigen binding regions of both parental antibodies.

The HBF antibodies can also be produced using genetic engineering techniques. In these procedures, DNA encoding the heavy and light chain variable regions of each of the parental antibodies are introduced into an appropriate host cell, preferably a lymphoid cell (e.g., a myeloma cell) . The transformed cell synthesizes, assembles, and secretes the heterobifunctional immunoglobulin proteins. After the HBF antibodies are produced using any of the above techniques, the HBF antibodies are characterized and assayed for both immunoreac- tivities. A preferred assay is a solid phase im unoassay wherein a positive signal results only if both immunoreactivities (platelet specificity and thrombolytic specificity) are present. The platelet component antigen is coated onto a solid phase and used as the capture antigen for the platelet specific HBF antibody. Test solutions containing the synthetic HBF antibodies or appropriate controls (parental antibodies alone or in combination) are incubated in with the solid phase. Subsequently, the solid phase is incubated in the presence of a radiolabelled thrombolytic agent. Specific binding of the thrombolytic agent by HBF antibodies is thereby determined.

The effect of the HBF antibodies of this invention on platelet aggregation can be tested. A sample is prepared in such a way that induces aggregation. Collagen can be introduced into a sample of whole blood to induce aggregation. Subsequently, an HBF antibody can be added to the sample containing collagen induced aggregated platelets while a control sample can be prepared in the same manner omitting the HBF antibody. The amount or degree of aggregation in both samples can be measured using an aggregometer (Helena Laboratories) .

The HBF antibodies of this invention can be used to block platelet aggregation while recruiting an endogenous or pharmalogically administered throm¬ bolytic agent to the site of a thrombus. The HBF antibody may be administered simultaneously with or subsequent to administration of a thrombolytic agent. The HBF antibody has thrombus inhibiting activity and it can also increase the efficiency of thro bolytic agents by attracting them to the thrombi.

The invention is illustrated further by the following examples.

Example 1

Crosslinking Technology

The crosslinking technology used to create the heterobifunctional antibodies of this example is that of the first embodiment of chemically joining the antigen binding regions of the parental antibodies as taught by Chang et al. J. Nucl. Med. , 27, 1041, (1986) and Glennie et al. , J. Immunol. , 139, 2367, (1987). Parental IgGl murine monoclonal antibodies (one specific for human platelet receptor glycoprotein GPIIb/IIIa and the other specific for human tissue-type plasminogen activator) were cleaved with pepsin yielding F(ab')_ fragments which were subsequently subjected to cysteine reduction in order to reduce selectively only the hinge region disulfide linkages while leaving the heavy-light and i trachain bonds intact.

Reduction was accomplished by concentrating 10 mg of each parental F(ab')₂ to 20 rtιg/ml in 50 mM sodium borate, 100 mM NaCl (pH 8.0). 1M L-cysteine (free base) in 10 mM sodium phosphate, 150 mM NaCl (pH 7.2) was added to each of the F(ab')₂ to yield a final concentration of 20 mM cysteine. Reduction was allowed to proceed at 37°C for 1 hour. The resulting Fab¹ was derivatized with BMI-1E. A 50 mM BMME stock was prepared in dimethylformamide, and the 7E3 Fab' solution was added over vortexing to enough 50 mM BMME to provide a 30:1 ratio of BMME to Fab¹. This mixture was allowed to react at room temperature for 10 minutes and was then desalted as above. The desalted BMME-derivatized 7E3 Fab' was

05 mixed with the anti-tPA Fab' and allowed to react at room temperature for 60 minutes. At that time, unreacted sulfhydryl groups were reacted with 5,5 '-dithiobis (N,N*) nitrobenzoic acid (DTNB) at a final concentration of 1 mM. The resulting product

10 was a heterobifunctional F(ab')₂ molecule possessing both of the parental functions (i.e., specificity for human platelet receptor glycoprotein and specificity for human tissue type plasminogen activator) .

15 Parental Antibody Selection

A murine monoclonal IgGl designated 7E3 has been characterized (see Coller, B.S., J. Clin. Invest, 96, 101, (1985)) which recognizes a conformational determinant formed by the

20 Ca -mediated association of the human platelet receptor glycoprotein GPIIb/IIIa. This antibody has been shown to inhibit platelet aggregation in vitro by inhibiting the binding of fibrinogen to GPIIb/IIIa (See, Coller, B.S., cited supra) and has

25 been shown to inhibit platelet function in vivo in a dog model when administered as F(ab*)₂ fragments (See, Coller et al. , Blood, 66, 1456, (1986)) . Several murine antibodies directed against human tPA have been purified and have been shown to have tPA n r_* fl —T

³⁰ binding affinities of at least a Ka of 10 M .

These IgGl antibodies have been tested for their ability to neutralize tPA activity in a standard chromogenic-substrate amidolysis assay in order to screen for antibodies which were non-neutralizing as candidates for the anti-tPA partner in the synthetic HBF antibody.

Purification of Heterobifunctional Antibodies

The chemical crosslinking reaction may result in several unwanted by-products as well as the desired HBF antibodies. These by-products could be either one of the parental F(ab')₂ species reformed through BMME coupling or through oxidative reformation of hinge-region disulfides, unreacted parental Fab' molecules, or other unrelated by-products. Thus, it was necessary to develop a purification method capable of separating the HBF antibodies from the parental F(ab')₂ and intermediate species. A two-stage purification method was developed which first resolves the lower molecular weight contaminants using gel filtration followed by high performance liquid chromatography (HPLC) using hydrophobic interaction chromatography (HIC) on a phenyl-derivatized packing. The HIC is capable of idiotype-specific separations.

The HIC is performed on a 7.5x75 mm column of Biorad TSK phenyi 5-PW equilibrated in 100 mM Na phosphate (pH 6.3) containing 1 M ( H₄)₂ SO.

(Buffer A) . The gel filtration pool is separated in this system using a linear gradient of 0-100% of Buffer B at a flow of 1 ml/min. Buffer B contains 100 mM Na phosphate at pH 6.3. The linear gradient is run from 5 to 55 minutes after 5 minutes of isocratic elution in Buffer A. Buffer B is held at 100% from 55 to 70 minutes. The gradient conditions may vary for a given set of isotype-matched parental antibodies.

Characterization by SDS-PAGE and Gel Filtration HPLC Two anti-platelet/anti-tPA HBF antibodies were synthesized. In each HBF, the anti-platelet partner was derived from the antibody 7E3, while the anti-TPA partner was derived from antibody 25G5 or antibody P4B6. This resulted into two different HBF antibodies (25G5 x 7E3; P4B6 x 7E3) sharing common properties. In both HBF antibodies, the

BMME-derivatized partner was 7E3 and the anti-tPA partner was reacted as the F(ab') fragment containing free sulfhydryls in the hinge region. However, a HBF antibody could have been produced using the anti-tPA partners as the BMME-derivatized partner and the 7E3 fragment containing free sulfhydryls in the hinge region.

Figure 1 is a photograph showing the SDS-PAGE analysis of a typical sequence of processing of the parental F(ab')₂ molecules through reduction, derivatization, and crosslinking, resulting in the appearance of a F(ab')₂ sized band which was not present in either of the reactant molecules. Figure 2 is a photograph showing the the F(ab')₂ sized band after the two-stage purification process on non-reducing SDS-PAGE. Figure 3 demonstrates that a non-disulfide, covalent crosslink is present in the HBF by the appearance of a Mr 50-60 kD band in SDS-PAGE in the presence of dithiothreitol. This band is absent in either of the fully reduced parental antibodies. Figure 4 shows the analysis of the 25G5x7E3 HBF antibody under native conditions by gel filtration HPLC which results in a single major peak with a Mr of approximately 115kD as determined by comparison to molecular weight calibrators.

Heterobifunctional Characterization Using Hydrophobic Interaction HPLC

The parental F(ab')₂ molecules used to create the HBF antibodies were determined to be baseline resolved in the HIC system. Thus, the presence of a peak with a retention time intermediate between the two parental F(ab')₂ molecules and which is characterized as predominantly F(ab') by SDS-PAGE is taken as evidence that a species comprised of half of each parental F(ab')₂ is present. Figure 5 shows that the 25G5x7E3 (anti-platelet x anti tPA) HBF reaction mixture chromatogram on HIC-HPLC had 4 major peaks. The HBF antibody was the third peak with a retention time of 43 minutes. The F(ab')₂ or Fab' molecules chromatographed with retention times significantly different from the HBF peak.

Anti-GPIIbllla x an i-tPA Heterobifunctional Immunoassay

The product HBF antibodies have been characterized in an immunoassay specifically designed to test for the presence of both parental immunoreactivities (platelet specificity and TPA specificity) . A positive signal results only if both immunoreactivities are present. The platelet GPIIb/IIIa receptor is coated on flexible polyvinyl chloride 96-well plates and used as the capture antigen for the 7E3 partner of the HBF antibody. Test solutions containing the synthetic HBF antibodies or appropriate controls (parental F(ab')₂ species alone and in combination) are incubated in the wells and subsequently incubated in the presence

125 of [ I] labelled tPA. Specific binding of the HBF molecules is observed and the radioactivity incorporated is detected by cutting out the washed wells and counting in a gamma counter. Figure 6 shows specific binding of the HBF antibodies to the antigen and that no signal above background is obtained in control wells containing either parent alone or in combination. The anti-tPA and anti-GPIIb/IIIa E1A data (Figure 6, bottom) show that the parental immunoreactivities are fully retained by the HBF molecule as compared to parental F(ab') in separate immunoasεays.

Inhibition of Platelet Aggregation

As demonstrated by Table 1 , the HBF antibody (25G5 x 7E3) showed dose-dependent inhibition of human platelet aggregation similar to that observed for parental 7E3 F(ab')₂ or Fab' indicating that this parental function was retained by the HBF. The slope of the line representative of aggregation is inversely proportional to the rate of platelet aggregation in this assay (i.e. as the slope of the line decreases, the effectiveness of the HBF antibody as a platelet aggregation inhibitor^' increases) . A preliminary result demonstrated that the HBF (25G5 x 7E3) was more effective than 7E3 Fab' alone in preventing platelet aggregation when exogenous tPA was added to the whole blood. These results suggest a possible synergism due to the combination of the HBF antibody and tPA.

Table I Human Platelet Aggregation Inhibition

Slo e

None 34

SUBSTITUTESHEET Eσuivalents

Those skilled in the art will know, or be able to ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.

These and all other equivalents are intended to be encompassed by the following claims.

Claims

1. A heterobifunctional antibody having a first specificity for a platelet and a second speci¬ ficity for a thrombolytic agent.

2. A heterobifunctional antibody of Claim 1, wherein the first specificity is for platelet glycoprotein Ilb/IIIa receptor complex.

3. A heterobifunctional antibody of Claim 2, wherein the first specificity is derived from the monoclonal antibody 7E3.

4. A heterobifunctional antibody of Claim 1, wherein the second specificity is for tissue plasminogen activator, streptokinase or uro- kinase.

5. A heterobifunctional antibody of claim 4, wherein the second specificity is for tissue plasminogen activator.

6. A heterobifunctional antibody of claim 5, wherein the second specificity is derived from the monoclonal antibody 25G5 or P4B6.

7. A heterobifunctional antibody comprising an antigen binding region of a first antibody specific for platelets linked to an antigen binding region of a second antibody specific for a thrombolytic agent.

8. A heterobifunctional antibody of Claim 7, wherein the antigen binding region of each antibody is an Fab' fragment.

9. ^' A heterobifunctional antibody of Claim 4, wherein the Fab' fragments are chemically crosslinked.

10. A heterobifunctional antibody of claim 9, wherein the Fab' fragments are chemically crosslinked using bis-maleimidomethyl ether.

ll. A heterobifunctional antibody of Claim 7, wherein the first antibody is specific for platelet glycoprotein to Ilb/IIIa receptor and the second antibody is specific for tissue plasminogen activator.

12. A heterobifunctional antibody comprising two chemically crosslinked Fab' antibody fragments of the same isotype, the first Fab¹ fragment being specific for platelet glycoprotein Ilb/IIIa receptor complex and the Fab' fragment being specific for tissue plasminogen ac¬ tivator.

13. A method of thrombolytic therapy, comprising administering to a patient having a thrombus or at risk of thrombus, a heterobifunctional antibody having the first specificity for platelet and a second specificity for a throm¬ bolytic agent.

14. A method of Claim 12, wherein the heterobifunc¬ tional antibody is administered in conjunction with the thrombolytic agent.

15. A method of Claim 13, wherein the heterobifunc- tional antibody is administered along with, or subsequent to, administration of the thrombo¬ lytic agent.

16. A method of Claim 14, wherein the first speci- ficty of the heterobifunctional antibody is for platelet glycoprotein Ilb/IIIa receptor comple .

17. A method of Claim 15, wherein the first speci¬ ficity of the heterobifunctional antibody is derived from the monoclonal antibody 7E3.

18. A method of Claim 14, wherein the second specificity of the heterd ifunctional antibody is for tissue plasminogen activator, strep- tokinase or urokinase.

19. A method of Claim 14, wherein the second specificity is derived from the monoclonal antibody 25G5 or P4B6.

20. A method of thrombotic therapy, comprising administering to a patient having a thrombus or at risk of thrombus formation, a heterobifunc- tional antibody comprising two chemically crosslinked Fab¹ antibody fragments of the same isotype, the first Fab' fragment being specific for platelet glycoprotein Ilb/IIIa receptor complex and the Fab' fragment being specific for tissue plasminogen activator.

21. A method of Claim 18, wherein the heterobifunc¬ tional antibody is administered in conjunction with tissue plasminogen activator.

22. A method of Claim 20, wherein the heterobifunc¬ tional antibody is administered along with, or subsequent to, administration of the thrombo¬ lytic agent.

23. A method of Claim 20, wherein the Fab' antibody fragments are chemically crosslinked using bis-maleimidyomethyl ether.

24. A pharmaceutical composition comprising a thrombolytically effective amount of a heterobifunctional antibody and a pharmaceutically acceptable vehicle.

25. A method of separating heterobifunctional antibodies from a mixture of heterobifunctional antibodies and other antibodies or antibody fragments, comprising: separating the heterobifunctional antibodies from the mixture according to molecular weight, separating the heterobifunctional antibodies from the mixture according to hydrophobicit .

26. A method of separating heterobifunctional antibodies according to Claim 25, wherein the heterobifunctional antibodies are separated from the mixture according to molecular weight using gel filtration chromatography.

27. A method of separating heterobifunctional antibodies according to Claim 25, wherein the heterobifunctional antibodies are separated from the mixture according to hydrophobicity using high pressure liquid chromatography on a phenyl derivatized packing.