EP0447489A1

EP0447489A1 - Human platelet-specific antibodies

Info

Publication number: EP0447489A1
Application number: EP19900901321
Authority: EP
Inventors: Herbert Lazarus; Barry S. Coller
Original assignee: Centocor Inc
Current assignee: Janssen Biotech Inc
Priority date: 1988-12-01
Filing date: 1989-11-29
Publication date: 1991-09-25
Also published as: WO1990006134A1

Abstract

On décrit des fragments humains d'immunoglobulines et des fragments humains d'immunoglobulines monoclonales qui sont spécifiques contre les plaquettes de sang. Lesdites immunoglobulines, ou fragments de celles-ci, sont de préférence spécifiques contre le récepteur de glycoprotéine IIb/IIIa dans sa forme complexée. Ces anticorps bloquent la liaison de ligand au récepteur, empêchant ainsi l'agrégation plaquettaire impliquée dans la formation de thrombi. Ces immunoglobulines sont utiles dans la thérapie anti-thrombotique,s eules ou en combinaison avec des agents thrombolytiques, ainsi que dans l'imagerie scintigraphique de thrombi.Human fragments of immunoglobulins and human fragments of monoclonal immunoglobulins that are specific against blood platelets are described. Said immunoglobulins, or fragments thereof, are preferably specific against the glycoprotein IIb / IIIa receptor in its complexed form. These antibodies block the ligand binding to the receptor, thus preventing the platelet aggregation involved in the formation of thrombi. These immunoglobulins are useful in anti-thrombotic therapy, alone or in combination with thrombolytic agents, as well as in scintigraphic imaging of thrombi.

Description

HUMAN PLATELET-SPECIFIC ANTIBODES Description Bachground of the Invention

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

Heart attack patients are typically treated with thrombolytic agents such as tissue plasminogen activator or streptokinase, which dissolve the fibrin component of clots. A major complication associated with fibrinolysis is reocclusion based on platelet aggregation which can result in further heart damage. Since glycoprotein

Ilb/IIIa (GPIIb/IIIa) receptors are known to be responsible for platelet aggregation, reagents that block these receptors are expected to reduce or prevent reocclusion following thrombolytic therapy and to accelerate the rate of thrombolysis.

One approach to blocking platelet aggregation involves monoclonal antibodies specific for GPIIb/IIIa receptors. A rαurine monoclonal antibody, designated 7E3, that inhibits platelet aggregation and appears useful in the treatment of human thrombotic diseases is described in published European Patent Application Nos. 205,207 and 206,532. Murine antibodies have characteristics that may severely limit their use in human therapy. They are foreign proteins, which may elicit immune reactions that reduce or destroy their therapeutic efficacy and/or evoke allergic or hypersensitivity reactions in patients. The need for readministration of such therapeutic modalities in thromboembolic disorders increases the likelihood of these types of immune reactions.

Summary of the Invention

This invention pertains to human platelet-specific monoclonal antibodies. The antibodies are specific for the GPIIb/IIIa receptor, or other platelet components. These antibodies bind to platelets, and can block platelet aggregation, and thus, are useful as antithrombotic agents, and to prevent or reduce reocclusion following thrombolysis. Human platelet-specific antibodies minimize some of the problems often associated with the immunogenicity of antibodies composed of nonhuman protein.

Detailed Description of the Invention

The present invention relates to human platelet- specific monoclonal antibodies. The antibodies are comprised entirely of human protein. These antibodies target platelet components, such as the GPIIb/IIIa receptor. The antibodies bind to platelets and thereby prevent platelet aggregation and thrombus formation.

The human antibodies invention are specific for platelet surface components. Preferred are specific for platelet GP Ilb/IIIa receptors; they bind to the

GPIIb/IIIa receptor and block ligand binding to the GPIIb/IIIa receptor complex. The preferred antibodies are specific for the complexed form of GPIIb/IIIa receptor. However, antibodies can be also specific for either the GPIIb or GPIIIa components. Alternatively, antibodies specific for other platelet antigens can also be employed. For example, human antibodies that bind to platelet granule membrane protein GMP-140 can be used.

In general, platelet- specific antibodies can be prepared by obtaining lymphoid cells from an individual who produces antibody against a platelet antigen. The lymphoid cells are fused to immortalizing cells to produce continuous hybrid cell lines. Hybrid cells producing antibody against the desired platelet antigen are selected and cloned.

In a preferred embodiment, human monoclonal antibody specific for GPIIb/IIIa can be prepared as follows.

Since GPIIb/IIIa is normally on all human platelets, humans are 'tolerant' to this heterodimer and do not mount an antibody response to it. Certain rare individuals (e.g. individuals with Glanzmann's throm- basthenia) do not express this complex on their platelets. Individuals who lack GPIIb/IIIa may mount an antibody response when exposed to GPIIb/IIIa. Such exposure would be likely to occur during the course of transfusions that include blood platelets. Transfusion might be employed for a variety of medically justified reasons. In the case of Glanzmann patients it would be used to treat hemorrhages caused by the inability of their defective (i.e. GPIIb/IIIa lacking) platelets to aggregate properly. Following transfusions such individuals would be expected to respond immuno- logically to the GPIIb/IIIa complex just as they would to any 'foreign' protein. They would mount a B cell

response with the appearance of specific antibody in their blood and with an amplification of antigen-specific B cells. A hybridorαa capable of secreting human monoclonal antibody specific to the human GPIIb/III complex can be made from B cells from such patients.

An individual is identified who has a serum antibody titer to GPIIb/IIIa. Such an individual might be one who lacks the heterodimer as in Glanzmann's thrombasthenia. This individual after having been exposed to normal

(GPIIb/IIIa-containing) platelets as a result of a

transfusion would be expected to develop an antibody response. Alternatively, a normal individual might be exposed to GPIIb/IIIa in an immunogenic fashion. This might take the form of repeated transfusions wherein some of the material might become partially denatured and hence more immunogenic or it might occur through the binding of a drug or other substance to the platelets causing modification of surface molecules and eliciting an antibody response. Additionally, individuals with autoimmune disease, such as idiopathic thrombocytopenic purpura might be suitable sources of antigen specific B lymphocytes.

B lymphocytes are obtained from the individual in the form of, for example, spleen, lymph nodes or peripheral blood obtained by venipuncture or pheresis. The lymphoid cells can be enriched by use of a one step gradient such as Ficoll-Hypaque. The recovered cells can be washed to thoroughly remove the gradient material which may be toxic. Unwanted or undesirable cell populations such as suppressor cells (CD8 ) or B cells making an unwanted isotype such as IgM are removed. This may be accomplished by complement mediated lysis, cell sorting using flow cytometry or affinity purification such as 'panning'.

Prior to fusion, the B cells can be stimulated with antigen, lymphokines and/or other mitogenic substances or substances that will induce the B cells to synthesize and secrete antibody.

The appropriately stimulated cells are then fused using polyethylene glycol or other fusogenic agents or devices. The fusion partner is a cell or hybrid of B cell lineage capable of supporting the synthesis and secretion of human antibodies.

Generally, the immortalizing cell line is a tumor cell, which endows the hybridoma with the ability to grow permanently in culture. This ensures a stable culture of antibody-producing hybridoma cells which can produce monoclonal antibodies in a continuous supply. The immortalizing cell may be a plasmacytoma cell, such as a myeloma cell. The myeloma cell can be human, non-human, or a heteromyeloma. Suitable human immortalizing cell lines include the HMMA2.11, HF2 cell line, and the U-266. A heteromyeloma is a myeloma hybrid formed by the fusion of cells of two different species. See Oestberg, U.S. Patent 4,634,664.

The cell fusions are accomplished by standard procedures. See, Kohler and Milstein, Natrure (London),256:495-497 (1975); Olsson and Kaplan, Proc. Natl. Acad.Sci USA 77:5429 (1980). The hybridomas are then screened for production of antibodies reactive with platelets or platelet component such as the GPIIb/IIIa receptor. The screening can be accomplished by an enzyme immunoassay. For example, purified GPIIb/IIIa can be bound to a solid phase. The solid phase can then be contacted with hybridoma supernatant and antibody binding to the GPIIb/IIIa- solid phase can be evaluated with enzyme-conjugated anti-human antibody. Hybridomas that secrete reactive antibodies are cloned.

Another method of forming the antibody-producing cells is by viral or oncogenic transformation. For example, human B-lymphocyte which produced a plate- specific antibody may be infected and transformed with a virus, such as the Epstein-Barr virus, to give an immortal antibody-producing cell. See, e.g., Kozbor and Roder (1983) Immunolpgy Today, 4(3) : 72-79. Or, the

B-lymphocyte may be transformed by a transforming gene or gene product.

Monoclonal antibodies are generally produced In large quantities by culturing hybridomas that produce anti-platelet antibody in vitro and isolating the secreted monoclonal antibodies from the cell culture medium.

The human platelet-specific antibodies of this invention are useful as antithrombotic therapeutic agents. The antibodies (or fragments thereof) can be used to inhibit platelet aggregation and thrombus formation. The antibodies can be used in any situation where thrombus formation or reformation is to be prevented. For example, the antibody alone can be used to prevent clotting in post- angioplas ty treatment, pulmonary embolism, deep vein thrombosis and coronary bypass surgery. The antibody can also be administered in conjunction which a thrombolytic agent, such as t i s sue plasminogen activator, streptokinase, single chain streptokinase, acyl-plasminogen-streptokinase activator complex, urokinase or the mutant variants of tissue plasminogen activator, streptokinase and urokinase, to prevent or reduce reocculusion that can occur after thrombolysis, and to accelerate clot lysis. The antibody or fragment can be administered before, along with, or subsequent to administration of the thrombolytic agent, in an amount sufficient to prevent platelet aggregation, which can result in reocclusion. The antibody is given parenterally, preferably intravenously, in a pharma- ceutically acceptable vehicle such as sterile saline. The antibody could be given multiple times or by a controlled release mechanism (e.g., by a polymer or patch delivery system).

During repeat therapy with anti-platelet antibodies drug-induced thrombocytopenia may occur; this may be a result of the body recognizing the antibody-coated platelets as foreign proteins, raising antibodies against them and then clearing them more rapidly than normal. The use of a human anti -platelet antibody may avoid this problem.

The platelet-specific human antibody of this invention is also useful for thrombus imaging. For this purpose, antibody fragments are generally preferred.

Antibody fragments such as Fab, Fab' and F(ab')2 can be produced by standard procedures. The fragments can be labeled directly, or through a coupled chelating agent such as diethylenetr iaminepentaacetic acid, with radio- isotopes such as ^{1 3 1} Iodine , ¹²⁵ I odine , ^{9 9m} Technetium or

Indium to produce radioimmunoscintigraphic agents. The radiolabeled antibody is administered to a patient suspected of having thrombus. After sufficient time to allow the labeled immunoglobulin to localize at the thrombus site, the signal generated by the label is detected by a photoscanning device such as a gamma camera. The detected signal is then converted to an image of the thrombus. The image makes it possible to locate the thrombus in vivo and to devise an appropriate therapeutic strategy.

The invention is further illustrated by the following examples. EXEMPLIFICATION

A donor was identified who had been diagnosed as having Glanzmann's thrombas thenia. She had no detectable

GPIIb/IIIa on her platelets. She had a transfusion history of over 100 blood transfusions. She had a demonstrable IgG anti-GPIIb/IIIa titer as determined by

EIA using purified GPIIb/IIIa as the solid phase.

The donor was lymphopheresed using a Fenwal CS3000

Blood Cell Separator. The cells were collected in an acid-citrate dextrose anticoagulant. A total of 125ml containing 1.6x10⁹ cells were obtained. A white cell differential analysis showed 93% lymphocytes. The cells were diluted 1:3 in Hanks' Balanced Salt Solution (HBSS) and layered over Ficoll-Paque. The cells recovered from the interface were washed two times in HBSS. Recovery by Coulter count was 1.5x10⁹. The cells were pooled into 4 groups (which were processed separately throughout the remainder of the experiment) and each pool was placed in a 75cm² tissue culture flask which had been pre-coated with 50μg/ml of goat anti-human IgM and 50μg/ml of mouse monoclonal anti-CD8 antibodies for 2hrs at 4°C. The cells were allowed to adhere to the antibody coated plates for 45 minutes with occasional gentle agitation at 4ºC. At the end of this 'panning' step 1.0x10⁹ cells (Coulter count) were recovered by gently removing the non-adherent cells. The cells were washed with HBSS and each group was seeded at 2x10⁶ cells/ml in 30ml in 75cm² tissue culture flasks (4 flasks/group, a total of 16 flasks). Each 500ml of medium ('modified' alpha MEM) was supplemented with Eagle's nonessential amino acids (100x, 5ml), sodium pyruvate (100mM, 5ml), glutamine (200mM, 5ml), fetal bovine serum (100ml), gentamycin (50mg/ml; 2.5ml), Pokeweed Mitogen (Gibco, 0.25ml), and GPIIb/IIIa adsorbed to fumed silica (final concentration of

GPIIb/IIIa, lμg/ml). The cells were incubated for 4 days at 37°C in a humidified atmosphere of 5% CO₂ in air.

On the fourth day, the stimulated lymphocytes were fused with the human myeloma analogue HMMA 2.11tg/o. The lymphocytes in each group were mixed with an equivalent number (1x10⁹) of HMMA cells. The cells were washed 2 times in HBSS, pH 7.8 and the resulting pellets were very slowly resuspended in 1.5ml of polyethylene glycol (PEG)

(46% w/v in HBSS pH 7.8, m.w. 8,000) over a period of 3 minutes with constant agitation. The fused cells were then allowed to remain in the PEG for an additional one minute. The cells were then slowly resuspended in HBSS containing 5% fetal bovine serum. Ten ml were added over a period of 3 minutes with constant agitation. Another 10ml were added in the next 1 minute. The cells were then centrifuged and resuspended in 'modified' alpha MEM supplemented as above except that Pokeweek Mitogen and silica adsorbed GPIIb/IIIa were omitted and HAT (hypoxanthine, aminoipterin, thymidine) was added. Each fusion was then distributed in 75cm² flasks (5),

50ml/flask, 1x10⁶ hybrid equivalents/flask. The cells were incubated for 48 hours at 37°C in a humidified atmosphere of 5% CO₂ in air. They were then redistributed into 100 flat bottom 96 well tissue culture plates and further incubated until they were ready to be screened for antibody production.

Initial screening consisted of identifying those hybrids which secreted IgG antibodies which bound to human platelet derived GPIIb/IIIa. Purified GPIIb/IIIa was allowed to adhere to 96 well polystyrene EIA plates (Costar) at a concentration of 2μg/ml overnight at 4°C. The plates were washed, blocked with 3% bovine serum albumin-1% normal goat serum for one hour, supernate was added and incubated for one hour, washed and then incubated with goat anti-human IgG conjugated to horseradish peroxidase, incubated for one hour and then developed with o-phenylenediamine. From the four fusions two hybrids were obtained which secreted antibodies detectable by this assay. The two positive hybrids designated Gimmel 51F11 and Gimmel 51G10 were subcultured, cloned and cryopreserved in liquid nitrogen. Equivalents

Those skilled in the art will recognized, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A human monoclonal platelet-specific antibody.

2. A human monoclonal antibody of Claim 1 that

specifically binds the glycoprotein Ilb/IIIa receptor complex of platelets.

3. An antigen-binding fragment of the human monoclonal antibody of Claim 1.

4. A radiolabeled antigen binding fragment of Claim 3.

5. A human monoclonal platelet- specific antibody

fragment Fab, Fab' or F(ab')₂.

6. A human, monoclonal antibody fragment of Claim 5 which is radiolabeled.

7. A human monoclonal antibody of Claim 6 wherein the radiolabel is selected from the group consisting of 99m_TC 111_In, 125_{τ and} 131_l.

8. A hybridoma that produces human monoclonal anti- platelet antibody, the hybridoma produced by the fusion of a human lymphocyte from an individual immunized against platelets and a human lymphoblastoid cell.

9. A hybridoma of Claim 8 wherein the lymphocyte is obtained from an individual immunized against the gp Ilb/IIIa receptor.

10. A hybridoma of Claim 8, wherein the lymphocyte is a peripheral blood lymphocyte.

11. A method of producing a human platelet- specific

monoclonal antibody, comprising:

a. obtaining lymphoid cells from an individual who produces antibody against a platelet antigen; b. fusing the lymphoid cells with an immobilizing cell to produce a hybrid cell; and

c. selecting and cloning hybrid cells that produce antibody against the platelet antigen.

12. A method of Claim 11, wherein the platelet antigen is GPIIb/IIIa.

13. A method of Claim 12, wherein the individual has

Glanzmann's thrombasthenia.

14. A method of antithrombotic therapy, comprising

administering to a patient having a thrombus, or at risk of thrombus formation, an anti-thrombotic amount of a human monoclonal antibody or antibody fragments specific for blood platelets.

15. A method of Claim 14 wherein the antibody or fragments are specific for the GPIIb/IIIa receptor.

16. A method of Claim 15 wherein the antibody fragment is an Fab, Fab' or F(ab')₂ fragment.

17. A method of antithrombotic therapy, comprising

administering to a patient having a thrombus or at risk for thrombus formation, a thrombolytic agent and a human monoclonal antibody or antibody fragment specific for platelets.

18. A method of Claim 17, wherein the human monoclonal antibody or antibody fragment is administered along with or subsequent to administration of the thrombolytic agent.

19. A method of Claim 17, wherein the thrombolytic agent is tissue plasminogen activator, streptokinase, single chain streptokinase, acyl-plasminogen- streptokinase activator complex, urokinase or the mutant variants of tissue plasminogen activator, streptokinase and urokinase.

20. A method of Claim 17, wherein the human monoclonal antibody or fragment is specific for glycoprotein Ilb/IIIa.

21. A method of Claim 17, wherein the antibody fragment is an Fab, Fab' or F(ab')₂ fragment.

22. A method of thrombus imaging, comprising:

a. administering to an individual suspected of

having a thrombus, a radiolabeled human platelet-specific human antibody or fragment thereof;

b. allowing the antibody or antibody- fragment to accumulate at a thrombus site;

c. detecting the signal generated by the radio- label by means of a photo scanning device; and d. converting the detected signal to an image of the thrombus.

23 A method of Claim 22, wherein a Fab, Fab' or F(ab') fragment is administered.

24. A method of Claim 22, wherein the radiolabel is

selected from the group consisting of ^99mTc, ¹¹¹In,

¹²⁵l and ¹³¹l.

25. A method of Claim 22, wherein the platelet-specific antibody or fragment is specific for the glycoprotein Ilb/IIIa receptor.