Therapeutic use of a monoclonal mouse igg antibody directed against human glycoprotein ib

Abstract

The present invention relates to a monoclonal mouse IgG antibody against human glycoprotein Ib (GPIb) for therapeutic use for inhibiting a GPIb-mediated activation of platelets, the monoclonal mouse IgG antibody comprising the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR 3 region of the variable domain of the heavy chain.

Description

[0001] 202400478 DESCRIPTION Antiplatelet Drug TECHNICAL FIELD The present invention relates to a monoclonal mouse IgG antibody against human glycoprotein Ib (GPIb) for therapeutic use in inhibiting GPIb-mediated platelet activation, wherein the monoclonal mouse IgG antibody comprises the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR3 region of the variable domain of the heavy chain. BACKGROUND Platelet aggregation is the clumping of platelets, which leads to primary (cellular) hemostasis and the closure of injured blood vessels. Platelet aggregation is activated by external signals. Typically, platelets circulating in the blood come into direct contact with cell walls that are not endothelial and are covered with von Willebrand factor (VWF).Platelets bind to this factor via a specific receptor consisting of a glycoprotein complex that includes glycoprotein Ib (GPIb). GPIb is a surface protein on platelets and is involved in blood coagulation. This protein is a component of the GPIb-V-IX complex on platelets. The GPIb-V-IX complex binds von Willebrand factor (VWF), thereby enabling platelet adhesion and the formation of platelet plugs at sites of vascular injury. GPIb slows down passing platelets and brings them into close contact with the exposed subendothelium or inflamed endothelium. According to current understanding, following a vascular injury, platelets roll onto the subendothelium via the interaction between the GPIb-V-IX integrin and the high-molecular-weight VWF of the subendothelium.The platelets are subsequently stabilized by binding to VWF and the collagen receptors GPIaIIa and GPVI via a second receptor, GPIIbIIIa. Downstream signal transduction pathways then release thromboxane A2, which further activates platelets autocrinely and paracrinely (Jourdi et al., 2021, Int. J. Mol. Sci, 22, 13079). This activation results, among other things, in a conformational change of the membrane-bound fibrinogen receptor integrin αIIbβ3, leading to easier binding of fibrinogen. The subsequent increased binding to fibrinogen leads, among other things, to strong cross-linking with other platelets. Following this, coagulation factors such as activated factor X and factor V bind to the platelet surface and catalyze the massive production of thrombin by converting prothrombin.This initiates secondary hemostasis, whereby thrombin, in addition to the strong activation of the platelet receptors PAR1 and PAR4, causes the cleavage of extracellular fibrinogen to fibrin, leading to the formation of a fiber network and the stabilization of blood clots. Platelet aggregation inhibition is a crucial approach to preventing abnormal platelet activation in pathophysiological conditions such as myocardial infarction, ischemia, or stroke. Typically, aspirin, which selectively inhibits the enzyme cyclooxygenase-1, is still used as the preferred platelet aggregation inhibitor in clinical practice, as many alternative platelet aggregation inhibitors are unsuitable for use in patients due to various safety, efficacy, and tolerability issues.Unforeseen cases of increased bleeding risk and recurrent arterial thrombosis have hampered the development of next-generation platelet aggregation inhibitors. Newer platelet aggregation inhibitors, designed to address some of these shortcomings, target enzymes (e.g., phosphodiesterase), receptors (e.g., prostaglandin receptor, thromboxane receptor), and glycoproteins (e.g., αIIbβ3, GPVI, von Willebrand factor (VWF), or GPIb) in or on platelets. The aim is to prevent unwanted clot formation after initial platelet activation (Yeung and Hoinstat, 2012, Journal of Blood Medicine, 3, 33-42). Inhibition of the GPIb receptor by binding inhibitory ligands is an attractive option for antithrombotic therapy. Jourdi et al., 2021 reports that in addition to the snake venom anfibatide, two antibodies (SZ2 and h6B4-Fab) are being investigated as direct GPIbα inhibitors.In addition to its binding site for VWF, the GPIb receptor has a variety of interaction sites for ligands. These ligands include thrombin, protein C, Mac-I, P-selectin, and thrombospondin (Clementson and Clementson, 2008, Thromb Haemost, 99, 473-479). This allows for the inhibition of non-VWF-dependent functionality of the GPIb receptor and thus its antithrombotic effect. However, only a few therapeutically unused antibodies have been described that are active in this interaction domain. For example, the antibody VM16d has been shown to inhibit thrombin-induced platelet aggregation at low concentrations (Mazurov et al., 1991, Thrombosis Research, 62, 673-684). Furthermore, the antibody 4H12 was described, which at nanomolar concentrations inhibits the binding of thrombin to platelets and does not inhibit ristocetin-induced binding of VWF to platelets (US 5,486,361).The present invention is therefore based on the objective of identifying and therapeutically utilizing GPIb ligands, i.e., antibodies, which on the one hand do not impair VWF binding, but on the other hand inhibit non-VWF-dependent and GPIb-mediated platelet activation, since such ligands can inhibit prothrombotic processes without leading to increased bleeding tendencies. BRIEF SUMMARY OF THE INVENTION This objective is achieved by the subject matter of the independent claims. Further advantageous aspects of the invention are reflected in the dependent claims.The invention relates initially to a monoclonal mouse IgG antibody against human glycoprotein Ib (GPIb) for therapeutic use in inhibiting GPIb-mediated platelet activation, wherein the monoclonal mouse IgG antibody comprises the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR3 region of the variable domain of the heavy chain. The therapeutic use of the antibody according to the invention advantageously allows for moderate inhibition of platelets. In contrast to the VM16d and 4H12 antibodies from the prior art, the antibody according to the invention hardly inhibits thrombin binding; that is, the inhibition of thrombin-mediated platelet activation via GPIb binding is largely absent. The antibody according to the invention thus exhibits properties that differ from those mentioned above.The antibody according to the invention differs advantageously from prior art anti-GPIb antibodies. The therapeutic use of the antibody leads to moderate inhibition of primary hemostasis (demonstrated by a significant prolongation of the in vitro bleeding time, no inhibition of VWF binding, and very little or no inhibition of thrombin binding) without causing excessive bleeding tendencies. Furthermore, the antibody according to the invention binds gain-of-function variants of GPIb, which in turn potentially exhibit structural properties that are only generated in vivo by shear stress and VWF binding in the GPIb molecule. In a preferred embodiment for therapeutic use, the monoclonal mouse IgG antibody comprises, in the variable domain of the heavy chain, a CDR 1 region of a sequence with SEQ ID No. 1, a CDR 2 region of a sequence with SEQ ID No. 2, and a CDR 3 region of a sequence with SEQ ID No. 3.In a further preferred embodiment of therapeutic use, the monoclonal mouse IgG antibody comprises in the variable domain of the light chain a CDR 1 region of a sequence with SEQ ID NO. 4, a CDR 2 region of a sequence with SEQ ID NO. 5, and a CDR 3 region of a sequence with SEQ ID NO. 6. In another preferred embodiment of therapeutic use, the monoclonal mouse IgG antibody comprises a variable domain of the heavy chain of the sequence SEQ ID NO. 7. In a further preferred embodiment of therapeutic use, the monoclonal mouse IgG antibody comprises a variable domain of the light chain of the sequence SEQ ID NO. 8. In a further preferred embodiment of the therapeutic use of the monoclonal mouse IgG antibody, the inhibition of GPIb-mediated activation of platelets does not inhibit the binding of von Willebrand factor (VWF) to GPIb.In a further preferred embodiment of the therapeutic use of the monoclonal mouse IgG antibody, the inhibition of GPIb-mediated platelet activation is accompanied, at most, by a weak inhibition of thrombin binding to GPIb. In a further preferred embodiment of the therapeutic use of the monoclonal mouse IgG antibody, the weak inhibition of thrombin binding to GPIb comprises an inhibition of the original binding by approximately 1% to 20%, preferably by no more than 10%. In a further embodiment of the therapeutic use of the monoclonal mouse IgG antibody, the inhibition of GPIb-mediated platelet activation is accompanied by no additional inhibition of thrombin binding to GPIb.In another embodiment of the therapeutic use of the mouse monoclonal IgG antibody, the therapeutic use includes the treatment or prevention of diseases associated with blood clots or thrombosis. In particularly preferred embodiments, these diseases include arterial thrombosis, chronic venous insufficiency, venous thromboembolism, pulmonary embolism, visual disturbances, myocardial infarction, ischemia, or stroke. BRIEF DESCRIPTION OF THE FIGURES Figure 1 shows the inhibition of thrombin binding to GPIb by anti-GPIb antibodies. The measured extinction (1) is shown on the ordinate, while the concentration of thrombin in µg / ml is plotted on the abscissa (2). The antibodies 100 / 05 (3), VM16d (4), and 4H12 (5) were measured. Figure 2 shows the prolongation of in vitro bleeding time by anti-GPIb antibodies that do not block the binding of VWF.The ordinate shows the closure time in a Platelet Function Analyzer (PFA) test (6), while the abscissa shows the concentration in the sample in µg / ml (7). The antibodies 100 / 05 (5), 4H12 (3), and VM16d (4) were measured. DETAILED DESCRIPTION OF EMBODIMENTS Although the present invention is described with respect to certain embodiments, this description is not to be construed as limiting. Before exemplary embodiments of the present invention are described in detail, definitions important for understanding the present invention are given. 202400478 8 As used in this description and in the appended claims, the singular forms of "a" and "an" also include the respective plural forms unless the context clearly indicates otherwise.In the context of the present invention, the terms "approximately" and "about" denote an accuracy range that a person skilled in the art will understand in order to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the stated numerical value of ±20%, preferably ±15%, more preferably ±10%, and even more preferably ±5%. It is understood that the term "comprising" is not restrictive. For the purposes of the present invention, the term "consisting of" or "essentially consisting of" is considered a preferred embodiment of the term "comprising." Where, in the following, a group is defined as comprising at least a certain number of embodiments, this shall also include a group that preferably consists only of those embodiments. Furthermore, the terms "(i)", "(ii)", "(iii)" or "(a)", "(b)", "(c)", "(d)" or "first", "second", "third", etc.and the like in the description or in the claims to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It is understood that the terms used in this way are interchangeable under suitable circumstances and that the embodiments of the invention described herein may be used in a different order than described here. If the terms refer to steps of a method, procedure, or use, there is no temporal or time-interval coherence between the steps; that is, the steps may be performed simultaneously, or there may be time intervals of seconds, minutes, hours, days, weeks, etc., between such steps, unless otherwise specified. It is understood that this invention is not limited to the specific methods, protocols, etc., described herein, as these may vary.It is understood that the terminology used herein serves only to describe certain embodiments and is not intended to limit the scope of the present invention, which is limited only by the appended claims. The drawings are to be regarded as schematic representations, and elements depicted in the drawings are not necessarily drawn to scale. Rather, the various elements are presented in such a way as to make their function and general purpose obvious to a person skilled in the art. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as they would normally be understood by a person skilled in the art. Regardless of the grammatical gender of a given term, persons of male, female, or other gender identities are included.202400478 10 As stated above, the present invention relates to a monoclonal mouse IgG antibody against human glycoprotein Ib (GPIb) for therapeutic use in inhibiting GPIb-mediated platelet activation, wherein the monoclonal mouse IgG antibody has the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR 3 region of the variable domain of the heavy chain. The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active parts of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that binds an antigen in an immune-specific manner. The immunoglobulin molecules according to the invention are preferably of the IgG type. They may, for example, belong to the class IgG1, IgG2, IgG3, or IgG4. It is particularly preferred that the antibody be a mouse antibody.An "antibody," as understood according to the present invention, is typically a protein with at least one or two variable regions of the heavy chain (abbreviated VH) and at least one or two variable regions of the light chain (abbreviated VL). The VH and VL regions can be further subdivided into regions of hypervariability, referred to as "complementarity-determining regions" (abbreviated CDR), and more conserved regions, referred to as "framework regions" (abbreviated FR). Antibodies generally comprise six complementarity-determining regions (CDRs); three of these are located in the variable region of the heavy chain (VH): CDRH1, CDRH2, and CDRH3, and three in the variable region of the light chain (VL): CDRL1, CDRL2, and CDRL3. The six CDRs define the paratope of the antibody, that is, the part of the antibody that binds to the target antigen.The VH region and the VL region consist of the scaffold regions (FR1, FR2, FR3, and FR4) on either side of each CDR, which form a scaffold for the CDRs to express the CDRs on the surface of the VH and VL regions. From the N-terminus to the C-terminus, the VH regions have the following structure: N-terminus-[FR1]-[CDRH1]-[FR2]-[CDRH2]-[FR3]-[CDRH3]-[FR4]-C-terminus; and VL regions comprise the following structure: N-terminus-[FR1]-[CDRL1]-[FR2]-[CDRL2]-[FR3]-[CDRL3]-[FR4]-C-terminus. There are various conventions for defining antibody CDRs and FRs, e.g., B. Kabat et al., 1991, Sequences of Proteins of Immunological Interest, or Chothia et al., 1987, J. Mol. Biol. 196:901-917. Further information is known to those skilled in the art or can be obtained from suitable literature sources such as http: / / www.bioinf.org.uk / abs / info.html#cdrid (Prof. Andrew CR Martin's group at UCL bioinf.org.uk) or the IMGT database (Brochet et al., 2008, Nucl. Acids Res.).36, W503-508). A "monoclonal" antibody as described herein is an antibody produced by a single cell line or clone, where this cell line or clone is derived from a single B lymphocyte and is directed against a single epitope. Monoclonal antibodies with defined specificity are typically produced using the hybridoma technology developed by Köhler and Milstein (Köhler and Milstein, 1976, Eur. J. Immunol., 6: 511-519). In this process, mice are immunized with a target protein or a cell expressing such a protein. Once an immune response is detected, e.g., when antibodies specific to the antigen are found in the mouse serum, the mouse spleen is removed and splenocytes are isolated. The splenocytes are then fused with suitable myeloma cells, e.g., B. with cells of the SP20 cell line. The hybridomas are selected and cloned by limited dilution.The hybridoma clones are then examined using established methods for cells that secrete antibodies capable of binding the target protein. The term "human glycoprotein Ib" refers to a human glycoprotein receptor on the surface of the platelet membrane, consisting of a heterodimer comprising an alpha and a beta chain, both chains linked by disulfide bonds. The alpha chain is encoded by the GP1BA gene and is further described under Uniport No. P07359. The beta chain is encoded by the GP1BB gene and is further described under Uniport No. P13224. "GPIb-mediated platelet activation" as described herein refers to platelet aggregation in which GPIb activates platelets via interactions with binding ligands. It is preferred that this activation occurs independently of an interaction between GPIb and von Willebrand factor (VWF).Von Willebrand factor, or VWF, is a large multimeric plasma glycoprotein crucial for maintaining hemostasis. It acts as both a carrier of antihemophilic factor and a platelet-vessel mediator in the blood coagulation system, primarily mediating the binding and adhesion of circulating platelets to sites of vascular injury. Mutations in this gene or deficiencies in this protein lead to von Willebrand disease (VWD). VWF is typically expressed by endothelial cells and megakaryocytes. It is synthesized as 250-kDa monomers, which undergo intracellular processing, glycosylation, multimerization, and propeptide removal, ultimately resulting in the formation of mature VWF multimers. The VWF monomer is a protein consisting of 2050 amino acids. Each monomer contains a number of specific domains with a particular function. The monomer contains, among other things...a D' / D3 domain that binds to coagulation factor VIII; and an A1 domain that binds to the platelet GPIb receptor, heparin, and possibly collagen. A canonical human version of VWF isoform 1 is deposited with UniProt under deposit number P04275-1. According to the present invention, the mouse IgG antibody comprises at least the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR3 region of the variable domain of the heavy chain (CDRH3). The term "consensus sequence," as used herein, refers to the sequence of amino acids which, in total, deviates least from a given set of corresponding pattern sequences. The amino acid sequence YVM is thus found in the CDRH3 region of all antibodies according to the present invention. The presence of this sequence ensures that the antibody binds to the target antigen.In preferred embodiments, the monoclonal mouse IgG antibody comprises in the variable domain of the heavy chain a CDR 1 region of a sequence with SEQ ID NO. 1, a CDR 2 region of a sequence with SEQ ID NO. 2, and a CDR 3 region of a sequence with SEQ ID NO. 3. These sequences are shown in Table 1 below: 202400478 14 Table 1: SEQ ID Sequence (N-Term to C-Term) Note NO: 1 DYGVN CDR1 heavy chain (CDR-H1) 2 MIWVDGSTDYNSALKS CDR2 heavy chain (CDR-H2) 3 DTMIKGHYVMDY CDR3 heavy chain (CDR-H3) In a further preferred embodiment, the monoclonal mouse IgG antibody comprises in the variable domain of the light chain a CDR 1 region of a sequence with SEQ ID NO. 4, a CDR 2 region of a sequence with SEQ ID NO. 5 and a CDR 3 region of a sequence with SEQ ID NO. 6.These sequences are shown in Table 2 below: Table 2: SEQ ID Sequence (N-Term to C-Term) Note NO: 4 GANANIYGALN CDR1 light chain (CDR-L1) 5 GATNLAD CDR2 light chain (CDR-L2) 6 QSVLTTPYT CDR3 light chain (CDR-L3) In another embodiment, the mouse monoclonal IgG antibody comprises in the variable domain of the heavy chain a CDR 1 region of a sequence with SEQ ID NO. 1, a CDR 2 region of a sequence with SEQ ID NO. 2 and a CDR 3 region of a sequence with SEQ ID NO. 3 and in the variable domain of the light chain a CDR 1 region of a sequence with SEQ ID NO. 4, a CDR 2 region of a sequence with SEQ ID NO. 5 and a CDR 3 region of a sequence with SEQ ID NO. 6. In a further preferred embodiment, the monoclonal mouse IgG antibody comprises a variable domain of the heavy chain of the sequence SEQ ID NO. 7.This sequence is shown in Table 3 below: Table 3: SEQ ID Sequence (N-Term to C-Term) Note NO: 7 QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVNW Variable domain VRQPPGKGLEWLGMIWVDGSTDYNSALKSRLSISKD heavy chain NSKSQVFLKMNSLQTDDTARYYCARDTMIKGHYVMD (VH); CDR YWGQGTSVTVSS Regions are underlined. In a further preferred embodiment, the monoclonal mouse IgG antibody comprises a variable domain of the light chain of the sequence SEQ ID NO. 8. This sequence is shown in Table 4 below: Table 4: SEQ ID Sequence (N-Term to C-Term) Note NO: 8 DIQMTQSPVSLSASVGETVTITCGANANIYGALNWY Variable domain QRKQGKSPQLLIYGATNLADGMSSRFSGSGSGRQFS light chain LKISGLHPDDVATYSCQSVLTTPYTFGGGTKLEIK (VL); CDR regions are underlined 202400478 16 In another embodiment, the mouse monoclonal IgG antibody comprises a variable heavy chain domain of sequence SEQ ID NO. 7 and a variable light chain domain of sequence SEQ ID NO. 8.According to the present invention, in the therapeutic use of the antibody described herein, the inhibition of GPIb-mediated platelet activation does not involve inhibition of von Willebrand factor (VWF) binding to GPIb. The term "inhibition of GPIb-mediated platelet activation," as used herein, refers to the prevention or, in certain embodiments, reduction of an interaction between GPIb and platelets that leads to platelet activation. GPIb has a multitude of ligand interaction sites, which, upon activation or binding of a ligand, can directly or indirectly lead to platelet activation, for example, of thrombin. One of the main ligands of GPIb is VWF, which typically leads to platelet activation. This binding of VWF to GPIb is not inhibited according to the therapeutic use of the monoclonal mouse IgG antibody.The monoclonal mouse IgG antibody against human glycoprotein Ib for therapeutic use according to the present invention advantageously does not bind to the VWF binding site of GPIb and thus does not inhibit the binding of VWF to GPIb. Tests for determining such binding inhibition or non-inhibition are known to those skilled in the art or can be found in the general literature. Additional information can be found in the examples. 202400478 17 In a preferred embodiment, the inhibition of GPIb-mediated platelet activation also results in at most a weak inhibition of thrombin binding to GPIb. Thrombin is a serine protease whose functional spectrum includes, among other things, the cleavage of fibrinogen to fibrin and fibrinopeptides. Thrombin is activated from the inactive form prothrombin via a cascade of activators, i.e., coagulation factors, and by prothrombinase.A canonical human version of prothrombin is deposited with UniProt under deposit number Q69EZ8. A canonical human version of thrombin is deposited with UniProt under deposit number P00734. Furthermore, thrombin is involved in platelet activation. The binding site for thrombin on GPIb plays a key role in the release of negatively charged phospholipids on the platelet surface and the formation of further thrombin. Platelet activation via thrombin includes, among other things, the activation of the PAR-1 receptor. It is assumed that platelet activation via thrombin occurs independently of activation via VWF.Within the scope of the present invention, "weak inhibition" of thrombin binding to GPIb is understood as a reduction of the original binding of thrombin to GPIb by a factor of less than 25%, preferably less than 20%, and more preferably in the range of 1% to 20%. In further embodiments, weak inhibition is an inhibition of the original binding of thrombin to GPIb of at most 10%. The term "original binding" as used herein refers to binding under physiological conditions in which the binding partners are present in their native form. A test of the binding or a measurement of the inhibition can be carried out using methods known to those skilled in the art in the field of sandwich ELISA development. Further information can be obtained from the general technical literature. Additional details can be found in the examples.In particular embodiments of the present invention, the inhibition of GPIb-mediated platelet activation does not additionally inhibit the binding of thrombin to GPIb. Without committing to a specific theory, it is assumed that the inventive approach leads to the binding of the antibody described herein to regions or epitopes of GPIb, thereby inhibiting or impairing the interaction of GPIb with additional ligands (which differ from VWF and thrombin) and thus potentially resulting in a prolonged closure time. In a further embodiment, the monoclonal mouse IgG antibody as described herein is used for the treatment or prevention of diseases associated with the formation of blood clots or thromboses.The inhibition of GPIb-mediated activation of platelets by an antibody as described herein leads, according to the invention, to an antithrombotic effect, so that blood clots are not formed. Diseases associated with the formation of blood clots or thromboses can include, for example, thromboembolism, splenic infarction due to thromboembolism, tumor disease resulting from thrombosis, stroke, arterial thrombosis and its consequences, chronic venous insufficiency (CVI), pulmonary embolism, heart attack (myocardial infarction), phlegmasia cerulea dolens, post-thrombotic syndrome (PTS), venous thromboembolism (VTE), recurrent thrombosis, visual disturbances, amaurosis (blindness), mesenteric infarction (bowel infarction) due to arterial thromboembolism, leukemias, lymphomas, ischemia, or renal infarction due to arterial thromboembolism.In particularly preferred embodiments, the therapeutic use according to the invention includes the treatment or prevention of thrombosis, myocardial infarction, ischemia, or stroke. For the treatment or prevention of the aforementioned diseases, the monoclonal mouse IgG antibody can be administered in the form of pharmaceutical compositions, wherein the pharmaceutical compositions additionally comprise, for example, pharmaceutically compatible carriers or, optionally, a pharmaceutical adjuvant. The term "pharmaceutical composition" used herein refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical composition of this invention comprises the monoclonal mouse IgG antibody as defined herein. Typically, the pharmaceutical composition includes suitable formulations of carriers, stabilizers, and / or excipients.For example, the pharmaceutical composition may be a composition for parenteral, transdermal, intraluminal, intra-arterial, intrathecal, and / or intranasal administration, or a composition administered by direct injection into tissue. Furthermore, the composition may be administered to a patient by infusion. The term "pharmaceutically acceptable," as used here, means that the substance has been approved for use in animals and, in particular, in humans. The term "carrier" refers to a diluent, excipient, or pharmaceutical vehicle by which the antibody is administered. Such a carrier is pharmaceutically acceptable, meaning that it is non-toxic to the recipient at the dosage and concentration used.It is preferably isotonic, hypotonic, or slightly hypertonic and has a relatively low ionic strength, such as that offered by a sucrose solution. Such pharmaceutical carriers can be sterile liquids like water and oils. Salt solutions, as well as aqueous dextrose and glycerol solutions, can also be used as liquid carriers. Suitable pharmaceutical excipients further include starch, glucose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium ions, dried skimmed milk, glycerol, propylene, glycol, water, ethanol, etc. If desired, the composition can also contain small amounts of wetting agents, emulsifiers, or pH buffers. In general, the ingredients can be administered either individually or mixed in unit-dose formulations.In a specific embodiment, the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition suitable for intravenous administration to humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. The term "pharmaceutical adjuvant" as used here refers to additional ingredients such as chloroquine, protic polar compounds such as propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or their derivatives, or aprotic polar compounds such as polysorbates (e.g., Tween®-20), dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile, or their derivatives.The pharmaceutical adjuvant may also be one or more surfactants, dispersants, buffers, stabilizers, or isotonic agents. The present invention also provides for any suitable pharmaceutical adjuvant known to those skilled in the art. The pharmaceutical composition of the present invention may also include a preservative such as butylparaben or ethylparaben. The term "administered" means the administration of a therapeutically effective dose of the pharmaceutical composition by any suitable route. "Therapeutically effective amount" means a dose that produces the effect for which it is administered to a patient. The precise dose depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques.Adjustments can and will be made to the systemic versus local administration, age, body weight, general health, sex, diet, time of administration, drug interactions, and disease severity. The following examples and figures serve for illustration. It is understood, therefore, that the examples and figures should not be interpreted as limiting. The person skilled in the art can clearly imagine further modifications to the principles set forth here.

[0002] 202400478 23 EXAMPLES EXAMPLE 1 Test of the inhibition of VWF or thrombin binding to GPIb by antibody 100 / 05 and similar antibodies. The inhibition of VWF binding to GPIb by antibody 100 / 05 is demonstrated using an ELISA. Antibody 100 / 05 has the CDR sequences as described in Tables 1 and 2, or the variable domains as described in Tables 3 and 4. The ELISA test was performed as follows: - Coating of an ELISA plate with anti-mouse IgG antibody - Addition of the mouse 100 / 05 antibody - Binding of the mouse 100 / 05 antibody to the anti-mouse IgG antibody - Binding of GPIb with gain-of-function mutations to the 100 / 05 antibody - Binding of VWF to the GPIb - Binding of a rabbit anti-VWF antibody-HRP conjugate - Detection of VWF binding by color reaction after addition of TMB substrate. An absorbance of 3.44 was measured, while the control reaction without GPIb addition showed an absorbance of 0.02.Thus, a strong binding of VWF and GPIb could be demonstrated. This strong binding was not inhibited by the mouse 100 / 05 antibody. 202400478 24 An analogous test was performed with thrombin. As can be deduced from Figure 1, antibody 100 / 05 (3) (and thus also any antibody that possesses its binding properties, i.e., any antibody that includes the CDRs as mentioned in Tables 1 and 2 or that includes the variable domains as mentioned in Tables 3 and 4), unlike the prior art antibody VM16d and antibody 4H12, hardly inhibits thrombin binding (see also Table 5).

[0003] 202400478 25 EXAMPLE 2 Platelet Function Test It was further investigated whether incubating donor blood with the antibodies 4H12, VM16d, and 100 / 05 leads to a prolongation of the closure time in the Platelet Function Analyzer (PFA). This was demonstrated for the incubation of donor blood with the antibodies 4H12, VM16d, and 100 / 05. The Platelet Function Analyzer measures the in vitro bleeding time. This is prolonged with other antiplatelet drugs, e.g., aspirin. In this experiment, it was shown that the prolongation of the closure time increases in the order VM16d - 100 / 05 - 4H12 (see also Figure 2). From this, it can be deduced that the 100 / 05 antibody causes at least moderate inhibition of platelets in vitro. A corresponding in vivo effect is therefore suggested. The inhibition of thrombin-mediated activation of platelets via GPIb binding is largely absent.Thus, antibody 100 / 05 exhibits properties that differ from known anti-GPIb antibodies, which also do not prevent VWF binding (see Table 5). It could therefore be shown that antibody 100 / 05 (and thus also all antibodies that possess its binding properties, i.e., antibodies that include the CDRs as listed in Tables 1 and 2 or that include the variable domains as listed in Tables 3 and 4) can be used as a therapeutic agent, since it causes moderate inhibition of primary hemostasis (with a significant prolongation of the in vitro bleeding time, no inhibition of VWF binding, and very little or no inhibition of thrombin binding) without causing excessive bleeding tendencies. Table 5 Antibody Inhibition Thrombin Prolongation of binding to GPIb PFA closure time 4H12 +++ +++ VM16d ++ + 100 / 05 +- ++ Table 5: Properties of anti-GPIb antibodies that do not inhibit VWF binding.

Claims

202400478 27 CLAIM 1. A monoclonal mouse IgG antibody against human glycoprotein Ib for therapeutic use in inhibiting GPIb-mediated platelet activation, wherein the monoclonal mouse IgG antibody comprises the amino acid consensus sequence tyrosine-valine-methionine (YVM) within the CDR 3 region of the variable domain of the heavy chain.

2. A monoclonal mouse IgG antibody for therapeutic use according to claim 1, wherein the monoclonal mouse IgG antibody comprises in the variable domain of the heavy chain a CDR 1 region of a sequence with SEQ ID NO. 1, a CDR 2 region of a sequence with SEQ ID NO. 2, and a CDR 3 region of a sequence with SEQ ID NO.

3.

3. A monoclonal mouse IgG antibody for therapeutic use according to claim 1 or 2, wherein the antibody comprises in the variable domain of the light chain a CDR 1 region of a sequence with SEQ ID NO. 4, a CDR 2 region of a sequence with SEQ ID NO.5 and a CDR 3 region of a sequence with SEQ ID NO.

6.

4. Monoclonal mouse IgG antibody for therapeutic use according to any one of claims 1 to 3, wherein the monoclonal mouse IgG antibody comprises a variable domain of the heavy chain of the sequence SEQ ID NO.

7.

5. Monoclonal mouse IgG antibody for therapeutic use according to any one of claims 1 to 4, wherein the. 202400478 28 monoclonal mouse IgG antibody comprising a variable domain of the light chain of the sequence SEQ ID NO.

8.

6. Monoclonal mouse IgG antibody for therapeutic use according to any one of claims 1 to 5, wherein, in the inhibition of GPIb-mediated activation of platelets, there is no inhibition of the binding of von Willebrand factor (VWF) to GPIb.

7. Monoclonal mouse IgG antibody for therapeutic use according to claim 6, wherein, in the inhibition of GPIb-mediated activation of platelets, there is additionally at most a weak inhibition of the binding of thrombin to GPIb.

8. A monoclonal mouse IgG antibody for therapeutic use according to claim 7, wherein the weak inhibition of thrombin binding to GPIb comprises an inhibition of the original binding by approximately 1% to 20%, preferably by no more than 10%. 9.A monoclonal mouse IgG antibody for therapeutic use according to claim 6, wherein, in the inhibition of GPIb-mediated activation of platelets, there is additionally no inhibition of thrombin binding to GPIb. A monoclonal mouse IgG antibody for therapeutic use according to any one of claims 1 to 9, wherein the therapeutic use comprises the treatment or prevention of diseases associated with blood clots or thrombosis, preferably arterial thrombosis, chronic venous insufficiency. 202400478 29 venous thromboembolism, pulmonary embolism, visual disturbances, heart attack, ischemia or stroke.

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