WO1996017622A1 - Anticorps inhibant la fixation de la thrombine sur les plaquettes - Google Patents

Anticorps inhibant la fixation de la thrombine sur les plaquettes Download PDF

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Publication number
WO1996017622A1
WO1996017622A1 PCT/US1994/013667 US9413667W WO9617622A1 WO 1996017622 A1 WO1996017622 A1 WO 1996017622A1 US 9413667 W US9413667 W US 9413667W WO 9617622 A1 WO9617622 A1 WO 9617622A1
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WIPO (PCT)
Prior art keywords
platelets
antibody
thrombin
binding
patient
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PCT/US1994/013667
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English (en)
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The International Foundation For The Advancement Of Thrombosis And Hemostasis Research, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by The International Foundation For The Advancement Of Thrombosis And Hemostasis Research, Inc. filed Critical The International Foundation For The Advancement Of Thrombosis And Hemostasis Research, Inc.
Priority to PCT/US1994/013667 priority Critical patent/WO1996017622A1/fr
Priority to AU12613/95A priority patent/AU1261395A/en
Priority to GB9711672A priority patent/GB2310857A/en
Publication of WO1996017622A1 publication Critical patent/WO1996017622A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention is related to monoclonal antibodies which interact with platelet cells to inhibit various functions associated with platelet activation.
  • the principal activity demonstrated by the monoclonal antibodies of the present invention is inhibition of the binding of thrombin to the glycoprotein lb on the surface of the platelet cells.
  • thrombin a coagulation serene protease, derived from prothrombin by cleavage by coagulation factor Xa.
  • Thrombin plays a key role in blood coagulation and thrombosis (pathological coagulation) .
  • Thrombin is a central bioregulatory enzyme in hemostasis. It catalyzes the conversion of fibrinogen to fibrin and is responsible for the activation of coagulation factors V, VIII and XIII. Thrombin may also interact with blood vessel walls causing vasoconstriction and may even mediate leukocyte adherence.
  • Thrombin is the most potent physiologic stimulus which induces platelet activation.
  • the reaction of thrombin with platelets represents an unusual agonist- receptor interaction.
  • stimulation of platelets requires the catalytic activity of thrombin, a major protein, the glycoprotein lb (GPIb) , has been isolated on the platelet surface to which thrombin binds.
  • GPIb glycoprotein lb
  • High affinity thrombin binding to GPIb induces platelet activation; this includes platelet shape change, ADP and serotonin secretion, alpha granule release, lysosomal release, the conversion of arachidonic acid to thromboxane A2 and irreversible platelet aggregation.
  • Thromboxane A2 acts as a potent vasoconstrictor and causes aggregation of platelets.
  • thrombin causes platelet activation and aggregation which is resistant to conventional thrombolytic therapy.
  • any agent which could interfere with high affinity thrombin binding to the GPIb on the platelet would be an important tool in treating patients with acute arterial thrombosis and could be efficacious in ameliorating the effects of thrombin in arterial thrombus formation and the resultant resistance of arterial thrombi to thrombolytic therapy.
  • Monoclonal antibodies have been identified which inhibit platelet functions induced by ristocetin or thrombin.
  • TM-60 inhibits binding of von Willebrand factor to platelets in the presence of ristocetin and inhibited the release of adenosine diphosphate (ADP) by thrombin.
  • ADP adenosine diphosphate
  • TM-60 inhibits binding of von Willebrand factor to platelets in the presence of ristocetin and inhibited the release of adenosine diphosphate (ADP) by thrombin.
  • ADP adenosine diphosphate
  • Another monoclonal antibody to human platelet GPIb designated antibody SZ-2, inhibited both ristocetin- and collagen- induced aggregation of platelets (C. Ruan et al., in Monoclonal Antibodies and Blood Platelets. INSERM Symposium No. 27, pp. 59-68, ed. J.L. McGregor, c. 1986 by Elsiver Science Publishers BV.).
  • a third antibody to GPIb is designated VM 16d and blocks thrombin-induced platelet-aggregation at low doses of thrombin, 0.05 u/ml.
  • LJ- IblO is able to inhibit at most only 50% of the total measurable thrombin binding to platelets. Interference of thrombin binding to platelets by LJ-IblO results in decreased fibrinogen binding to platelets and also inhibits the thrombin-mediated calcium flux across the platelet membrane and release of ADP from the cells. The antibody LJ-IblO only partially inhibited thrombin- induced platelet aggregation.
  • the antibody F124H12 (4H12) is similar to the LJ-IblO antibody, but demonstrates advantageous properties over that and the other previously described antibodies to GPIb. 4H12 completely inhibits the high affinity binding of thrombin to GPIb, as shown for LJIb-10. As a result, 4H12 completely inhibits the binding of fibrinogen to platelets that results from thrombin activation of the cells. 4H12 also completely inhibits the other physiologic responses of platelets to thrombin.
  • Platelets incubated with 4H12 prior to thrombin exposure retain their unactivated shape, do not exhibit a flux of calcium across the membrane and do not release ADP, serotonin, lysosomes or alpha granules, do not convert arachidonate to thromboxane, and most importantly, the platelets do not aggregate.
  • the invention is characterized by the example of the monoclonal antibody F124H12, which specifically binds to the oc chain of the GPIb. By means of this interaction, the antibody totally inhibits the binding of thrombin to normal human platelets.
  • the antibody 4H12 has also been used in studies of the role played by thrombin in maintaining the adhesion of platelets to ⁇ ubendothelial surfaces in the vascular system. 4H12, either as intact antibodies, or as Fab or (Fab') 2 fragments, inhibits platelet adhesion to subendothelial surfaces at high shear rates. This inhibition occurs despite the observation that 4H12 does not inhibit ristocetin- or botrocetin-induced binding of von Willebrand factor to platelet cells.
  • this antibody has, by in vitro and ex vivo studies, an excellent potential for being an effective antithrombotic agent by i) inhibiting thrombin binding to platelets, and ii) by inhibiting platelet adhesion to subendothelial surfaces.
  • Figure l shows the time course of binding of human alpha thrombin to platelets purified free of plasma proteins.
  • Figure 2 illustrates a Scatchard analysis of thrombin binding to human platelets purified free of plasma proteins.
  • Figure 3 illustrates the results of a study showing the complete inhibition of high and partial inhibition of moderate affinity thrombin binding to purified platelets by monoclonal antibody 4H12.
  • Figure 4 shows the immunoprecipitation of the platelet membrane glycoprotein GPIb by the antibody 4H12.
  • Figure 5A and 5B shows inhibition of thrombin stimulation of ATP release from platelets and platelet aggregation by antibody 4H12.
  • Figure 5A shows the result of a control experiment using the irrelevant antibody 122C11.
  • Figure 5B shows the result obtained using antibody 4H12. ( , ATP release; , platelet aggregation)
  • Figure 6A-6D show the dose dependence of inhibition of thrombin-induced platelet aggregation by the antibody 4H12 (6A, 0 ⁇ g/ml antibody, 6B, 25 ⁇ g/ml, 6C, 3.35 ⁇ g/ml, 6D, 0.87 ⁇ g/ml)
  • the 4H12 antibody is important in inhibiting the interaction of platelets with thrombin.
  • the interaction of platelets with thrombin result in a platelet shape change, release of alpha granule constituents and the expression of adhesive proteins on the surface, dense body release reaction, platelet aggregation, and platelet adherence to the subendothelial surface; however, the antibody 4H12 inhibits all of these reactions.
  • These reactions are of central importance in the formation of thrombi on areas of diseased blood vessel surface, in areas of thrombin formation or in areas of high shear force such as a partial obstruction of a coronary artery.
  • the antibody 4H12 In vitro, the antibody 4H12 totally interrupts the sequence of events leading to platelet activation that are mediated by thrombin binding and thus can inhibit the effects of thrombin on platelet release and platelet aggregation. This capability has been explored in flowing and static systems.
  • This antibody has the ability to totally inhibit thrombin interaction with platelets and the effects of thrombin on platelets. This presents a major advance in the field of monoclonal antibodies which inhibit platelet function. It has been postulated and proven that thrombin is generated in areas of thrombosis. Thrombin has a high affinity to bind to platelet GPIb and to cause the reactions described above.
  • the antibody 4H12 has the ability to totally inhibit the effects of thrombin on human platelets.
  • the antibody 4H12 displays the following characteristics which have not been demonstrated by previous monoclonal antibodies: i) inhibition of nanomolar concentrations of thrombin to platelets; ii) total inhibition of thrombin-induced platelet aggregation; iii) inhibition of >90% of thrombin-induced Von Willebrand factor or fibrinogen binding to platelets; iv) inhibition of platelet adhesion to the subendothelial layer of arterial walls under shear flow.
  • EXAMPLE I ISOLATION OF HYBRIDOMAS SECRETING ANTIBODIES WHICH INHIBIT THROMBIN BINDING TO PLATELETS
  • mice were immunized with purified human platelets, separated from human plasma on arabinogalactan gradients. After six weeks, spleens were removed and the cells dissociated in culture. The immunized spleen cells were hybridized with mouse myeloma cells (SP2-0-Agl4) and distributed in microtiter dishes in the manner well-known in the art. Cultures which demonstrated proliterative growth were cloned by a limiting dilution method. Two separate immunization and fusion experiments were performed. Thus established hybridoma cell lines were grown in the peritoneal cavity of BALB/c mice and the ascites fluid was obtained therefrom.
  • SP2-0-Agl4 mouse myeloma cells
  • samples of ascites fluid from 250 clones pooled from both fusions were screened by incubating 8 x 10 7 arabinogalactan purified human platelets with 25-50 ⁇ l of ascites fluid in a total volume of 0.4 ml of buffer. To this mixture, 1 x 10 4 cpm of 12S I radiolabelled thrombin were added. The samples were washed to remove unbound thrombin and the amount of radioactivity bound to the cell fraction was quantitated using a gamma counter (TRACOR Analytic) .
  • a gamma counter gamma counter
  • F124H12 (called hereinafter 4H12)
  • F81A11 (called hereinafter 1A11)
  • the hybidoma 4H12 was deposited under the terms and conditions of the Budapest Treaty at the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD on February 7, 1992 and was assigned the accession number HB 10972.
  • Figure 1 shows a time course of the binding of thrombin to arabingalactan purified human platelets. 12i I-labelled thrombin was added to the platelets in a modified HEPES buffer, pH 7.35. Half-maximal binding occurs within one minute after the addition of thrombin to purified platelets and reaches saturation at 20 to 25 minutes after the addition of thrombin.
  • Figure 2 is a Scatchard analysis of binding of thrombin to human platelets. Platelets (8 x 10 7 ) were titrated with increasing amounts of thrombin (0.4 ⁇ M to 1.23 mM) . This analysis demonstrates the existence of two different binding sites for thrombin on the platelet surface.
  • the solid lines represent the binding isotherms calculated by the LIGAND computer program.
  • One asymptote defines a class of binding sites of high affinity and low capacity with approximately 1,000 receptors with a kD of 3 nM.
  • the second asymptote depicts a second class of receptors of low affinity and high capacity.
  • lane 1 is the glycoprotein which is immunoprecipitated in a nonreduced state.
  • This protein has a molecular weight of 172 kD and after reduction using dithiothreitol (lane 2) it has a molecular weight of approximately 143 kD (GPIb ⁇ ) .
  • GPIb ⁇ molecular weight of approximately 143 kD
  • Human platelets were purified on LAREX gradients. The platelets were separated from all other cellular elements and plasma proteins by centrifugation on a discontinuous gradient, 10%-20% LAREX. The platelets were washed once and then suspended in phosphate buffered saline (PBS) . The platelets were diluted in buffer to a count of 2 x 10 5 cells/ ⁇ l. 400 ⁇ l of purified platelets were placed in a test tube to which varying concentrations of monoclonal antibody was added. The monoclonal antibody concentration varied from 0.05 ⁇ g/ml to 50 ⁇ g/ml. The antibody and platelets were incubated for 5 minutes, at which time thrombin was added to a concentration of 0.01 U/ml.
  • PBS phosphate buffered saline
  • the aggregation curve was observed for at least 7 minutes at 37°C.
  • PBS was added in place of the monoclonal antibody solution.
  • the turbidity value observed in the control reaction was considered to be 100% aggregation.
  • a sample containing PBS in place of monoclonal antibody was incubated at 37°C for 7 minutes, followed by the addition of thrombin.
  • the value obtained in this "blank" experiment was considered 0% aggregation.
  • the reduction in aggregation attributable to the incubation with the antibody is defined as the percentage of reduction in aggregation compared to the no antibody sample.
  • Figure 5 illustrates the effect of antibody 4H12 on the thrombin-induced aggregation of platelets and also shows that 4H12 inhibits ATP release from platelets upon stimulation by thrombin.
  • Figure 5A results using an irrelevant, control antibody 122C11, are shown.
  • Figure 5B illustrates the same experiment performed using the antibody 4H12. The total inhibition of both ATP release and platelet aggregation by antibody 4H12 is clearly evident from comparison of the two figures.
  • Figure 6 illustrates the dose-dependence of the inhibition of platelet aggregation by antibody 4H12. Aggregation of 8 x 10 7 platelets stimulated by 0.1 unit of thrombin was assayed as described above using various amounts of the antibody. Total inhibition of platelet aggregation was observed at the lowest antibody dose tested, 0.87 ⁇ g/ml.
  • the IC 50 for thrombin-stimulated platelet aggregation was found to be 1.8 ⁇ M for antibody 4H12. In a similar manner, the IC 50 for thrombin-stimulated platelet aggregation for antibody lall was found to be 1.7 ⁇ M.
  • the antibody 4H12 at a concentration of 5 ⁇ g/ml, inhibits 31% of platelet adhesion to the endothelial cell layer. Similarly, inhibition of 51% of the adhesion was seen at 10 ⁇ g/ml, 56% at 20 ⁇ g/ml and 65% at 40 ⁇ g/ml. Of great importance, no platelet thrombi were formed on the subendothelial surface in the presence of the antibody.
  • F(ab) ' 2 fragments of the 4H12 antibody are similarly displayed by F(ab) ' 2 fragments of the 4H12 antibody.
  • the IC 50 for inhibition of thrombin and ristocetin-induced platelet aggregation by F(ab) ' 2 fragments of antibody 4H12 are 1.5 ⁇ M and >20 ⁇ M, respectively.
  • F(ab) ' 2 fragments of antibodies are made by limited proteolysis of the antibody using methods well known in the art.
  • the monoclonal antibodies of the present invention can be formulated into pharmaceutical compositions by use of any of the various additives commonly employed in the art. Typical of such additives are carriers and excipients, diluents and the like.
  • the antibodies can be formulated by dilution in a sterile saline solution for administration by injection.
  • Administration of the antibodies of the present invention can be performed by the routes typical in the art.
  • intravenous injection would be an effective route of administration.
  • Dosages to be employed would be expected to be typical for administration of monoclonal antibodies to a patient, in the range of 0.05 to 20 mg/kg as a unit dose.
  • the preparation of monoclonal antibodies for administration by intravenous injection, as well as by many other routes, is considered well-known in the art.

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Abstract

L'invention concerne des anticorps monoclonaux se fixant spécifiquement sur la glycoprotéine Ib. Ces anticorps inhibent complètement la fixation de la thrombine sur les plaquettes, inhibant ainsi totalement l'activation des plaquettes par la thrombine. Les anticorps inhibent également complètement l'agrégation plaquettaire et inhibent d'autre part notablement l'adhésion des plaquettes à une surface artérielle sous-endothéliale dans un système de modèles ex vivo.
PCT/US1994/013667 1994-12-05 1994-12-05 Anticorps inhibant la fixation de la thrombine sur les plaquettes WO1996017622A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/US1994/013667 WO1996017622A1 (fr) 1994-12-05 1994-12-05 Anticorps inhibant la fixation de la thrombine sur les plaquettes
AU12613/95A AU1261395A (en) 1994-12-05 1994-12-05 Antibodies which inhibit the binding of thrombin to platelets
GB9711672A GB2310857A (en) 1994-12-05 1994-12-05 Antibodies which inhibit the binding of thrombin to platelets

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Application Number Priority Date Filing Date Title
PCT/US1994/013667 WO1996017622A1 (fr) 1994-12-05 1994-12-05 Anticorps inhibant la fixation de la thrombine sur les plaquettes

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7112661B1 (en) 1998-10-30 2006-09-26 The Research Foundation Of State University Of New York Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein Ib alpha
EP2186829A1 (fr) * 2008-11-14 2010-05-19 Canadian Blood Services Anticorps contre GPIbalpha
CN107531797A (zh) * 2016-02-05 2018-01-02 江苏恒瑞医药股份有限公司 凝血酶抗体、其抗原结合片段及医药用途
CN108341874A (zh) * 2018-03-08 2018-07-31 华东理工大学 靶向小鼠血小板膜糖蛋白GPIbα的肿瘤转移抑制剂及其鉴定方法

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BLOOD, Volume 70, Number 5, issued 1987, PARKER et al., "Fibrin Monomer Induces Binding of Endogenous Platelet Von Willebrand Factor to the Glycocalicin Portion of Platelet Glycoprotein IB", pages 1589-1594. *
BLOOD, Volume 71, Number 5, issued 1988, GEORGE et al., "Thrombin Decreases Von Willebrand Factor Binding to Platelet Glycoprotein IB", pages 1253-1259. *
BLOOD, Volume 83, Number 12, issued 1994, MICHELSON et al., "The Activation-induced Decrease in the Platelet Surface Expression of the Glycoprotein Ib-IX Complex is Reversible", pages 3562-3573. *
BLOOD, Volume 84, Number 1, issued 1994, LAROSA et al., "Neutrophil Cathepsin G Modulates the Platelet Surface Expression of the Glycoprotein (GP) Ib-IX Complex by Proteolysis of the Von Willebrand Factor Binding Site on GPIb-alpha and by a Cytoskeletal-mediated Redistribution of the Remainder of the Complex", pages 158-168. *
THROMBIN HAEMOSTASIS, Volume 67, Number 6, issued 1992, PENG et al., "Characterization of Three Alboaggregins Purified from Trimeresurus-Albolabris Venom", pages 702-707. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7112661B1 (en) 1998-10-30 2006-09-26 The Research Foundation Of State University Of New York Variable heavy chain and variable light chain regions of antibodies to human platelet glycoprotein Ib alpha
EP2186829A1 (fr) * 2008-11-14 2010-05-19 Canadian Blood Services Anticorps contre GPIbalpha
US8323652B2 (en) 2008-11-14 2012-12-04 Canadian Blood Services Antibodies against GPIbα
CN107531797A (zh) * 2016-02-05 2018-01-02 江苏恒瑞医药股份有限公司 凝血酶抗体、其抗原结合片段及医药用途
CN107531797B (zh) * 2016-02-05 2021-07-02 江苏恒瑞医药股份有限公司 凝血酶抗体、其抗原结合片段及医药用途
CN108341874A (zh) * 2018-03-08 2018-07-31 华东理工大学 靶向小鼠血小板膜糖蛋白GPIbα的肿瘤转移抑制剂及其鉴定方法

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GB9711672D0 (en) 1997-08-06
GB2310857A (en) 1997-09-10
AU1261395A (en) 1996-06-26

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