RU2773819C2 - Protein bound with human glycoprotein, composition, pharmaceutical composition, expression vector - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, namely to the production of a humanized protein specifically binding human glycoprotein VI (hGPVI); it can be used in medicine. The produced protein is an antigen-binding fragment of an antibody, where a variable region of a heavy chain contains an amino acid sequence SEQ ID NO:7, and a variable region of a light chain contains a sequence SEQ ID NO:8 or SEQ ID NO:9.

EFFECT: produced protein can be used for the treatment of a cardiovascular disease or event associated with thrombosis.

9 cl, 14 dwg, 1 tbl, 5 ex

Description

The field of technology to which the invention relates

The present invention relates to novel antibodies against human glycoprotein VI and their use in the treatment of cardiovascular diseases.

Prerequisites for the creation of the invention

Acute disorders of the coronary and cerebral circulation are currently the main cause of death in the world. In addition, the overall incidence of relapses and deaths over the 6-month period following treatment after acute coronary syndrome is still 8-15%.

In the case of acute ST-segment elevation coronary syndrome, mechanical treatment with coronary angioplasty and stent insertion is highly effective in order to urgently restore blood flow in the coronary artery, but this does not prevent morbidity/mortality in about 15% of patients over the next 6 months. Treatment with thrombolytics, which is based on the long-term relationship of fibrinolytics, anticoagulants and antiplatelet agents, gives even less encouraging results. Indeed, despite improvement in thrombosis management, morbidity/mortality at 6 months is similar to that seen in non-ST elevation acute coronary syndrome.

With regard to ischemic cerebrovascular accidents, their treatment is still very limited due to the usually late care of most patients and the risk of hemorrhage associated with currently available antithrombotic therapy.

Thus, there is still a clinical need to improve the treatment of cardiovascular diseases and, in particular, for new molecules with improved properties compared to available molecules. The problem to be solved is to obtain molecules with high efficiency in relation to pathological thrombosis, but without the risk of bleeding.

роль в тромбообразовании, происходящем в пораженном сосуде, чем при физиологическом гемостазе, необходимом для ограничения кровотечения в здоровом сосуде. Это относится к тромбоциту гликопротеин VI, который, как продемонстрировано на животных, играет определенную роль в экспериментальном тромбозе, включая инсульт, ремоделировании сосудов и крайне важен при тромбозе артерий, пораженных атеросклерозом.To meet this requirement, the target must play

role in thrombosis occurring in the affected vessel than in the physiological hemostasis necessary to limit bleeding in a healthy vessel. This refers to platelet glycoprotein VI, which has been shown in animals to play a role in experimental thrombosis, including stroke, vascular remodeling, and is critical in thrombosis of atherosclerotic arteries.

Unlike αIIbβ3 integrin antagonists, which are currently used in the treatment of thrombosis and inhibit the phase of final platelet activation, i.e. platelet aggregation, and from platelet recruitment antagonists (P2Y12 adenosine diphosphate receptor inhibitors and aspirin), GPVI is involved in several steps in platelet formation: initiation through interaction with an injured vessel wall, amplification through initial platelet activation leading to secondary agonist secretion, integrin activation, and procoagulant activity platelets, growth and stabilization through interaction with fibrin (Mammadova et al., Blood 2015). Thus, GPVI antagonists can prevent not only platelet aggregation, but also the release of secondary agonists, as well as the secretion of growth factors and cytokines, leading to the development of vascular lesions. Finally, GPVI expression is limited to platelets and megakaryocytes and therefore represents a highly specific target for thrombosis treatment.

Accordingly, GPVI antagonists have been developed for the treatment of cardiovascular diseases.

WO 2001/000810 and WO 2003/008454 describe a soluble recombinant GPVI protein that is a fusion protein between the extracellular domain of GPVI and the Fc domain of human Ig. This soluble recombinant GPVI protein competes with the GPVI platelet for collagen binding. Promising results with this soluble GPVI protein were first obtained in a mouse model of thrombosis, but these results have not been confirmed. In addition, this approach has structural, functional and pharmacological disadvantages. First, this compound is a high molecular weight chimeric protein (~160 kDa molecular weight). The target of GPVI-Fc is collagen found at the site of vascular injury, the amount and availability of which is poorly predictable, and therefore the amount of product administered is a potential disadvantage of using GPVI-Fc. Another disadvantage may be the risk of immunization against neoepitopes potentially found in the fusion protein.

Neutralizing monoclonal antibodies directed against human GPVI are also described.

For example, EP 1224942 and EP 1228768 describe a rat anti-GPVI monoclonal antibody JAQ1 that specifically binds mouse GPVI for the treatment of thrombosis. The JAQ1 antibody induces irreversible uptake of the GPVI receptor by mouse platelets.

Patent EP 1538165 describes another rat anti-GPVI monoclonal antibody (hGP 5C4), the Fab fragment of which was found to have a pronounced inhibitory effect on the main physiological functions of platelets induced by collagen stimulation: Fab hGP 5C4 completely prevented stimulation of markers of collagen-mediated physiological activation PAC-I and CD62P-selectin, and Fab hGP 5C4 effectively inhibited human platelet aggregation ex vivo without any intrinsic activity. However, 5C4 is a rat antibody and therefore has a very limited therapeutic potential.

WO 2005/111083 describes 4 anti-GPVI mouse monoclonal antibodies OM1, OM2, OM3 and OM4 that have been found to inhibit collagen GPVI binding, collagen-induced secretion and in vitro formation of thromboxane A2 (TXA2), and collagen-induced aggregation. ex vivo platelets after intravenous injection in cynomolgus monkeys. OM4 also appears to inhibit thrombosis in a rat model of thrombosis.

WO 2001/000810 also describes various anti-GPVI mouse monoclonal antibodies named 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, 1P10.2, 4L7.3, 7H4.6, 9O12. 2, 7H14.1 and 9E18.2, as well as several scFv fragments of human phage antibodies named A9, A10, C9, A4, C10, B4, C3 and D11. Several of these antibodies and scFv fragments have been found to inhibit collagen GPVI binding, including antibodies 8M14.3, 3F8.1, 9E18.3, 3J24.2, 6E12.3, 1P10.2, 4L7.3, 7H4.6 and 9O12.2, and scFv fragments A10, A4, C10, B4, C3 and D11. In addition, 9O12.2 Fab fragments were found to completely block collagen-induced platelet aggregation and secretion, block fibrin-induced platelet aggregation, inhibit the procoagulant activity of collagen- or fibrin-stimulated platelets and platelet adhesion to collagen or fibrin under static conditions, and weaken platelet adhesion. and prevent thrombus formation under arterial flow conditions.

WO 2008/049928 describes a 9O12.2-derived scFv fragment consisting of the VH and VL domains of the monoclonal antibody 9O12.2 linked via a (Gly ₄ Ser) ₃ peptide.

However, none of the currently known anti-GPVI antibodies has been shown to be effective in vivo for the prevention and/or treatment of cardiovascular disease. In particular, most of the reported anti-GPVI antibodies are not suitable for the development of an antithrombotic agent for human medical use, especially because of their animal origin.

In particular, some human anti-human GPVI scFv phage antibodies have been reported to have inhibitory activity, but their affinity appears to be low. Moreover, crosslinking of GPVI on the surface of platelets with a divalent or multivalent ligand, such as whole IgG 9O12.2, results in platelet activation through dimerization of GPVI and crosslinking of GPVI to the low affinity Fc receptor (FcγRIIA). On the contrary, 9O12.2 monovalent Fab and scFv fragments have inhibitory activity.

However, these fragments cannot be used therapeutically due to their size and animal origin, making them immunogenic in human patients. In addition, scFv fragments have a short half-life, which limits their therapeutic potential.

Thus, there is still a need to neutralize GPVI antagonists without being immunogenic in humans.

US 2006/0088531 describes a human 10B12 scFv fragment with a human _GPVI binding KD of about 7.9×10 ^-7 . M. Smethurst et al. 2004 also described an epitope associated with 10B12 in Ig-like domain 1 (D1) of type C2 of human GPVI. This epitope contains residues R58, K61, R66, K79, and R80 of human GPVI (numbered according to accession number Q9HCN6 in the open protein sequence database UniProtKB).

и др., 2006, также описан человеческий scFv-фрагмент 1С3 с низкой аффинностью в отношении GPVI (5,4×10^-7 М), который не блокировал ни индуцированную коллагеном агрегацию тромбоцитов, ни связывание GPVI коллагена, но усиливал ингибирующий эффект антитела 10В12. Эпитоп 1С3 в GPVI содержит аминокислоту I168, и также теоретически допускается, что этот эпитоп может охватывать область между остатками S164 и S182, которая является высококонсервативной при переносе человеку от мыши.At

et al., 2006, also described a human 1C3 scFv fragment with low affinity for GPVI (5.4×10 ^-7 M), which did not block either collagen-induced platelet aggregation or collagen GPVI binding, but enhanced the inhibitory effect of the 10B12 antibody. . The 1C3 epitope in GPVI contains the amino acid I168, and it is also theoretically possible that this epitope may cover the region between residues S164 and S182, which is highly conserved when transferred to humans from the mouse.

Thus, there is a need for a humanized antibody (i.e., an antibody without human immunogenicity) with improved affinity, potency, and half-life over known antagonists as a means of effectively and adequately preventing and/or treating human cardiovascular disease. In addition, these antagonists should preferably be easily purified.

Anti-GPVI antibodies inducing a depleted GPVI phenotype have been described (WO 2006/118350, WO 2011/073954, EP 2363416). In particular, WO 2006/118350 describes an antibody against GPVI of all mammals. Described is a GPVI epitope bound by this antibody and corresponding to loops 9 and 11 of the Ig-like domain 2 type C2 of IgGVI, which correspond to amino acid residues 136-142 and 158-162, respectively.

However, GPVI depletion is undesirable because it cannot be controlled and is irreversible (ie, it lasts for the lifetime of platelets or even longer due to GPVI depletion in megakaryocytes).

Therapy requires a rapid and safe antiplatelet effect. Therefore, antibodies should be developed that do not induce a depleted GPVI phenotype and preferably do not induce platelet depletion in vivo (ie, with a reversible effect).

Applicant has developed an anti-GPVI antibody that binds a novel, undescribed conformational epitope. This anti-GPVI antibody has high affinity for human GPVI and blocks the interaction of GPVI with its ligands (including collagen and fibrin) without reducing platelets or depleting GPVI in vivo. Thus, the present invention relates to an improved humanized neutralizing antibody specific for human GPVI.

Brief summary of the invention

The present invention provides an isolated humanized human glycoprotein VI (hGPVI) binding protein, wherein the protein binds a conformational epitope comprising:

at least one amino acid residue from amino acid residues 114-142 of hGPVI (SEQ ID NO: 13) or from a sequence at least 60% identical over amino acid residues 114-142 of hGPVI (SEQ ID NO: 13); and

at least one amino acid residue from amino acid residues 165-187 of hGPVI (SEQ ID NO: 13) or from a sequence at least 60% identical over amino acid residues 165-187 of hGPVI (SEQ ID NO: 13).

In one embodiment, the conformational epitope bound by the isolated humanized protein of the invention contains at least one amino acid residue from hGPVI amino acid residues 121-135 (SEQ ID NO: 13) or from a sequence at least 60% identical over amino acid residues 121-135 hGPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169-183 of hGPVI (SEQ ID NO: 13) or from a sequence at least 60% identical over amino acid residues 169-183 of hGPVI (SEQ ID NO: 13) .

The present invention also relates to an isolated humanized human glycoprotein VI (hGPVI) binding protein, wherein the protein has an _hGPVI binding KD of less than 15 nM as measured by surface plasma resonance using 960-1071 RU of soluble human GPVI and using PBS pH 7, 4 as a moving buffer. In one embodiment, the isolated humanized protein of the invention does not induce a GPVI depletion phenotype in vivo.

In one embodiment, the protein of the invention is an antibody molecule selected from the group consisting of whole antibody, humanized antibody, single chain antibody, Fv, Fab; or an antibody fragment selected from the group consisting of a unitbody, a domain antibody, and a nanobody; or a monomeric mimetic antibody selected from the group consisting of affitelo, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, finomer, and versatelo.

In one embodiment, an antibody of the invention is monovalent.

In one embodiment, the heavy chain variable region contains at least one of the following hypervariable regions (CDRs):

VH-CDR1: GYTFTSYNMH (SEQ ID NO: 1);

VH-CDR2: GIYPGNGDTSYNQKFQG (SEQ ID NO: 2); and

VH-CDR3: GTVVGDWYFDV (SEQ ID NO: 3)

or any CDR having an amino acid sequence at least 60% identical to SEQ ID NOs: 1-3.

In one embodiment, the light chain variable region contains at least one of the following CDRs:

VL-CDR1: RSSQSLENSNGNTYLN (SEQ ID NO: 4);

VL-CDR2: RVSNRFS (SEQ ID NO: 5); and

VL-CDR3: LQLTHVPWT (SEQ ID NO: 6)

or any CDR having an amino acid sequence at least 60% identical to SEQ ID NOs: 4-6.

In one embodiment, the heavy chain variable region contains at least one of the above CDRs and the light chain variable region contains at least one of the above CDRs.

In one embodiment, the heavy chain variable region contains the following CDRs: GYTFTSYNMH (SEQ ID NO: 1), GIYPGNGDTSYNQKFQG (SEQ ID NO: 2) and GTVVGDWYFDV (SEQ ID NO: 3) and the light chain variable region contains the following CDRs: RSSQSLENSNGNTYLN (SEQ ID NO: 4), RVSNRFS (SEQ ID NO: 5) and LQLTHVPWT (SEQ ID NO: 6) or any CDR having an amino acid sequence at least 60% identical to said SEQ ID NO: 1-6.

In one embodiment, the amino acid sequence encoding the heavy chain variable region is SEQ ID NO: 7 and the amino acid sequence encoding the light chain variable region is SEQ ID NO: 8, or any amino acid sequence at least 60% identical SEQ ID NO: 7 or 8.

In one embodiment, the amino acid sequence encoding the heavy chain variable region is SEQ ID NO: 7 and the amino acid sequence encoding the light chain variable region is SEQ ID NO: 9, or any amino acid sequence at least 60% identical SEQ ID NO: 7 or 9.

The present invention also relates to a composition containing a human GPVI binding protein as described above.

The present invention further relates to a pharmaceutical composition comprising a human GPVI binding protein as described above and at least one pharmaceutically acceptable excipient.

In one embodiment, the pharmaceutical composition of the invention is for the treatment of or for use in the treatment of a GPVI-associated condition.

In one embodiment, said GPVI-associated condition is a cardiovascular disease selected from arterial and venous thrombosis, restenosis, acute coronary syndrome, and ischemic cerebrovascular disease.

The present invention also relates to the use of an anti-human GPVI antibody of the invention for the detection of GPVI in a biological sample.

Another object of the invention is to provide an expression vector containing at least one of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, or any nucleic acid sequence at least 60% identical to said SEQ ID NO: 10-12.

Detailed description of the invention

The term "glycoprotein VI (GPVI)" means a membrane platelet glycoprotein that is involved in platelet-collagen interactions. GPVI is a transmembrane collagen receptor that is expressed on the surface of platelets. In one embodiment, the human GPVI amino acid sequence is SEQ ID NO: 13 (accession number BAA89353.1) or any amino acid sequence at least about 90% identical to SEQ ID NO: 13, preferably at least about 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identical to SEQ ID NO: 13.

The extracellular domain of GPVI consists of two C2-type Ig-like domains, namely D1 and D2, linked by a hinged interdomain region. In one embodiment, D1 contains amino acid residues 21-109 of SEQ ID NO: 13. In one embodiment, the hinge interdomain region between D1 and D2 contains amino acid residues 110-113 of SEQ ID NO: 13. In one embodiment, D2 contains amino acid residues 114-207 SEQ ID NO: 13.

The term "about" preceding any numerical value is 10% more or less than the specified value.

Antibody or immunoglobulin

As used herein, the term "immunoglobulin" means a protein having a combination of two heavy and two light chains, whether or not it has any corresponding specific immunoreactivity. The term "antibodies" refers to nodes that have significant known specific immunoreactivity for an antigen of interest (eg, human GPVI). The term "anti-GPVI antibodies" means antibodies that exhibit immunospecificity for the human GPVI protein. As explained below, "specificity" for human GPVI does not preclude cross-reactivity with species homologues of GPVI. Antibodies and immunoglobulins contain light and heavy chains with or without a covalent bond between the chains. The basic structures of immunoglobulin in vertebrate systems are relatively well understood. The generic term "immunoglobulin" encompasses five different classes of antibodies that are distinguishable by biochemical methods. Antibodies of all five classes are included in the scope of the present invention; the following description will focus on the IgG class of immunoglobulin molecules. With regard to IgG, immunoglobulins contain two identical polypeptide light chains with a molecular weight of about 23,000 daltons and two identical heavy chains with a molecular weight of about 53,000-70,000 daltons. All four chains are linked by disulfide bonds to form a Y-configuration in which the light chains demarcate the heavy chains starting at the "Y" mouth and continuing through the variable region. Antibody light chains are classified into kappa and lambda classes ([κ], [λ]). The heavy chain of each class can be linked to a kappa light chain or a lambda chain. Typically, the light and heavy chains are covalently linked to each other, and the "tail" regions of both heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulins are formed by hybridomas, B cells, or engineered host cells. In the heavy chain, the amino acid sequences run from the N-terminus at the bifurcated ends of the Y-configuration to the C-terminus at the bottom of each chain. It is known to those skilled in the art that heavy chains are classified into gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε) classes, which have several subclasses (eg, γ1-γ4). It is the nature of this chain that determines the "class" of an antibody as IgG, IgM, IgA, IgD, or IgE, respectively. Immunoglobulin subclasses (isotypes), e.g. IgG1, IgG2, IgG3, IgG4, IgA1, etc. are well characterized and are known to confer functional specialization. Modified versions of each of these classes and isotypes are readily recognizable to those of ordinary skill in the context of the present disclosure and are therefore within the scope of the present invention. As stated above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind epitopes in antigens. In other words, the light chain variable domain (VL domain) and the heavy chain variable domain (VH domain) of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen-binding site at the end of each "Y" arm. More specifically, the antigen binding site is defined by three hypervariable regions (CDRs) in each of the VH and VL chains.

isolated antibody

As used herein, the term "isolated antibody" means an antibody that has been isolated and/or reconstituted from any component of its natural environment. Contaminant components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous components. In preferred embodiments, the antibody is purified: (1) to reach an antibody concentration of greater than 80, 85, 90, 91, 92, 93, 94, 95 wt % or greater, as determined by the Lowry method, most preferably to greater than 99 wt %; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary beaker sequencer; or (3) until homogeneity is achieved by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or preferably silver staining. An isolated antibody includes the antibody in situ in recombinant cells due to the absence of at least one component of the antibody's natural environment. Typically, however, an isolated antibody is obtained by purification in at least one step.

Affinity options

K_A GPVI человека,

скорость ассоциации с GPVI человека или меньшая скорость диссоциации с GPVI человека или изменение схемы перекрестной реактивности с гомологами GPVI животных помимо человека. Варианты аффинности обычно имеют одно или несколько изменений CDR в аминокислотной последовательности по сравнению с эталонным антителом против GPVI. Такие замены могут приводить к замене исходной аминокислоты, присутствующей в заданном положении в CDR, отличающимся аминокислотным остатком, которым может являться встречающийся или не встречающийся в природе аминокислотный остаток. Замены аминокислот могут являться консервативными или неконсервативными.As used herein, the term "affinity variant" means a variant antibody that contains one or more amino acid sequence changes compared to a reference anti-GPVI antibody, wherein the affinity variant has an altered affinity for human GPVI protein compared to the reference antibody. Typically, affinity variants have improved affinity for human GPVI compared to a reference anti-GPVI antibody. An improvement may be a lower K _D GPVI of a person,

K _A GPVI human,

a rate of association with human GPVI, or a lower rate of dissociation from human GPVI, or a change in cross-reactivity pattern with non-human animal GPVI homologues. Affinity variants typically have one or more CDR amino acid sequence changes compared to a reference anti-GPVI antibody. Such substitutions may result in the replacement of the original amino acid present at a given position in the CDR with a different amino acid residue, which may or may not be a naturally occurring amino acid residue. Amino acid substitutions may be conservative or non-conservative.

Binding site

The term "binding site" is used to mean the region of a protein that is responsible for the selective binding of a target antigen of interest (eg, human GPVI). Binding domains or regions contain at least one binding site. Examples of binding domains include the variable domain of an antibody. The protein of the invention may contain a single antigen-binding site or multiple (eg, two, three or four) antigen-binding sites. However, it is preferred that the protein of the invention contains a single antigen-binding site.

Conservative amino acid substitution

The term "conservative amino acid substitution" as used herein means a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are known in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Thus, a non-essential amino acid residue in an immunoglobulin polypeptide can be replaced by another amino acid residue from the same side chain family. In another embodiment, the amino acid chain may be replaced with a structurally similar chain that differs in the order and/or composition of the side chain family members.

Chimeric protein

As used herein, the term "chimeric" protein means a protein in which the first amino acid sequence is linked to a second amino acid sequence to which it is not naturally linked. Amino acid sequences may normally exist in separate proteins that are combined into a fusion protein, or may normally exist in the same protein but have a new location in the fusion protein. The chimeric protein can be created, for example, by chemical synthesis or by creating and transforming a polynucleotide in which the peptide regions are encoded in the desired relationship.

hypervariable region

As used herein, the term "hypervariable region" or "CDR" refers to non-contiguous antigen-binding sites of antibodies found in the variable region of both heavy and light chain polypeptides. CDRs have been identified according to the following rules established by Kabat et al. (1991) and Chotia and Lesk (1987):

CDR-L1:

Beginning - approximately remainder 24

The preceding residue is always Cys

The next remainder is always Trp. Usually TRP-TYR-GLN, but also TRP-LEU-GLN, TRP-PHE-GLN, TRP-TYR-LEU

Length from 10-17 residues

CDR-L2:

Start - always 16 residues after the end of L1

The preceding residues are usually ILE-TYR, but also, VAL-TYR, ILE-LYS, ILE-PHE The length is always 7 residues.

CDR-L3:

The beginning is always 33 residues after the end of L2.

The preceding residue is always Cys.

Subsequent residues are always PHE-GL Y-XXX-GLY (SEQ ID NO: 21)

Length from 7-11 residues

CDR-H1:

Onset is about residue 26 (always 4 residues after CYS) [Chothia/AbM definition]; according to the definition of Kabat, it starts 5 residues later

Preceding residues are always CYS-XXX-XXX-XXX (SEQ ID NO: 22)

The next balance is always TRP. Usually TRP-VAL, but also TRP-ILE, TRP-ALA

Length 10-12 residues (AbM determination); the definition of Chothia excludes the last 4 residues

CDR-H2: start is always 15 residues after the end of CDR-H1 as defined by Kabat/AbM)

Preceding residues are usually LEU-GLU-TRP-ILE-GLY (SEQ ID NO: 23), but with a number of variations

Subsequent residues are LYS/ARG-LEU/ILE/VAL/PHE/THP ALA-THR/SEMLE/ALA

Length 16-19 residues according to the definition of Kabat (according to the definition of AbM ends 7 residues earlier)

CDR-H3:

Start - always 33 residues after the end of CDR-H2 (always 2 residues after CYS)

Preceding residues are always CYS-XXX-XXX (usually CYS-ALA-ARG)

Subsequent residues are always TRP-GLY-XXX-GLY (SEQ ID NO: 24)

Length 3-25 remnants

CH2 domain

The term "CH2 domain" as used herein refers to the region of the heavy chain molecule that typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it does not form a close pair relationship with another domain. In contrast, between the two CH2 domains of the intact native IgG molecule, there are two N-linked branched carbohydrate chains. It has been suggested that the carbohydrate may provide a replacement for domain pairing and help stabilize the CH2 domain (Burton, Molec. Immunol. 22 (1985) 161-206).

Derivative

When used, the term "derivative" of said protein (eg, anti-GPVI antibody or antigen-binding fragment thereof) means the origin of the protein. In one embodiment, the protein or amino acid sequence derived from a particular parent protein is a CDR sequence or a sequence associated therewith. In one embodiment, the amino acid sequence derived from a particular parent protein is not contiguous. For example, in one embodiment, the parent antibody derivative is one, two, three, four, five, or six CDRs. In one embodiment, the protein or amino acid sequence derived from a particular parent protein or amino acid sequence is an amino acid sequence that is substantially identical to the parent amino acid sequence, or a region thereof, of at least 3-5 amino acids, at least 5- 10 amino acids, at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or whose origin from the original sequence may otherwise be recognized by one of skill in the art. In one embodiment, one or more CDR sequences derived from the parent antibody are altered to produce variant CDR sequences, affinity variants, while the variant CDR sequences retain binding activity for GPVI.

Diabody

As used herein, "diabodies" refers to small antibody fragments obtained by constructing sFv fragments (see sFv section) with short linkers (about 5-10 residues) between the VH and VL domains, resulting in interchain rather than intrachain pairing. binding of V domains, resulting in a bivalent fragment, that is, a fragment having two antigen-binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, in which the VH and VL domains of both antibodies are present in different polypeptide chains. Diabodies are described in more detail, for example, in EP 0404097; WO 1993/011161; and Holliger et al., Proc. Natl. Acad. Sci., 90: 6444-6448 (1993).

Bioengineering

As used herein, the term "bioengineered" means the manipulation of nucleic acid molecules or polypeptides by synthetic methods (eg, with recombinant methods, in vitro peptide synthesis methods, by enzymatic or chemical coupling of peptides, or by any combination of these methods). Antibodies of the invention are preferably bioengineered antibodies, including, for example, humanized and/or chimeric antibodies, and antibodies designed to improve one or more properties such as antigen binding, stability/half-life, or effector function.

epitope

As used herein, the term "epitope" refers to the specific arrangement of amino acids in a protein or proteins that an antibody binds. Epitopes often consist of reactive surface groupings of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural characteristics as well as specific charge characteristics. Epitopes may be linear or conformational, ie. involving two or more amino acid sequences in different regions of the antigen, which may optionally be adjacent.

wireframe region

The term "framework region" or "FR region" as used herein refers to amino acid residues that are part of the variable region but not part of the CDR (eg CDR as defined by Kabat/Chothia). Therefore, the variable region framework is about 100-120 amino acids long, but contains only those amino acids that are outside the CDR. As a specific example of a heavy chain variable region and CDRs as defined by Kabat/Chothia, framework 1 may correspond to a variable region domain spanning amino acids 1-25; framework region 2 may correspond to a variable region domain spanning amino acids 36-49; framework 3 may correspond to a variable region domain spanning amino acids 67-98, and framework 4 may correspond to a variable domain spanning amino acids 110 to the end of the variable region. The light chain framework regions are similarly separated by each of the light chain variable region CDRs. In naturally occurring antibodies, the six CDRs present in each monomeric antibody are short, non-contiguous amino acid sequences that have a specific arrangement to form an antigen-binding site when the antibody assumes its three-dimensional configuration in an aqueous environment. Remains of the heavy and light chain variable regions show less intermolecular variability in amino acid sequence and are referred to as framework regions. The framework regions largely adopt a beta sheet conformation, with the CDRs forming loops that connect and in some cases form part of the beta sheet structure. Thus, these framework regions form a cellular scaffold that allows the placement of the six CDRs in the correct orientation through interstrand non-covalent interactions. The antigen-binding site formed by positioned CDRs defines a surface complementary to an epitope of an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to the immunoreactive antigenic epitope. The position of the CDR can be easily recognized by a person skilled in the art.

Fragment

The term "fragment" is used to mean a portion or region of an antibody or antibody chain containing fewer amino acid residues than an intact or complete antibody or antibody chain. The term "antigen-binding fragment" means a protein fragment of an immunoglobulin or antibody that binds an antigen or competes with an intact antibody (i.e., the intact antibody of which it is derived) for antigen binding (i.e., specific binding of human GPVI). As used herein, "fragment" of an antibody molecule means antigen-binding fragments of antibodies, e.g., antibody light chain variable domain (VL), antibody heavy chain variable domain (VH), single chain antibody (scFv), F(ab')2 fragment, Fab fragment, An Fd fragment, an Fv fragment, a single domain antibody (DAb) fragment, an incomplete (univalent) antibody, a diabody, or any antigen-binding molecule formed by combination, assembly, or conjugation of such antigen-binding fragments. Fragments can be obtained, for example, by chemical or enzymatic processing of an intact or complete antibody or antibody chain, or by recombinant methods.

fv

The term "Fv" as used herein refers to the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in close non-covalent association. When these two domains are folded, six hypervariable loops (three each of H and L chains) are formed, which contribute to antigen binding and confer antigen-binding specificity to the antibody. However, even one variable domain (or half of the Fv containing only three antigen-specific CDRs) has the ability to recognize and bind an antigen, albeit at a lower affinity than the entire binding site.

Heavy chain region

The term "heavy chain region" as used herein refers to amino acid sequences that are derived from the constant domains of an immunoglobulin heavy chain. A protein having a heavy chain region contains at least one of the following: a CH1 domain, a hinge domain (eg, an upper, middle, and/or lower hinge region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. In one embodiment, a binding molecule of the invention may comprise an immunoglobulin heavy chain Fc region (eg, a hinge region, a CH2 domain, and a CH3 domain). In another embodiment, the binding molecule of the invention lacks at least a constant domain region (eg, all or part of the CH2 domain). In some embodiments, at least one, preferably all, constant domains are human immunoglobulin heavy chain derivatives. For example, in one preferred embodiment, the heavy chain region contains the entire human hinge domain. In other preferred embodiments, the heavy chain region comprises the entire human Fc region (eg, human immunoglobulin hinge, CH2 and CH3 domain sequences). In some embodiments, the constant domains that form the heavy chain region are from different immunoglobulin molecules. For example, the heavy chain region of a protein may contain a CH2 domain derived from an IgG1 molecule, a hinge region derived from an IgG3 or IgG4 molecule. In other embodiments, constant domains are chimeric domains containing regions of different immunoglobulin molecules. For example, the hinge may comprise a first region derived from an IgG1 molecule and a second region derived from an IgG3 or IgG4 molecule. As indicated above, one of skill in the art will appreciate that the constant domains of the heavy chain region can be modified to differ in amino acid sequence from a naturally occurring immunoglobulin (wild-type) molecule. In other words, the described proteins according to the invention may contain changes or modifications in one or more heavy chain constant domains (CH1, hinge, CH2 or CH3) and/or in the light chain constant domain (CL). Examples of modifications include additions, deletions or substitutions of one or more amino acids in one or more domains.

Hinge area

The term "hinge region" as used herein refers to the region of the heavy chain molecule that connects the CH1 domain to the CH2 domain. This hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently of each other. The hinge regions can be subdivided into three distinct domains: superior, middle and inferior hinge domains (Roux et al., J. Immunol. 1998 161:4083).

The terms "hypervariable loop" and "hypervariable region" are not strictly synonymous because hypervariable loops (HVs) are characterized based on structure, while hypervariable regions (CDRs) are characterized based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, USA, 1983), although HV and CDR limits may differ in some VH and VL domains. The CDRs of the VH and VL domains can generally be characterized according to the Kabat/Chothia definition (see above). In one embodiment, the CDRs of the VL and VH domains may comprise the following amino acids: residues 24-39 (CDRL1), 55-61 (CDRL2) and 94-102 (CDRL3) in the light chain variable domain and residues 26-35 (CDRH1 ), 50-66 (CDRH2) and 99-109 (CDRH3) in the heavy chain variable domain. Thus, HVs may be included in the respective CDRs, and references to "hypervariable loops" of the VH and VL domains should also be considered to refer to the respective CDRs, and vice versa, unless otherwise indicated. The more highly conserved regions of the variable domains are referred to as the framework region (FR), as described below. The native heavy and light chain variable domains each contain four FRs (FR1, FR2, FR3, and FR4, respectively), mostly in a beta-pleated configuration connected by three hypervariable loops. The hypervariable loops in each chain are held in close proximity to each other by the FR and, with the hypervariable loops from the other chain, promote the formation of an antibody antigen-binding site. Structural analysis of antibodies revealed a relationship between the sequence and shape of the binding site formed by hypervariable regions (Chothia et al., J. Mol. Biol. 227: 799-817 (1992)); Tramontano et al., J. Mol. Biol, 215: 175-182 (1990)). Despite their high sequence variability, five of the six loops have only a small set of main chain conformations, referred to as "canonical structures". These conformations are primarily determined by the length of the loops, and secondly by the presence at certain positions in the loops and in the framework regions of key residues that determine the conformation through their packing, hydrogen bonding, or the ability to adopt unusual backbone conformations.

Humanization

As used herein, the term "humanized" refers to chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', or other antigen-binding antibody subsequences) that contain a minimal murine immunoglobulin-derived sequence. For example, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region (CDR) of the recipient are replaced by residues from the CDR of the parent antibody (donor antibody) while maintaining the desired specificity, affinity, and binding capacity of the parent antibody.

Humanizing replacements

As used herein, "humanizing substitutions" means amino acid substitutions in which an amino acid residue present at a specific position in the VH or VL domain of a non-human animal antibody (e.g., a mouse antibody) against GPVI is replaced with an amino acid residue that is at an equivalent position in a reference human VH or VL domain. The reference human VH or VL domain may be the VH or VL domain encoded by the human germline, in which case the substituted residues may be referred to as "humanizing substitutions". Humanizing substitutions can be made in said framework regions and/or CDRs of an anti-GPVI antibody.

High homology to human proteins

An antibody containing a heavy chain variable domain (VH) and a light chain variable domain (VL) is considered to have high homology to human proteins if the amino acid sequences of the combined VH and VL domains are at least 70, 75, 80, 85, 90.95% or more identical to the most closely matched sequences of the VH and VL domains of the human germline cell line. Antibodies with high homology to human proteins can include antibodies containing the VH and VL domains of native antibodies of animals other than humans with a fairly high percentage of identity to human germline sequences, as well as bioengineered, in particular, humanized versions of such antibodies, as well as "entirely human antibodies. In one embodiment, the amino acid sequence identity or sequence homology of the VH domain of a highly homologous human protein antibody and one or more human VH domains can be 75%, 80%, or more across the FR1, FR2, FR3, and FR4 framework regions. In other embodiments, the amino acid sequence identity or sequence homology of the VH domain of a protein of the invention and the most closely matched human germline VH domain sequence can be 85% or more, 90% or more, 95% or more, 97% or more, or up to 99% or even 100%. In one embodiment, the VH domain of an antibody with high homology to a human protein may contain one or more (e.g., 1 to 20) amino acid sequence mismatches across the FR1, FR2, FR3, and FR4 framework regions compared to the most closely matched VH sequence. - human domain. In another embodiment, the sequence identity or sequence homology of the VL domain of an antibody with high homology to a human protein and one or more human VL domains can be 80% or more across the FR1, FR2, FR3, and FR4 framework regions. In other embodiments, the amino acid sequence identity or sequence homology of the VL domain of a protein of the invention and the most closely matched human germline VL domain sequence can be 85% or more 90% or more, 95% or more, 97% or more, or up to 99% or even 100%.

In one embodiment, the VL domain of an antibody with high homology to a human protein may contain one or more (e.g., 1 to 20, preferably 1 to 10, more preferably 1 to 5) amino acid sequence mismatches throughout the FR1, FR2 framework regions. , FR3 and FR4 compared to the most closely matched sequence of the human VL domain. Prior to analyzing the percent sequence identity of an antibody with high homology to the human protein and human germline VH and VL domains, canonical folds can be determined that allow the identification of a family of human germline segments with the same combination of H1 and H2 or L1 and L2 canonical folds ( and L3). Subsequently, a member of the human germline family that has the highest degree of sequence homology relative to the variable region of the antibody of interest is selected to assess sequence homology. Determination of the canonical classes of hypervariable loops L1, L2, L3, H1 and H2 according to Chothia can be carried out by means of bioinformatics, publicly available at www.bioinf.org.uk/abs/chothia.html.page. The results of the calculations contain the requirements for the key balances in the data file. In these data files, positions of key residues are shown with the allowed amino acids at each position. The sequence of the variable region of the antibody of interest is entered as input and is first aligned with the consensus sequence of the antibody to be assigned a number according to the Kabat/Chothia numbering scheme. The canonical fold analysis uses a set of key residue templates generated by the automated method developed by Martin and Thornton (Martin et al., J. Mol. Biol. 263: 800-815 (1996)). If a specific human V cell germline segment is known that uses the same combination of canonical folds for H1 and H2, or for L1 and L2 (and L3), the best matching family member in terms of sequence homology can be determined. Bioinformatics tools can determine the percentage identity of the amino acid sequences of the VH and VL domains of the antibody of interest and the corresponding sequences encoded by the human germline, but manual sequence alignment can also be used. Human immunoglobulin sequences can be identified in several protein databases such as the VBase database (https://vbase.mrc-cpe.cam.ac.uk/) or the Pluckthun/Honegger database (https://www.bioc.unizh .ch/antibody/Sequences/Germlines). To compare human sequences to the V regions of the VH or VL domains in the antibody of interest, a sequence alignment algorithm such as that available from sites such as www.expasy.ch/tools/#align can be used, but manual alignment can also be performed. with a limited set of sequences. Light and heavy chain sequences of human germline families with the same combination of canonical folds and with the highest degree of homology to framework regions 1, 2 and 3 of each chain are selected and compared to the variable region of interest; FR4 is also checked for correspondence to the JH and JK or JL regions of the human germline. It should be noted that when calculating the overall percent sequence homology, FR1, FR2 and FR3 residues are evaluated using the most closely matched sequence from a human germline family with an identical combination of canonical folds. Only residues that differ from the best match or other members of the same family with the same combination of canonical folds are evaluated (excluding any primer-encoded differences). However, for the purposes of humanization, residues in framework regions that are identical to members of other human germline cell families that do not share the same combination of canonical folds may be considered "human" despite being scored "negative" under the most stringent conditions. described above. This assumption is based on a "mix and match" approach to humanization, whereby each of FR1, FR2, FR3, and FR4 is individually compared to its most closely matched human germline sequence, whereby the humanized molecule contains a combination of different FRs, as done by Qu et al. (Qu et al., Clin. Cancer Res. 5: 3095-3100 (1999)) and Ono et al. (Ono et al., Mol. Immunol. 36: 387-395 (1999)) . The boundaries of individual framework regions can be defined using the IMGT numbering scheme, which is a variant of the Chothia numbering scheme (Lefranc et al., Nucleic acid res 27: 209-212 (1999); https://im.gt.cines.fr). Antibodies with high homology to a human protein may contain hypervariable loops or CDRs having canonical human folds or similar to human folds, as detailed below. In one embodiment, at least one hypervariable loop or CDR in the VH domain or VL domain of an antibody with high human protein homology can be derived from or isolated from the VH domain or VL domain of an antibody from non-human animals, but have a predicted or actual canonical fold structure substantially identical to the canonical fold structure that occurs in human antibodies. It is well known in the art that although the primary amino acid sequences of the hypervariable loops present in both the VH domains and the VL domains encoded by the human germline are by definition highly variable, all hypervariable loops except CDR H3 of the VH domain , have only a few special structural conformations called canonical folds (Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Tramontano et al., Proteins 6: 382-94 (1989)), which depend both on the length of the hypervariable loop and on the presence of so-called canonical amino acid residues (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)). The actual canonical structures of hypervariable loops in intact VH or VL domains can be determined by structural analysis (eg, X-ray crystallography), but the canonical structure can also be predicted based on key amino acid residues characteristic of a particular structure (as described in more detail below). Essentially, the specific residue schema that defines each canonical structure forms a "signature" that allows recognition of the canonical structure in the hypervariable loops of the VH or VL domain of the unknown structure; accordingly, canonical structures can be predicted based on the primary amino acid sequence alone. The predicted canonical hypervariable loop fold structures of any given VH or VL domain sequence in an antibody with high homology to a human protein can be analyzed using algorithms publicly available at www.bioinf.org.uk/abs/chothia.html, www.biochem.ucl .ac.uk/~martin/antibodies.html and www.bioc.unizh.ch/antibody/Sequences/Germlines/Vbase_hVk.html. These tools allow you to align the desired sequences of VH or VL domains of VH and sequences of human VH or VL domains with known canonical structure and predict the canonical structure of hypervariable loops of the desired sequence. With respect to the VH domain, H1 and H2 loops can be judged to have a canonical fold structure "substantially identical" to the canonical fold structure known to occur in human antibodies when at least the first, preferably both, of the following criteria are met .

1. The same distance, determined by the number of residues, to the most exact matching canonical human structural class.

2. At least 33%, preferably at least 50% identity to the key amino acid residues described for the respective canonical human H1 and H2 structural classes (for the purposes of the above analysis, H1 and H2 loops are considered separately and compared each of them with the most closely matching canonical human structural class). The above analysis is based on the prediction of the canonical structure of the H1 and H2 loops of the antibody of interest. If the actual structures of the H1 and H2 loops in the antibody of interest are known, for example, according to X-ray crystallography, the H1 and H2 loops in the antibody of interest can also be assessed as having a canonical fold structure "substantially identical" to the canonical fold structure known to occurs in human antibodies if the loop length differs from the length of the most accurate matching human canonical structural class (usually by +1 or +2 amino acids), but the actual structure of the H1 and H2 loops in the antibody of interest matches that of the human canonical fold. Key amino acid residues of the first and second hypervariable loops of human VH domains (H1 and H2) found in canonical human structural classes are described in Chothia et al., J. Mol. Biol. 227: 799-817 (1992), the contents of which are hereby incorporated by reference in their entirety. In particular, Table 3, which is specifically incorporated herein by reference, on page 802 of Chothia et al., lists the preferred amino acid residues at key positions in canonical H1 structures found in human germline cells, and Table 4 on page 803, which is also specifically incorporated herein by reference, lists preferred amino acid residues at key positions in the canonical H2 CDR structures found in human germline cells. In one embodiment, both H1 and H2 in the VH domain of an antibody with high homology to a human protein have a predicted or actual canonical fold structure substantially identical to the canonical fold structure that occurs in human antibodies. Antibodies with high homology to a human protein may contain a VH domain in which the H1 and H2 hypervariable loops form a combination of canonical fold structures identical to the combination of canonical structures known to occur in at least one VH domain of a human germline cell . It has been observed that in the VH domains encoded by the human germline, only certain combinations of the canonical H1 and H2 fold structures actually occur. In one embodiment, the H1 and H2 in the VH domain of antibodies with high homology to a human protein can be derived from the VH domain of animals other than humans, but still form a combination of predicted or actual canonical fold structures that is identical to the combination of canonical structures known to occurs in the human germline or VH domain with a somatic mutation. In non-limiting embodiments, the H1 and H2 in the VH domain, antibodies with high human protein homology can be derived from the VH domain of non-human animals and form one of the following combinations of canonical folds: 1-1, 1-2, 1-3, 1- 6, 1-4, 2-1, 3-1 and 3-5. An antibody with high homology to a human protein may comprise a VH domain having high homology/sequence identity to human VH sequences and containing hypervariable loops with structural homology to human VH. It may be advantageous that the canonical folds present in H1 and H2 in the VH domain of an antibody with high homology to a human protein and their combination be "correct" for the human VH cell germline sequence most closely matching the VH domain of an antibody with high homology human protein in terms of the overall identity of the primary amino acid sequences. As an example, if the most exact sequence match is with the human VH3 germline domain, it may be advantageous for H1 and H2 to form a combination of canonical folds, which also occurs naturally in the human VH3 domain. This may be particularly important in the case of antibodies with high human protein homology originating in animals other than humans, for example, antibodies containing VH and VL domains derived from traditional camelid antibodies, especially antibodies containing humanized camel VH and VL domains. Thus, in one embodiment, the VH domain sequences of an anti-GPVI antibody with high homology to a human protein may be 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more or up to 99% or even 100% identical or homologous to the human VH domain throughout the FR1, FR2, FR3 and FR4 framework regions, and H1 and H2 in the same antibody are derived from the VH domain of non-human animals but form a combination predicted or actual canonical fold structures, identical to the combination of canonical folds known to occur naturally in the same human VH domain. In other embodiments, the L1 and L2 loops in the VL domain of an antibody with high human protein homology are derived from the VL domain of non-human animals and each has a predicted or actual canonical fold structure substantially identical to the canonical fold structure that occurs in human antibodies. As in VH domains, hypervariable loops of VL domains of both V-lambda and V-kappa types can have a limited number of conformations or canonical structures, determined in part by length, as well as by the presence of key amino acid residues at certain canonical positions. Within an antibody of interest with high human protein homology, loops L1, L2, and L3 derived from the VL domain of non-human animals, e.g. is known to occur in human antibodies when at least the first, preferably both, of the following criteria are met.

1. The same distance, determined by the number of residues, to the most exact matching canonical human structural class.

2. At least 33%, preferably at least 50% identity to the key amino acid residues described for the respective canonical V-kappa or V-lambda human structural classes L1 and L2 (for the purposes of the above loop analysis L1 and L2 are considered separately and each of them is compared with the most closely matching canonical human structural class). The above analysis is based on the prediction of the canonical structure of the L1, L2 and L3 loops of the antibody of interest. If the actual structures of the L1, L2, and L3 loops in the antibody of interest are known, for example, according to X-ray crystallography, the L1, L2, or L3 loops in the antibody of interest can also be judged to have a canonical fold structure "substantially identical" to the canonical fold structure, which is known to occur in human antibodies if the loop length differs from the length of the most accurate matching human canonical structural class (usually by +1 or +2 amino acids), but the actual loop structure matches that of the human canonical fold. Key amino acid residues found in the CDRs of human V-lambda chain domains and human V-kappa chain domains in human canonical structural classes are described in Morea et al. Methods, 20: 267-279 (2000) and Martin et al., J. Mol . Biol, 263: 800-815 (1996). The structural set of the human V-lambda chain domain is also described in Tomlinson et al., EMBO J. 14: 4628-4638 (1995), and the structural set of the V-kappa chain domain is described in Williams et al., J. Mol. Biol., 264: 220-232 (1996). The contents of all of these documents are incorporated by reference into this application. L1 and L2 in the VL domain of an antibody with high homology to a human protein can form a combination of predicted or actual canonical folds that is identical to the combination of canonical folds known to occur in the VL domain of human germline cells. In non-limiting embodiments, L1 and L2 in the VL domain of an antibody with high homology to a human protein may form one of the following combinations of canonical folds: 11-7, 13-7 (A, B, C), 14-7 (A, B), 12-11, 14-11 and 12-12 (as defined by Williams et al., J. Mol. Biol. 264: 220-32 (1996) available at https://www.bioc.uzh.ch/antibody /Sequences/Germlines/VBase_hVL.html). In non-limiting embodiments, L1 and L2 in the V kappa domain may form one of the following combinations of canonical folds: 2-1, 3-1, 4-1, and 6-1 (as defined by Tomlinson et al., EMBO J. 14: 4628 -38 (1995) available at https://www.bioc.uzh.ch/antibody/Sequences/Germlines/VBase_hVK.html).

In yet another embodiment, all three L1, L2 and L3 in the VL domain of an antibody with high homology to a human protein may have a predominantly human structure. Preferably, the VL domain of an antibody with high homology to a human protein has high homology/sequence identity to the human VL domain, and the hypervariable loops in the VL region also have structural homology to the human VL domain.

In one embodiment, the sequences of the VL domain of an anti-GPVI antibody with high homology to a human protein may be 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, or up to 99% or even 100% identical to the human VL domain throughout the FR1, FR2, FR3 and FR4 framework regions, and the L1 hypervariable loop and the L2 hypervariable loop can also form a combination of predicted or actual canonical fold structures identical to the combination of canonical folds, which is known to naturally occur in such a human VL domain. Of course, it is envisaged that VH domains having high homology/sequence identity to the human VH domain, as well as structural homology to the hypervariable loops of the human VH domain, are combined with VL domains having high homology/sequence identity to the human VL domain, as well as structural homology to the hypervariable loops of the human VL domain, in order to obtain antibodies with high homology to the human protein, containing pairwise linked VH/VL domains with maximum sequence homology and structural homology to pairwise linked human VH/VL domains encoded by the human germline.

Immunospecificity, specificity, or specific binding In the context of the present invention, an antibody is considered "immunospecific", "specific", or "specifically binding" to an antigen if it reacts with the antigen at a detectable level, preferably with an affinity constant K _A of about 10 ⁶ M ^-1 or more, about 10 ⁷ M ^-1 or more, about 10 ⁸ M ^-1 or more, about 1.5×10 ⁸ M ^-1 or more, about 10 ⁹ M ^-1 or more, about 5×10 ⁹ M ^{- 1} or more. The affinity of an antibody for its cognate antigen is also usually expressed as the dissociation constant K _D , and in some embodiments, the antibody specifically binds the antigen if the K _D is 10 ^-6 M or less, 10 ^-7 M or less, 1.5 x 10 ^-8 M or less, 10 ^-8 M or less, 5×10 ^-9 M or less, or 10 ^-9 M or less. Antibody affinity can be easily determined by conventional methods, such as those described in Scatchard G et al. (The attractions of proteins for small molecules and ions. Ann NY Acad Sci 1949, 51: 660-612). The properties of antibodies to bind antigens, cells or their tissues can usually be determined and assessed by immunodetection methods, including, for example, ELISA, immunofluorescence assay methods such as immunohistochemistry (IHC) and/or fluorescence-excited cell sorting (FACS), or surface plasmon resonance ( SPR, BIAcore).

isolated nucleic acid

As used herein, "isolated nucleic acid" means a nucleic acid substantially separated from other DNA sequences of the genome, as well as proteins or complexes, such as ribosomes and polymerases, that naturally accompany the native sequence. The term encompasses a nucleic acid sequence that has been extracted from its natural environment and includes isolates and chemically synthesized analogs of recombinant or cloned DNA, or analogs biologically synthesized by heterologous systems. Advantageously pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences that are later manually added to the isolated nucleic acid by humans.

The term "polypeptide" is used in its conventional meaning, ie. means a sequence of less than 100 amino acids. A polypeptide generally refers to a monomeric entity. The term "protein" refers to a sequence of more than 100 amino acids and/or a multimeric entity. The proteins of the invention are not limited to a specific product length. This term does not include post-expression modifications of the protein, for example, as a result of glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, whether or not occurring in nature. The protein may be an entire protein or a subsequence thereof. Specific proteins of interest in the context of the present invention are amino acid subsequences containing CDRs and capable of binding an antigen. An "isolated protein" is a protein that has been identified and isolated and/or extracted from a component of its natural environment. In preferred embodiments, the isolated protein is purified: (1) to a concentration of greater than 80, 85, 90, 95% by weight of Lowry protein, most preferably greater than 96, 97, 98, or 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a rotary beaker sequencer; or (3) until homogeneity is achieved by SDS-PAGE under reducing or non-reducing conditions and using Coomassie blue or preferably silver staining. The isolated protein includes the protein in situ in recombinant cells due to the absence of at least one component of the antibody's natural environment. Typically, however, an isolated antibody is obtained by at least one purification step.

Identity

The term "identity" or "identical", used in relation to the relationship between two or more amino acid sequences, means the degree of relationship between amino acid sequences, determined by the number of matches in the chains of two or more amino acid residues. "Identity" is a measure of the percentage of identical matches in the lesser of two or more sequences with gap alignments (if they exist), as determined by a particular mathematical model or computer program (ie, "algorithms"). The identity of the associated amino acid sequences can be easily calculated by known methods. Such methods include, without limitation, those described in Computational Molecular Biology, edited by Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, edited by Smith, D. W., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, edited by Griffin, A.M. and Griffin, H.G., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to obtain the highest match of the tested sequences. Identity determination methods are described in publicly available computer programs. Preferred computer software methods for determining the identity of two sequences include the GCG software package, including GAP (Devereux et al., Nucl. Acid. Res. 2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al., NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith-Waterman algorithm can also be used to determine identity.

Modified antibody

As used herein, the term "modified antibody" means synthetic forms of antibodies that are modified in such a way that they do not occur naturally, such as antibodies that contain at least two heavy chain regions but not two complete heavy chains (such as, for example, domain-deleted antibodies or mini-antibodies); multispecific forms of antibodies (eg, bispecific, trispecific, etc.) modified to bind two or more different antigens or different epitopes on the same antigen; heavy chain molecules attached to scFv molecules; and the like. ScFv molecules are known in the art and are described, for example, in US Pat. same antigen). In another embodiment, the modified antibody of the invention is a fusion protein comprising at least one heavy chain region devoid of a CH2 domain and comprising a protein binding domain comprising a binding region of one member of a receptor-ligand pair.

Mammal

As used herein, the term "mammal" means any mammal, including humans, farm animals, zoo animals, competitive animals, or pets such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. The mammal is preferably a primate, more preferably a human.

monoclonal antibody

As used herein, the term "monoclonal antibody" means an antibody derived from a population of predominantly homogeneous antibodies, ie. the individual antibodies contained in a population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. In addition, unlike polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The term "monoclonal" should not be interpreted as requiring the production of an antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be produced by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be by recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, for example, patent US 4816567). "Monoclonal antibodies" can also be isolated from phage antibody libraries by the methods described, for example, in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol, 222: 581-597 (1991).

native sequence

As used herein, "native sequence nucleotide" means a polynucleotide that has the same nucleotide sequence as a naturally occurring polynucleotide. Accordingly, a "native sequence" protein is a protein that has the same amino acid sequence as a protein (eg, antibody) of natural origin (eg, from animals of any species). Such native sequence polynucleotides and proteins may be naturally occurring or may be produced by recombinant or synthetic methods. As used herein, the term polynucleotide "variant" means a polynucleotide that typically differs from the polynucleotide specifically described in the invention by one or more substitutions, deletions, additions, and/or insertions. Such variants may occur naturally or may be synthesized, for example, by modifying one or more of the polynucleotide sequences of the invention and evaluating one or more of the biological activities of the described encoded proteins and/or by using any of a number of methods well known in the art. As used herein, the term "variant" of a protein means a protein that typically differs from the specifically described protein by one or more substitutions, deletions, additions, and/or insertions. Such variants may occur naturally or may be synthesized, for example, by modifying one or more protein sequences according to the invention and evaluating one or more indicators of the biological activity of the described protein and/or by using any of a number of methods well known in the art. Modifications can be made to the structure of the polynucleotides and proteins of the present invention to provide a functional molecule that encodes a protein variant or derivative with the desired characteristics. When it is desired to change the amino acid sequence of a protein to create an equivalent or even improved variant or region of the protein according to the invention, one skilled in the art will typically change one or more codons of the DNA coding sequence. For example, some amino acids can be replaced by other amino acids in the structure of a protein without a noticeable loss of the ability to bind other proteins (eg, antigens) or cells. Since the biological functional activity of a protein is determined precisely by its binding capacity and nature, several amino acid sequence substitutions can be made in the protein sequence and, of course, in the underlying DNA coding sequence to obtain a protein with similar properties. Thus, it is contemplated that the amino acid sequences of the disclosed compositions, or the corresponding DNA sequences that encode said proteins, may be altered in various ways without significant loss of their biological utility or activity. In many cases, the protein variant contains one or more conservative substitutions. A "conservative substitution" is a substitution in which an amino acid is replaced with another amino acid with similar properties, whereby one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the protein to remain substantially unchanged. As indicated above, amino acid substitutions are generally based on the relative similarity of the amino acid side chain substituents, eg, their hydrophobicity, hydrophilicity, charge, size, and the like. Examples of substitutions taking into account some of the above characteristics are well known to those skilled in the art and include arginine and lysine; glutamate and aspartate; series and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups with similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and series, threonine, phenylalanine and tyrosine. Other groups of amino acids that may contain conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. A variant may also contain, or alternatively contains, non-conservative changes. In one preferred embodiment, protein variants differ from the native sequence by a substitution, deletion, or addition of five or fewer amino acids. Variants can also (or alternatively) be modified, for example by deletion or addition of amino acids that have minimal effect on the immunogenicity, secondary structure, and hydropathy of the protein.

Pharmaceutically acceptable excipient

As used herein, the term "pharmaceutically acceptable excipient" means any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The excipient does not cause an adverse, allergic or other undesirable reaction when administered to an animal, preferably a human. In the case of human administration, preparations must comply with the requirements of sterility, non-pyrogenicity and the general standards of safety and purity established by regulatory bodies, such as, for example, the FDA or EMA.

Specificity

The term "specificity" is used to mean the ability to specifically bind (eg, mount an immune response) to a given target, eg GPVI. A protein may be monospecific and contain one or more binding sites that specifically bind to a target, or a protein may be multispecific and contain two or more binding sites that specifically bind to the same or different targets. In one embodiment, an antibody of the invention is specific for multiple targets. For example, in one embodiment, the multispecific binding molecule of the invention binds GPVI and a second molecule.

Single chain Fv also abbreviated as sFv or scFv

The terms "single chain Fv", "sFv" or "scFv" as used herein refer to antibody fragments that contain the VH and VL domains of antibodies combined into a single amino acid chain. The sFv amino acid sequence preferably further comprises a peptide linker between the VH and VL domains which allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, volume 113, edited by Rosenburg and Moore, Springer, New York, pp. 269-315 (1994); Borrebaeck 1995.

Subject

The term "subject" is used to mean a mammal, preferably a human. In one embodiment, the subject may be a "patient", i.e., a warm-blooded animal, more preferably a human, who is awaiting or receiving medical attention, or has been/is/will be the subject of a medical procedure or is being monitored for the development of a disease.

Synthetic

The term "synthetic" as used in reference to proteins includes proteins containing an amino acid sequence that does not occur in nature. For example, non-naturally occurring proteins are modified forms of naturally occurring proteins (for example, containing a mutation such as an addition, substitution, or deletion) or proteins containing a first amino acid sequence (which is not necessarily naturally occurring) linked in a linear amino acid sequence to a second an amino acid sequence (which is not necessarily naturally occurring) to which it is not naturally associated.

The term "therapeutically effective amount" means the level or amount of a substance that, without significant negative or adverse side effects, targets the target to (1) delay or prevent the onset of a GPVI-associated disease; (2) slowing or stopping the progression, exacerbation, or worsening of one or more symptoms of a GPVI-associated disease; (3) improving symptoms of GPVI-associated disease; (4) reducing the severity or incidence of GPVI-associated disease; or (5) curing a GPVI-associated disease. A therapeutically effective amount may be administered prior to the onset of a GPVI-associated disease for the purpose of prophylaxis or prevention. Alternatively, or additionally, a therapeutically effective amount may be administered after the onset of a GPVI-associated disease for the purpose of treatment.

Variable region or variable domain

The term "variable" is used to mean that certain regions of the variable VH and VL domains have significant sequence differences among antibodies and are involved in the binding and specificity of each particular antibody for its target antigen. However, variability is unevenly distributed among the variable domains of antibodies. It is concentrated in three segments, called "hypervariable loops", in each VL domain and VH domain, which are part of the antigen binding site. The first, second and third hypervariable loops of the V-lambda light chain domain are called L1(λ), L2(λ) and L3(λ) and can be characterized as containing residues 24-33 (L1(λ) consisting of 9, 10 or 11 amino acid residues), 49-53 (L2(λ) consisting of 3 residues) and 90-96 (L3(λ) consisting of 6 residues) in the VL domain (Morea et al., Methods 20: 267-279 (2000)). The first, second and third hypervariable loops of the V-kappa light chain domain are called L1(κ), L2(κ) and L3(κ) and can be characterized as containing residues 25-33 (L1(κ) consisting of 6, 7, 8, 11, 12 or 13 residues), 49-53 (L2(κ) consisting of 3 residues) and 90-97 (L3(κ) consisting of 6 residues) in the VL domain (Morea et al., Methods 20: 267-279 (2000)). The first, second and third hypervariable loops of the VH domain are called H1, H2 and H3 and can be characterized as containing residues 25-33 (H1 consisting of 7, 8 or 9 residues), 52-56 (H2 consisting of 3 or 4 residues) and 91-105 (H3, highly variable in length) in the VH domain (Morea et al., Methods 20: 267-279 (2000)). Unless otherwise indicated, the terms L1, L2, and L3, respectively, refer to the first, second, and third hypervariable loops of the VL domain, including hypervariable loops derived from both V-kappa and V-lambda isotypes. The terms H1, H2, and H3, respectively, refer to the first, second, and third hypervariable loops of the VH domain, including hypervariable loops derived from any of the known heavy chain isotypes, including gamma, epsilon, delta, alpha, or mu. Each of the hypervariable loops L1, L2, L3, H1, H2 and H3 may be part of a "hypervariable region" or "CDR" as described above.

Valence

The term "valency" as used herein refers to the number of potential target binding sites in a protein. Each target binding site specifically binds one target molecule or a specific region of the target molecule. When a protein contains multiple target binding sites, each target binding site may specifically bind the same or different molecules (eg, may bind different ligands or different antigens or different epitopes on the same antigen). The binding molecules in question preferably have at least one binding site specific for the human GPVI molecule. The proteins described are preferably monovalent.

Treatment or relief

As used herein, the terms "treatment" or "relief" refer to both therapeutic treatment and prophylactic or preventive measures; wherein the objective is to prevent or slow down (weaken) the target pathological condition or disorder. Those in need of treatment include those who are already suffering from the disorder, as well as those who are predisposed to the disorder or who are to be prevented from having the disorder. A subject or mammal is successfully "treated" with a target pathological condition or disorder if, after receiving a therapeutic amount of a protein according to the present invention, the patient exhibits an observable and/or measurable decrease or absence of one or more of the following: a decrease in the number of pathogenic cells; a decrease in the percentage of the total number of cells that are pathogenic; and/or alleviating to some extent one or more symptoms associated with a particular disease or condition; reduction in morbidity and mortality; and improving the quality of life. The above parameters for assessing the success of treatment and the positive dynamics of the disease are easily measurable by standard methods familiar to physicians.

The present invention relates to isolated humanized human GPVI binding proteins, preferably isolated humanized anti-human GPVI antibodies. In one embodiment, the isolated protein of the invention is purified.

In one embodiment, the protein of the invention binds the extracellular domain of GPVI.

In one embodiment, the protein of the invention binds the Ig-like domain 2 type C2 (D2) of human GPVI.

Thus, in one embodiment, a protein of the invention binds an epitope containing at least one amino acid residue from amino acid residues 114-207 of human GPVI (SEQ ID NO: 13) or from a sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical throughout amino acid residues 114-207 of human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least one amino acid residue from amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119- 185, more preferably 120-184, even more preferably 121-183 human GPVI (SEQ ID NO: 13) or from a sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical over amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119-185, more preferably 120-184, even more preferably 121-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, 50 or more amino acid residues from amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119- 185, more preferably 120-184, even more preferably 121-183 human GPVI (SEQ ID NO: 13) or from a sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical over amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119-185, more preferably 120-184, even more preferably 121-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least one amino acid residue from amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119- 137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical over amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25, 26, 27, 28 amino acid residues from amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90 %, 95%, 96%, 97%), 98%, 99%) over amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least one amino acid residue from amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119- 135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical over amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21 amino acid residues from amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121- 135 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope comprises at least one amino acid residue from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165- 187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22 amino acid residues from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 of human GPVI (SEQ ID NO: 13) or from the sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, an isolated protein of the invention binds a conformational epitope.

In one embodiment, said conformational epitope contains at least one amino acid residue of human GPVI.

In one embodiment, said conformational epitope comprises at least one amino acid residue of the Ig-like domain 2 type C2 (D2) of human GPVI.

In one embodiment, said conformational epitope contains at least one amino acid residue from 114-207 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least one amino acid residue from amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119 -185, more preferably 120-184, even more preferably 121-183 Human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably from 118-186, more preferably 119-185 , more preferably 120-184, even more preferably 121-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid residues from amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably 118-186, more preferably 119 -185, more preferably 120-184, even more preferably 121-183 Human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 114-187, preferably 115-187, more preferably 116-187, more preferably 117-187, more preferably from 118-186, more preferably 119-185 , more preferably 120-184, even more preferably 121-183 Human GPVI (SEQ ID NO: 13) .

In one embodiment, said conformational epitope comprises at least one amino acid residue from amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119 -137, more preferably 120-136, even more preferably 121-135 Human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably from 118-138, more preferably 119-137 , more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 amino acid residues from amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138 , more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 114-142, preferably 115-141, more preferably 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least one amino acid residue from amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119 -135, more preferably 120-135, even more preferably 121-135 Human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80% 90%, 95% , 96%, 97%, 98%, 99% over amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably from 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acid residues from amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, more more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98% , 99% over amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121 -135 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope comprises at least one amino acid residue from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187 , preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acid residues from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 of human GPVI (SEQ ID NO: 13) or from sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%), 98%, 99%) over amino acid residues 165-187, preferably 166- 186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains:

at least one amino acid residue from amino acid residues 114-142, preferably 115-141, more preferably from 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70% 75%, 80%, 90%, 95%, 96%, 97%, 98% , 99% over amino acid residues 114-142, preferably 115-141, more preferably from 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, even more preferably 121-135 human GPVI (SEQ ID NO: 13); and

at least one amino acid residue from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 of human GPVI (SEQ ID NO: 13) or from the sequence, at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187, preferably from 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains:

at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28 amino acid residues from amino acid residues 114-142, preferably 115-141, more preferably from 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120 -136, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75% 80%, 90%, 95%, 96%, 97% , 98%, 99% over amino acid residues 114-142, preferably 115-141, more preferably from 116-140, more preferably 117-139, more preferably 118-138, more preferably 119-137, more preferably 120-136, more preferably 121-135 human GPVI (SEQ ID NO: 13); and

at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acid residues from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical , 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168- 184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains:

at least one amino acid residue from amino acid residues 114-135, preferably 115-135, more preferably from 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98 %, 99% over amino acid residues 114-35, preferably 115-135, more preferably from 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13); and

at least one amino acid residue from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 of human GPVI (SEQ ID NO: 13) or from the sequence, at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 Human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains:

at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 amino acid residues of amino acid residues 114-135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%), 98%, 99% over amino acid residues 114- 135, preferably 115-135, more preferably 116-135, more preferably 117-135, more preferably 118-135, more preferably 119-135, more preferably 120-135, even more preferably 121-135 human GPVI (SEQ ID NO: 13); and

at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 amino acid residues from amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168-184, even more preferably 169-183 human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical , 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 165-187, preferably 166-186, more preferably 167-185, more preferably 168- 184, even more preferably 169-183 human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least one amino acid residue from amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75% , 80% 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169-183 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 169-183 of human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, a protein of the invention binds a conformational epitope containing at least one amino acid residue from amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or from a sequence of at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169-183 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 169-183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope contains at least one amino acid residue from amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75% , 80% 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169-180 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 169-180 of human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, a protein of the invention binds a conformational epitope containing at least one amino acid residue from amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or from a sequence of at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and at least one amino acid residue from amino acid residues 169-180 of human GPVI (SEQ ID NO: 13) or from a sequence at least 60% identical, 70%, 75%, 80%, 90%, 95 %, 96%, 97%, 98%, 99% over amino acid residues 169-180 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope consists of:

amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and

amino acid residues 169-183 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 169-183 of human GPVI (SEQ ID NO: 13).

Thus, in one embodiment, a protein of the invention binds a conformational epitope consisting of:

amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and

amino acid residues 169-183 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 169-183 of human GPVI (SEQ ID NO: 13).

In one embodiment, said conformational epitope consists of:

amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and

amino acid residues 169-180 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90, 95, 96, 97, 98, 99% throughout the amino acids human GPVI residues 169-180 (SEQ ID NO: 13).

Thus, in one embodiment, a protein of the invention binds a conformational epitope consisting of:

amino acid residues 121-135 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% over amino acid residues 121-135 of human GPVI (SEQ ID NO: 13); and

amino acid residues 169-180 of human GPVI (SEQ ID NO: 13) or sequence at least 60% identical, 70%, 75%, 80%, 90, 95, 96, 97, 98, 99% throughout the amino acids human GPVI residues 169-180 (SEQ ID NO: 13).

Another object of the invention is to provide an isolated protein that binds human GPVI, said protein having a binding K _D of human GPVI, preferably soluble human GPVI, of 15 nM or less, preferably 10 nM or less, more preferably 5 nM or less.

In one embodiment, the isolated protein of the invention has a human GPVI binding k _off of about 8x10 ^-4 sec ^-1 or less, preferably about 6x10 ^-4 sec ^-1 or less, more preferably about ^{4x10 -4} sec ^-1 or less.

In one embodiment, the isolated protein of the invention has a k _on human GPVI of at least about 5×10 ^-4 M ^-1 sec ^-1 , preferably at least about 5.5×10 ^-4 M ^-1 sec ^-1 , more preferably at least about 5.9×10 ^-4 M ^-1 sec ^-1 , even more preferably at least about 6×10 ^-4 sec ^-1 .

In one embodiment, K _D may be determined by surface plasmon resonance (SPR, BIAcore) using immobilized soluble GPVI at about 700 to about 1600 resonance units (RU) (corresponding to about 8.5 to about 11.3 fmol /mm ² ), preferably from about 850 to about 1200 RU, more preferably from about 950 to about 1075 RU and/or using PBS pH 7.4 as working buffer and/or using version 3.0 of the BIAevaluation analysis software data. In one embodiment, the soluble GPVI corresponds to the extracellular domain of GPVI, the C-terminus of which is fused to the hFc sequence via a linker (such as, for example, a Gly-Gly-Arg linker). This soluble GPVI may be referred to as soluble GPVI-Fc.

The following describes a method for determining the affinity of a protein according to the invention to soluble GPVI.

The inventive protein binding of soluble human GPVI was analyzed by surface plasmon resonance using the BIAcore 2000 system (Usila, Sweden).

Soluble GPVI-Fc is immobilized at about 700 to about 1600 RU (corresponding to about 8.5 to 11.3 fmol/mm ² ), preferably about 850 to about 1200 RU, more preferably about 950 to about 1075 RU and even more preferably from about 960 to about 1071 RU on the carboxymethyldextran sensor chip CM5 by the amine coupling method (Wizard procedure). The protein is then passed over immobilized GPVI-Fc in PBS pH 7.4 (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.42 at 25.4°C) at a rate of 20 μl/h at 25°C. FROM. Kinetic constants (k _on , k _off ) and affinity are determined using version 3.0 of the BIAevaluation software by fitting data to a coupling model. PBS pH 7.4 is running buffer.

In one embodiment, said protein is an antibody molecule selected from the group consisting of whole antibody, humanized antibody, single chain antibody, dimeric single chain antibody, Fv, Fab, F(ab)'2, defucosylated antibody, bispecific antibody, diabody, triatebody, and tetrabody.

In another embodiment, said protein is an antibody fragment selected from the group consisting of a unitbody, a domain antibody, and a nanobody.

In another embodiment, said protein is a mimic antibody selected from the group consisting of affinity antibody, affin, affin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, finomer, versatelo, and duocalin.

A domain antibody is well known in the art and refers to the smallest functional antibody binding units corresponding to the variable regions of antibody heavy or light chains.

A nanobody is well known in the art and refers to a therapeutic protein derived from an antibody that has the unique structural and functional properties of naturally occurring heavy chain antibodies. These heavy chain antibodies may contain one variable domain (VHH) and two constant domains (CH2 and CH3).

Unitel is well known in the art and refers to an antibody fragment that lacks the hinge region of IgG4 antibodies. Deletion of the hinge region results in a molecule predominantly half the size of traditional IgG4 antibodies, which has a univalent binding region instead of the bivalent binding region of IgG4 antibodies.

Affitelo is well known in the art and refers to affinity proteins based on a protein domain of 58 amino acid residues derived from one of the IgG binding domains of Staphylococcus A protein.

DARPins (Designed Ankyrin Repeat Proteins) are well known in the art and refer to a DRP (Designed Repeat Protein) mimetic antibody designed to exploit the binding abilities of non-antibody proteins.

Anticalins are well known in the art and refer to another antibody mimic technology whose specificity is inherited from lipocalins. Anticalins can also be in a dual targeting protein format called duocalin.

Avimers are well known in the art and are another technology to mimic antibodies.

Versatela are well known in the art and refer to another antibody mimic technology. They are small proteins with a molecular weight of 3-5 kDa and a cysteine content of >15% that form a scaffold with a high density of disulfides instead of the hydrophobic core that typical proteins have.

In one embodiment, the protein of the invention is monovalent and is preferably selected from a whole monovalent antibody, a humanized monovalent antibody, a single chain antibody, an Fv, a Fab, or an antibody fragment selected from the group consisting of a single body, a domain antibody, and a nanobody; or from a monomeric mimetic antibody selected from the group consisting of affitelo, affilin, affitin, adnectin, atrimer, evasin, DARPin, anticalin, avimer, finomer, and versatelo.

In another embodiment, said protein is an immunoconjugate containing an antibody or fragment thereof conjugated to a therapeutic agent.

In another embodiment, said protein is a conjugate containing a protein of the invention conjugated to an imaging agent. Said protein can be used, for example, in imaging applications.

In one embodiment, said protein is a monoclonal antibody.

In another embodiment, said protein is a polyclonal antibody.

Another object of the invention is to provide an anti-hGPVI antibody or antigen-binding fragment thereof, wherein the heavy chain variable region contains at least one of the following CDRs:

VH-CDR1: GYTFTSYNMH (SEQ ID NO: 1);

VH-CDR2: GIYPGNGDTSYNQKFQG (SEQ ID NO: 2); and

VH-CDR3: GTVVGDWYFDV (SEQ ID NO: 3).

The numbering and definition of CDRs are given according to Kabat/Chothia.

Another object of the invention is to provide an anti-hGPVI antibody or antigen-binding fragment thereof, wherein the light chain variable region contains at least one of the following CDRs:

VL-CDR1: RSSQSLENSNGNTYLN (SEQ ID NO: 4);

VL-CDR2: RVSNRFS (SEQ ID NO: 5); and

VL-CDR3: LQLTHVPWT (SEQ ID NO: 6).

The numbering and definition of CDRs are given according to Kabat/Chothia.

In one embodiment, an anti-hGPVI antibody or antigen-binding fragment thereof according to the invention comprises:

at least one of the following heavy chain CDRs: GYTFTSYNMH (SEQ ID NO: 1), GIYPGNGDTSYNQKFQG (SEQ ID NO: 2) and GTVVGDWYFDV (SEQ ID NO: 3) and/or at least one of the following CDRs in the light chain: RSSQSLENSNGNTYLN (SEQ ID NO: 4), RVSNRFS (SEQ ID NO: 5) and LQLTHVPWT (SEQ ID NO: 6).

In another embodiment, the heavy chain of an anti-hGPVI antibody or antigen-binding fragment contains three CDRs: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

In another embodiment, the light chain of an anti-hGPVI antibody or antigen-binding fragment thereof contains three CDRs: SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

In another embodiment, the heavy chain of an anti-hGPVI antibody, or antigen-binding fragment thereof, contains three CDRs: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and its light chain contains three CDRs: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6.

In another embodiment, the heavy chain of an anti-hGPVI antibody, or antigen-binding fragment thereof, contains three CDRs: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and its light chain contains three CDRs: SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, optionally, one, two, three or more amino acids in any of these sequences may be replaced by another amino acid.

According to the invention, any of the heavy and light chain CDRs 1, 2 and 3 can be characterized as having an amino acid sequence of at least 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97% , 98%, 99% identical to the specific CDR or sets of CDRs listed in the corresponding SEQ ID NO.

In another embodiment, the hGPVI antibody, or antigen-binding fragment thereof, is selected from the group consisting of an antibody having:

(i) heavy chain CDRs 1, 2 and 3 (VH-CDR1, VH-CDR2, VH-CDR3) containing the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively; and

(ii) light chain CDRs 1, 2 and 3 (VL-CDR1, VL-CDR2, VL-CDR3) containing the amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively;

while one, two, three or more amino acids in any of these sequences may optionally be replaced by another amino acid.

In one embodiment, an anti-GPVI antibody or antigen-binding fragment thereof according to the invention comprises a heavy chain variable region comprising or consisting of the sequence of SEQ ID NO: 7.

In one embodiment, an anti-GPVI antibody or antigen-binding fragment thereof according to the invention comprises a light chain variable region comprising or consisting of the sequence of SEQ ID NO: 8.

In one embodiment, an anti-GPVI antibody or antigen-binding fragment thereof according to the invention comprises a light chain variable region comprising or consisting of the sequence of SEQ ID NO: 9.

In another embodiment, the anti-GPVI antibody (ACT017 antibody) or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the sequence of SEQ ID NO: 7 and a light chain variable region comprising or consisting of the sequence of SEQ ID NO: eight.

In another embodiment, the anti-GPVI antibody (ACT006 antibody) or antigen-binding fragment thereof comprises a heavy chain variable region comprising or consisting of the sequence of SEQ ID NO: 7 and a light chain variable region comprising or consisting of the sequence of SEQ ID NO: 9.

According to the invention, one, two, three or more amino acids of the heavy chain variable regions or light chain variable regions described above may be replaced by another amino acid.

In another embodiment, an antibody of the invention comprises heavy and light chain variable regions having amino acid sequences homologous to the amino acid sequences of the disclosed ACT017 antibody, wherein the antibodies retain the desired functional properties of the protein of the invention.

In another embodiment, an antibody of the invention comprises heavy and light chain variable regions having amino acid sequences homologous to the amino acid sequences of the disclosed ACT006 antibody, wherein the antibodies retain the desired functional properties of the protein of the invention.

In accordance with the invention, the heavy chain variable region contains sequences 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 7.

According to the invention, the light chain variable region contains sequences 60%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 8 or SEQ ID NO:9.

In any of the antibodies of the invention, such as ACT017 or ACT006, said variable region and CDR sequences may contain conservative sequence modifications. Conservative sequence modifications refer to amino acid modifications that do not significantly affect or change the binding characteristics of an antibody containing an amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard methods known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are typically substitutions in which an amino acid residue is replaced with an amino acid residue having a side chain with similar physicochemical properties. Said variable region and CDR sequences may contain one, two, three, four or more amino acid insertions, deletions or substitutions. Where substitutions are made, conservative modifications are preferred substitutions. Families of amino acid residues having similar side chains are known in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (eg, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine). Thus, one or more amino acid residues in the CDR regions of an antibody of the invention may be replaced with other amino acid residues from the same side chain family, and the altered antibody may be tested for retention of function (i.e., described properties) by the tests described.

In one embodiment, the invention also provides an antibody that predominantly binds the same epitope as an ACT017 antibody or an ACT006 antibody. In the present invention, an antibody that predominantly binds the same epitope as an ACT017 antibody or an ACT006 antibody is referred to as an ACT017-like or ACT006-like antibody, respectively.

In some embodiments, anti-hGPVI antibodies containing VH and VL domains or their CDRs may contain CH1 and/or CL domains whose amino acid sequence is wholly or predominantly human. When the antigen-binding protein of the invention is an antibody for therapeutic use in humans, all or at least a portion of the antibody constant region will typically have wholly or predominantly the human amino acid sequence. Therefore, the amino acid sequence of one or more or any combination of the CH1 domain, hinge region, CH2 domain, CH3 domain, and CL domain (and CH4 domain, if present) may be wholly or predominantly human. The CH1 domain, the hinge region, the CH2 domain, the CH3 domain and the CL domain (and the CH4 domain if present) may preferably be wholly or predominantly of the human amino acid sequence. When applied to the constant region of a humanized or chimeric antibody or antibody fragment, the term "predominantly human" means that the amino acid sequence is at least 70% or at least 80% or at least 90%, or, at least 95%, or at least 97%, or at least 99% identical to a human constant region. The term "human amino acid sequence" in this context means the amino acid sequence encoded by the human immunoglobulin gene, which includes germline genes, rearranged genes, and somatically mutated genes. The invention also provides proteins containing human sequence constant domains that have been altered by one or more amino acid additions, deletions, or substitutions from a human sequence, except for those embodiments that explicitly require the presence of a "fully human" hinge region. . The presence of a "fully human" hinge region in the anti-hGPVI antibodies of the invention can be beneficial both in minimizing immunogenicity and in optimizing the stability of the antibody. It is believed that one or more amino acid substitutions, insertions or deletions can be made in the heavy and/or light chain constant region, especially within the Fc region. Amino acid substitutions may result in the replacement of the substituted amino acid with another naturally occurring amino acid or a non-naturally occurring or modified amino acid. Other structural modifications are also contemplated, such as, for example, changes in the glycosylation pattern (eg, by adding or deleting N- or O-linked glycosylation sites). Depending on the intended use of the antibody, it may be desirable to modify the antibody of the invention in terms of its Fc receptor binding properties, for example, to modulate effector function. For example, a cysteine residue(s) may be introduced into the Fc region, thereby allowing the formation of an interchain disulfide bond in the region. The thus obtained homodimeric antibody may have an improved effector function. See Caron et al., J. Exp. Med. 176: 1191-1195 (1992) and Shopes, B.J. Immunol. 148: 2918-2922(1992).

Another object of the invention is to provide an isolated nucleic acid sequence encoding the heavy chain variable region of SEQ ID NO: 7. Said nucleic acid sequence is preferably SEQ ID NO: 10:

Another object of the present invention is to provide an isolated nucleic acid sequence encoding the light chain variable region of SEQ ID NO: 8. Said nucleic acid sequence is preferably SEQ ID NO: 11:

Another object of the invention is to provide an isolated nucleic acid sequence encoding the light chain variable region of SEQ ID NO: 9. Said nucleic acid sequence is preferably SEQ ID NO: 12

Another object of the invention is to provide an expression vector containing nucleic acid sequences encoding an anti-GPVI antibody of the invention. In one embodiment, the expression vector according to the invention contains at least one of the sequences of SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12 or any nucleic acid sequence of at least 60%, 70 %, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 10-12.

In one embodiment, the implementation of the vector according to the invention contains the sequence of SEQ ID NO: 10 and the sequence encoding the constant region of the heavy chain. A non-limiting example of a sequence encoding a heavy chain constant region is SEQ ID NO: 14.

In one embodiment, the implementation of the vector according to the invention contains the sequence of SEQ ID NO: 11 or SEQ ID NO: 12 and a sequence encoding a light chain constant region. A non-limiting example of a sequence encoding a light chain constant region is SEQ ID NO: 15.

In one embodiment, the implementation of the vector according to the invention contains a sequence encoding a signal peptide. Non-limiting examples of signal peptide sequences include, without limitation, SEQ ID NO: 16 and SEQ ID NO: 17.

In one embodiment, the implementation of the vector according to the invention contains SEQ ID NO: 10, a sequence encoding a heavy chain constant region (such as, for example, SEQ ID NO: 14), and a signal peptide sequence. An example of such a vector is the vector containing SEQ ID NO: 18. SEQ ID NO: 18 further contains cloning sites.

In one of the embodiments of the invention, the vector according to the invention contains SEQ ID NO: 8, a sequence encoding a light chain constant region (for example, SEQ ID NO: 15), and a signal peptide sequence. An example of such a vector is the vector containing SEQ ID NO: 19. SEQ ID NO: 19 further contains cloning sites.

In one embodiment, the implementation of the vector according to the invention contains SEQ ID NO: 9, a sequence encoding a light chain constant region (such as, for example, SEQ ID NO: 15), and a signal peptide sequence. An example of such a vector is the vector containing SEQ ID NO: 20. SEQ ID NO: 20 further contains cloning sites.

Another object of the invention is to create an isolated host cell containing said vector. Said host cell can be used for recombinant production of antibodies according to the invention. In one embodiment, the host cells may be prokaryotic, yeast, or eukaryotic cells, preferably mammalian cells, such as, for example, monkey kidney cell lines CV1 transformed with SV40 (COS-7, ATCC CRL 1651); human kidney embryonic cell lines (293 cells subcloned for suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary/-DHFR cells (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Sertoli mouse cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); mouse myeloma cells SP2/0-AG14 (ATCC CRL 1581, ATCC CRL 8287) or NSO (No. 85110503 in the HPA culture collection); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); Buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells; FS4 cells; a human hepatoma cell line (Hep G2); and a PERC-6 cell line from DSM. Expression vectors suitable for use in each of these host cells are also generally known in the art. It should be noted that the term "host cell" generally refers to a cultured cell line. Explicitly excluded from the definition of "host cell" is the human organism as a whole, into which an expression vector encoding an antigen-binding protein of the invention has been introduced.

Another object of the invention is to provide a method for producing an anti-hGPVI antibody or an antigen-binding fragment thereof, which comprises culturing host cells containing an isolated polynucleotide sequence encoding an anti-hGPVI antibody under conditions applicable to expressing an anti-hGPVI antibody and recovering the anti-hGPVI antibody after expression. This recombinant method can be used for large-scale production of antibodies against hGPVI according to the invention, including monoclonal antibodies intended for use in therapy, diagnostics in vitro, ex vivo, in vivo. These methods are known to those skilled in the art.

In one embodiment, the protein of the invention may be purified by chromatography, preferably affinity chromatography, more preferably L-agarose protein affinity chromatography.

Accordingly, in one embodiment, the protein of the invention comprises a protein binding domain L (PpL). Methods for transferring PpL binding activity to proteins of the invention are described in Muzard et al., Analytical Biochemistry 388, 331-338, 2009 and Lakhrif et al., MAbs. 2016; 8 (2): 379-88, which are incorporated herein by reference.

Fragments and derivatives of antibodies according to the present invention (according to the term "antibody" or "antibodies" used in this application, unless otherwise indicated or the context clearly implies otherwise), preferably AST017-like or ACT006-like antibody can be obtained known from the art ways. "Fragments" are part of an intact antibody, usually an antigen-binding site or variable region. Examples of antibody fragments include Fab, Fab', Fab'-SH, F(ab')2 and Fv fragments; diabody; fragment of any antibody, which is a protein having a primary structure consisting of one contiguous sequence of contiguous amino acid residues (referred to as a "single chain antibody fragment" or "single chain protein"), including, without limitation (1) single chain Fv molecules, (2) single chain proteins containing only one light chain variable domain, or a fragment thereof, which contains three light chain variable domain CDRs without a corresponding heavy chain portion, and (3) single-chain proteins containing only one heavy chain variable region, or a fragment thereof, containing three variable region CDRs heavy chain without the corresponding part of the light chain; and multispecific antibodies formed from antibody fragments. Fragments of antibodies according to the present invention can be obtained by standard methods. For example, Fab or F(ab')2 fragments can be prepared by protease digestion of isolated antibodies according to conventional techniques. It should be noted that immunoreactive fragments can be modified by known methods; for example, in order to slow down clearance in vivo and obtain a more desirable pharmacokinetic profile, the fragment can be modified with polyethylene glycol (PEG). Methods for binding and site-specific conjugation of PEG to a Fab' fragment are described, for example, in Leong et al., Cytokines 16 (3): 106-119 (2001) and Delgado et al., Br. J. Cancer 73 (2): 175-187 (1996), the contents of which are incorporated herein by reference.

Alternatively, the DNA encoding an antibody of the invention, preferably an ACT017-like or an ACT006-like antibody, can be modified to encode an antibody fragment of the invention. The modified DNA is then inserted into an expression vector and used to transform or transfect the appropriate cell, which then expresses the desired fragment.

One of the objectives of the invention is to provide a composition containing at least one of the proteins described above according to the invention.

Another object of the invention is to provide a pharmaceutical composition containing at least one of the proteins described above according to the invention and at least one pharmaceutically acceptable excipient.

Pharmaceutically acceptable excipients that may be used in these compositions include, without limitation, ion exchangers, alumina, aluminum stearate, lecithin, whey proteins such as human serum albumin, buffering agents such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances (e.g. sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes , block polymers of polyethylene and polyoxypropylene, polyethylene glycol and lanolin.

Another object of the invention is to provide a medicament containing at least one of the proteins described above according to the invention.

In one embodiment, said protein is an anti-hGPVI antibody, or an antigen-binding fragment thereof, that inhibits GPVI signaling and/or GPVI ligand signaling. As used herein, the term "inhibit" means that the protein is capable of blocking, restoring, preventing, or neutralizing the interaction of GPVI with its ligands, GPVI signaling, and/or activation of molecules from the GPVI signaling pathway.

Examples of GPVI ligands include, without limitation, collagen, fibrin, fibronectin, vitronectin, and laminins.

In one embodiment, said protein is an anti-hGPVI neutralizing antibody.

In one embodiment, said protein inhibits GPVI binding of its ligand (such as, for example, collagen, fibrin, or any other GPVI ligand capable of inducing downstream signaling and platelet activation).

In one embodiment, said protein inhibits and/or prevents platelet aggregation in response to a GPVI ligand, such as, for example, collagen. In one embodiment, said protein inhibits and/or prevents platelet adhesion to a GPVI ligand, such as, for example, collagen.

In one embodiment, said protein inhibits and/or prevents the formation of GPVI-dependent thrombin in response to a GPVI ligand, such as, for example, fibrin. In one embodiment, said protein inhibits and/or prevents platelet-catalyzed thrombin formation in response to collagen and/or tissue factor.

In one embodiment, said protein inhibits and/or prevents platelet recruitment by a GPVI ligand (such as, for example, fibrin) by GPVI.

In one embodiment, the protein of the invention induces platelet saturation in whole blood or platelet-rich plasma at a concentration of about 0.1 to about 10 μg/ml, preferably about 0.5 to about 5 μg/ml, and more preferably about 1 up to about 2 μg/ml (corresponding to about 2 to 200 nM, preferably about 10 to about 100 nM, more preferably about 20 to about 40 nM).

In one embodiment, a protein of the invention inhibits collagen-induced platelet aggregation when used at a concentration of at least about 15 μg/mL, preferably at least about 10 μg/mL, more preferably at least about 5 μg. /ml The protein of the invention preferably completely inhibits collagen-induced platelet aggregation when used at such concentrations.

In one embodiment, the IC50 of the protein of the invention for inhibiting collagen-induced platelet aggregation is about 0.5 to about 10 µg/ml, preferably about 1 to about 6 µg/ml, more preferably about 2 to about 3.2 µg /ml

In one embodiment, the concentration of the protein according to the invention, which reduces by 50% the rate of collagen-induced platelet aggregation, is from about 0.5 to about 5 μg/ml, preferably from about 1 to about 3 μg/ml, more preferably about 2 μg/ml. ml.

In one embodiment, the protein concentration of the invention that reduces collagen-induced platelet aggregation is about 0.5 to about 10 µg/mL, preferably about 1 to about 6 µg/mL, more preferably about 3.2 µg/mL.

In one embodiment, the protein of the invention does not induce GPVI depletion when administered in vivo, for example at a dose of 0.01 to 500 mg.

In one embodiment, the protein of the invention does not induce a decrease in the number of platelets, ie thrombocytopenia, when administered in vivo, for example at a dose of 0.01 to 500 mg.

The present invention also relates to a protein, composition, pharmaceutical composition or medicament as described above for the treatment or use in the treatment of a disease, disorder or condition associated with GPVI.

In another embodiment, the described protein, composition, pharmaceutical composition or drug is used to modulate interactions between leukocytes and platelets and between platelets and endothelium in inflammation and/or thrombosis. Thus, in one embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat inflammation and/or thrombosis.

In another embodiment, the described protein, composition, pharmaceutical composition or drug is used to modulate, preferably to prevent platelet aggregation and degranulation.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat disorders associated with abnormal or aberrant megakaryocyte and/or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation, and/or function. Examples of these disorders include, without limitation, bleeding disorders (such as, for example, bleeding tendency and/or prolonged bleeding), such as thrombocytopenia, such as, for example, idiopathic thrombocytopenic purpura (ITP) or immune thrombocytopenia.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat thrombotic disorders (such as, for example, thrombotic occlusion of the coronary arteries), hemorrhagic disorders, diseases characterized by quantitative or qualitative platelet dysfunction, and diseases characterized by endothelial dysfunction. These diseases include, without limitation, diseases of the coronary arteries and cerebral arteries.

In another embodiment, the protein, composition, pharmaceutical composition, or drug described above is used to treat cerebrovascular diseases, including stroke and ischemia, venous thromboembolic diseases (such as, for example, diseases associated with swelling, pain and ulceration of the legs, pulmonary embolism, abdominal venous thrombosis), thrombotic microangiopathy, vascular purpura.

In another embodiment, the disclosed protein, composition, pharmaceutical composition, or drug is used to treat symptoms associated with platelet dysfunction and/or disease (such as, for example, bleeding disorders). In particular, the protein, composition, pharmaceutical composition or drug described above can be used to modulate symptoms associated with ITP such as purpura and serious bleeding disorders.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat coronary artery disease (such as, for example, cardiovascular disease, including unstable angina, myocardial infarction, acute myocardial infarction, ischemic heart disease, coronary artery revascularization, coronary restenosis). arteries, ventricular thromboembolism, atherosclerosis, coronary artery disease (eg, arterial occlusive lesions), plaque formation, cardiac ischemia, including complications associated with coronary bypass procedures such as percutaneous angioplasty of the coronary arteries (balloon angioplasty)). With respect to coronary procedures, such treatment may be carried out by administering a protein according to the invention before, during or after the procedure. In one of the preferred embodiments, such an introduction can be used to prevent acute cardiac ischemia after angioplasty.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat disorders resulting from any damage to blood vessels that can lead to platelet aggregation. Such vascular lesions include, but are not limited to, vessel wall injury, such as vascular injury that results in an exposed, highly thrombogenic surface within an otherwise intact blood vessel, e.g., vessel wall injury resulting in the release of ADP, thrombin and/or adrenaline, fluid shear stress , which occurs at the site of narrowing, cracks and / or ruptures of blood vessels in areas of atherosclerotic plaques, and injuries resulting from balloon angioplasty or atherectomy.

Further, in some embodiments, it is preferred that the protein of the invention does not interfere with other platelet traits or functions, such as agonist-induced platelet reshaping (e.g., GPIb-vWF mediated platelet activation), release of platelet granule contents, activation of signal transduction pathways, or induction of calcium mobilization upon platelet activation by agonists that do not interact with GPVI.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat disorders associated with aberrant signal transduction in response to GPVI ligands (including, without limitation, collagen, fibrin, fibronectin, vitronectin, and laminins) or other extracellular matrix proteins.

In another embodiment, the described protein, composition, pharmaceutical composition or drug is used to treat disorders associated with aberrant levels of expression and/or activity of GPVI in cells that normally express GPVI or in cells that do not express GPVI. For example, a protein of the invention can be used to modulate disorders associated with aberrant expression of GPVI in cancer (eg, tumor) cells that do not normally express GPVI. Such disorders may include, for example, disorders that are associated with the migration and metastasis of tumor cells.

In another embodiment, the described protein, composition, pharmaceutical composition or drug is used to modulate platelet immunoregulatory functions.

In another embodiment, the described protein, composition, pharmaceutical composition or drug is used to treat diseases of the liver, bone marrow and peripheral blood.

In another embodiment, the described protein, composition, pharmaceutical composition, or drug is used to treat platelet-mediated disorders by modulating inflammatory responses, including, without limitation, chronic or prolonged inflammation associated with infection, arthritis, fibrosis, or disorders in which platelets modulate function. cells, including without limitation the proliferation and/or spread of cancer cells.

Other examples of diseases, disorders, or conditions associated with GPVI include, without limitation, cardiovascular disease and/or cardiovascular events such as, for example, arterial thrombosis, including atherothrombosis, ischemic events, acute coronary syndrome, myocardial infarction (heart attack), acute ischemia brain (stroke), percutaneous coronary intervention, stent thrombosis, ischemic restenosis, ischemia (acute and chronic), diseases of the aorta and its branches (such as aortic aneurysm, thrombosis), peripheral arterial disease, venous thrombosis, acute phlebitis and pulmonary embolism, cancer-related thrombosis (Trousseau's symptom), inflammatory thrombosis, and thrombosis associated with infection.

In one embodiment, the pharmaceutical composition or medicament of the invention is for the treatment or for use in the treatment of arterial or venous thrombosis, restenosis, acute coronary syndrome or cerebrovascular accident due to atherosclerosis, preferably thrombosis.

Another object of the invention is to provide a method for treating a disease, disorder or condition associated with GPVI, comprising administering to a subject in need thereof a protein, composition, pharmaceutical composition or drug according to the present invention.

A subject in need thereof is preferably administered a therapeutically effective amount of a protein according to the invention.

It is clear that the total daily dosage of the protein according to the invention, compositions, pharmaceutical compositions or drugs according to the present invention is determined by the attending physician on the basis of a reasonable medical opinion. The specific therapeutically effective dosage for any particular patient will depend on a variety of factors including the type of disease and its severity; the activity of the particular protein used; the particular composition used, age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the particular protein used; duration of treatment; drugs used in combination or simultaneously with the specific protein used; and similar factors well known in medicine. For example, it is well known to start with lower doses of a compound than are required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

However, the daily dosage of protein can vary widely from 0.01 to 500 mg per day for an adult. The compositions preferably contain about 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg an active ingredient for symptomatic dosage adjustment for the patient to be treated. The medicament usually contains from about 0.01 mg to about 1000 mg of the active ingredient, preferably from 1 mg to about 500 mg of the active ingredient.

In one embodiment, the subject is suffering, preferably the subject is diagnosed with a disease, disorder or condition associated with GPVI, preferably with a cardiovascular disease and/or event.

In another embodiment, the subject is at risk of developing a disease, disorder or condition associated with GPVI, preferably a cardiovascular disease and/or event. Examples of risk factors include, without limitation, family history (such as, for example, genetic predisposition), ethnicity, age, exposure to tobacco, high blood pressure (hypertension), high cholesterol, obesity, physical inactivity, diabetes (particularly type 2 diabetes) , unhealthy food and harm from drinking alcohol.

If used for administration to a subject, the composition will be formulated for administration to a subject. The compositions of the present invention may be administered parenterally, by inhalation aerosol, rectally, nasally, or via an implanted reservoir. As used herein, the term "administration" includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intrawound and intracranial injection or infusion methods. Examples of forms suitable for injection include, but are not limited to, solutions such as, for example, sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms useful for preparing solutions or suspensions by adding a liquid prior to use, such as, for example, a powder. , liposomal forms, and the like.

Sterile injectable forms of the compositions of the present invention may be in the form of an aqueous or oleaginous suspension. These suspensions may be formulated by methods known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic, parenterally acceptable diluent or solvent. Acceptable vehicles and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils have traditionally been used as the solvent or suspending medium. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives, as well as natural pharmaceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated forms, can be used in the preparation of injectable materials. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant such as carboxymethyl cellulose or similar dispersants commonly used in pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other common surfactants such as Tweens, Spans, and other emulsifiers or bioavailability enhancers that are widely used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms can also be used for formulation purposes.

The modes of administration and dosage of the protein in the pharmaceutical compositions of the present invention can be determined by methods known for these products, for example, in accordance with the instructions of the manufacturers. For example, the protein present in the pharmaceutical composition of the present invention may be supplied at a concentration of about 1 to about 100 mg/ml, such as, for example, 1 mg/ml, 5 mg/ml, 10 mg/ml, 50 mg/ml or 100 mg/ml. In one embodiment, the protein is supplied at a concentration of about 10 mg/ml in 100 mg (10 ml) or 500 mg (50 ml) disposable tubes. In one embodiment, the implementation of the pharmaceutical composition according to the invention may contain the protein according to the invention in PBS with a pH of 7.2-7.7. It should be appreciated that these regimens are exemplary and that the optimal schedule and regimen may be adapted to the affinity and tolerability of the particular antibody in the pharmaceutical composition, which should be determined in clinical trials.

In one embodiment, a protein of the invention may be used in vitro or in vivo to identify samples, tissues, organs, or cells that express GPVI.

Examples of assays in which a protein of the invention may be used include, without limitation, ELISA, double antibody ELISA, RIA, FACS, tissue immunohistochemistry, Western blot, and immunoprecipitation.

In one embodiment, the sample is a biological sample. Examples of biological samples include, without limitation, bodily fluids, preferably blood, more preferably serum, plasma, synovial fluid, bronchoalveolar lavage fluid, sputum, lymph, ascitic fluids, urine, amniotic fluid, abdominal fluid, cerebrospinal fluid, pleural fluid, pericardial fluid and alveolar macrophages, tissue lysates, biopsies and extracts obtained from diseased tissues.

In one embodiment, the term "sample" refers to a sample taken from a human prior to any analysis.

In another embodiment, a protein of the invention may be labeled for diagnostic or detection purposes. Labeling means that a compound has at least one attached element, isotope, or chemical compound to enable detection of the compound. Examples of labels include, without limitation, isotopic labels such as radioactive or heavy isotopes; magnetic, electrical or thermal marks; and colored or luminescent dyes. Examples of luminescent dyes include, without limitation, lanthanide complexes, quantum dots, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl coumarins, pyrene, malachite green, stilbene, Lucifer yellow, Cascade blue, Texas red, Alexa dyes, and cyanine dyes.

Another object of the invention is to provide a kit containing at least one protein according to the invention.

By "kit" is meant any article (eg, package or container) containing at least one reagent, ie, for example, an antibody for specifically detecting GPVI expression. The kit may be offered, supplied or sold as a kit for carrying out the methods of the present invention. In addition, any or all of the kit's reagents may be supplied in containers that protect them from the outside environment, such as sealed containers. The kits may also contain an package leaflet describing the kit and how to use it.

The present invention further relates to a method for inhibiting GPVI receptor function and downstream signaling to thereby treat a GPVI associated condition, said method comprising administering to a subject a protein of the invention.

In one embodiment, the method of inhibiting GPVI receptor function and downstream signaling does not affect platelet count, platelet surface expression of GPVI, or bleeding time.

In one embodiment, the method for inhibiting GPVI receptor function and downstream signaling is effective and reversible.

The present invention also relates to a method for inhibiting the binding of GPVI to its ligands (preferably, but not exclusively, collagen) to thereby treat a GPVI-associated condition, said method comprising administering to the subject a protein of the invention.

In one embodiment, a method for inhibiting GPVI binding of its ligands does not affect platelet count, platelet surface expression of GPVI, or bleeding time.

In one embodiment, the method for inhibiting the binding of GPVI to its ligands is effective and reversible.

The present invention further relates to a method for inhibiting and/or preventing platelet and collagen adhesion to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention also relates to a method for inhibiting and/or preventing collagen-induced platelet aggregation to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention further relates to a method for inhibiting and/or preventing platelet activation, in particular platelet aggregation, in response to collagen to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention further relates to a method for inhibiting and/or preventing the formation of thrombin in response to collagen and/or tissue factor, thereby treating a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention further relates to a method for inhibiting GPVI binding of fibrin to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention further relates to a method for inhibiting and/or preventing platelet recruitment by fibrin by GPVI to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

The present invention further relates to a method for inhibiting and/or preventing GPVI-dependent thrombin formation in response to fibrin to thereby treat a GPVI-associated condition, said method comprising administering to a subject a protein of the invention.

In one embodiment, administration of a protein of the invention to a subject does not cause depletion of GPVI in vivo.

In one embodiment, administering a protein of the invention to a subject does not cause a decrease in platelet count. Thus, in one embodiment, administration of a protein of the invention to a subject does not cause thrombocytopenia.

In one embodiment, administration of a protein of the invention to a subject does not cause an increase in bleeding time.

In one embodiment, the method of the invention comprises administering to a subject a therapeutically effective amount of a protein of the invention.

Brief description of the drawings

In FIG. 1 is a photograph of a blue Coomassie stained gel showing a protein according to the invention. MM: molecular weight marker, FT: flux through the fraction, PX: purified protein (eluted from protein L-agarose column); CM: conditioned culture medium.

In FIG. 2 is a graph illustrating the isothermal binding curves of two humanized Fabs of the invention. Placed purified GPVI-Fc on microtiter plates and added purified proteins in increasing concentrations. After washing, bound proteins were detected using an alkaline phosphatase-conjugated anti-human Fab specific antibody, and hydrolysis of the alkaline phosphatase substrate at 485 nm was measured.

In FIG. 3 shows a curve plotted from flow cytometry using Alexa488-conjugated ACT017 antibody. AST017 was conjugated with A488 according to the manufacturer's instructions. A488-ACT0017 conjugate was added at increasing concentrations to anti-clotting whole blood or platelet-rich plasma obtained from a healthy volunteer. After 20 minutes at room temperature, the samples were diluted and immediately analyzed by flow cytometry. The average platelet fluorescence is shown depending on the concentration of AKT017 conjugated with A488. Data from one of three representative experiments is presented.

In FIG. 4 is a curve illustrating platelet aggregation measured in platelet rich plasma (PRP). Within 10 minutes, PRP was pre-incubated with increasing concentrations of purified ACT017 at 37°C without stirring until platelet aggregation was initiated by collagen (1 μg.ml ^-1 ). Continued incubation was prolonged at 37°C with stirring and continuous recording of changes in light transmission. Data from one of three representative experiments are presented.

In FIG. 5 is a graph illustrating the ability of ACT017 to inhibit collagen-induced platelet aggregation quantified by the rate and intensity of the response. We measured the intensity and rate of platelet aggregation at each concentration of ACT017. Residual responses were calculated as the ratio of responses in the presence and absence of ACT017×100 and plotted against AST017 concentration using a non-linear regression curve of competition success (logarithm (inhibitor) of relative response) (three parameters) from Graph Pad Prism software (5.0 ). Shown are curves from one of three representative experiments.

In FIG. 6 shows a combination of graphs illustrating collagen-induced platelet aggregation. The maximum degree of platelet aggregation induced by the addition of collagen (1 μg/ml) to platelet-rich plasma (PRP) of mice treated with vehicle or increasing doses of ACT017 was recorded.

In FIG. 7 shows a combination of graphs illustrating the expression of GPVI in platelets. Blood obtained before injection and 30 min after injection of vehicle or increasing doses of AST was incubated with FITC-conjugated monoclonal antibody 3J24 against GPVI. Left panel: Average fluorescence of platelets from mice treated with vehicle (0 mg/kg ACT017) or AST017 at various doses. Middle and right panels: evolution of GPVI expression in individual animals treated with vehicle or ACT017 (4 mg/kg) from pre-injection to 30 minutes post-injection.

In FIG. 8 is a graph illustrating the blood platelet count of mice after administration of ACT017 (4 mg/kg) or vehicle (0 mg/kg ACT017).

In FIG. 9 shows a combination of graphs illustrating bleeding time (A) and blood loss (B) in mice 30 min after administration of vehicle (Ctrl), AST017 (4 mg/kg) or clopidogrel, an adenosine diphosphate receptor antagonist P2Y12 in platelets (10 mg/kg ).

In FIG. 10 shows a combination of graphs illustrating the mean intensity ± standard error of the mean (SEM) (A) and rate (B) of collagen-induced platelet aggregation measured 30 minutes and 2 hours after the end of the 15-minute incremental infusion of ACT017 (1, 2 , 4.8 mg/kg) cynomolgus monkeys (n=4).

In FIG. 11 shows platelet counts in cynomolgus monkeys measured before any treatment (time = 0) or 24 hours after infusion with vehicle or AST017 at increasing doses (1, 2, 4, 8 mg/kg).

In FIG. 12 shows a combination of graphs illustrating bleeding time measured in 4 subjects before treatment (time = 0) and 30 minutes and 2 hours after infusion of ACT017 (1, 2, 4, 8 mg/kg) or vehicle to cynomolgus monkeys.

In FIG. 13 shows a combination of graphs illustrating the mean intensity ± standard deviation (SD) (A) and rate (B) of collagen-induced platelet aggregation measured at different times after initiation of a one-hour infusion of two doses of AST017 (2 and 8 mg/kg) to cynomolgus monkeys (n =8).

In FIG. 14 is a graph illustrating the expression level of GPVI measured by flow cytometry in cynomolgus monkey platelets (n=8) at various times after the start of a one-hour infusion of ACT017 at a dose of 2 mg/kg (MFI: mean fluorescence intensity).

Examples

The present invention is further illustrated by the following examples.

Example 1. Obtaining ACT017 and ACT006

AST017 and AST006 antibodies (Fab fragments) were obtained in the CHO-S cell line (Invitrogen) by transient transfection under standard conditions according to the manufacturer's instructions.

CHO-S cells were split and seeded immediately before transfection in 10 ml of serum-free growth medium at a density of 0.5-1.0×10 ⁶ cells/ml. The cells were then transfected with a vector containing the sequence encoding the light chain and heavy chain of the antibody of the invention. Culture supernatants were recovered 4-5 days after transfection, when cell viability was less than 80%.

In the case of ACT017, cells were transfected using a vector containing SEQ ID NO: 19 and SEQ ID NO: 18, wherein SEQ ID NO: 19 contains sequences encoding the constant and variable regions of the ACT017 light chain fused to the signal peptide sequence and cloning sites , a SEQ ID NO: 18 contains sequences encoding AST017 heavy chain constant and variable regions fused to a signal peptide sequence and cloning sites.

In the case of ACT006, cells were transfected using a vector containing SEQ ID NO: 20 and SEQ ID NO: 18, wherein SEQ ID NO: 20 contains sequences encoding ACT006 light chain constant and variable regions fused to a signal peptide sequence and cloning sites , a SEQ ID NO: 18 contains sequences encoding AST006 heavy chain constant and variable regions fused to a signal peptide sequence and cloning sites.

For purification, the conditioned cell medium transiently transfected with the humanized Fab cDNA was placed on a protein L-agarose column (PIERCE protein L-agarose, cat. no. 20510017) according to the manufacturer's instructions. The Fab was eluted using 0.1 M glycine pH 2.5 and fractions pooled on 1 M Tris pH 11 to neutralize the pH. After dialysis on PBS, the Fab concentration was determined by measuring the absorbance at 280 nm and 320 nm corrected for light scattering and using the absorbance value of 1.5 determined according to the sequence for 0.1% solutions (A _{280 nm} ^{0.1 %} ). The protein concentration was confirmed by the BCA method. Fab purity was assessed based on SDS-PAGE and Coomassie blue staining.

As shown in FIG. 1, under non-reducing conditions, the purified humanized Fab migrated as the 42 kDa major band according to its amino acid sequence. After the reduction of the disulfide bridge, a doublet with a molecular weight of 23-24 kDa was observed, corresponding to the heavy and light chains.

Example 2 Binding of GPVI-Fc

Soluble GPVI-Fc was prepared as follows: the open reading frame of the predicted extracellular domain of GPVI was subjected to PCR amplification from the Kozak sequence to the first methionine in asparagine 269 just before the predicted transmembrane sequence. The PCR fragment was ligated into the host vector pCDM8 containing the genomic sequence of the human IgG1 Fc domain, as a result of which the extracellular portion at the C-terminus of the hGPVI cDNA was fused via a 3-alanine linker to the hFc sequence. The sequenced DNA construct was transfected into HEK 293T cells. GPVI-Fc was purified from the conditioned cell medium by affinity chromatography on protein A-agarose according to the manufacturer's instructions.

Placed GPVI-Fc in PBS (2 μg/ml, 100 per well) on microtiter plates and kept overnight at 4°C. Within 120 minutes, non-specific binding sites were saturated with 100 μl of 1% BSA in PBS. The plates were then incubated for 120 minutes at increasing concentrations of antibody preparations (100 μl in PBS containing 0.1% BSA and 0.1% Tween 20). After 4 wash cycles, plates were incubated for 120 minutes with alkaline phosphatase-conjugated mouse anti-human Fab antibody (100 μl in PBS containing 0.1% BSA and 0.1% Tween 20). Finally, 100% substrate solution (p-nitrophenyl phosphate) was added to each well over 5 minutes, and the colorimetric reaction was stopped with 25 µl of 3 M NaOH before absorbance reading at 405 nm. Alternatively, antibody-bound GPVI was detected using peroxidase-conjugated L-protein, and O-phenylenediamine dihydrochloride (OPD) as substrate, followed by reading at 485 nm.

In FIG. 2 shows isothermal binding curves of two humanized Fabs according to the invention of immobilized GPVI-Fc. Analysis of the curves made it possible to calculate a half-saturation constant of 1 nM for AST006 and 0.5 nM for AST017.

Example 3 Surface Plasmon Resonance (BIAcore)

The binding of ACT017, AST006 and Fab 9012 of soluble human GPVI was analyzed by surface plasmon resonance using the BIAcore 2000 system (Uppsala, Sweden).

Soluble GPVI-Fc (prepared as described in Example 2) was immobilized in 10 mM acetate buffer pH 6 at a dose of 960-1071 RU (corresponding to about 6.4-7.15 fmol/mm ² ) on a CM5 carboxy-sensor chip. methyldextran by the amine coupling method (Wizard procedure). The protein was then passed over immobilized GPVI-Fc in PBS pH 7.4 (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH 7.42 at 25.4°C) at a rate of 20 μl/h at 25°C. FROM. Kinetic constants (K _A , K _D , k _on , k _off ) and affinity were determined using version 3.0 of the BIAevaluation software by fitting the data to a coupling model. PBS pH 7.4 is running buffer.

The results are shown in Table 1.

Thus, the antibodies of the invention (ACT017 and ACT006) have an increased affinity for soluble GPVI compared to the monoclonal Fab 9012, which is described in the prior art and tested under the same experimental conditions.

Example 4

biological data

Materials and methods

human platelets

Blood was collected by venipuncture from healthy volunteers who had not taken medication for 10 days in syringes with 3.2% sodium citrate (Vacutainer Beckton Dickinson, Les Ponts-de-Clais, France). All blood donors gave voluntary and informed written consent to participate in this study, which complies with the ethical standards of the Declaration of Helsinki.

flow cytometry

The humanized ACT017 Fab was conjugated to Alexa488 (Molecular Probes) as recommended by the manufacturer. Separation was carried out using a conjugated antibody purification kit (Molecular Probes) and the concentration was determined at 280 and 494 nm. A488-conjugated humanized Fab at various concentrations was incubated at room temperature in the dark with human platelets in whole blood or platelet-rich plasma for 20 minutes. After dilution in PBS, the cells were analyzed in a flow cytometer with cell sorting with fluorescence excitation (FACS LSR II BD).

Platelet aggregation

Received platelet rich plasma (PRP) after centrifugation (120×g, 15 minutes; 20°C) and immediately used. Platelet aggregation was measured using the APACT® aggregator, which was initiated by adding 1 μg.ml ^-1 collagen type I (Horm collagen, Nycomed, Germany) in PRP containing various concentrations of humanized Fab, with continuous recording. The rate and intensity of aggregation were measured.

Humanized GPVI Mice

A gp6 mutant mouse strain was generated, as previously reported, by introducing the human gp6 gene sequence into exon 1 of the mouse gp6 gene ATG (Mangin P et al., Humanized Glycoprotein VI (GPVI) Mouse Model to Assess the Antithrombotic Efficacies of Anti- GPVI Agents J Pharmacol Exp Ther 2012; 341(1): 156-63 doi: 10.1124/jpet.111.189050 Epub 2012, Jan 11). The mice were viable and capable of reproduction and did not have any hematological defects. Approximately 3700 copies of human GPVI were found on the surface of platelets.

Platelet count

Collected whole blood in syringes with EDTA (6 mm) after dissection of the tail of mice. The platelet count was determined using a SCIL Vet abc™ veterinary hematology analyzer (Scil Animal Care, Oltsheim, France) tuned to mouse parameters.

Ex vivo platelet aggregation

Blood was collected in sodium citrate syringes prior to injection and 30 minutes after injection of increasing doses of ACT017 or an equivalent volume of vehicle and centrifuged PRP. The platelet count was corrected to 300 10 ⁹ ⋅l ^-1 and platelet aggregation initiated by 1 μg ml ^-1 collagen type I was continuously recorded in a four-channel CARAT TX4 aggregator.

GPVI expression

Blood samples were collected in EDTA syringes prior to injection and 30 minutes after injection of increasing doses of AST017 or an equivalent volume of vehicle and incubated with FITC-conjugated anti-GPVI monoclonal antibody 3J24 (10 μg ml ^-1 ), which recognizes human GPVI at an epitope differing from the AST017 epitope, and measured fluorescence on a Beckman Coulter Gallios Flow cytometer.

Length of tail bleeding and blood loss

Cut the end of the tail of the anesthetized mice with a scalpel in the transverse direction (3 mm) and immediately immersed in a test tube with 13 ml of 0.9% isotonic saline at a temperature of 37°C. Within 30 minutes, the bleeding was observed and measured, which, if necessary, was manually stopped after 10 minutes to prevent death. The volume of blood lost was measured based on the hemoglobin content.

Javanese macaques

Used for research 8 cynomolgus macaques that had not previously received any treatment. Platelet aggregation was measured essentially as described for human platelets using collagen at a concentration of 2.5 mg⋅ml ^-1 . The number of platelets in blood protected from clotting with EDTA was measured. The duration of bleeding was measured in awake monkeys on the surface of the forearm according to standard clinical procedure using a 0.5 cm Surgicutt™ device to measure the duration of bleeding using. GPVI expression was measured essentially as described above using a commercially available FITC-conjugated anti-human GPVI monoclonal antibody (clone 1G5, Biocytex) that cross-reacts with cynomolgus monkey GPVI.

results

In vitro binding of human platelets by ACT017 antibody

In FIG. 3 shows a typical Alexa488-conjugated human platelet ACT017 antibody binding curve as measured by flow cytometry. Saturation of whole blood and PRP with platelets is achieved at an antibody concentration of 1-2 μg/ml (20-40 nM).

In Vitro Inhibition of Collagen-Induced Human Platelet Aggregation

In FIG. 4 shows typical aggregation curves obtained after pre-incubation of human PRP with increasing concentrations of AST. Under these conditions, ACT017 completely inhibited collagen-induced platelet aggregation at a concentration of ≥5 µg/mL.

The ability of AST017 to inhibit collagen-induced platelet aggregation was quantified by the rate and intensity of the response, as shown in FIG. 5. The mean IC50 of intensity and rate for three different donors was 3.2±2.5 and 2±0.7 µg⋅ml ^-1 , respectively, with total inhibition in both cases observed at a concentration of 6.7±2, 9 μg⋅ml ^-1 which is in good agreement with the results of platelet binding.

Thus, these results demonstrate that the proteins of the invention are potent inhibitors of collagen-induced platelet aggregation.

For the purpose of ex vivo analysis of the effects of ACT017 on collagen-induced platelet aggregation, platelet count and GPVI expression, humanized GPVI mice were injected intravenously with ACT017 (1.2 or 4 mg/kg) or vehicle. Blood was collected 30 minutes after injection for analysis of collagen-induced platelet aggregation, platelet count, and GPVI expression.

As shown in FIG. 6, complete inhibition of collagen-induced platelet aggregation was observed when mice received ACT017 at a dose of ≥0.5 mg/kg.

With regard to GPVI expression, there was no statistical difference in mean platelet fluorescence between samples obtained before and after treatment, and between samples taken from mice treated with vehicle (0 mg/kg AST017) or AST017 at various doses (Fig. 7, left panel). The middle and right panels in Fig. 7 illustrate the evolution of GPVI expression from pre-injection to 30 minutes post-injection in selected animals treated with vehicle or 4 mg/kg ACT017.

These results indicate that ACT017 does not cause GPVI depletion in vivo.

In addition, the platelet count was measured in mice treated with vehicle (0 mg/kg ACT017) or AST017 (4 mg/kg). As shown in FIG. 8, no statistical difference between the two groups was confirmed (mean ± standard deviation: 847.7 ± 110.5 vs. 842 ± 115.5).

Bleeding time and blood loss, measured by tail cutting of mice 30 min after administration of ACT017 (4 mg/kg), did not change compared to control (vehicle-injected) mice (FIG. 9). In comparison, mice treated with clopidogrel (10 mg/kg the day before and two hours before the experiment) significantly increased the duration of bleeding, as well as blood loss.

This result demonstrates that AST017 does not cause a decrease in platelet count, i.e. thrombocytopenia in vivo.

Additionally, the effect of AST017 when administered to primates other than humans was characterized. Eight cynomolgus monkeys participated in the study. First, increasing doses of AST017 (1, 2, 4, 8 mg/kg) were administered intravenously over 15 minutes. Blood was collected 30 minutes and 2 hours after administration. Platelet aggregation was reversibly inhibited in a dose-dependent manner (FIG. 10). Increasing the dose from 1 to 2 and from 2 to 4 mg/kg increased the effect, and the 8 mg/kg dose had no additional effect compared to the 4 mg/kg dose. The platelet count measured 24 hours after the injection did not change compared to the values obtained before the injection (FIG. 11). After administration of vehicle or 2, 4, or 8 mg/kg of AST017 (FIG. 12), no significant increase in bleeding time was observed. Then, after washout, cynomolgus monkeys received ACT017 (2 or 8 mg/kg) as a one hour perfusion. Platelet aggregation and GPVI expression were measured at various times after the start of treatment: (after 1, 1.5, 2, 4 and 7 hours) (FIGS. 13 and 14). In macaques treated with 8 mg/kg, collagen-induced platelet aggregation was reversibly inhibited and the duration of effect increased compared to the 2 mg/kg dose (FIG. 13). The expression of GPVI in platelets remained stable at different times after the start of treatment compared with pre-treatment (Fig. 14).

Together, these results confirm that AST017 administration to non-human primates effectively and reversibly inhibits GPVI function without affecting platelet count, platelet surface GPVI expression, or bleeding time.

Example 5 Epitope Mapping

Materials and methods

In order to reconstruct the epitopes of target molecules, ie human GPVI, a library of peptides was synthesized. An amino-functionalized polypropylene support was prepared by grafting a proprietary hydrophilic polymer composition followed by reaction with tert-butyloxycarbonyl-hexamethylenediamine (BocHMDA) using dicyclohexylcarbodiimide (DCC) with N-hydroxybenzotriazole (HOBt) and then Boc cleavage using trifluoroacetic acid (TFA). Standard Fmoc peptide synthesis was used to synthesize peptides on an amino-functionalized solid support using custom JANUS fluid handlers (Perkin Elmer).

The synthesis of structural simulations was carried out using Pepscan's proprietary Chemically Linked Peptides on Scaffolds (CLIPS) technology. CLIPS technology allows structuring peptides into single loops, double loops, triple loops, leaf folds, helical folds, and combinations thereof. CLIPS templates were conjugated to cysteine residues. Multiple cysteine side chains were conjugated in peptides with one or two CLIPS templates. For example, a 0.5 mM solution of P2 CLIPS (2,6-bis(bromomethyl)pyridine) was dissolved in ammonium bicarbonate (20 mM, pH 7.8)/acetonitrile (1:3 by volume). This solution was added to the peptide matrices. The CLIPS template linked the side chains of the two cysteines present in the solid phase linked peptides of the peptide arrays (455 well plate with 3 µl wells). Peptide matrices completely covered with the solution were gently shaken for 30-60 minutes. Finally, the peptide matrices were washed extensively with copious amounts of H ₂ O and sonicated for 30 minutes in a buffer solution containing 1% SDS/0.1% beta-mercaptoethanol in PBS (pH 7.2) at 70°C followed by sonication sonicated in H ₂ O for another 45 minutes. T3 CLIPS containing the peptides but with three cysteines was prepared in a similar manner.

AST017 antibody binding was tested for each of the synthesized peptides by PEPSCAN-based ELISA. The peptide matrices were incubated with the primary antibody solution (overnight at 4°C). After washing for one hour, the peptide arrays were incubated with a 1/1000 dilution of the appropriate peroxidase-antibody conjugate (SBA, goat antibody-human HRP conjugate, Southern Biotech, 2010-05) at 25°C. After washing, the peroxidase substrate 2,2'-azino-di-3-ethylbenzothiazoline sulfonate (ABTS) and 20 μl/ml 3% H ₂ O ₂ were added. After one hour, the color development was measured. Presented the development of color in a quantitative form using a charge-coupled device (CCD) camera and an image processing system.

The values determined using the CCD camera ranged from 0 to 3000 mA, which is similar to the readings of a standard ELISA reader for 96-well plates. The results were quantified and stored in the Peplab database.

In order to check the quality of the synthesized peptides, a separate set of positive and negative control peptides was synthesized in parallel. They were screened with antibody 57.9 (Posthumus et al., J. Virology, 1990, 64: 3304-3309).

We analyzed the intensity profiles for each set of peptides (linear, helical, looped, discontinuous) and assessed the systematic binding.

results

After several rounds of condition optimization, ACT017 was shown to systematically recognize a discontinuous epitope consisting of peptide sequencing fragments GPAVSSGGDVTLQCQ and TVTAAHSGTYRCYSF, of which GPAVSSGGDVTLQCQ is the dominant part of the recognition site, since CLIPS-restricted peptides containing this sequence can also be bound by ACT017; however, there is no detectable binding to linear peptides containing this sequence.

Without wishing to be bound by theory, it is believed that the TVTAAHSGTYRCYSF peptide sequencing fragment likely provides additional structural binding context.

A peptide sequencing fragment of GPAVSSGGDVTLQCQ in mouse GPVI is significantly modified by several non-synonymous mutations; in contrast, it is highly conserved in primates other than humans. Without being bound by any theory, this is believed to explain why ACT017 binds human GPVI and non-human primate GPVI, but not mouse GPVI.

Without wishing to be bound by any theory, it is believed that ACT017 binding of the identified epitope should impair collagen binding of the Ig-like domain 1 C2 (D1) of GPVI and/or GPVI dimerization and subsequent high affinity to collagen.

--->

Sequence listing

<110> AKTIKOR BIOTEK [FR]

BILALDE, Philip; GENRO-PERRUS, Martinet

<120> Protein associated with human glycoprotein

<140> PCT/EP2016/068778

<170> BiSSAP 1.3.6

<210> SEQ ID NO: 1

<211> 10

<212> PRT

<213> Artificial sequence

<223> VH-CDR1

<400>

Gly Tyr Thr Phe Thr Ser Tyr Asn Met His

1 5 10

<210> SEQ ID NO: 2

<211> 17

<212> PRT

<213> Artificial sequence

<223> VH-CDR2

<400>

Gly Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe Gln

1 5 10 15

gly

<210> SEQ ID NO: 3

<211> 11

<212> PRT

<213> Artificial sequence

<223> VH-CDR3

<400>

Gly Thr Val Val Gly Asp Trp Tyr Phe Asp Val

1 5 10

<210> SEQ ID NO: 4

<211> 16

<212> PRT

<213> Artificial sequence

<223> VL-CDR1

<400>

Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> SEQ ID NO: 5

<211> 7

<212> PRT

<213> Artificial sequence

<223> VL-CDR2

<400>

Arg Val Ser Asn Arg Phe Ser

fifteen

<210> SEQ ID NO: 6

<211> 9

<212> PRT

<213> Artificial sequence

<223> VL-CDR3

<400>

Leu Gln Leu Thr His Val Pro Trp Thr

fifteen

<210> SEQ ID NO: 7

<211> 120

<212> PRT

<213> Artificial sequence

<223> Heavy chain variable region

<400>

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Gly Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Val Val Gly Asp Trp Tyr Phe Asp Val Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> SEQ ID NO: 8

<211> 113

<212> PRT

<213> Artificial sequence

<223> Light chain variable region

<400>

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Leu

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Thr

100 105 110

Arg

<210> SEQ ID NO: 9

<211> 113

<212> PRT

<213> Artificial sequence

<223> Light chain variable region

<400>

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala

35 40 45

Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Leu

85 90 95

Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg

<210> SEQ ID NO: 10

<211> 360

<212> DNA

<213> Artificial sequence

<223> Heavy chain variable region

<400>

caggttcagc tggttcagtc aggggctgag gtgaagaagc ctggagcctc agtgaaggtg 60

tcctgcaagg cttctggcta cacatttacc agttacaata tgcactggggt aagacaggct 120

cctggacagg gcctggaatg gatgggaggt atttatccag gaaatggtga tacttcctac 180

aatcagaagt tccagggccg agttactatg actcgggaca cttccacctc tacagtgtac 240

atggagctca gcagcctgag atctgaggac accgcggtct attactgtgc aagaggcacc 300

gtggtcggcg actggtactt cgatgtgtgg ggccaaggca ccctggtcac cgtgagcagt 360

<210> SEQ ID NO: 11

<211> 339

<212> DNA

<213> Artificial sequence

<223> Heavy chain variable region

<400>

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagagtgacc 60

atcacctgta gaagtagtca gagccttgag aacagcaacg gaaacaccta cctgaattgg 120

taccagcaga agccaggtaa ggctccaaag ctgctgatct acagagtttc caaccgattc 180

tctggtgtgc caagcagatt cagcggtagc ggtagcggta ccgacttcac cttcaccatc 240

agcagcctcc agccagagga catcgccacc tactactgcc tccagctgac tcatgtccca 300

tggaccttcg gtcagggcac caaggtggag atcacccgg 339

<210> SEQ ID NO: 12

<211> 339

<212> DNA

<213> Artificial sequence

<223> Light chain variable region

<400>

gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggtga cagagtgacc 60

atcacctgta gtgccagtca gagccttgag aacagcaacg gaaacaccta cctgaattgg 120

taccagcaga agccaggtaa ggctccaaag ctgctgatct acagagtttc caaccgattc 180

tctggtgtgc caagcagatt cagcggtagc ggtagcggta ccgacttcac cctcaccatc 240

agcagcctcc agccagagga cttcgccacc tactactgcc tccagctgac tcatgtccca 300

tggaccttcg gtcagggcac caaggtggag atcaaacgc 339

<210> SEQ ID NO: 13

<211> 339

<212> PRT

<213> Homo sapiens

<400>

Met Ser Pro Ser Pro Thr Ala Leu Phe Cys Leu Gly Leu Cys Leu Gly

1 5 10 15

Arg Val Pro Ala Gln Ser Gly Pro Leu Pro Lys Pro Ser Leu Gln Ala

20 25 30

Leu Pro Ser Ser Leu Val Pro Leu Glu Lys Pro Val Thr Leu Arg Cys

35 40 45

Gln Gly Pro Pro Gly Val Asp Leu Tyr Arg Leu Glu Lys Leu Ser Ser

50 55 60

Ser Arg Tyr Gln Asp Gln Ala Val Leu Phe Ile Pro Ala Met Lys Arg

65 70 75 80

Ser Leu Ala Gly Arg Tyr Arg Cys Ser Tyr Gln Asn Gly Ser Leu Trp

85 90 95

Ser Leu Pro Ser Asp Gln Leu Glu Leu Val Ala Thr Gly Val Phe Ala

100 105 110

Lys Pro Ser Leu Ser Ala Gln Pro Gly Pro Ala Val Ser Ser Gly Gly

115 120 125

Asp Val Thr Leu Gln Cys Gln Thr Arg Tyr Gly Phe Asp Gln Phe Ala

130 135 140

Leu Tyr Lys Glu Gly Asp Pro Ala Pro Tyr Lys Asn Pro Glu Arg Trp

145 150 155 160

Tyr Arg Ala Ser Phe Pro Ile Ile Thr Val Thr Ala Ala His Ser Gly

165 170 175

Thr Tyr Arg Cys Tyr Ser Phe Ser Ser Arg Asp Pro Tyr Leu Trp Ser

180 185 190

Ala Pro Ser Asp Pro Leu Glu Leu Val Val Thr Gly Thr Ser Val Thr

195 200 205

Pro Ser Arg Leu Pro Thr Glu Pro Ser Ser Val Ala Glu Phe Ser

210 215 220

Glu Ala Thr Ala Glu Leu Thr Val Ser Phe Thr Asn Lys Val Phe Thr

225 230 235 240

Thr Glu Thr Ser Arg Ser Ile Thr Thr Ser Pro Lys Glu Ser Asp Ser

245 250 255

Pro Ala Gly Pro Ala Arg Gln Tyr Tyr Thr Lys Gly Asn Leu Val Arg

260 265 270

Ile Cys Leu Gly Ala Val Ile Leu Ile Ile Leu Ala Gly Phe Leu Ala

275 280 285

Glu Asp Trp His Ser Arg Arg Lys Arg Leu Arg His Arg Gly Arg Ala

290 295 300

Val Gln Arg Pro Leu Pro Pro Leu Pro Pro Leu Pro Gln Thr Arg Lys

305 310 315 320

Ser His Gly Gly Gln Asp Gly Gly Arg Gln Asp Val His Ser Arg Gly

325 330 335

Leu Cys Ser

<210> SEQ ID NO: 14

<211> 321

<212> DNA

<213> Artificial sequence

<223> Heavy chain constant region

<400>

gcctccacca agggtccctc agtcttccca ctggcaccct cctccaagag cacctctggt 60

ggcacagctg ccctgggctg cctggtcaag gactacttcc cagaaccagt gactgtgtca 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc ctgctgtctt gcagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtcgagcct 300

aagtcatgcg acaagactca c 321

<210> SEQ ID NO: 15

<211> 318

<212> DNA

<213> Artificial sequence

<223> Light chain constant region

<400>

actgtggctg caccaagtgt gttcatcttc ccacctagcg atgagcagtt gaaatctgga 60

actgcctctg tcgtgtgcct cctgaacaac ttctacccac gggaggccaa ggtacagtgg 120

aaggtggata acgccctcca atccggtaac tcccaggaga gtgtcacaga gcaagatagc 180

aaggacagca cctacagcct cagcagcacc ctgactctga gcaaagcaga ctacgagaag 240

cacaaggtct acgcctgcga agtcacccat cagggcctga gttcccctgt cacaaagagc 300

ttcaaccggg gagagtgt 318

<210> SEQ ID NO: 16

<211> 66

<212> DNA

<213> Artificial sequence

<223> Signal peptide

<400>

atggatatgc gtgtaccagc tcaactactt ggacttctat tgctttggct tcgtggtgct 60

agategt 66

<210> SEQ ID NO: 17

<211> 57

<212> DNA

<213> Artificial sequence

<223> Signal peptide

<400>

atggactgga cttggagaat cctattcttg gttgctgcag ctacaggtgc tcattca 57

<210> SEQ ID NO: 18

<211> 762

<212> DNA

<213> Artificial sequence

<223> Heavy chain

<400>

gcggccgcca ccatggactg gacttggaga atcctattct tggttgctgc agctacaggt 60

gctcattcac aggttcagct ggttcagtca ggggctgagg tgaagaagcc tggagcctca 120

gtgaaggtgt cctgcaaggc ttctggctac acatttacca gttacaatat gcactgggta 180

agacaggctc ctggacaggg cctggaatgg atgggaggta tttatccagg aaatggtgat 240

acttcctaca atcagaagtt cggggccga gttactatga ctcgggacac ttccacctct 300

acagtgtaca tggagctcag cagcctgaga tctgaggaca ccgcggtcta ttactgtgca 360

agaggcaccg tggtcggcga ctggtacttc gatgtgtggg gccaaggcac cctggtcacc 420

gtgagcagtg cctccaccaa gggtccctca gtcttcccac tggcaccctc ctccaagagc 480

acctctggtg gcacagctgc cctgggctgc ctggtcaagg actacttccc agaaccagtg 540

actgtgtcat ggaactcagg cgccctgacc agcggcgtgc acaccttccc tgctgtcttg 600

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 660

acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 720

gtcgagccta agtcatgcga caagactcac tgatgaggat cc 762

<210> SEQ ID NO: 19

<211> 742

<212> DNA

<213> Artificial sequence

<223> Light chain

<400>

gacgtcacca tggatatgcg tgtaccagct caactacttg gacttctatt gctttggctt 60

cgtggtgcta gatgtgacat ccagatgacc cagagcccaa gcagcctgag cgccagcgtg 120

ggtgacagag tgaccatcac ctgtagaagt agtcagagcc ttgagaacag caacggaaac 180

acctacctga attggtacca gcagaagcca ggtaaggctc caaagctgct gatctacaga 240

gtttccaacc gattctctgg tgtgccaagc agattcagcg gtagcggtag cggtaccgac 300

ttcaccttca ccatcagcag cctccagcca gaggacatcg ccacctacta ctgcctccag 360

ctgactcatg tcccatggac cttcggtcag ggcaccaagg tggagatcac ccggactgtg 420

gctgcaccaa gtgtgttcat cttcccacct agcgatgagc agttgaaatc tggaactgcc 480

tctgtcgtgt gcctcctgaa caacttctac ccacgggagg ccaaggtaca gtggaaggtg 540

gataacgccc tccaatccgg taactcccag gagagtgtca cagagcaaga tagcaaggac 600

agcacctaca gcctcagcag caccctgact ctgagcaaag cagactacga gaagcacaag 660

gtctacgcct gcgaagtcac ccatcagggc ctgagttccc ctgtcacaaa gagcttcaac 720

cggggagagt gttgatgata tc 742

<210> SEQ ID NO: 20

<211> 742

<212> DNA

<213> Artificial sequence

<223> Light chain

<400>

gacgtcacca tggatatgcg tgtaccagct caactacttg gacttctatt gctttggctt 60

cgtggtgcta gatgtgacat ccagatgacc cagagcccaa gcagcctgag cgccagcgtg 120

ggtgacagag tgaccatcac ctgtagtgcc agtcagagcc ttgagaacag caacggaaac 180

acctacctga attggtacca gcagaagcca ggtaaggctc caaagctgct gatctacaga 240

gtttccaacc gattctctgg tgtgccaagc agattcagcg gtagcggtag cggtaccgac 300

ttcaccctca ccatcagcag cctccagcca gaggacttcg ccacctacta ctgcctccag 360

ctgactcatg tcccatggac cttcggtcag ggcaccaagg tggagatcaa acgcactgtg 420

gctgcaccaa gtgtgttcat cttcccacct agcgatgagc agttgaaatc tggaactgcc 480

tctgtcgtgt gcctcctgaa caacttctac ccacgggagg ccaaggtaca gtggaaggtg 540

gataacgccc tccaatccgg taactcccag gagagtgtca cagagcaaga tagcaaggac 600

agcacctaca gcctcagcag caccctgact ctgagcaaag cagactacga gaagcacaag 660

gtctacgcct gcgaagtcac ccatcagggc ctgagttccc ctgtcacaaa gagcttcaac 720

cggggagagt gttgatgata tc 742

<210> SEQ ID NO: 21

<211> 4

<212> PRT

<213> Artificial sequence

<223> CDR-L3 - residue after

<221> site

<222> 3

<223> X - any amino acid

<400>

Phe Gly Xaa Gly

one

<210> SEQ ID NO: 22

<211> 4

<212> PRT

<213> Artificial sequence

<223> CDR-H1 - residue after

<221> site

<222> 2

<223> X - any amino acid

<221> site

<222> 3

<223> X- any amino acid

<221> site

<222> 4

<223> X - any amino acid

<400>

Cys Xaa Xaa Xaa

one

<210> SEQ ID NO: 23

<211> 5

<212> PRT

<213> Artificial sequence

<223> CDR-H2 - residues before

<400>

Leu Glu Trp Ile Gly

fifteen

<210> SEQ ID NO: 24

<211> 4

<212> PRT

<213> Artificial sequence

<223> CDR-H3 - residue after

<221> site

<222> 3

<223> X-any amino acid

<400>

Trp Gly Xaa Gly

one

<---

Claims (9)

1. An isolated humanized protein that specifically binds human glycoprotein VI (hGPVI) and is a monovalent antigen-binding fragment of an antibody, wherein the heavy chain variable region of said fragment contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region of said fragment contains the sequence of SEQ ID NO: 8 or SEQ ID NO: 9. 2. An isolated humanized protein specifically binding human glycoprotein VI (hGPVI) according to claim 1, wherein said protein has an hGPVI binding Kd of less than 15 nM as measured by surface plasmon resonance using 960-1071 RU of soluble human GPVI and using PBS at pH 7.4 as a running buffer, and the isolated humanized protein does not induce the GPVI depletion phenotype in vivo. 3. An isolated humanized protein specifically binding human glycoprotein VI (hGPVI) according to claim 1, which is an antigen-binding fragment selected from the group consisting of Fv or Fab. 4. The use of a composition containing a therapeutically effective amount of an isolated humanized protein that specifically binds human glycoprotein VI (hGPVI), according to any one of paragraphs. 1-3 for the treatment of a cardiovascular disease or event associated with thrombosis. 5. The use of a pharmaceutical composition for the treatment of a cardiovascular disease or event associated with thrombosis, containing an isolated humanized protein that specifically binds human glycoprotein VI (hGPVI), according to any one of paragraphs. 1-3 in a therapeutically effective amount and at least one pharmaceutically acceptable excipient. 6. The use of a pharmaceutical composition according to claim 5, wherein the cardiovascular disease or thrombotic event is selected from arterial and venous thrombosis, restenosis, acute coronary syndrome, ischemic cerebrovascular disease, cerebrovascular disease, venous thromboembolism and vascular purpura . 7. The use of a pharmaceutical composition according to claim 5, wherein the cardiovascular disease or thrombotic event is selected from coronary artery disease and cerebral atherosclerosis. 8. Use of a pharmaceutical composition according to claim 5, wherein the cardiovascular disease or thrombotic event is selected from the group consisting of atherothrombosis, ischemic events, acute coronary syndrome, myocardial infarction, stroke, percutaneous coronary intervention, stent thrombosis, ischemic restenosis , acute ischemia, chronic ischemia, diseases of the aorta and its branches, peripheral arterial disease, venous thrombosis, acute phlebitis and pulmonary embolism, cancer-associated thrombosis, inflammatory thrombosis and infection-associated thrombosis. 9. An expression vector containing a nucleotide sequence encoding an isolated humanized protein that specifically binds human glycoprotein VI (hGPVI) according to any one of paragraphs. 1-3, wherein said vector contains a nucleotide sequence encoding the heavy chain variable region of SEQ ID NO: 10, or any sequence at least 90% identical to SEQ ID NO: 10, and a nucleotide sequence encoding the light chain variable region of SEQ ID NO: 11 or SEQ ID NO: 12, or any sequence at least 90% identical to SEQ ID NO: 11-12.

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