Anti-GPVI antibody and its functional fragment

Abstract

This invention relates to antibody molecules and functional fragments thereof that can bind to human glycoprotein VI (GPVI), processes for their production, and their therapeutic applications.

Description

[Technical Field]

[0001] This invention relates to antibody molecules and functional fragments thereof that can bind to human glycoprotein VI (GPVI), processes for their production, and their therapeutic applications. [Background technology]

[0002] Platelet adhesion and aggregation at the site of vascular injury are essential for normal hemostasis and maintaining vascular integrity. 1~3 Pathological deviations from hemostasis are thrombosis, which are associated with numerous pathologies including cardiovascular diseases (such as myocardial infarction, stroke, lower limb ischemia, and venous thromboembolism) as well as cancer, infections, or inflammatory diseases. They are a leading cause of death and severe disability worldwide and continue to be a major global health burden.

[0003] Thrombosis is the uncontrolled formation of blood clots in blood vessels, for example, at the site of atherosclerotic plaque rupture. This can lead to vascular occlusion and potentially life-threatening cardiovascular conditions such as myocardial infarction or ischemic stroke. 4、5 Therefore, acetylsalicylic acid, P2Y 12 Antiplatelet agents, such as ADP receptor blockers or glycoprotein (GP) IIb / IIIa inhibitors, have become essential treatments for the effective prevention or treatment of arterial thrombosis. However, because these drugs target mechanisms common to hemostasis, their use leads to an increased risk of uncontrolled bleeding complications correlated with their inherent hemostatic and antithrombotic effects. It is most evident that their use should be limited, and that in patients with multiple diseases, dual platelet inhibition or concomitant anticoagulation is necessary. 2、6、7、26 Therefore, there is a need for new, safe, and efficient treatments that do not involve systemic side effects such as bleeding. However, developing new antithrombotic treatment regimens that focus on reducing the risk of bleeding without sacrificing drug efficacy remains challenging.

[0004] Glycoprotein VI (GPVI) is an activated platelet receptor for collagen / fibrin (fibrinogen), and its absence and functional inhibition are important antithrombotic targets for preventing experimental thrombosis and thromboinflammatory disease states without impairing hemostasis. In particular, both the absence and functional inhibition of GPVI provide protection from thrombotic inflammatory disease mechanisms such as pathological thrombosis and (super) acute ischemic stroke in several mammalian model organisms without causing major bleeding complications. 8 Thus, functional inhibition of GPVI is a promising and appropriate strategy for treating thrombosis and related diseases. 9-13 GPVI is a transmembrane signaling receptor of approximately 65 kDa for collagen and fibrin, and is expressed only in platelets and megakaryocytes. It associates non-covalently with the immunoreceptor tyrosine-based activation motif (ITAM)-containing Fc receptor common γ subunit (FcRγ chain), which functions as the signaling subunit of the receptor complex.

[0005] The ectodomain of GPVI consists of two Ig-like domains, namely D1 and D2, followed by a putative intrinsically disordered region proximal to the transmembrane domain. Based on X-ray crystallography, collagen binding is attributed to D1, while GPVI dimerization is mainly mediated by D2 and a part of the subsequent disordered region. 14,15 Different approaches for pharmacologically targeting GPVI-mediated platelet activation have been reported, including antibody (IgG)-mediated GPVI immunodepletion, the competitive inhibitor Revacept (dimeric GPVI-Fc fusion protein), and GPVI blockade by Fab fragments derived from monovalent IgG. These studies have shown significant benefits of these different anti-GPVI strategies in experimental models of thrombosis and thromboinflammation. 16 17 18

[0006] 9、19、20 21、22 20、23、24、25、54

[0007] ​​​​​​​To date, several antibodies and antibody fragments have been reported, and their GPVI binding sites have been mapped to various different interfaces within residues 58–187, but nothing beyond that has been discovered. 31 Some of these antibodies and antibody fragments can block the GPVI-collagen interaction, some induce GPVI loss, and they generally exhibit a weak affinity for GPVI, making them unsuitable for clinical investigation. 26 The function of blocking the first reported anti-GPVI antibody is that of mouse GPVI, which recognizes human GPVI but does not block its function. 33 It was JAQ1 (rat IgG) that was triggered by this. 32、55、56 However, in vivo administration of JAQ1 IgG in mice induces GPVI via an Fc-dependent mechanism. 19 This results in a persistent GPVI knockout phenotype and long-term antithrombotic protection. 9 Subsequent research suggested that a similar mechanism of GPVI depletion could occur in humans. 14、48、20 GPVI depletion is uncontrollable and irreversible (i.e., it persists for the lifetime of platelets, or even longer, due to GPVI depletion on megakaryocytes). In the art, other anti-GPVI antibodies that induce the GPVI depletion phenotype have been described. 57、58、59 In general, whole-bivalent IgG can potentially crosslink platelet membrane GPVI with low-affinity FcγRIIA receptors, leading to platelet activation and potentially inducing GPVI deficiency due to internalization or loss. 26 .

[0008] One antibody fragment with a slightly high but still moderate binding affinity to GPVI is a humanized monovalent GPVI blocker Fab (ACT017, glenzosimab) with a KD of 4.1 nM, based on the parental mouse antibody fragment, and a KD of 17 nM. D 9O12 Fab That is 26、60、61The mapping of the ACT017 binding site to GPVI demonstrated recognition of a discontinuous epitope consisting of two extensions of hGPVI located in the D2 domain, 114-142 and 165-187 (numbered by Q9HCN6). 31 .

[0009] ACT017, 9012 Fab Initial research using monovalent parent mouse antibody fragments 26 In that context, it is a non-human primate. 29 In this study, and in mouse lines expressing human GPVI instead of mouse GPVI, GPVI was efficiently inhibited ex vivo. 23 Initial data from a Phase II clinical trial using a humanized GPVI blocker Fab (ACT017, glenzosimab) suggest that treatment with an anti-GPVI Fab fragment is effective in the acute phase of ischemic stroke. 26、27 It has been shown that it may be effective as an add-on therapy in reducing the thromboinflammatory response that drives infarct progression. In contrast, Revacept, another GPVI inhibitor that has recently entered clinical trials, has not been able to reduce the incidence of myocardial injury in patients with stable ischemic heart disease. 22 Furthermore, other groups have also reported that this indirect approach is less effective compared to direct functional inhibition of GPVI with antibodies in mouse and human platelets. 46、47 .

[0010] However, 9O12 in mice Fab The in vivo half-life (at a dose of 4 mg / kg, approximately 2.5 hours, intravenously) 23,29 ), as well as the in vivo half-life of ACT017 (glenzosimab) in humans (approximately 10 hours at doses greater than 16 mg / kg, intravenously 30 ) is relatively short, and at least part of it is 9012 Fab This can be explained by the somewhat moderate binding affinity of both ACT017 and ACT017 to GPVI (K D (17.08 nM and 4.1 nM respectively) 26、29、30 The moderate binding affinity and relatively short plasma half-life were parallelized in mice by complete clearance of the antibody fragment within 24 hours after injection.23 In primates, 9O12 in circulating platelets Fab The rapid decrease in the detection of binding to 9O12 on circulating platelets was parallelized. Fab The levels decreased to less than 50% of the peak level, and within 24 hours after injection, 9012 Fab Positive circulating platelets decrease by more than half. 29 .

[0011] Other GPVI antibodies, including four mouse monoclonal anti-GPVI antibodies OM1, OM2, OM3, and OM4, have been described in prior art. These antibodies have been found to inhibit GPVI binding to collagen, collagen-induced secretion, and thromboxane A2 (TxA2) formation in vitro, and collagen-induced platelet aggregation ex vivo after intravenous injection into cynomolgus monkeys. OM4 also appeared to inhibit thrombus formation in a rat thrombosis model. 62 These antibodies have a smaller K than, for example, ACT017. D It has been reported that it has a value, but this is greater than 0.7 nM. Furthermore, each patent document states that K D The measurements were performed using bivalent IgG antibodies, and it has been reported that monovalent fragments generally have somewhat reduced affinity compared to the corresponding intact IgG. 62 .

[0012] Therefore, there is still a need for improved human GPVI inhibitors as antiplatelet agents to efficiently prevent or treat arterial or venous thrombosis and thromboinflammatory disease conditions in at-risk individuals, while maintaining a low bleeding risk. GPVI inhibitors should have at least high affinity for human GPVI (e.g., at least K D It should have a concentration of <700 pM) and preferably a long plasma half-life. Ideally, such a GPVI inhibitor should be characterized by long-lasting persistence on circulating platelets and be effective in inhibiting thrombus formation, as evaluated in vitro and / or in vivo. [Overview of the project]

[0013] The inventors of this application have identified the dissociation equilibrium constant (K D We identified a specific anti-GPVI antibody and its functional fragment that exhibits extremely high binding affinity to human GPVI at concentrations below 700 pM and as low as 195 pM (and therefore approximately 21 times lower than ACT017). Furthermore, the novel anti-GPVI antibody and functional fragment unexpectedly bound to a novel epitope on human GPVI containing the proposed GPVI dimerized residue; and in vitro, it bound to humanized mouse (hGP6) GPVI. tg / tg Mouse (human and hGP6) tg / tg In mouse platelets, it has a long plasma half-life and potently inhibits GPVI function; (iii) and in vivo, it provides sustained GPVI blockade (e.g., over 50% receptor occupancy at 48 hours post-injection and over 35% occupancy at 72 hours post-injection into circulating platelets), as well as serious thrombosis protection (e.g., effective in inhibiting thrombus formation as evaluated in vitro and / or in vivo), but hGP6 tg / tg It does not have a significant effect on tail hemorrhage time in mouse models. Based on these results, it is reasonable to expect that the anti-GPVI antibody and functional fragment of the present invention will achieve the desired level of GPVI inhibition in humans at relatively lower doses and / or longer durations compared to, for example, ACT017. The monovalent form of the antibody and its functional fragment excludes Fc-dependent thrombocytopenia and / or GPVI depletion, and the Fab fragment avoids the possible Fc-mediated action of full-length IgG via platelet-expressed Fc gamma receptor IIA (FcγRIIA, CD32). Therefore, the present invention provides an anti-GPVI antibody and functional fragment with very high affinity for human GPVI, enabling sustained and safe inhibition of human GPVI function to circulating platelets with a very low bleeding risk, possibly through an unorthodox mechanism of action.

[0014] The present invention provides antibody molecules and functional fragments thereof that bind to human GPVI with very high affinity and can block its function in vitro and in vivo with a low risk of bleeding. Accordingly, the present invention relates to the subject matter defined in the following items (1) to (133). (1) An antibody or functional fragment thereof capable of binding to human glycoprotein VI (GPVI), comprising (i) a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 9, or SEQ ID NO: 64, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3 L (ii) a domain and a V including a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 13, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6 H An antibody or a functional fragment thereof containing a domain. (2) An antibody or functional fragment thereof capable of binding to human glycoprotein VI (GPVI), comprising (i) a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 64, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3 L (ii) a domain and a V including a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6 H An antibody or a functional fragment thereof containing a domain. (3) The antibody or functional fragment thereof described in item (1) or (2), wherein in the amino acid sequence of Sequence ID No. 9, residue X is A, R, N, D, Q, E, G, H, I, L, K, M, F, S, T, W, Y, or V. (4) An antibody or functional fragment thereof according to any one of items (1) to (3), wherein residue X in the amino acid sequence number 9 is A. (5) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X in the amino acid sequence number 9 is R. (6) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is N in the amino acid sequence number 9. (7) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is D in the amino acid sequence number 9. (8) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X in the amino acid sequence number 9 is Q. (9) An antibody or functional fragment thereof according to any one of items (1) to (3), wherein residue X in the amino acid sequence number 9 is E. (10) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is G in the amino acid sequence number 9. (11) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is H in the amino acid sequence number 9 of the sequence. (12) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is I in the amino acid sequence number 9 of the sequence. (13) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is L in the amino acid sequence number 9. (14) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is K in the amino acid sequence number 9. (15) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is M in the amino acid sequence number 9. (16) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is F in the amino acid sequence number 9 of the sequence. (17) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is S in the amino acid sequence number 9. (18) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is T in the amino acid sequence number 9 of the sequence. (19) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is W in the amino acid sequence number 9. (20) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is Y in the amino acid sequence number 9. (21) An antibody or functional fragment thereof according to any one of (1) to (3), wherein residue X is V in the amino acid sequence number 9 of the sequence. (22)(i) A V region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3 L (ii) a domain and a V including a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6 H An antibody or functional fragment thereof, including the domain, as described in item (1). (23) The antibody or functional fragment thereof described in item (1), wherein in the amino acid sequence of sequence number 13, residue X at position 5 (X5) is D or E; and / or residue X at position 6 (X6) is G or A. (24) A monovalent antibody or functional fragment thereof as described in any of the preceding items. (25) A functional fragment as described in any one of the preceding items, which is a fragment antigen-binding (Fab), F(ab'), Fv, disulfide-linked variable fragment (dsFv), monovalent IgG, or single-stranded variable fragment (scFv). (26) A functional fragment described in any of the preceding items, which is a fragment antigen-binding (Fab). (27) An antibody or functional fragment thereof that specifically binds to human GPVI, as described in any one of the preceding items. (28) An antibody or functional fragment thereof, as described in any one of the preceding items, which does not significantly bind to a protein structurally closely related to human GPVI. (29) An antibody or functional fragment capable of binding to human GPVI at a binding epitope corresponding to amino acids V178-E192 and / or S223-P237 of SEQ ID NO: 36, in particular the antibody or functional fragment described in any one of the preceding items. (30) The antibody according to item (29), wherein the conjugated epitope is a discontinuous epitope comprising or consisting of amino acids V178-E192 and S223-P237 of SEQ ID NO: 36. (31) An antibody or functional fragment described in any one of the preceding items, which is capable of binding to human GPVI at a GPVI residue that is proposed to be involved in dimerization. (32) An antibody or functional fragment described in any of the preceding items, whose binding to human GPVI interrupts or interferes with GPVI dimerization. (33) An antibody or functional fragment thereof that binds to essentially the same binding epitope as the antibody or functional fragment of any one of the preceding items. (34) The antibody or functional fragment has a dissociation equilibrium constant (K) of less than 1 μM, preferably less than 900 pM, more preferably less than 800 pM, even more preferably less than 700 pM, even more preferably less than 600 pM, even more preferably less than 500 pM, even more preferably less than 400 pM, even more preferably less than 350 pM, even more preferably less than 300 pM, even more preferably less than 250 pM, even more preferably less than 200 pM, even more preferably about 195 pM or less, and even more preferably about 175 pM or less. D An antibody or functional fragment described in any one of the preceding items, which binds to human GPVI. (35) Dissociation equilibrium constant (K) less than 195 pM D An antibody or functional fragment described in any one of the preceding items, which binds to human GPVI. (36) Dissociation equilibrium constant (K DAn antibody or functional fragment of item (34) or (35) determined by biolayer interferometry (BLI), surface plasmon resonance (SPR) technique (e.g., in a BIACORE instrument), or ELISA, preferably BLI (e.g., using an Octet instrument and Fortebio software). (37) Dissociation equilibrium constant (K D ) is determined using biolayer interferometry (BLI) (for example, using an Octet instrument), preferably K D The determination of the antibody or functional fragment described in any one of items (34) to (36) is carried out as described in the examples in Section 1.16. (38) Dissociation equilibrium constant (K D ) preferably using an Octet instrument at 25°C, preferably at a constant orbital flow rate of, for example, 1000 rpm; using an antigen (human GPVI) loading concentration for antigen immobilization on a biosensor of 1.2 μg / ml; using an association time of 900 seconds and a dissociation time of 1200 seconds; using 3-fold serial dilutions to determine an antibody screening range having seven concentrations in the range of 10 to 0.014 nM, and using a 1:1 fitting model, the antibody or functional fragment as described in any one of the preceding items. (39) An antibody or functional fragment having an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35, more preferably 16, 17, 18, 32, 33, and 34, or comprising a V L An antibody or functional fragment, including the domain, as described in any one of the preceding items. (40) An antibody or functional fragment having an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 32, 33, 34, and 35, preferably 32, 33, and 35, more preferably 32, 33, and 34, or comprising a V L An antibody or functional fragment described in any one of the preceding items, including the domain. (41) An antibody or functional fragment having an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, or even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 29, 30, and 31, preferably 16, 17, 18, and 19, more preferably 16, 17, and 18, or comprising a V L An antibody or functional fragment, including the domain, as described in any one of the preceding items. (42) An antibody or functional fragment having an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs. 21, 22, 23, 24, and 25, preferably 21, 22, and 23, or comprising a V HAn antibody or functional fragment, including the domain, as described in any one of the preceding items. (43) An antibody or functional fragment containing or comprising the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, preferably 16, 17, 18, 19, 32, 33, 34, or 35, more preferably 16, 17, 18, 32, 33, or 34. L An antibody or functional fragment, including the domain, as described in any one of the preceding items. (44) An antibody or functional fragment comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, or 35, preferably 32, 33, 34, or 35, more preferably 32, 33, or 34. L An antibody or functional fragment, including the domain, as described in any one of the preceding items. (45) An antibody or functional fragment comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 19, 29, 30, or 31, preferably 16, 17, 18, or 19, more preferably 16, 17, or 18. L An antibody or functional fragment, including the domain, as described in any one of the preceding items. (46) The antibody or functional fragment contains or consists of the amino acid sequence shown in SEQ ID NOs: 21, 22, 23, 24, or 25, preferably 21, 22, 23, 24, or 25. H An antibody or functional fragment, including the domain, as described in any one of the preceding items. (47)V L Domain and V HThe domains are: SEQ ID NOs: 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23, 26 and 24, 26 and 25, SEQ ID NOs: 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 23, 19 and 24, 19 and 25, 29 and 21, 29 and 22, 29 and 23, 29 and 24, or 29 and 25; preferably 32 and 22, 32 and 23, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 16 and 22, 16 and 23, 1 6 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, or 18 and 23, more preferably 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, 34 and 23, 16 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23; more preferably 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, 34 and 23, 17 and 21 , 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23; more preferably 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, or 18 and 22; more preferably 33 and 21, 33 and 22, 34 and 22, 17 and 21, 17 and 22, or 18 and 22, comprising or consisting of a pair of amino acid sequences as shown in any one of the preceding items, as described in any one of the preceding items. (48)V L Domain and V HThe domains are: Atlases 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23 , 26 and 24, 26 and 25, 27 and 21, 27 and 22, 27 and 23, 27 and 24, 27 and 25, 28 and 21, 28 and 22, 28 and 23, 28 and 24, or 28 and 25, preferably 32 and 22, 32 and 23, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 26 and 22, 26 and 23, 26 and 25, 27 and 21, 27 and 22, or 27 and 23; more preferably 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, 34 and 23, 26 and 22, 27 and 21, 27 and 22, or 27 and 23; even more preferably 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 21, 34 and 22, 27 and 2 An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a pair of amino acid sequences represented in 1, 27 and 22, or 27 and 23; more preferably 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 21, 34 and 22, 27 and 21, or 27 and 22; even more preferably 33 and 21, 33 and 22, 34 and 22, or 27 and 22. (49)V L Domain and V HThe domains are sequence numbers 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23, 26 and 24, or 26 and 25; preferably 32 and 22, 32 and 23, 32 and 25, 33 and 21, 33 and 22, 33 and 24, 34 and 25, 34 and 21, 34 and 2 An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a pair of amino acid sequences segmented in 2, or 34 and 23; more preferably 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, or 34 and 23; even more preferably 33 and 21, 33 and 22, 33 and 23, 34 and 21, or 34 and 22; even more preferably 33 and 21, 33 and 22, or 34 and 22. (50)V L Domain and V HThe domains are sequence numbers 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 22, 19 and 23, 19 and 24, 19 and 25, 29 and 21, 29 and 22, 29 and 23, 29 and 24, 29 and 25, 30 and 21, 30 and 22, 30 and 23, 30 and 24, 30 and 25, 30 and 21, 30 and 22, 30 and 23, 30 and 24, 30 and 25, 31 and 21, 31 and 22, 31 and 23, 31 and 24, 31 and 25; preferably 16 and 22, 16 and 23, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 2 2, 18 and 23, 29 and 22, 29 and 23, 29 and 25, 30 and 21, 30 and 22, or 30 and 23; more preferably 16 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 18 and 23, 29 and 22, 30 and 21, 30 and 22, or 30 and 23; even more preferably 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 18 An antibody or functional fragment according to any one of items (1) to (47), comprising or consisting of a pair of amino acid sequences shown in and 23, 30 and 21, 30 and 22, or 30 and 23; more preferably 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 30 and 21, or 30 and 22; even more preferably 17 and 21, 17 and 22, 18 and 22, or 30 and 22. (51)V L Domain and V HThe domains are sequence numbers 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 22, 19 and 23, 19 and 24, 19 and 25, 29 and 21, 29 and 22, 29 and 23, 29 and 24, or 29 and 25; preferably 16 and 22, 16 and 23, 16 and 25, 17 and 21, 17 and 22, 17 and 24, 17 and 25, 18 and 21, 18 and 22, or 18 and 23; more preferably An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a pair of amino acid sequences shown in 16 and 22, 18 and 21, 18 and 22, 18 and 23; more preferably 16 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23; even more preferably 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23; even more preferably 17 and 21, 17 and 22, 17 and 23, 18 and 21, or 18 and 22; even more preferably 17 and 21, 17 and 22, or 18 and 22. (52) An antibody or functional fragment having an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 17, or comprising a V L Domain; and a V comprising or consisting of an amino acid sequence having sequence identity of at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% with respect to the amino acid sequence shown in SEQ ID NO: 21. HAn antibody or functional fragment according to any one of the preceding items, comprising a domain. (53) The antibody or functional fragment comprises, or consists of, an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 33 for the V L domain; and an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% sequence identity to the amino acid sequence shown in SEQ ID NO: 21 for the V H domain, of the antibody or functional fragment according to any one of the preceding items. (54) The antibody or functional fragment comprises, or consists of, a V L domain comprising the amino acid sequence shown in SEQ ID NO: 17, and a V H domain, of the antibody or functional fragment according to any one of items (1)-(47) and (50)-(52). (55) The antibody or functional fragment comprises, or consists of, a V L domain comprising the amino acid sequence shown in SEQ ID NO: 33, and a V H domain, of the antibody or functional fragment according to any one of items (1)-(49). (56) The antibody or functional fragment comprises, or consists of, a V L domain comprising the amino acid sequence shown in SEQ ID NO: 15, and / or a V Hcomprising a domain and optionally V L domain and / or V H The antibody or functional fragment according to any one of items (1) to (42), wherein the domain comprises 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution with respect to the sequences shown in SEQ ID NO: 15 and SEQ ID NO: 20, respectively. (57) The antibody or functional fragment according to any one of the preceding items, which is immunoglobulin G (IgG) or a functional fragment thereof, preferably IgG1 or a functional fragment thereof. (58) The antibody or functional fragment, wherein L the light chain comprises a V domain and H the heavy chain comprises a V domain, or consists of or comprises the antibody or functional fragment according to any one of the preceding items. (59) The antibody or functional fragment, wherein L in addition to the V domain, preferably L a constant domain (C L ) that is C-terminal to the V domain, the light chain comprising, and H in addition to the V domain, preferably H a constant domain (C H ) that is C-terminal to the V domain, the heavy chain comprising, or consisting of or comprising the antibody or functional fragment according to any one of the preceding items. (60) The antibody or functional fragment comprises a light chain comprising a constant domain derived from the human IgK light chain constant domain (allotype Km3), preferably, for example, as shown in SEQ ID NO: 60, the total number of amino acids different from the amino acid sequence of the human IgK light chain constant domain (allotype Km3) is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2, and / or the antibody or functional fragment comprises a heavy chain comprising a constant domain derived from the human IgG1 heavy chain CH1 constant domain (allotype G1m17,1), preferably, for example, as shown in SEQ ID NO: 61, the total number of amino acids different from the amino acid sequence of the human IgG1 heavy chain C H 1 constant domain (allotype G1m17,1) is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2, the antibody or functional fragment according to item (58) or (59). (61)CL An antibody or functional fragment according to any one of items (59) to (60), wherein the domain comprises or consists of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% sequence identity with respect to the amino acid shown in SEQ ID NO: 60. (62)C H An antibody or functional fragment according to any one of items (59) to (60), wherein the domain comprises or consists of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% sequence identity with respect to the amino acid shown in SEQ ID NO: 61. (63) The light chain comprises or consists of an N-terminal light chain signal peptide containing the amino acid sequence shown in SEQ ID NO: 62, and / or contains or consists of the amino acid sequence shown in SEQ ID NO: 60, preferably V L The C-terminal light chain constant domain (C L An antibody or functional fragment as described in any one of items (58) to (62), including ). (64) The heavy chain comprises or consists of an N-terminal heavy chain signal peptide containing the amino acid sequence shown in SEQ ID NO: 63, and / or contains or consists of the amino acid sequence shown in SEQ ID NO: 61, preferably V H The heavy chain constant domain (C) is the C-terminus of the domain. H An antibody or functional fragment as described in any one of items (58) to (63), including ). (65) A sequence comprising, or consisting of, an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58. A monovalent, preferably Fab, antibody or functional fragment according to any one of the preceding items, comprising or comprising a heavy chain, which comprises an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99.5% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, and 48. (66) A light chain comprising or consisting of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99.5% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41; and a heavy chain comprising or consisting of an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99%, and even more preferably at least 99.5% sequence identity with respect to a sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, and 48; and a monovalent, preferably Fab, antibody or functional fragment according to any one of the preceding items. (67) An antibody or functional fragment according to any one of the preceding items, comprising a light chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48. (68) An antibody or functional fragment according to any one of the preceding items, comprising or comprising a light chain comprising the amino acid sequence shown in SEQ ID NOs. 55, 56, 57, and 58, preferably one of SEQ ID NOs. 55, 56, and 57, and a heavy chain comprising or comprising the amino acid sequence shown in SEQ ID NOs. 44, 45, 46, 47, and 48, preferably one of SEQ ID NOs. 44, 45, and 46. (69) An antibody or functional fragment according to any one of items (1) to (67), comprising a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NOs. 38, 39, 40, and 41, preferably one of SEQ ID NOs. 38, 39, and 40, and a heavy chain comprising or consisting of the amino acid sequence shown in SEQ ID NOs. 44, 45, 46, 47, and 48, preferably one of SEQ ID NOs. 44, 45, and 46. (70) An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a light chain comprising the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48, and being monovalent, preferably a fragment antigen-binding (Fab). (71) An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a light chain comprising the amino acid sequence shown in one of SEQ ID NOs. 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48, and being monovalent, preferably a fragment antigen-binding (Fab). (72) An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a light chain comprising the amino acid sequence shown in one of SEQ ID NOs. 55, 56, 57, and 58, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48, and being monovalent, preferably a fragment antigen-binding (Fab). (73) An antibody or functional fragment according to any one of the preceding items, comprising or consisting of a light chain comprising the amino acid sequence shown in one of SEQ ID NOs. 55, 56, and 57, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs. 44, 45, and 46, and being monovalent, preferably a fragment antigen-binding (Fab). (74) A monovalent, preferably fragment antigen-binding (Fab) antibody or functional fragment according to any one of items (1) to (70), comprising a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, and 51, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48. (75) A monovalent, preferably fragment antigen-binding (Fab) antibody or functional fragment according to any one of items (1) to (70), comprising a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 38, 39, 40, and 41, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48. (76) A monovalent, preferably fragment antigen-binding (Fab) antibody or functional fragment according to any one of items (1) to (70), comprising a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 38, 39, and 40, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, and 46. (77) (preferably monovalent, more preferably Fab, and) Sequence IDs 55 and 44, 55 and 45, 55 and 46, 55 and 47, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 57 and 47, 57 and 48, 58 and 44, 58 and 45, 58 and 46, 58 and 47, 58 and 48, 52 and 44, 52 and 45, 52 and 46, 52 and 47, 52 and 48, 53 and 44, 53 and 45, 53 and 46, 53 and 47, 53 and 48, 54 and 44, 54 and 45, 54 and 46, 54 and 47, or 54 and 48; preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 52 and 44, 52 and 45, 52 and 46, 53 and 44, 53 and 45, or 53 and 46; more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, 57 and 46, 52 and 45, 53 and 44, 53 and 45, or 53 and 46; even more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, 57 and 46, 53 and 44, 53 and 45, or 5 An antibody or functional fragment according to any one of items (1) to (70), comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in 3 and 46; more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, 53 and 45; even more preferably 56 and 44, 56 and 45, 57 and 45, or 53 and 45; even more preferably 56 and 44. (78) (preferably monovalent, more preferably Fab, and) Sequence IDs 55 and 44, 55 and 45, 55 and 46, 55 and 47, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 57 and 47, 57 and 48, 58 and 44, 58 and 45, 58 and 46 , 58 and 47, or 58 and 48; preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, or 57 and 46; more preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48 An antibody or functional fragment according to any one of items (1) to (70), comprising or consisting of the amino acid sequences shown in 57 and 44, 57 and 45, or 57 and 46; more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, or 57 and 46; more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, or 57 and 45; more preferably 56 and 44, 56 and 45, 56 and 46, or 57 and 45; more preferably 56 and 44, 56 and 45, or 57 and 45, and even more preferably 56 and 44. (79) (preferably monovalent, more preferably Fab, and) SEQ ID NOs: 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46, 41 and 47, 41 and 48, 49 and 44, 49 and 45, 49 and 46, 49 and 47, 49 and 48, 50 and 44, 50 and 45, 50 and 46, 50 and 47, 50 and 48, 51 and 44, 51 and 45, 51 and 46, 51 and 47, or 51 and 48; preferably 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 49 and 44, 49 and 45, 49 and 46, 50 and 44, 50 and 45, or 50 and 46; more preferably 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 40 and 46, 49 and 45, 50 and 44, 50 and 45, or 50 and 46; even more preferably 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 40 and 46, 50 and 44, 50 and 45, or 5 An antibody or functional fragment according to any one of items (1) to (70), comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in 0 and 46; more preferably 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 50 and 44, or 50 and 45; more preferably 39 and 44, 39 and 45, 40 and 45, or 50 and 45; even more preferably 39 and 44. (80) (preferably monovalent, more preferably Fab, and) SEQ ID NOs: 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46 , 41 and 47, or 41 and 48; preferably 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, or 40 and 46; and preferably 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48 An antibody or functional fragment according to any one of items (1) to (70), comprising or consisting of the amino acid sequences shown in , 40 and 44, 40 and 45, or 40 and 46; more preferably 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46; more preferably 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 45; more preferably 39 and 44, 39 and 45, 39 and 46, or 40 and 45; more preferably 39 and 44, 39 and 45, or 40 and 45, and more preferably 39 and 44. (81) Sequence IDs 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46, 41 and 47, 41 and 48, 49 and 44, 49 and 45, 4 An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequences shown in 9 and 46, 49 and 47, 49 and 48, 50 and 44, 50 and 45 and 46, 50 and 47, 50 and 48, 51 and 44, 51 and 45, 51 and 46, 51 and 47, or 51 and 48, monovalent, preferably Fab. (82) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NOs: 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 49 and 45, 49 and 48, 50 and 44, 50 and 45, or 50 and 46, monovalent, preferably Fab. (83) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NOs. 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, or 40 and 46, monovalent, preferably Fab. (84) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NOs. 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46, monovalent, preferably Fab. (85) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46, monovalent, preferably Fab. (86) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, 40 and 44, or 40 and 45, monovalent, preferably Fab. (87) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 39 and 44, 39 and 45, or 40 and 45, monovalent, preferably Fab. (88) An antibody or functional fragment according to any one of items (1) to (70), comprising a pair of light chains and heavy chains having the amino acid sequences shown in SEQ ID NOs. 39 and 44, monovalent, preferably Fab. (89) An antibody or functional fragment according to any one of items (1) to (66), comprising or consisting of a pair of light chains and heavy chains, which include or consist of the amino acid sequences shown in SEQ ID NOs. 37 and 43, and is monovalent, preferably Fab. (90) The antibody or functional fragment according to any one of the preceding items, which can sustain inhibition of human GPVI in plasma for at least 12 hours, more preferably at least 24 hours, even more preferably at least 36 hours, even more preferably at least 48 hours, even more preferably at least 60 hours, even more preferably at least 72 hours, even more preferably at least 84 hours, and even more preferably at least 96 hours. (91) The antibody or functional fragment described in any one of the preceding items, preferably in monovalent form, for example as Fab, can extend binding to circulating platelets, preferably with a GPVI receptor epitope occupancy of more than 50%, for at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, and even more preferably at least 60 hours, after intravenous administration of, for example, 4 mg / kg of the anti-GPVI antibody or functional fragment, preferably with a GPVI receptor epitope occupancy of more than 50%, when normalized with a negative control (e.g., control Fab that is not specific to GPVI), for at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, and even more preferably at least 60 hours, after intravenous administration of, for example, 4 mg / kg of the anti-GPVI antibody or functional fragment, for at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, and even more preferably at least 60 hours, as determined by flow cytometry analysis in ex vivo diluted blood, for example, as described in the examples of Section 1.10. (92) The antibody or functional fragment described in any one of the preceding items, preferably in monovalent form, for example as Fab, can extend binding to circulating platelets, preferably at least 20% GPVI receptor epitope occupancy, for at least 36 hours, preferably at least 48 hours, more preferably at least 60 hours, more preferably at least 72 hours, even more preferably at least 84 hours, even more preferably at least 96 hours, after intravenous administration of, for example, 4 mg / kg of the anti-GPVI antibody or functional fragment, preferably at least 20% GPVI receptor epitope occupancy, when normalized with a negative control (e.g., control Fab that is not specific to GPVI), for at least 36 hours, preferably at least 48 hours, more preferably at least 60 hours, more preferably at least 72 hours, even more preferably at least 84 hours, even more preferably at least 96 hours, as determined by flow cytometry analysis in ex vivo diluted blood, for example, as described in the examples of Section 1.10. (93) An antibody or functional fragment according to any one of the preceding items that can completely inhibit CRP and / or collagen-induced aggregation of washed human platelets at a concentration of 10 μg / ml or less, preferably 5 μg / ml or less, more preferably 2 μg / ml or less, and even more preferably 1 μg / ml or less, as determined using a standard light transmission agglutination assay, as described in the examples of Section 1.7. (94) Washed mouse hGP6 for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 60 hours after intravenous administration of a dose of 4 mg / kg to a humanized mouse model (hGP6tg / tg mouse) for GPVI, as determined using a standard light transmission agglutination assay, as described in the examples in Section 1.7. tg / tg An antibody or functional fragment according to any one of the preceding items that can completely inhibit platelet CRP and / or collagen-induced aggregation. (95) An antibody or functional fragment as described in any one of the preceding items that can completely inhibit human platelet adhesion (platelet surface coverage) and thrombus formation (relative thrombus volume) on a collagen-coated surface at a concentration of 5 μg / ml, as determined by using a fluid adhesion assay in heparinized human blood under flow conditions (preferably using a shear rate of 1000 s⁻¹), for example as described in the examples of Section 1.5, and optionally, complete inhibition refers to a reduction of at least 60%, preferably at least 65%, more preferably at least 70%, of platelet surface coverage and / or a reduction of at least 80%, preferably at least 85%, more preferably at least 87%, and even more preferably at least 90%, of relative thrombus volume, compared to a negative control. (96) An antibody or functional fragment according to any one of the preceding items, which can reduce the relative thrombus volume on a collagen-coated surface (preferably coated with 200 μg / ml) by at least 50%, preferably at least 60%, 65%, 70%, 80%, 85%, 90%, or 95% compared to a negative control in a fluid adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹). (97) Humanized mouse model for GPVI (hGP6 tg / tg After intravenous administration of 4 mg / kg to mice, under flow conditions (preferably 1000s) -1 An antibody or functional fragment according to any one of the preceding items that can reduce the relative thrombus volume on a collagen-coated surface (preferably coated with 200 μg / ml) by at least 50%, preferably at least 60%, compared to a negative control, in a fluid adhesion assay (for example, as described in the examples of Section 1.5) in heparinized human blood (using the shear rate of ), for at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 60 hours. (98) An antibody or functional fragment as described in any one of the preceding items, which can completely inhibit thrombus formation (relative thrombus volume), phosphatidylserine (PS) exposure, and fibrin deposition on a surface coated with collagen and tissue factor (preferably at 200 μg / ml and 500 pM for collagen and tissue factor, respectively) in citrated and remineralized human blood under flow (preferably using a shear rate of 1000 s⁻¹) as determined in a fluid adhesion assay adapted for coagulation (for example, as described in the examples in Section 1.6), and optionally, complete inhibition refers to a reduction of at least 90%, preferably at least 95%, in relative thrombus volume / phosphatidylserine (PS) exposure / fibrin deposition compared to a negative control. (99) An antibody or functional fragment according to any one of the preceding items, which, when used at a concentration of 10 μg / ml, can be conjugated to a fibrinogen-coated surface in a diffusion assay on washed human platelets (for example, as described in the examples in Section 1.8), and can reduce the portion of stage 4 (fully diffused) platelets by at least 40%, preferably at least 50%, and increase the portion of stage 2 (filamentous platelets) by a similar degree compared to a negative control. (100) Determined using a fluid adhesion assay, for example, as described in the examples in Section 1.5, under flow (preferably 1000s) -1 An antibody or functional fragment as described in any one of the preceding items can completely inhibit human platelet adhesion (platelet surface coverage) and thrombus formation (relative thrombus volume) on a collagen-coated surface (preferably coated at 200 μg / ml) in heparinized human blood (using a shear rate of ), and optionally, complete inhibition refers to a reduction of at least 80%, preferably at least 85%, of platelet surface coverage and / or at least 90%, preferably at least 95%, of relative thrombus volume compared to a negative control. (101) Using a fluorescently labeled antibody that is preferably specifically capable of binding to activated integrin αIIbβ3 and / or specifically capable of binding to P-selectin, as determined by, for example, flow cytometry analysis in ex vivo diluted blood, preferably in monovalent form, preferably as Fab, as described in the examples of Section 1.10, for example, a humanized mouse model (hGP6) against GPVI. tg / tg An antibody or functional fragment as described in any one of the preceding items, which, after intravenous administration of a dose of 4 mg / kg to mice, causes significantly impaired CRP-induced activation of circulating platelets for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, or at least 96 hours, where “significantly impaired” preferably means a reduction of more than 50% in the fluorescence signal compared to a negative control when using circulating platelets sampled at at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, or at least 96 hours after administration. (102) Preferably monovalent, preferably Fab, an antibody or functional fragment according to any one of the preceding items, which can inhibit the dimerization of GPVI, while optionally not cleaving or completely cleaving ligand binding to GPVI. (103) A monovalent antibody or functional fragment according to any one of the preceding items, which can inhibit GPVI-induced activation of platelets while optionally preserving the initial adhesion function of the GPVI receptor. (104) Preferably, a humanized mouse model of occlusive arterial thrombosis (hGP6) for GPVI, as described in the examples in Section 1.11. tg / tg An antibody or functional fragment as described in any one of the preceding items, which can provide sustained protection from obstructive thrombosis, such as arterial thrombosis, as determined using a mouse. (105) Optionally, a humanized mouse model of occlusive arterial thrombosis for GPVI (hGP6 tg / tg An antibody or functional fragment as described in any one of the preceding items, which can inhibit the formation of a stable thrombus at the site of arterial injury by reducing platelet activation at sites of exposed extracellular matrix in blood vessels and / or potent inhibition of local platelet-dependent coagulation, preferably as described in the examples in Section 1.11, as determined using a mouse. (106) An antibody or functional fragment described in any one of the preceding items, having significantly higher potency in inhibiting GPVI function compared to ACT017 (glenzosimab), as determined by i) an agglutination assay using a standard light transmission agglutination assay, for example, as described in the examples of Section 1.7; ii) a fluid adhesion assay (for example, as described in the examples of Section 1.5), using heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹); and / or iii) one hour after intravenous administration, determined based on CRP-induced activation of circulating platelets by flow cytometry analysis in ex vivo diluted blood using a fluorescently labeled antibody capable of specifically binding to activated integrin αIIbβ3. (107) When normalized with a negative control (e.g., a control Fab that is not specific to GPVI), the antibody or functional fragment described in any of the preceding items, in monovalent form, preferably as Fab, can increase binding to circulating platelets compared to ACT017 (glenzosimab), for example, by flow cytometry analysis in ex vivo diluted blood, for example, as described in the example of 1.10, using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, for example, using a fluorescently labeled Fab-specific antibody, as measured 1 hour after intravenous administration of ACT017 (glenzosimab). (Preferably, an increase of 1.5-fold or more, more preferably 1.7-fold or more, 1.8-fold or more, or 1.9-fold or more in GPVI receptor epitope binding. (108) When normalized with a negative control (e.g., a control Fab that is not specific to GPVI), for example, an anti-GPVI antibody or functional fragment, or an antibody or functional fragment as described in any of the preceding items, can increase binding to circulating platelets compared to ACT017, for example, by flow cytometry analysis in ex vivo diluted blood, for example, as described in the example of 1.10, using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, for example, using a fluorescently labeled Fab-specific antibody, as measured 3 hours after intravenous administration of ACT017 (glenzosimab), for example. (109) Using a fluorescently labeled antibody preferably capable of (specifically) binding to activated integrin αIIbβ3, as determined, for example by flow cytometry analysis in ex vivo diluted blood, preferably in monovalent form, preferably as Fab, as described in the examples of Section 1.10, for example, a humanized mouse model (hGP6) against GPVI. tg / tg An antibody or functional fragment as described in any one of the preceding items, which results in significantly impaired CRP-induced activation of circulating platelets 1 hour after intravenous administration of a dose of 4 mg / kg to mice (whereas ACT017 (glenzosimab) does not cause significantly impaired CRP-induced activation), and optionally, “significantly impaired” means a reduction of fluorescence signal of more than 85%, preferably more than 90%, more than 95%, more than 97%, more than 98%, more than 99%, or more than 99.5%, compared to a negative control, when circulating platelets sampled 24 hours after administration. (110) The antibody or functional fragment according to any one of the preceding items, wherein the antibody or functional fragment has more than 2 times, preferably more than 4 times, more than 6 times, more than 8 times, or more than 10 times higher potency than ACT017 (glenzosimab) in vitro, as measured by a standard light transmission agglutination assay at a concentration of preferably 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, and optionally the potency factor is determined as the ratio of the maximum agglutination percentage of the antibody or functional fragment-treated sample to the maximum agglutination percentage of the ACT017-treated sample. (111) An antibody or functional fragment according to any one of the preceding items, having more than 2 times, preferably more than 4 times, more than 6 times, more than 8 times, or more than 10 times higher potency than ACT017 (glenzosimab) in inhibiting collagen and / or CRP-induced aggregation of human platelets washed in vitro, as measured using a standard light transmission agglutination assay, as described in the examples of Section 1.7, and optionally, the potency factor is determined as the ratio of the maximum agglutination percentage of the antibody or functional fragment-treated sample to the maximum agglutination percentage of the ACT017-treated sample. (112) In heparinized human blood under flow conditions (preferably using a shear rate of 1000 s⁻¹), an antibody or functional fragment described in any one of the preceding items can significantly reduce (e.g., more than 1.5 times, preferably more than 2 times, 2.5 times, or more than 3 times) the platelet surface coverage on a collagen-coated surface (preferably coated with 200 μg / ml) compared to a negative control, in a flow adhesion assay (e.g., as described in the examples of Section 1.5) at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml (whereas ACT017 (glenzosimab) treated samples do not result in a (significant) reduction). (113) An antibody or functional fragment according to any one of the preceding items, which can reduce platelet surface coverage on a collagen-coated surface (preferably coated with 200 μg / ml) by more than 1.5 times, preferably 2 times, 2.5 times, or more than 3 times, compared to an ACT017 (glenzosimab) treated sample, in a flow adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹). (114) An antibody or functional fragment according to any one of the preceding items, which can reduce the relative thrombus volume on a collagen-coated surface (preferably coated with 200 μg / ml) by more than 2 times, preferably more than 4 times, 6 times, 8 times, or 10 times, compared to an ACT017 (glenzosimab) treated sample, in a flow adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹. (115) An antibody or functional fragment thereof that binds to an epitope on human GPVI that is essentially the same as an antibody exhibiting one or more of the characteristics mentioned in (1) to (114) above. (116) A nucleic acid encoding an antibody or functional fragment as described in any one of the preceding items. (117) A vector or plasmid containing the nucleic acid described in item (116). (118) Cells containing the nucleic acid described in item (116), or the vector or plasmid described in item (117). (119) A method for preparing an antibody or functional fragment according to any one of items (1) to (115), comprising culturing the cells described in item (118) in a culture medium under conditions that enable the expression of nucleic acids encoding an antibody or functional fragment, and recovering the antibody or functional fragment from the cells or the culture medium. (120) The method according to item (119), wherein the cells are Chinese hamster ovary (CHO) cells, and a nucleic acid codon-optimized for expression in CHO cells is transiently expressed from a vector or plasmid, and recovery is accompanied by affinity chromatography. (121) A pharmaceutical composition comprising an antibody or functional fragment described in any one of items (1) to (115), and optionally a pharmaceutically acceptable carrier and / or excipient. (122) An antibody or functional fragment described in any one of items (1) to (115) for use in a method of treating or preventing a GPVI-related condition in a subject. (123) The GPVI-related condition is a thromboinflammatory disease, and the antibody or functional fragment for use as described in item (122). (124) An antibody or functional fragment for use as described in item (122), wherein the GPVI-related condition is cardiovascular disease. (125) An antibody or functional fragment for use as described in item (123) or (124), wherein the cardiovascular disease is selected from thrombosis and thrombotic disorders (arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic disease, thrombotic occlusion of the coronary arteries, thrombotic microangiopathy, cancer-associated thrombosis (Trousseau syndrome), immunothrombosis and thrombosis associated with infection (e.g., cerebral malaria)), restenosis, acute coronary syndrome, ischemic cerebrovascular disease, cerebrovascular disease, and vascular purpura, coronary artery disease and cerebral artery disease, ischemic events, acute coronary syndrome, myocardial infarction (heart attack), acute cerebrovascular isovascular disease (stroke), percutaneous coronary intervention, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches (e.g., aortic aneurysm, thrombosis), peripheral artery disease, acute phlebitis, and pulmonary embolism. (126) The cardiovascular disease is thrombosis or thrombotic disorder, preferably selected from arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic disease, coronary artery thromboembolic occlusion, thrombotic microangiopathy, cancer-associated thrombosis (Trousseau syndrome), immunothrombosis and thrombosis associated with infection (e.g., cerebral malaria), and the antibody or functional fragment for use as described in item (123) or (124). (127) An antibody or functional fragment for use as described in item (124), wherein the cardiovascular disease is selected from arterial or venous thrombosis, restenosis, acute coronary syndrome, or atherosclerosis, preferably a cerebrovascular event resulting from arterial or venous thrombosis. (128) The GPVI-related condition is inflammation or thrombotic inflammation, preferably an infectious disease [e.g., cerebral malaria], arthritis, autoimmune disease [e.g., celiac disease, post-infectious IBS, diabetes mellitus type 1, Henoch-Schönlein purpura (HSP), sarcoidosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), alopecia areata and multiple sclerosis (MS), fibrosis, acute respiratory disorder] Antibodies or functional fragments for use as described in item (122) or (123), selected from platelet-mediated disorders of cellular function, including but not limited to: ARDS syndrome, ischemia-reperfusion injury (IRI) of various organs (liver, colon, etc.), antiphospholipid syndrome (APS), deep vein thrombosis, thrombophlebitis and vasculitis, transfusion-associated acute lung injury (TRALI), transplant rejection, pre-eclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, sickle cell disease, bacterial and viral infections leading to ischemic restenosis, sepsis, major trauma, and cancer cell proliferation and / or dissemination. (129) An antibody or functional fragment for use as described in item (122), wherein the GPVI-associated condition is cancer, preferably skin cancer (especially malignant cutaneous melanoma), colon cancer, breast cancer, ovarian cancer, lung cancer, or metastatic cancer. (130) An antibody or functional fragment for use as described in item (122) for a GPVI-related condition in which the condition is a disorder relating to the proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function of abnormal or ectopic megakaryocytes and / or platelets. (131) An antibody or functional fragment for use as described in any one of items (122) to (130), the subject being a human subject. (132) An antibody or functional fragment for use according to any one of items (122) to (131), wherein the method comprises administering the antibody or functional fragment intravenously to a human subject. (133) A method for treating or preventing a GPVI-related condition, preferably one of the GPVI-related conditions defined in items (123) to (130), comprising administering to a patient in need an effective amount of an anti-GPVI antibody or a functional fragment thereof, as defined in any of the pharmaceutical compositions of items (1) to (115) or item (121). [Brief explanation of the drawing]

[0015] [Figure 1]Emf6.1Fab inhibits GPVI-induced aggregation, thrombosis, and diffusion of human platelets. (A) Aggregation response of washed human platelets treated with 10 μg / mL Emf6.1 IgG or control IgG. Gray arrows indicate agonist addition. (B-C) Evaluation of platelet adhesion (B) and aggregate formation (C) to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized human blood treated with 10 μg / mL Emf6.1 IgG or control IgG. (D-H) Evaluation of platelet adhesion (E), thrombosis (F), phosphatidylserine exposure (G), and fibrin deposition (H) to Horm collagen (200 μg / mL) + tissue factor (500 pM) under flow (1000 s-1) in recalcified human blood treated with 10 μg / mL Emf6.1Fab or control Fab. Values ​​are mean ± SD (n=4). Unpaired, Mann-U-Whitney U test. **P<0.01, ***P<0.001. (D) Representative images are shown, scale 50 μm. (I) Agglutination response of washed human platelets treated with 10 μg / mL Emf6.1Fab or control Fab by light transmission agglutination assay (n=4). (J~K) Washed human platelets were diffused on fibrinogen (100 μg / mL) for 45 minutes at 37°C. DIC images were taken (100× objective lens) (J), and the stage abundances were determined. Stage 1: adhesion, Stage 2: filopodia formation, Stage 3: lamellipodia formation, Stage 4: completely diffused platelets (K). Values ​​are mean ± SD (n=8), unpaired, two-sided Student's t-test ***P<0.001. [Figure 2]Emf6.1Fab inhibits GPVI-induced aggregation, thrombus formation, and diffusion of hGP6tg / tg platelets. (A-C) Evaluation of platelet adhesion (B) and aggregate formation (C) to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized hGP6tg / tg blood treated with either 5 μg / mL Emf6.1Fab or control Fab. Values ​​are mean ± SD (n=5). Unpaired, Mann-U-Whitney U test. **P<0.01, ***P<0.001. (A) Representative images are shown, scale 50 μm. (D) Aggregation response of washed hGP6tg / tg platelets treated with 10 μg / mL Emf6.1Fab or control Fab by light transmission agglutination assay (n=5); gray arrows indicate agonist addition. (E~F) Washed hGP 6tg / tg platelets were diffused on fibrinogen (100 μg / mL) for 45 minutes at 37°C. DIC images were taken (100× objective lens, 30 μm scale) (E), and the stage abundances were determined. Stage 1: Adhesion, Stage 2: Pedicular formation, Stage 3: Lamellipodia formation, Stage 4: Completely diffused platelets (F). Values ​​are mean ± SD (n=3). Unpaired, Mann-U-Whitney U test. *P<0.05, **P<0.01. [Figure 3] Emf6.1Fab efficiently blocks GPVI function in hGP6tg / tg mice. hGP6tg / tg animals (n=5) were treated with 4 mg / kg bwEmf6.1Fab or control Fab. (A-B) Platelet count (A) and size (B) were determined at the indicated time points after treatment using an automated cell counter. (C-D) GPVI exposure was tested using Emf2 IgG-FITC (C), while epitope saturation by Emf6.1 was tested using Emf3 IgG-FITC (D). (E-H) Degranulation (α-P-selectin-FITC) (EF) and activation of platelet αIIbβ3 integrin (JON / A-PE) (G, H) in response to CRP (E, G) or thrombin (F, H) were determined at the indicated time points after treatment by flow cytometry. Data are presented as mean ± SD, and significance is expressed as *p<0.05 and ***p<0.001 (two-way ANOVA) (n=5) for the indicated groups. [Figure 4] In vivo sustained GPVI inhibition by Emf6.1Fab. (AC) Agglutination response of washed platelets to CRP (A), collagen (B), or thrombin (C) from hGP6tg / tg mice intravenously treated with 4 mg / kg bwEmf6.1Fab or control Fab, by light transmission agglutination assay (n=6); arrows indicate agonist addition. (D-F) Evaluation of platelet adhesion (E) and aggregate formation (F) to Horm collagen (200 μg / mL) under heparinized blood flow (1000 s-1) from hGP6tg / tg mice treated with either 4 mg / kg bwEmf6.1Fab or control Fab at different time points after injection. Values ​​are mean ± SD. Two-way ANOVA *P<0.05, ***P<0.001. [Figure 5] Emf6.1Fab treatment (4 mg / kg bw) is highly protective in model arterial thrombosis without affecting bleeding time (A-B). Arterial thrombosis was assessed for up to 30 minutes after control Fab or Emf6.1Fab treatment in mechanical injury in a model of aortic thrombosis. (A) Time to vascular occlusion is shown in each circle, representing one animal. ***P<0.001, Fisher's exact test used. (B) Representative blood flow traces from control Fab and Emf6.1Fab-treated mice are shown. (C) Hemostasis was assessed using tail bleeding time, with each circle representing one mouse. [Figure 6]Emf6.1 binds to a novel GPVI epitope. Mapping of the Emf6.1-binding epitope in the human GPVI ectodomain. (A) A 15-mer duplicate peptide covering the human GPVI ectodomain (residues 24-267) was displayed in peptide-microarray format. Emf6.1 IgG binding was visualized with an HRP-labeled anti-mouse IgG secondary antibody. The mapping identified two distinct epitopes, 201V-E215 and 246S-P260. (B) Emf6.1 binding is neutralized in the presence of soluble GPVI peptide fragments 201V-E215 and 246S-P260. (C) Surface representation of human GPVI (PDB-ID 2gi7). The Emf6.1 binding epitope overlaps with the proposed dimerization site (D2 region) of GPVI, but does not overlap with the ligand binding site (shown for collagen, PDB-ID 5ou9). [Figure 7] Alignment of humanized variants. (A-B) Emf6.1 VL sequence (VL0) is aligned with four humanized VL variants (A), and Emf6.1 VH sequence (VH0) is aligned with five humanized VH variants (B). VL0 is the mouse sequence, VL1-4 are humanized variants, VH0 is the mouse sequence, and VH1-5 are humanized variants. CDRs are underlined. Key residues important for the VH / VL interface and canonical loop structure are maintained as much as possible in the humanized variants using the CDRx platform. (C) Homology of humanized variants to mouse VL (i.e., VL0). (D) Homology of humanized variants to mouse VH (i.e., VH0). [Figure 8]EMA601 inhibits GPVI function. (A-C) Evaluation of platelet adhesion (B) and aggregate formation (C) to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized hGP6tg / tg blood treated with 5 μg / mL EMA601 or control Fab. Values ​​are mean ± SD (n=3). Unpaired, Mann-U-Whitney U test. *P<0.05, **P<0.01. (A) Representative images are shown, scale is 50 μm. (D) Aggregation response of washed hGP6tg / tg platelets treated with 5 μg / mL EMA601 or control Fab by light transmission agglutination assay (n=3); arrows indicate agonist addition. (E-F) Washed hGP6tg / tg platelets were diffused on fibrinogen (100 μg / mL) for 45 minutes at 37°C. DIC images were taken (100× objective lens, 30μM scale) (E) and the stage abundance was determined. Stage 1: adhesion, Stage 2: filopodia formation, Stage 3: lamellipodia formation, Stage 4: completely diffused platelets (F). Values ​​are mean ± SD (n=3). Unpaired, Mann-U-Whitney U test. *P<0.05, **P<0.01. (G~K) Evaluation of platelet adhesion (H), thrombus formation (I), phosphatidylserine exposure (J), and fibrin deposition (K) to Horm collagen (200μg / mL) + tissue factor (500pM) under flow (1000s-1) in recalcified human blood treated with 10μg / mL EMA601 (gray) or control Fab (black). Values ​​are mean ± SD (n=4). Unpaired, Mann-U-Whitney U test. ***P<0.001. (G) A representative image is shown, with a scale of 50 μm. [Figure 9]Emf6.1Fab inhibits GPVI-induced activation and aggregation of human platelets. (A) Evaluation of platelet adhesion (bar graph next to the image) and aggregate formation (bar graph on the right) to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized human blood treated with either 10 μg / mL (2nd row), 5 μg / mL (3rd row), 2 μg / mL (4th row), or 1 μg / mL (bottom row) of Emf6.1Fab or control Fab (top row). Values ​​are mean ± SD (n=4). Unpaired, Mann-U-Whitney U test. *P<0.05, **P<0.001. Representative images are shown on the left, scale 50 μm. (B) Aggregation response (n=4) of washed human platelets treated with 5, 2, or 1 μg / mL of Emf6.1Fab or control Fab by light transmission agglutination assay. [Figure 10] Emf6.1Fab inhibits GPVI-induced activation and aggregate formation of hGP6tg / tg platelets. (A-B) Degranulation (α-P-selectin-FITC) (A) and activation of platelet αIIbβ3 integrin (JON / A-PE) (B) in hGP6tg / tg platelets treated with 10 μg / mL Emf6.1Fab or control Fab were determined by flow cytometry when activated by the indicated agonists (n=5). Unpaired, Mann-U-Whitney U test. ***P<0.001. (C) Agglutination response of washed hGP6tg / tg platelets pre-incubated with either 2 μg / mL Emf6.1Fab or control Fab by light transmission agglutination assay (n=4). Arrows indicate the addition of agonists. (D) Evaluation of platelet adhesion and aggregate formation to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized hGP 6tg / tg blood treated with either 2 μg / mL Emf6.1Fab or control Fab. Left: Representative images are shown, scale is 50 μM. Right: Surface area coverage and thrombus volume data at the end of perfusion are presented as mean ± SD (n=4). Unpaired, Mann-U-Whitney U test ***P<0.001. [Figure 11]EMA601 inhibits GPVI function in human platelets. (A) Evaluation of platelet adhesion and aggregate formation to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized human blood pre-incubated with control Fab (10 μg / mL) or EMA601 (20, 10, or 1 μg / mL). Left: Representative fluorescence and bright-field images at the end of the experiment are shown, scale 50 μM. Right: Surface area coverage and thrombus volume data at the end of perfusion are presented as mean ± SD (n=3). Unpaired, Mann-U-Whitney U test. *P<0.05, **P<0.01. (B) Aggregation response (n=4) of washed human platelets pre-incubated for 10 minutes with 10 μg / mL (left) or 1 μg / mL (right) EMA601 or control Fab. [Figure 12]Face-to-face comparison of EMA601 and ACT017 (glenzosimab) in vitro and in vivo. (A-B) Diluted heparinized blood from hGP6tg / tg mice was pre-incubated with either EMA601 or ACT017 at the indicated concentrations. Epitope saturation was tested by flow cytometry using Emf3FITC for (A) EMA601 and JAQ1FITC (n=4) for (B) ACT017. (C) Diluted heparinized blood from hGP6tg / tg mice was pre-incubated with either EMA601 or ACT017 at the indicated concentrations, and bound Fab was detected using fluorescently labeled anti-human IgG-Fab antibody (n=4). (D-E) Aggregation tracing and quantification of washed human platelets pre-treated with either EMA601 or ACT017 at the indicated concentrations and stimulated with collagen or CRP (D). (E~G) Evaluation of platelet adhesion (F) and aggregate formation (G) to Horm collagen (200 μg / mL) under flow (1000 s-1) in heparinized human blood treated with EMA601, ACT017, or control Fab at the indicated concentrations. Scale bar 50 μm. (H~I) hGP6tg / tg mice (n=3) were intravenously treated with 4 mg / kg bwEMA601, ACT017, or control Fab (n=3). One hour after treatment, bound Fab was detected ex vivo by flow cytometry using a fluorescently labeled anti-human IgG-Fab antibody (H), and the activity of platelet αIIbβ3 integrin in response to 0.5 μg / mL CRP was measured using JON / A-PE(I). Data are presented as mean ± SD, and significance is expressed as *p<0.05, **p<0.01, and ***p<0.001 (standard one-way ANOVA) for the indicated groups. [Figure 13]Emf6.1Fab treatment protects mice in a tMCAO model of ischemic stroke. A: Infarct volume in the mouse brain (shown in cross-section) measured by triphenyltetrazolium chloride (TTC) staining in hGp6tg / tg mice treated with two doses of 4 mg / kg bw control fab (Ctrl.) or Emf6.1-fab 24 hours after tMCAO. Representative 2 mm control brain slices stained with TTC (top row) and hGp6tg / tg mice treated with Emf6.1-Fab (bottom row) on day 1 after tMCAO. The checkered areas represent brain regions not stained with TTC, meaning that the tissue in this region was affected by infarction. The non-checkered areas represent brain regions stained with TTC and therefore were not affected by infarction. B: Infarct volume in the brain of hGp6tg / tg mice treated with two doses of 4 mg / kg bw control-fab (Ctrl.) or Emf6.1-fab 24 hours after tMCAO (n=9 mice per group). The x-axis represents samples from the two groups (left: control fab-treated hGp6tg / tg mice, right: Emf6.1-fab-treated hGp6tg / tg mice), while the y-axis represents infarct volume in mm³. Each dot represents a data point, i.e., data from one mouse. In addition, the median, as well as the 25th and 75th percentiles forming the bottom and top lines of the rectangle, are marked. Finally, the minimum and maximum values ​​for each sample type are also shown by lines. Statistical significance, p<0.05, was analyzed by Student's t-test. [Figure 14] All EMA601(HC1LC2) variants in which the cysteine ​​of Kabat L55 is replaced by different amino acid inhibitors of GPVI in human platelets. Agglutination response of washed human platelets, shown by light transmission agglutination assay (n=4), after pre-incubation for 5 minutes at 0.5 μg / mL or 10 μg / mL of each HC1LC2 variant or control Fab. Platelets were stimulated with 10 μg / mL collagen (left) or 0.5 μg / mL CRP. All variants inhibited GPVI more efficiently than glenzosimab (last panel). [Modes for carrying out the invention]

[0016] The present invention relates to antibodies or functional fragments thereof that can bind to human GPVI. In the context of this application, the term “antibody” is used as a synonym for “immunoglobulin” (Ig), which is defined as a protein belonging to class IgG, IgM, IgE, IgA, or IgD (or any subclass thereof), and includes all conventionally known antibodies and their functional fragments. In the context of the present invention, a “functional fragment” of an antibody / immunoglobulin, also referred to as a “functional antibody fragment,” is defined as an antigen-binding fragment, or another derivative of a parent antibody that essentially maintains one or more of the properties of such a parent antibody as referred to in (1) to (114) above.

[0017] The "antigen-binding fragment" of an antibody / immunoglobulin is defined as a fragment that holds an antigen-binding region (e.g., the variable region of IgG). The "antigen-binding region" of an antibody is typically found in one or more hypervariable regions of the antibody, i.e., the CDR-1, -2, and / or -3 regions. The "antigen-binding fragment" of the present invention includes Fab fragments, F(ab')2 fragments, and F(ab') fragments. The "functional fragment" of the present invention includes scFv, Fv, dsFv, monovalent IgG, diabodies, triabodies, tetrabodies, and Fc fusion proteins. The antibody or functional fragment of the present invention may be part of a bifunctional construct or a polyfunctional construct.

[0018] "Fab," also referred to as "F(ab)," "fragment antigen binding," or "Fab fragment," as used herein, includes both a heavy chain and a light chain, each comprising a variable domain and a constant domain (i.e., a variable heavy domain ("V") H 」)+steady weight 1 domain(「C H 1") and variable light domains ("V L 」)+steady-state light domain(「C LThis refers to a monovalent antigen-binding fragment that contains () but lacks a fragment crystallizable region (Fc region). Generally, the light and heavy chains in a Fab are linked by disulfide bonds. A Fab may contain additional N-terminal and / or C-terminal sequences, such as the N-terminal signal peptide and / or C-terminal portion of the hinge region, but not such residues in the hinge region that are involved in the formation of disulfide bonds.

[0019] F(ab')2, also called the "F(ab')2 fragment," is divalent and contains two antigen-binding regions (i.e., two fragment antigen-binding regions), retaining part of a hinge region linked by one or more disulfide bonds. (Fab')2 typically lacks most, but not all, of the Fc region. Reduction of the F(ab')2 fragment produces two monovalent Fab' fragments, also called the "F(ab') fragment." Since Fab' is derived from F(ab')2, it may contain a small portion of the Fc region. F(ab')2, F(ab'), or Fab are C H 1 domain and C L The intermolecular disulfide interactions between the domains can be manipulated to minimize or completely eliminate them.

[0020] The "Fv" fragment is one of V H It includes the region, and one is V L The antigen-binding site consists of two chains containing the region, but the constant region of Fab (C H 1 and C L ) lacks and there is no disulfide bond linking the two chains. "Monovalent IgG" preferably refers to a functional fragment containing one full-length light chain and one full-length heavy chain, and therefore only one fragment antigen-binding region. "dsFv" refers to V linked by constructed interchain disulfide bonds. H and V L It is a disulfide-linked variable fragment containing a chain. "scFv" is a variable light ("V" L ) domain and variable weight ("V H It is a single-stranded Fv fragment in which the ) domain is linked by peptide crosslinking.

[0021] A "diabody" is a dimer consisting of two fragments (referred to herein as diabody-forming fragments) each having a variable region connected together via a linker or the like, and is typically two V L and two V H Includes. The diamond body forming fragment is V L and V H , V L and V L , V H and V H For example, preferably V H and V L It includes a fragment consisting of the following. In the diabody-forming fragment, the linker connecting the variable regions is not particularly limited, but preferably short enough to avoid non-covalent bonds between variable regions within the same fragment. The length of such a linker can be appropriately determined by those skilled in the art, but is typically 2 to 14 amino acids, preferably 3 to 9 amino acids, and particularly 4 to 6 amino acids. In this case, V encoded on the same fragment L and V H V on the same chain L and V H The links are connected via linkers short enough to avoid the formation of a single-stranded variable region fragment, thus avoiding non-covalent bonding between them and allowing the formation of a dimer with another fragment. The dimer can be formed via either covalent or non-covalent bonding between the diabody-forming fragments, or both.

[0022] Furthermore, diabody-forming fragments can be linked together via linkers to form single-chain diabodies (sc(Fv)2). By linking diabody-forming fragments using long linkers of approximately 15-20 amino acids, non-covalent bonds can be formed between diabody-forming fragments on the same chain, thereby forming dimers. Based on the same principle as diabody preparation, polymerized antibodies such as trimers or tetramers can also be prepared by linking three or more diabody-forming fragments.

[0023] The antibody or functional fragment may be monovalent or polyvalent, for example, bivalent, such as a full-length IgG molecule. According to a preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent. According to another preferred embodiment of the present invention, the antibody or functional fragment thereof is a monovalent functional fragment of an antibody. The monovalent antibody fragment comprises a fragment antigen-binding (Fab), F(ab'), Fv, a disulfide-linked variable fragment (dsFv), monovalent IgG, and a single-stranded variable fragment (scFv). Preferred functional fragments of the present invention are Fab, Fv, dsFv, scFv, and a diabody. A particularly preferred functional antibody fragment of the present invention is the antigen-binding fragment Fab. According to another preferred embodiment of the present invention, the antibody or functional fragment, preferably a functional antibody fragment, is polyvalent, for example, bivalent, but contains only one GPVI antigen-binding region.

[0024] The term "human glycoprotein VI" or "human GPVI" is known to those skilled in the art and refers to a platelet membrane glycoprotein involved in platelet-collagen interaction. GPVI is a transmembrane collagen receptor expressed on the surface of platelets. In one embodiment, human GPVI refers to an amino acid having UniProt accession number Q9HCN6 (preferably Q9HCN6-1 having NCBI accession number NP_057447), or any amino acid sequence having at least about 90% identity with the amino acid sequence of Q9HCN6-1, Q9HCN6-2, or Q9HCN6-3, preferably a protein having at least about 91, 92, 93, 94, 95, 96, 97, 98, 99% or more identity with the amino acid sequence of Q9HCN6-1, Q9HCN6-2, or Q9HCN6-3.

[0025] Preferably, the antibody or functional fragment of the present invention binds specifically to GPVI. As used herein, the antibody or functional fragment "specifically recognizes" or "specifically binds" to human GPVI if the antibody or functional fragment can distinguish human GPVI from one or more reference molecules. Preferably, IC for binding to each of the reference molecules. 50 The value is IC for coupling to GPVI. 50The value is at least 1,000 times greater. In its most common form (and where no defined designation is mentioned), “specific binding” refers to the ability of an antibody or functional fragment to distinguish human GPVI from unrelated biomolecules, as determined, for example, according to specificity assay methods known in the art. Such methods include, but are not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR), ELISA, Western blotting, and immunofluorescence-based assays such as immunohistochemistry (IHC). For example, a standard BLI, SPR, or ELISA assay can be performed. Typically, the determination of binding specificity is performed by using a set of about 3 to 5 unrelated biomolecules, such as powdered milk, BSA, and transferrin, rather than a single reference biomolecule.

[0026] The binding epitope on GPVI to which the antibody or functional fragment of the present invention binds is not particularly limited. According to one embodiment of the present invention, the antibody or functional fragment binds to human GPVI near or at the position of the proposed GPVI dimerization residue. According to a preferred embodiment of the present invention, the antibody or functional fragment binds to human GPVI at a binding epitope that at least partially overlaps with the proposed GPVI dimerization residue. The proposed GPVI dimerization residue can be found in the ectodomain of GPVI, preferably referring to residues D173-T180 of SEQ ID NO: 36 (SEQ ID NO: 36 corresponds to a portion of the ectodomain of a GPVI isoform identified as isoform 1 by UniProt and as isoform 2 by NCBI), which corresponds to residues D196-T203 of GPVI isoforms 1-3 in UniProt (i.e., Q9HCN6-1, Q9HCN6-2, and Q9HCN6-3). According to another embodiment of the present invention, the binding of an antibody or functional fragment to human GPVI interrupts or interferes with GPVI dimerization. According to a preferred embodiment of the present invention, the antibody or functional fragment binds to human GPVI with a binding epitope comprising, or consisting thereof, amino acids V178-E192 and / or S223-P237 of SEQ ID NO: 36. According to another preferred embodiment of the present invention, the antibody or functional fragment binds to a discontinuous binding epitope comprising, or consisting thereof, amino acids V178-E192 and S223-P237 of SEQ ID NO: 36.

[0027] The antibody or functional fragment of the present invention is V L Domain and V H Includes domain. V L The domain includes the CDR1 region (CDRL1), the CDR2 region (CDRL2), the CDR3 region (CDRL3), and the framework region. H The domain includes the CDR1 region (CDRH1), the CDR2 region (CDRH2), the CDR3 region (CDRH3), and the framework region.

[0028] The term “CDR” or “complementarity-determining region” refers to one of six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions for the six CDRs is provided by Kabat EA et al., (1991) “Sequences of proteins of immunological interest.” NIH Publication 91-3242. Another commonly used system for defining / identifying CDRs is provided by Lefranc et al., (2003) “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and lg superfamily V-like domains”. Dev.Comp.Immunol., 27, 55-77. The CDR sequences of this antibody or functional fragment were identified using the IMGT and Kabat antibody numbering systems, i.e., as combinations of IMGT / Kabat CDR sequences.

[0029] The antibody or functional fragment of the present invention L The domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 9, or SEQ ID NO: 64, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3. In a preferred embodiment, the V of the antibody or functional fragment of the present invention L The domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 9, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3. In another preferred embodiment, the V of the antibody or functional fragment of the present invention LThe domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3. In another preferred embodiment, the V of the antibody or functional fragment of the present invention L The domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3. In another embodiment, the V of the antibody or functional fragment of the present invention L The domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 64, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3.

[0030] In amino acid sequence number 9, residue X is preferably alanine (A), arginine (R), asparagine (N), aspartic acid (D), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), serine (S), threonine (T), tryptophan (W), tyrosine (Y), or valine (V). In a particular embodiment of the present invention, residue X in amino acid sequence number 9 is A. In another particular embodiment of the present invention, residue X in amino acid sequence number 9 is R. In yet another particular embodiment of the present invention, residue X in amino acid sequence number 9 is N. In yet another particular embodiment of the present invention, residue X in amino acid sequence number 9 is D. In yet another particular embodiment of the present invention, residue X in amino acid sequence number 9 is Q. In yet another particular embodiment of the present invention, residue X in amino acid sequence number 9 is E. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is G. In amino acid sequence number 9, residue X is H. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is I. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is L. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is K. In yet another specific embodiment of the present invention, in an embodiment of the present invention, residue X is M. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is F. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is S. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is T. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is W. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is Y. In yet another specific embodiment of the present invention, in amino acid sequence number 9, residue X is V.

[0031] The antibody or functional fragment of the present invention HThe domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 13, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6. SEQ ID NO: 13 basically corresponds to the amino acid sequence of SEQ ID NO: 5, however, the residues at positions 5 and 6 are preferably selected from A, R, N, D, Q, E, G, H, I, L, K, M, F, S, T, W, Y, and V. In a particular embodiment, in SEQ ID NO: 13, the residue at position 5 is D or E, and the residue at position 6 is G or A. In a preferred embodiment of the present invention, V of the antibody or functional fragment of the present invention H The domain includes a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6.

[0032] In an alternative embodiment (Kabat only), the antibody or functional fragment of the present invention comprises (i) a CDR1 region having the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 2, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 3. L (ii) a domain and a V including a CDR1 region having the amino acid sequence shown in SEQ ID NO: 10, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 14. H Includes the domain.

[0033] In another alternative embodiment, the antibody or functional fragment of the present invention comprises (i) a CDR1 region having the amino acid sequence shown in SEQ ID NO: 7, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 8, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 3. L(ii) a domain and a V including a CDR1 region having the amino acid sequence shown in SEQ ID NO: 11, a CDR2 region having the amino acid sequence shown in SEQ ID NO: 12, and a CDR3 region having the amino acid sequence shown in SEQ ID NO: 6 H Includes the domain.

[0034] In another embodiment, the antibody or functional fragment of the present invention contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, even more preferably at least 94%, even more preferably at least 95%, even more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, and even more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35, more preferably 16, 17, 18, 32, 33, and 34, or a V L Includes a domain. In yet another embodiment, the antibody or functional fragment of the present invention contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 92%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and more preferably at least 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 26, 27, 28, 32, 33, 34, and 35, preferably 32, 33, and 35, more preferably 32, 33, and 34, or a V consisting of the same. L Includes the domain.

[0035] In yet another embodiment, the antibody or functional fragment of the present invention contains or comprises an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 29, 30, and 31. LIncludes a domain. In another embodiment, the antibody or functional fragment of the present invention contains or comprises an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, and 19, preferably 16, 17, and 18. L Includes a domain. In further embodiments, the antibody or functional fragment of the present invention includes or comprises an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, and 25, preferably 21, 22, and 23. H Includes the domain.

[0036] According to a preferred embodiment, the antibody or functional fragment of the present invention comprises or consists of an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid sequence shown in SEQ ID NO: 17. L A V comprising or consisting of an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the domain and the amino acid sequence shown in SEQ ID NO: 21. H Includes the domain.

[0037] According to one embodiment, the antibody or functional fragment of the present invention contains or comprises the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, preferably 16, 17, 18, 19, 32, 33, 34, or 35, more preferably 16, 17, 18, 32, 33, or 34. L The domain is included. According to another embodiment, the antibody or functional fragment of the present invention contains or consists of the amino acid sequence shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, or 35, preferably 32, 33, 34, or 35, more preferably 32, 33, or 34. L Includes the domain.

[0038] In yet another embodiment, the antibody or functional fragment of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 19, 29, 30, or 31. L Includes a domain. In another embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, or 19. L Includes a domain. In another embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NOs: 29, 17, 30, or 31. L Includes a domain. In preferred embodiments, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 32, 33, or 34. L Includes a domain. In another preferred embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 32, 33, or 34. L Includes a domain. In yet another preferred embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 16, 17, or 18. L Includes a domain. In a particularly preferred embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 17. L Includes a domain. In another particularly preferred embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 33. L Includes the domain.

[0039] In further embodiments, the antibody or functional fragment of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NOs: 21, 22, 23, 24, or 25. H Includes a domain. In preferred embodiments, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 21, 22, or 23. HIncludes a domain. In a particularly preferred embodiment, the antibody or functional fragment of the present invention includes or consists of the amino acid sequence shown in SEQ ID NO: 21. H Includes the domain.

[0040] According to certain preferred embodiments of the present invention, V L Domain and V H The domains are: Atlases 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23, 26 and 24, 26 and 25, 27 and 21, 27 and 22, 27 and 23, 27 and 24, 27 and 25, 28 and 21, 28 and 22, 28 and 23, 28 and 24, 28 and 25, 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 22, 19 and 23, 19 and 24, or 19 and 25 (i.e., V L is a sequence that includes or consists of the sequence shown in sequence number 32, and V H This includes or consists of the sequence shown in Sequence ID No. 21, or V L is a sequence that includes or consists of the sequence shown in sequence number 32, and V H (For example, it includes or consists of the sequence shown in Sequence ID No. 22), preferably including or consisting of a pair of amino acid sequences shown in 33 and 21, 33 and 22, 34 and 22, 17 and 21, 17 and 22, or 18 and 22.

[0041] According to another particular preferred embodiment of the present invention, V L Domain and V HThe domains are: Atlases 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23, 26 and 24, 26 and 25 , comprising or consisting of a pair of amino acid sequences arranged in 27 and 21, 27 and 22, 27 and 23, 27 and 24, 27 and 25, 28 and 21, 28 and 22, 28 and 23, 28 and 24, or 28 and 25, preferably 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, 34 and 23, 26 and 22, 27 and 21, 27 and 22, or 27 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain contains or consists of a pair of amino acid sequences that are arranged in sequence numbers 32 and 21, 32 and 22, 32 and 23, 32 and 24, 32 and 25, 33 and 21, 33 and 22, 33 and 23, 33 and 24, 33 and 25, 34 and 21, 34 and 22, 34 and 23, 34 and 24, 34 and 25, 35 and 21, 35 and 22, 35 and 23, 35 and 24, 35 and 25, 26 and 21, 26 and 22, 26 and 23, 26 and 24, or 26 and 25, preferably 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22, or 34 and 23.

[0042] According to yet another specific preferred embodiment of the present invention, V L Domain and V HThe domain contains or consists of a pair of amino acid sequences shown in SEQ ID NOs: 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 22, 19 and 23, 19 and 24, 19 and 25, 29 and 21, 29 and 22, 29 and 23, 29 and 24, 29 and 25, 30 and 21, 30 and 22, 30 and 23, 30 and 24, 30 and 25, 31 and 21, 31 and 22, 31 and 23, 31 and 24, or 31 and 25 (i.e., V L is a sequence that includes or consists of the sequence shown in sequence number 16, and V H This includes or consists of the sequence shown in Sequence ID No. 21, or V L is a sequence that includes or consists of the sequence shown in sequence number 16, and V H (This includes, for example, the sequence shown in Sequence ID No. 22.) According to another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of pairs of amino acid sequences of SEQ ID NOs: 16 and 22, 16 and 23, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 29 and 22, 29 and 23, 29 and 25, 30 and 21, 30 and 22, or 30 and 23. According to another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 16 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 29 and 22, 30 and 21, 30 and 22, or 30 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V HThe domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 18 and 23, 30 and 21, 30 and 22, or 30 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, 30 and 21, or 30 and 22. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain contains or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, 18 and 22, or 30 and 22.

[0043] According to a further specific preferred embodiment of the present invention, V L Domain and V H The domain contains or consists of a pair of amino acid sequences shown in SEQ ID NOs: 16 and 21, 16 and 22, 16 and 23, 16 and 24, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, 18 and 23, 18 and 24, 18 and 25, 19 and 21, 19 and 22, 19 and 23, 19 and 24, 19 and 25 (i.e., V L is a sequence that includes or consists of the sequence shown in sequence number 16, and V H This includes or consists of the sequence shown in Sequence ID No. 21, or V L is a sequence that includes or consists of the sequence shown in sequence number 16, and V H (This includes or consists of the sequence shown in Sequence ID No. 22). According to another particular preferred embodiment of the present invention, V L Domain and V HThe domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 16 and 22, 16 and 23, 16 and 25, 17 and 21, 17 and 22, 17 and 23, 17 and 24, 17 and 25, 18 and 21, 18 and 22, or 18 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 16 and 22, 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, 17 and 23, 18 and 21, or 18 and 22. According to yet another particular preferred embodiment of the present invention, V L Domain and V H The domain contains or consists of a pair of amino acid sequences shown in SEQ ID NOs: 17 and 21, 17 and 22, or 18 and 22.

[0044] According to a particularly preferred embodiment of the present invention, V L Domain and V H The domain comprises or consists of a pair of amino acid sequences shown in Sequence IDs 33 and 21. According to another particularly preferred embodiment of the present invention, V L Domain and V H The domain contains or consists of the pair of amino acid sequences shown in Sequence IDs 17 and 21.

[0045] In another embodiment, the antibody or functional fragment of the present invention is V LThe domain comprises or consists of the amino acid sequences shown in SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, respectively, instead of SEQ ID NO: 16, SEQ ID NO: 18, and SEQ ID NO: 19 as described in any of the embodiments described above.

[0046] In further embodiments, the antibody or functional fragment of the present invention comprises or consists of the amino acid sequence shown in SEQ ID NO: 15. L A V containing or consisting of a domain and / or the amino acid sequence shown in SEQ ID NO: 20 H Includes the domain.

[0047] In another specific embodiment, the antibody of the present invention is an immunoglobulin or a functional fragment thereof, preferably immunoglobulin G (IgG) or a functional fragment thereof. Suitable subclasses of IgG for the present invention are not limited to IgG1, IgG2, IgG3, and IgG4. Preferably, IgG is subclass 1, i.e., an IgG1 molecule.

[0048] Typically, the antibody or functional fragment of the present invention is V L Light chain including domain, and V H It includes a heavy chain containing a domain. According to a particular embodiment of the present invention, the antibody or functional fragment is V L Light chain including domain, and V H It consists of a heavy chain containing a domain. According to a preferred embodiment of the present invention, the antibody or functional fragment is monovalent, for example, Fab, and the above V L A V comprising or consisting of an amino acid sequence specified in any of the embodiments for the domain. L Light chain including domain, and the above V H A V comprising or consisting of an amino acid sequence specified in any of the embodiments for the domain. H It has a heavy chain containing a domain.

[0049] According to a preferred embodiment of the present invention, the light chain is V L In addition to the domain, preferably V L Light chain constant domain located at the C-terminus of the domain (CL ) includes. According to a further preferred embodiment of the present invention, the heavy chain is V H In addition to the domain, preferably V H A heavy chain constant domain (C) located at the C terminus of the domain. H ) includes.

[0050] According to a particular embodiment, the antibody or functional fragment of the present invention comprises a light chain derived from a human immunoglobulin kappa (IgK) light chain (preferably allotype Km3). According to another particular embodiment, the antibody or functional fragment of the present invention comprises a light chain comprising a constant domain derived from a human IgK light chain constant domain (preferably allotype Km3). According to yet another particular embodiment, the antibody or functional fragment of the present invention comprises a light chain constant domain comprising a human IgK light chain constant domain (preferably allotype Km3) or a light chain constant domain comprising the same. According to yet another particular embodiment, the antibody or functional fragment of the present invention comprises a heavy chain comprising a constant domain derived from a human IgG1 heavy chain CH1 constant domain (preferably allotype G1m17,1). According to yet another particular embodiment, the antibody or functional fragment of the present invention comprises a heavy chain comprising a constant domain comprising a human IgG1 heavy chain CH1 constant domain (preferably allotype G1m17,1) or a heavy chain comprising a constant domain comprising the same.

[0051] In yet another specific embodiment, the antibody or functional fragment of the present invention includes a light chain comprising a constant domain derived from a human IgK light chain constant domain (allotype Km3), preferably a light chain in which the total number of amino acids different from the amino acid sequence of the human IgK light chain constant domain (allotype Km3) shown in, for example, SEQ ID NO: 60 is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2, and / or a heavy chain comprising a constant domain derived from a human IgG1 heavy chain CH1 constant domain (allotype G1m17,1), preferably a heavy chain in which the total number of amino acids different from the amino acid sequence of the human IgG1 heavy chain CH1 constant domain (allotype G1m17,1) shown in, for example, SEQ ID NO: 61 is less than 10, 9, 8, 7, 6, 5, 4, 3, or 2.

[0052] According to a preferred embodiment of the present invention, the light chain constant domain (C L ) comprises an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid shown in SEQ ID NO: 60. According to another preferred embodiment of the present invention, the light chain constant domain (C L ) consists of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, or 99% sequence identity with the amino acid shown in SEQ ID NO: 60.

[0053] According to a preferred embodiment of the present invention, the heavy chain constant domain (C H ) comprises an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the amino acid shown in SEQ ID NO: 61. According to another preferred embodiment of the present invention, the light chain constant domain (C H ) consists of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, or 99% sequence identity with the amino acid shown in SEQ ID NO: 61.

[0054] According to a further preferred embodiment of the present invention, the light chain comprises or consists of an N-terminal light chain signal peptide comprising the amino acid sequence shown in SEQ ID NO: 62, and / or comprising or consisting of the amino acid sequence shown in SEQ ID NO: 60, preferably V L The C-terminal light chain constant domain (C L ) includes. According to another preferred embodiment of the present invention, the heavy chain comprises or consists of an N-terminal heavy chain signal peptide comprising the amino acid sequence shown in SEQ ID NO: 63, and / or comprising or consists of the amino acid sequence shown in SEQ ID NO: 61, preferably V H The heavy chain constant domain (C) is the C-terminus of the domain. H ) contains the N-terminal signal peptide and / or constant region. L and / or VH They may be positioned on the light chain and / or heavy chain so as to be directly linked to the domain, or they may be linked via a linker peptide. Preferably, the N-terminal signal peptide and the constant region are located such that they are V L and V H They are positioned on the light and heavy chains so as to be directly linked to the domain.

[0055] According to a preferred embodiment of the present invention, the antibody or functional fragment is preferably monovalent, more preferably Fab, and (a) the above V L V contains or consists of an amino acid specified in any of the embodiments of the domain. L Domain, above C L A C comprising or consisting of an amino acid specified in any of the domain embodiments. L (b) The domain, and optionally a light chain comprising or consisting of the N-terminal light chain signal peptide specified above, and (b) the above V H V contains or consists of an amino acid specified in any of the embodiments of the domain. H Domain, above C H A C comprising or consisting of an amino acid specified in any of the domain embodiments. H The domain and optionally include the N-terminal heavy chain signal peptide specified above, or a heavy chain comprising such signal peptide.

[0056] The light chain and heavy chain are V L and V H The domain may include further amino acid sequences at the N-terminus or C-terminus, such as other signal peptides, binding peptides, linker peptides, or other functional protein domains. Furthermore, the light and heavy chains may include further molecules covalently or otherwise bound to the N-terminus or C-terminus, such as other signal molecules, binding sites, (crosslinking) linker molecules, or other functional sites.

[0057] In preferred embodiments of the present invention, the antibody or functional fragment thereof comprises or comprises a light chain containing or consisting of the amino acid sequence shown in SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, preferably SEQ ID NOs: 38, 39, 40, 41, 55, 56, 57 and 58, more preferably one of SEQ ID NOs: 38, 39, 40, 55, 56 and 57, and a heavy chain containing or consisting of the amino acid sequence shown in SEQ ID NOs: 44, 45, 46, 47 and 48, preferably one of SEQ ID NOs: 44, 45 and 46. In another preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or comprises a light chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs. 52, 53, 54, 55, 56, 57, and 58, preferably one of SEQ ID NOs. 52, 53, and 54, and a heavy chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs. 44, 45, 46, 47, and 48, preferably one of SEQ ID NOs. 44, 45, and 46. In yet another preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or comprises a light chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs. 38, 39, 40, 41, 49, 50, and 51, preferably one of SEQ ID NOs. 38, 39, and 40, and a heavy chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs. 44, 45, 46, 47, and 48, preferably one of SEQ ID NOs. 44, 45, and 46.

[0058] According to preferred embodiments of the present invention, the antibody or functional fragment thereof is a monovalent functional antibody fragment, preferably a fragment antigen-binding (Fab), F(ab'), Fv, a disulfide-linked variable fragment (dsFv), a monovalent IgG, or a single-stranded variable fragment (scFv). A particularly preferred functional antibody fragment of the present invention is a monovalent Fab. In a preferred embodiment of the present invention, the functional fragment is a monovalent functional antibody fragment comprising, or consisting of, a light chain containing, an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, or 41, and a heavy chain containing, or consisting of, an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, or 48.

[0059] In another preferred embodiment of the present invention, the functional fragment is a monovalent functional antibody fragment having a light chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, preferably SEQ ID NOs: 38, 39, 40, 41, 55, 56, 57 and 58, more preferably one of SEQ ID NOs: 38, 39, 40, 55, 56 and 57, and a heavy chain comprising or consisting thereof an amino acid sequence shown in SEQ ID NOs: 44, 45, 46, 47 and 48, preferably one of SEQ ID NOs: 44, 45 and 46. In another preferred embodiment of the present invention, the functional fragment is a monovalent functional antibody fragment having a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48. In another preferred embodiment of the present invention, the functional fragment is a monovalent functional antibody fragment having a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 38, 39, 40, 41, 49, 50, and 51, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48.

[0060] In a more preferred embodiment of the present invention, the functional fragment is a monovalent, for example, fragment antigen-binding (Fab), comprising (a) a light chain comprising or having an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, or 41, and (b) a heavy chain comprising or having an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%t, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 44, 45, 46, 47, or 48. In another preferred embodiment of the present invention, the functional fragment is monovalent, preferably Fab, comprising (a) a light chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, and 51, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48. According to yet another preferred embodiment of the present invention, the functional fragment is monovalent, preferably Fab, comprising (a) a light chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, and 41, and (b) a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48. According to another preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a light chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48.According to another preferred embodiment, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or comprising a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 52, 53, 54, 55, 56, 57, and 58, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48. According to yet another preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting thereof a light chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 55, 56, 57, and 58, and a heavy chain comprising or consisting thereof the amino acid sequence shown in one of SEQ ID NOs. 44, 45, 46, 47, and 48. In yet another preferred embodiment, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a light chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs. 55, 56, and 57, and a heavy chain comprising or consisting of the amino acid sequence shown in one of SEQ ID NOs. 44, 45, and 46.

[0061] In certain preferred embodiments of the present invention, the antibody or its functional fragment (preferably monovalent, more preferably Fab) is SEQ ID NOs: 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46, 41 and 47, 41 and 48, 55 and 44, 55 and 45, 55 and 46, 55 and 47, 55 and 48, 56 and 44, 5 6 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 57 and 47, 57 and 48, 58 and 44, 58 and 45, 58 and 46, 58 and 47, or 58 and 48, comprising or consisting of a pair of light and heavy chains (i.e., the light chain has the sequence shown in SEQ ID NO: 38 and the heavy chain has the sequence shown in SEQ ID NO: 44, or the light chain has the sequence shown in SEQ ID NO: 38 and the heavy chain has the sequence shown in SEQ ID NO: 45, etc.).

[0062] In another particular preferred embodiment of the present invention, the antibody or functional fragment thereof (preferably monovalent, more preferably Fab) is SEQ ID NOs: 55 and 44, 55 and 45, 55 and 46, 55 and 47, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 57 and 47, 57 and 48, 58 and 44, 58 and 45, 5 8 and 46, 58 and 47, 58 and 48, 52 and 44, 52 and 45, 52 and 46, 52 and 47, 52 and 48, 53 and 44, 53 and 45, 53 and 46, 53 and 47, 53 and 48, 54 and 44, 54 and 45, 54 and 46, 54 and 47, or 54 and 48, preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 52 and 44, 52 and 45, 52 and 46, 53 and 44, 53 and 45, or 53 and 46, more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, 57 and 46, 52 and 45, 53 and 44, 53 and 45, or 53 and 46, even more preferably 56 and 44, 56 and 45, 56 and It comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in 46, 57 and 44, 57 and 45, 57 and 46, 53 and 44, 53 and 45, or 53 and 46, more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, 53 and 44, or 53 and 45, even more preferably 56 and 44, 56 and 45, 57 and 45, or 53 and 45.

[0063] In another particular preferred embodiment of the present invention, the antibody or its functional fragment is SEQ ID NOs: 55 and 44, 55 and 45, 55 and 46, 55 and 47, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, 57 and 46, 57 and 47, 57 and 48, 58 and 44, 58 and 45, 58 and 46, 5 8 and 47, or 58 and 48, preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 and 44, 57 and 45, or 57 and 46, more preferably 55 and 45, 55 and 46, 55 and 48, 56 and 44, 56 and 45, 56 and 46, 56 and 47, 56 and 48, 57 The amino acid sequences shown in 44, 57 and 45, or 57 and 46, more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, or 57 and 46, more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45, or 57 and 46, more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, or 57 and 45, more preferably 56 and 44, 56 and 45, 56 and 46, or 57 and 45, more preferably 56 and 44, 56 and 45, or 57 and 45, and more preferably 56 and 44, or a pair of light and heavy chains made therefrom, are monovalent, preferably Fab.

[0064] In yet another particular preferred embodiment of the present invention, the antibody or functional fragment thereof (preferably monovalent, more preferably Fab) is SEQ ID NOs: 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46, 41 and 47, 41 and 48, 49 and 44, 49 and 45, 49 and 46, 49 and 47, 49 and 4 8, 50 and 44, 50 and 45, 50 and 46, 50 and 47, 50 and 48, 51 and 44, 51 and 45, 51 and 46, 51 and 47, or 51 and 48, or a pair of light and heavy chains consisting of or comprising the amino acid sequences shown in these sequences (i.e., the light chain contains or consists of the sequence shown in SEQ ID NO: 38, and the heavy chain contains or consists of the sequence shown in SEQ ID NO: 44, or the light chain has the sequence shown in SEQ ID NO: 38, and the heavy chain has the sequence shown in SEQ ID NO: 45, etc.). In another particular preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 49 and 44, 49 and 45, 49 and 46, 50 and 44, 50 and 45, or 50 and 46. In yet another particular preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 40 and 46, 49 and 45, 50 and 44, 50 and 45, or 50 and 46.In yet another particular preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 40 and 46, 50 and 44, 50 and 45, or 50 and 46. In yet another particular preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains comprising or consisting of the amino acid sequence shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, 50 and 44, or 50 and 45. In yet another particular preferred embodiment of the present invention, the antibody or functional fragment thereof is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, comprising or consisting of the amino acid sequence shown in SEQ ID NOs. 39 and 44, 39 and 45, 40 and 45, or 50 and 45.

[0065] In further specific preferred embodiments of the present invention, the antibody or functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 38 and 44, 38 and 45, 38 and 46, 38 and 47, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, 40 and 46, 40 and 47, 40 and 48, 41 and 44, 41 and 45, 41 and 46, 41 and 47, or 41 and 48 (i.e., the light chain has the sequence shown in SEQ ID NO: 38 and the heavy chain has the sequence shown in SEQ ID NO: 44, or the light chain has the sequence shown in SEQ ID NO: 38 and the heavy chain has the sequence shown in SEQ ID NO: 45). In another particular preferred embodiment of the present invention, the functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, or 40 and 46. In yet another particular preferred embodiment of the present invention, the functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 38 and 45, 38 and 46, 38 and 48, 39 and 44, 39 and 45, 39 and 46, 39 and 47, 39 and 48, 40 and 44, 40 and 45, or 40 and 46. In yet another particular preferred embodiment of the present invention, the functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46.

[0066] In yet another particular preferred embodiment of the present invention, the antibody or functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, 40 and 44, or 40 and 45. In yet another particular preferred embodiment of the present invention, the antibody or functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 39 and 44, 39 and 45, 39 and 46, or 40 and 45. In yet another embodiment of the present invention, the functional fragment (preferably monovalent, more preferably Fab) comprises a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs: 39 and 44, 39 and 45, or 40 and 45.

[0067] In certain preferred embodiments of the present invention, the antibody or functional fragment thereof comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 39 and 45, and is monovalent, preferably Fab. In another certain preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 39 and 46, and is monovalent, preferably Fab. In another certain preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 40 and 44, and is monovalent, preferably Fab. In another certain preferred embodiment of the present invention, the antibody functional fragment comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 40 and 45, and is monovalent, preferably Fab. In a particularly preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 39 and 44, and is monovalent, preferably Fab. In another particularly preferred embodiment of the present invention, the antibody or functional fragment thereof comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NOs. 56 and 44, and is monovalent, preferably Fab.

[0068] According to a further embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is selected from A, R, N, D, Q, E, G, H, I, L, K, M, F, S, T, W, Y, and V. In a particular embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is A. In another particular embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is R. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is N. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is D. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 53, 54, 55, 56, 57, and 58, residue X is Q. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is E. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 55, 56, 57, and 58, residue X is G. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 55, 56, 57, and 58, residue X is H. In yet another specific embodiment of the present invention, in the amino sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is I.In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 55, 56, 57, and 58, residue X is L. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is K. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is M. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is F. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 53, 54, 55, 56, 57, and 58, residue X is S. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is T. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is W. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is Y. In yet another specific embodiment of the present invention, in the amino acid sequences shown in SEQ ID NOs: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 55, 56, 57, and 58, residue X is V.

[0069] According to a particular embodiment of the present invention, V contains or consists of the amino acid sequences shown in SEQ ID NOs: 32, 33, 34, and 35. LIn any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is A. In another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is R. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is N. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is D. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is Q. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is E. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is G. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is H. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. LIn any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is I. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is L. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is K. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is M. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is F. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is S. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is T. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs. 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is W. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. LIn any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is Y. In yet another specific embodiment of the present invention, V comprises or consists of the amino acid sequence shown in SEQ ID NOs: 32, 33, 34, and 35. L In any of the above embodiments relating to an antibody or functional fragment thereof having a light chain containing, residue X is V.

[0070] In a particular embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is A. In another particular embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is R. In yet another particular embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is N. In yet another particular embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is D. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is Q. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is E. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is G. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is H. In yet another specific embodiment of the present invention, in any of the above embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is I.In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is L. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is K. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is M. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is F. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is S. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is T. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is W. In yet another specific embodiment of the present invention, in any of the aforementioned embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is Y. In yet another specific embodiment of the present invention, in any of the above embodiments relating to an antibody or functional fragment thereof having a light chain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58, residue X is V.

[0071] In further embodiments, the present invention binds to essentially the same binding epitope as defined in any of the embodiments described above, but to a different CDR sequence and / or V as defined in any of the embodiments described above. L and V H The target is antibodies or functional fragments thereof, including sequences and / or light and heavy chain sequences.

[0072] affinity The antibody or functional fragment of the present invention has very high affinity for human GPVI. D The term "dissociation equilibrium constant" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction. Typically, the antibody or functional fragment of the present invention has a dissociation equilibrium constant (K) of less than about 1 μM, preferably less than 900 pM, more preferably less than 800 pM, even more preferably less than 700 pM, even more preferably less than 600 pM, even more preferably less than 500 pM, even more preferably less than 400 pM, even more preferably less than 350 pM, even more preferably less than 300 pM, even more preferably less than 250 pM, even more preferably less than 200 pM, for example, less than or equal to about 195 pM, or less than or equal to about 175 pM. D ) binds to human GPVI. In a particularly preferred embodiment, the antibody or functional fragment of the present invention has a dissociation equilibrium constant (K) of 195 pM or less. D It binds to human GPVI.

[0073] K D This can be easily determined using conventional techniques such as biolayer interferometry (BLI), surface plasmon resonance (SPR), or ELISA. According to a particular embodiment, the dissociation equilibrium constant (K D ) is determined using BLI, SPR technology (e.g., in a BIACORE instrument), or ELISA.

[0074] Preferably, the dissociation equilibrium constant (K D ) is determined using BLI, for example in Octet instruments, and using Fortebio software. In particular, K DThe determination of is preferably carried out as described in Section 1.16 of the Examples. According to another specific embodiment, the dissociation equilibrium constant (K D The antibody screening range is determined at 25°C by BLI using, for example, an Octet instrument, preferably at a constant orbital flow rate of, for example, 1000 rpm; an antigen (human GPVI) loading concentration of 1.2 μg / ml for antigen immobilization on the biosensor, an association time of 900 seconds and a dissociation time of 1200 seconds, an antibody screening range having seven concentrations in the range of 10 to 0.014 nM using 3-fold serial dilutions, and a 1:1 interaction / fitting model.

[0075] Functional properties of antibodies or functional antibody fragments The antibodies or functional fragments of the present invention have high efficacy in inhibiting human GPVI on platelets in plasma due to their very high affinity for human GPVI and high stability in plasma. In certain embodiments, the antibodies or functional fragments of the present invention can sustain the inhibition of human GPVI in plasma for at least 12 hours, preferably at least 24 hours, more preferably at least 36 hours, even more preferably at least 48 hours, even more preferably at least 60 hours, even more preferably at least 72 hours, even more preferably at least 84 hours, and even more preferably at least 96 hours. The antibodies or functional fragments of the present invention specified in any of the embodiments herein preferably have one or more further functional properties as described below.

[0076] Typically, the antibody or functional fragment of the present invention, preferably in a monovalent form, for example as Fab, enables long-term binding to GPVI on circulating platelets but does not affect the in vivo platelet count, size, or GPVI surface level on circulating platelets. The binding of the antibody or functional fragment to human GPVI on circulating platelets, and the GPVI surface level on circulating platelets, can be determined, for example, as described in the examples in Section 1.10. The platelet count and size can be determined, for example, as described in the examples in Section 1.9.

[0077] In one embodiment of the present invention, preferably in monovalent form, for example as Fab, the antibody or functional fragment of the present invention, when normalized with a negative control (e.g., a control Fab that is not specific to GPVI), can be used to test for GPVI in humanized mice (hGP6) at a dose of, for example, 4 mg / kg. tg / tg Long-term binding to circulating platelets is possible, for example, with a GPVI receptor epitope occupancy of more than 50% for at least 12, 24, 36, 48, or 60 hours at a dose of 4 mg / kg, determined by flow cytometry analysis of blood diluted ex vivo, for example, using competitive binding with different fluorescently labeled anti-GPVI antibodies or functional fragments, and by using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy.

[0078] In another embodiment of the present invention, preferably in monovalent form, for example as Fab, the antibody or functional fragment of the present invention is used to humanize mouse (hGP6) against GPVI at a dose of 2 mg / kg, for example. tg / tg Long-term binding to circulating platelets is possible, for example, at a dose of 2 mg / kg for at least 8, 12, 16, 20, 28, or 28 hours, with a GPVI receptor epitope occupancy of over 50%, when normalized with a negative control (e.g., a control Fab that is not specific to GPVI), as determined by, for example, flow cytometry analysis of blood diluted ex vivo after intravenous administration to a mouse model, and by, for example, using competitive binding with different fluorescently labeled anti-GPVI antibodies or functional fragments, and by, for example, using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy.

[0079] In another embodiment of the present invention, preferably in monovalent form, for example as Fab, the antibody or functional fragment of the present invention is used to humanize mouse (hGP6) against GPVI at a dose of 4 mg / kg, for example. tg / tgAfter intravenous administration to a mouse model, for example, as determined by flow cytometry analysis in blood diluted ex vivo, using, for example, competitive binding with different fluorescently labeled anti-GPVI antibodies or functional fragments and using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy, for example, at a dose of 2 mg / kg for at least 36, 48, 60, 72, 84, or 96 hours, when normalized with a negative control (e.g., control Fab not specific for GPVI), enables long-term binding to circulating platelets with at least 20% GPVI receptor epitope occupancy.

[0080] In another embodiment of the invention, the antibody or functional fragment of the invention, preferably in monovalent form, e.g., Fab, is, for example, at a dose of 4 mg / kg, in a humanized mouse (hGP6 tg / tg mouse) after intravenous administration, for example, as evaluated at 12, 24, 36, 48, 60, 72, 84, 96, 120, or 144 hours, as determined by flow cytometry analysis in blood diluted ex vivo, for example, as described in the examples of Section 1.10, does not affect the GPVI surface level on circulating platelets.

[0081] In another embodiment of the invention, the antibody or functional fragment of the invention, preferably in monovalent form, e.g., Fab, is, for example, at a dose of 4 mg / kg, in a humanized mouse (hGP6 tg / tg mouse) after intravenous administration, for example, as evaluated at 12, 24, 36, 48, 60, 72, 84, 96, 120, or 144 hours, and as tested ex vivo in sampled blood, for example, as described in the examples of Section 1.9, does not affect the platelet number and size on circulating platelets in vivo.

[0082] Typically, the antibodies or functional fragments of the present invention can completely inhibit CRP (collagen-related peptide) and collagen-induced aggregation of washed human platelets. The CRP or collagen-induced aggregation of washed human platelets can be determined using standard light transmission aggregation measurement methods, as described, for example, in the examples of Section 1.7. According to certain embodiments, the antibodies or functional fragments of the present invention can completely inhibit CRP and collagen-induced aggregation of washed human platelets at a concentration of 10 μg / ml or less, preferably 5 μg / ml or less, more preferably 2 μg / ml or less, and even more preferably 1 μg / ml or less, as determined using standard light transmission aggregation measurement methods, as described, for example, in the examples of Section 1.7.

[0083] According to another specific embodiment, the antibodies or functional fragments of the present invention can completely inhibit CRP and / or collagen-induced aggregation of washed mouse hGP6 platelets for at least 12, 24, 48, or 60 hours after intravenous administration at a dose of 4 mg / kg to a humanized mouse model (hGP6tg / tg mouse) humanized for GPVI, for example, as described in the examples of Section 1.7, as determined using standard light transmission aggregation measurement methods. tg / tg platelets, as described, for example, in the examples of Section 1.7.

[0084] According to another specific embodiment, the antibody or functional fragment of the present invention has more than 2 times, preferably >4 times, >6 times, >8 times, or more than 10 times higher potency than ACT017 (glenzosimab) at concentrations of preferably 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, and measured using a standard light transmission agglutination assay, for example, as described in the examples of Section 1.7, in inhibiting GPVI function in human platelets in vitro. According to another specific embodiment, the antibody or functional fragment of the present invention has more than 2 times, preferably >4 times, >6 times, >8 times, or more than 10 times higher potency than ACT017 (glenzosimab) at concentrations of preferably 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, and measured using a standard light transmission agglutination assay, for example, as described in the examples of Section 1.7, in inhibiting collagen and / or CRP-induced aggregation of washed human platelets in vitro. Optionally, the change in potency is determined as the ratio of the maximum agglutination percentage of the antibody or functional fragment-treated sample to the maximum agglutination percentage of the glenzosimab-treated sample.

[0085] Typically, the antibodies or functional fragments of the present invention can completely inhibit human platelet adhesion (determined as platelet surface coverage) and thrombus formation (determined as relative thrombus volume) on a collagen-coated surface (preferably coated at 200 μg / ml) in heparinized human blood under flow conditions, using a flow adhesion assay.

[0086] According to a particular embodiment, the antibody or functional fragment of the present invention is determined using a fluid adhesion assay, for example, as described in the examples of Section 1.5, under flow conditions (preferably 1000s). -1In heparinized human blood (using a shear rate of ), human platelet adhesion (platelet surface coverage) and / or thrombus formation (relative thrombus volume) on a collagen-coated surface (preferably coated at 200 μg / ml) can be completely inhibited at a concentration of 5 μg / ml. According to certain preferred embodiments of the present invention, “complete inhibition” means a reduction of at least 60%, 65%, or 70% of platelet surface coverage and / or a reduction of at least 80%, 85%, 86%, 87%, 88%, 89%, or 90% of relative thrombus volume compared to a negative control (e.g., control Fab, when a functional fragment in the form of anti-GPVI Fab is used), as described, for example, in the examples of Section 1.5.

[0087] According to another specific embodiment, the antibody or functional fragment of the present invention can reduce the relative thrombus volume on a collagen-coated surface (preferably coated with 200 μg / ml) by at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% compared to a negative control (e.g., control Fab, when a functional fragment in the form of anti-GPVI Fab is used) in a flow adhesion assay, for example, as described in the examples of Section 1.5, in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹). tg / tg After intravenous administration of a dose of 4 mg / kg to mice, in heparinized human blood under flow conditions (preferably using a shear rate of 1000 s⁻¹), the relative thrombus volume on the collagen-coated surface (preferably coated with 200 μg / ml) can be reduced by at least 50%, 55%, 60%, or 60% compared to a negative control in a flow adhesion assay, for example, as described in the examples in Section 1.5, for at least 12, 24, 48, or 60 hours.

[0088] According to another specific embodiment, the antibody or functional fragment of the present invention can significantly reduce (e.g., more than 1.5 times, preferably more than 2, 2.5, or more than 3 times) platelet surface coverage on a collagen-coated surface (preferably coated at 200 μg / ml) in a flow adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹) compared to a negative control, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml (whereas ACT017 (glenzosimab) treated samples do not result in a (significant) reduction).

[0089] According to another specific embodiment, the antibody or functional fragment of the present invention can reduce platelet surface coating on a collagen-coated surface (preferably coated at 200 μg / ml) by more than 1.5 times, preferably 2 times, 2.5 times, or more than 3 times, compared to an ACT017 (glenzosimab) treated sample, in a flow adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹). According to another specific embodiment, the antibody or functional fragment of the present invention can reduce the relative thrombus volume on a collagen-coated surface (preferably coated with 200 μg / ml) by more than 2 times, preferably more than 4 times, 6 times, 8 times, or more than 10 times, compared to a glenzosimab-treated sample, in a flow adhesion assay (e.g., as described in the examples of Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹).

[0090] Typically, the antibody or functional fragment of the present invention is determined using a coagulation-adapted fluid adhesion assay (as described in the examples in Section 1.6, for example) under flow (preferably 1000s) -1Using the shear rate, citrate oxidation and remineralization of human blood can completely inhibit platelet deposition, thrombosis, phosphatidylserine (PS) exposure, and / or fibrin deposition on surfaces coated with collagen and tissue factor (preferably at 200 μg / ml and 500 pM for collagen and tissue factor, respectively).

[0091] According to a particular embodiment, the antibody or functional fragment of the present invention is determined by a fluid adhesion assay adapted for coagulation, for example, as described in the examples of Section 1.6, under flow conditions (preferably 1000s) -1 Using a shear rate, citrate oxidation and remineralization of human blood can completely inhibit thrombus formation (determined as relative thrombus volume), phosphatidylserine (PS) exposure, and fibrin deposition on a surface coated with collagen and tissue factor (preferably at 200 μg / ml and 500 pM for collagen and tissue factor, respectively) at a concentration of 10 μg / ml. According to certain specific embodiments of the present invention, “complete inhibition” means a reduction of at least 85%, 90%, 93%, 95%, 97%, or 99% of relative thrombus volume / phosphatidylserine (PS) exposure / fibrin deposition compared to a negative control, as described, for example, in the examples of Section 1.6.

[0092] Typically, when used on washed human platelets that can be conjugated to a fibrinogen-coated surface, the antibodies or functional fragments of the present invention can, in a diffusion assay, significantly reduce the portion of stage 4 (fully diffused) platelets while similarly increasing the portion of stage 2 (filamentous platelets), as described, for example, in the examples of Section 1.8.

[0093] According to certain embodiments, when used at a concentration of 10 μg / ml in a diffusion assay on washed human platelets that can be conjugated to a fibrinogen-coated surface (as described in the examples in Section 1.8, for example), the antibody or functional fragment of the present invention can reduce the portion of stage 4 (fully diffused) platelets by at least 30%, 35%, 40%, 45%, 50%, or 55% compared to a negative control, and increase the portion of stage 2 (filamentous platelets) by a similar degree.

[0094] Typically, the antibody or functional fragment of the present invention (preferably in monovalent form, e.g., as Fab) is preferably used with a fluorescently labeled antibody that can specifically bind to activated integrin αIIbβ3 and / or a fluorescently labeled antibody that can specifically bind to P-selectin, as determined, for example, by flow cytometry analysis in ex vivo diluted blood, for example, as described in the examples of Section 1.10, for example, a humanized mouse model (hGP6) against GPVI. tg / tg Intravenous administration of a dose of 4 mg / kg to mice induces significantly impaired CRP-induced activation of circulating platelets for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, or at least 96 hours. In this context, “significantly impaired” preferably means a reduction of more than 50% in the fluorescence signal when using circulating platelets sampled at at least 12, 24, 36, 48, 60, 72, 84, or 96 hours after administration compared to a negative control, as determined, for example, in the example of 1.10.

[0095] According to certain embodiments, the antibody or functional fragment of the present invention, preferably as Fab in monovalent form, can increase binding to circulating platelets compared to glenzosimab, as measured 1 hour after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment, or ACT017 (glenzosimab), using, for example, a fluorescently labeled Fab-specific antibody and mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, as determined by, for example, flow cytometry analysis in ex vivo diluted blood, as described in Example 1.10 (preferably an increase of 1.5-fold or more, more preferably 1.7-fold or more, 1.8-fold or more, or 1.9-fold or more in GPVI receptor epitope binding).

[0096] According to another specific embodiment, the antibody or functional fragment of the present invention, preferably as Fab in a monovalent form, can increase binding to circulating platelets compared to glenzosimab, as measured 3 hours after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment, or glenzosimab, using, for example, a fluorescently labeled Fab-specific antibody and mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding (preferably a 2-fold, more preferably 2.5-fold, 3-fold, 3.5-fold, or 3.75-fold increase in GPVI receptor epitope binding).

[0097] According to another specific embodiment, the antibody or functional fragment of the present invention, preferably in monovalent form and more preferably as Fab, is preferably a fluorescently labeled antibody that can specifically bind to activated integrin αIIbβ3, as determined, for example, by flow cytometry analysis in ex vivo diluted blood, and is used, for example, as described in the examples of Section 1.10, for example, a humanized mouse model (hGP6) against GPVI. tg / tg In mice, intravenous administration of 4 mg / kg induced significantly impaired CRP-induced activation of circulating platelets 1 hour later, whereas ACT017 (glenzosimab) did not induce significantly impaired CRP-induced activation. Optionally, "significantly impaired" refers to a reduction in fluorescence signal of more than 85%, preferably more than 90%, 95%, 97%, 98%, 99%, or 99.5% compared to a negative control.

[0098] According to certain embodiments, the antibody or functional fragment of the present invention, preferably in a monovalent form (e.g., as Fab), can inhibit the dimerization of GPVI while optionally not quenching or completely quenching ligand binding to GPVI. Typically, the antibody or functional fragment of the present invention, preferably in a monovalent form (e.g., Fab), can inhibit GPVI-induced activation of platelets while optionally preserving the initial adhesion function of the GPVI receptor.

[0099] Typically, the antibody or functional fragment of the present invention is used, for example, as described in the examples in Section 1.11, in a humanized mouse with occlusive arterial thrombosis (hGP6) against GPVI. tg / tg As determined using mouse models, it may provide sustained protection against occlusive thrombosis, such as arterial thrombosis.

[0100] According to certain embodiments, the antibody or functional fragment of the present invention can optionally be used in a mouse model of occlusive arterial thrombosis (hGP6) that is humanized against GPVI. tg / tgAs determined in mice, for example, as described in the examples in Section 1.11, the formation of a stable thrombus at the site of arterial injury can be inhibited, preferably by reducing platelet activation at sites of exposed extracellular matrix within blood vessels and / or by potent inhibition of local platelet-dependent coagulation.

[0101] Typically, the in vivo use of the antibody or functional fragment of the present invention, preferably Fab, is associated with a low bleeding risk by not significantly increasing bleeding time, as determined, for example, by using a tail hemorrhage assay, as described in the examples in Section 1.12.

[0102] In certain embodiments, the antibody or functional fragment of the present invention has high target selectivity, i.e., it can distinguish GPVI from structurally closely related proteins. Preferably, IC of structurally closely related proteins. 50 The IC of GPVI is determined by a competitive BLI, ELISA, or SPR assay. 50 The value is at least 1,000 times, preferably at least 5,000 times, and more preferably at least 10,000 times greater than the given value.

[0103] Typically, the antibody or functional fragment of the present invention, preferably Fab, has high stability. Stability can be evaluated by different methodologies. The "melting temperature" of Fab is T mIt can be determined by differential scanning fluorimetry (DSF). Stability can also be evaluated by long-term binding to GPVI on circulating platelets, as determined, for example, in the examples of Section 1.10. In one embodiment, the antibody or functional fragment of the present invention is preferably capable of long-term binding to circulating platelets with a GPVI receptor epitope occupancy of more than 50% when administered in vivo to a human subject, for example, when administered at a dose in the range of 0.01 to 500 mg, normalized with a negative control (e.g., a control antibody or functional fragment that is not specific to GPVI), for at least 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, or 96 hours, as determined in the examples of Section 1.10.

[0104] In one embodiment, the antibody or functional fragment of the present invention does not induce a decrease in platelet count, i.e., thrombocytopenia, when administered in vivo, for example, when preferably administered to a human subject at a dose in the range of 0.01 to 500 mg. In another embodiment, the antibody or functional fragment of the present invention does not induce a decrease in the surface level of GPVI on platelets when administered in vivo, for example, when preferably administered to a human subject at a dose in the range of 0.01 to 500 mg.

[0105] Antibodies and functional fragments Certain preferred embodiments of the present invention relate to functional fragments of the antibodies described herein. Functional fragments include, but are not limited to, Fab, F(ab’), F(ab’)2, Fv, monovalent IgG, dsFv, scFv, diabody, triabody, and tetrabody. Preferably, the functional fragment is monovalent and includes, but is not limited to, Fab, F(ab’), Fv, monovalent IgG, dsFv, and scFv. In a particularly preferred embodiment, the functional fragment is fragment antigen binding (Fab). More preferably, the non-CDR sequences of Fab are human sequences.

[0106] Preferably, the antibody or its functional fragment is a monoclonal antibody or a monoclonal antibody fragment. As used herein, the term “monoclonal antibody” is not limited to antibodies produced by hybridoma technology. The term “monoclonal antibody” refers to an antibody derived from a single clone, including any eukaryote, prokaryote, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display techniques, or combinations thereof. (Harlow and Lane, “Antibodies, A Laboratory Manual,” CSH Press 1988, Cold Spring Harbor NY)

[0107] Other embodiments, including embodiments relating to the in vivo use of anti-GPVI antibodies or functional fragments thereof in humans, may use chimeric, primate-like, humanized, or human monovalent antibodies and antibody fragments. In preferred embodiments, the antibody or functional fragment thereof is a human or humanized antibody or functional fragment thereof, more preferably a monoclonal human, humanized antibody or functional fragment thereof, and even more preferably a monovalent monoclonal human or humanized antibody or functional fragment thereof.

[0108] As used herein, the terms “chimeric” antibody or antibody fragment refer to an antibody having a variable sequence derived from a non-human immunoglobulin, such as a rat or mouse antibody, and a human immunoglobulin constant region typically selected from a human immunoglobulin template. Methods for generating chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science 229(4719):1202-7; Oi et al, 1986, BioTechniques 4:214-221; Gillies et al., 1985, J.Immunol.Methods 125:191-202; U.S. Patents No. 5,807,715, No. 4,816,567, and No. 4,816,397, which are incorporated herein by reference in their entirety.

[0109] In some embodiments, the anti-GPVI antibody or its functional fragment is a humanized antibody or its functional fragment. Various recombination methodologies are available to those skilled in the art to make non-human (e.g., mouse) antibodies more human-like by generating immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, F(ab'), F(ab')2, or other target binding sites of the antibody) containing minimal sequences derived from such non-human immunoglobulins. Generally, the resulting recombinant antibody contains substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR region corresponding to that of a non-human immunoglobulin, and all or substantially all of the FR region being a human immunoglobulin sequence, particularly the human immunoglobulin consensus sequence. CDR-transplanted antibodies are antibody molecules that have one or more complementarity-determining regions (CDRs) from antibodies originally produced in non-human species that bind to a desired antigen and framework (FR) region from a human immunoglobulin molecule (EP239400, PCT Publication WO91 / 09967, U.S. Patents 5,225,539, 5,530,101 and 5,585,089). Often, in a process called "humanization," framework residues within the human framework region are further substituted with corresponding residues from a CDR donor antibody to modify, and preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, such as modeling of CDR-framework residue interactions to identify framework residues important for antigen binding and sequence comparison to identify abnormal framework residues at specific locations. See, for example, Riechmann et al., 1988, Nature 332:323-7 and Queen et al., U.S. Patent Nos. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370 (each incorporated in its entirety by reference).Antibodies can be further humanized using a variety of additional techniques known in the art, including, for example, veneering or resurfacing (EP592106, EP519596, Padlan, 1991, MoI.Immunol, 28:489-498, Studnicka et al, 1994, Prot.Eng.7:805-814, Roguska et al, 1994, Proc.Natl.Acad.Sci.91:969-973) and chain shuffling (U.S. Patent No. 5,565,332), all of which are incorporated herein by reference in their entirety. CDR implantation or humanized antibodies may also include immunoglobulin constant regions (Fc), typically at least a portion of the immunoglobulin constant regions (Fc) of a selected human immunoglobulin template.

[0110] In some embodiments, the humanized antibody or functional fragment thereof is prepared as described in Queen et al., U.S. Patents No. 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370.

[0111] In some embodiments, the anti-GPVI antibody or functional fragment thereof is a human antibody or functional fragment. A fully “human” anti-GPVI antibody may be desirable for the therapeutic treatment of human patients. As used herein, “human antibody or functional fragment” includes antibodies having the amino acid sequence of a human immunoglobulin, isolated from a human immunoglobulin library or from a transgenic animal for one or more human immunoglobulins, and including antibodies that do not express endogenous immunoglobulins. Human antibodies can be prepared using an antibody library derived from human immunoglobulin sequences by various methods known in the art, including the phage display method described above. See U.S. Patents No. 4,444,887 and No. 4,716,111, and PCT Publications WO98 / 46645, WO98 / 50433, WO98 / 24893, WO98 / 16654, WO96 / 34096, WO96 / 33735, and WO91 / 10741, which are incorporated herein by reference in their entirety. Human antibodies can also be produced using transgenic mice that can express human immunoglobulin genes but cannot express functional endogenous immunoglobulins. For example, see PCT Publications WO98 / 24893, WO92 / 01047, WO96 / 34096, WO96 / 33735, U.S. Patents 5,413,923, 5,625,126, 5,633,425, 5,569,825, 5,661,016, 5,545,806, 5,814,318, 5,885,793, 5,916,771, and 5,939,598, which are incorporated herein by reference in their entirety. Fully human antibodies that recognize selected epitopes can be produced using a technique called “guided selection”. This approach uses selected non-human monoclonal antibodies, such as mouse antibodies, to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al, 1988, Biotechnology 12:899-903).

[0112] In some embodiments, the anti-GPVI antibody or its functional fragment is derivatized. For example, the derivatized antibody or functional fragment is modified by, but is not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or conjugation to a cellular ligand or other protein (see below for a discussion of antibody conjugates). Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, or the metabolic synthesis of tunicamycin. Furthermore, the derivative may contain one or more non-classical amino acids.

[0113] In yet another embodiment, the anti-GPVI antibody has one or more amino acids inserted into one or more of its hypervariable regions, as described, for example, in US2007 / 0280931.

[0114] Antibody conjugate In some embodiments, the anti-GPVI antibody or functional fragment is modified, for example, by covalent bonding of any type of molecule (e.g., an effector molecule) to the antibody, resulting in an antibody / antibody fragment conjugate where the covalent bond does not hinder binding to GPVI. Techniques for conjugating the effector portion to the antibody are well known in the art (e.g., Hellstrom et al., Controlled Drug Delivery, 2 nd See Ed., at pp. 623-53 (Robinson et al., eds., 1987), Thorpe et al., 1982, Immunol. Rev. 62: 119-58, and Dubowchik et al., 1999, Pharmacology and Therapeutics 83: 67-123).

[0115] In one example, an antibody or its functional fragment is optionally fused at its N-terminus or C-terminus to the amino acid sequence (or a portion thereof; preferably at least 10, 20, or 50 amino acids of the protein) of another protein via a covalent bond (e.g., a peptide bond). In one embodiment, the antibody or its functional fragment is ligated to another protein at the C-terminus of the constant domain of the antibody or functional fragment. Such fusions can be prepared using recombinant DNA procedures, as described, for example, in WO86 / 01533 and EP0392745. In another example, the effector molecule can increase the half-life in vivo. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO2005 / 117984.

[0116] In some embodiments, an anti-GPVI antibody or a functional fragment thereof can be bound to a poly(ethylene glycol) (PEG) moiety. For example, if the antibody is an antibody fragment, the PEG moiety can be bound via any available amino acid side chain or terminal amino acid functional group located within the antibody fragment, such as any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may be naturally present within the antibody fragment or can be manipulated within the fragment using recombinant DNA methods. See, for example, U.S. Patent No. 5,219,996. Multiple sites can be used to link two or more PEG molecules. Preferably, the PEG moiety is covalently linked via a thiol group of at least one cysteine ​​residue located within the antibody fragment. When a thiol group is used as a binding site, appropriately activated effector moieties, such as thiol-selective derivatives like maleimide and cysteine ​​derivatives, can be used.

[0117] In another example, the anti-GPVI antibody conjugate is a modified Fab or F(ab') fragment, which is PEGylated, i.e., has PEG(poly(ethyleneglycol)) covalently bound to it, according to the method disclosed, for example, in EP0948544. See also Poly(ethyleneglycol)Chemistry, Biotechnical and Biomedical Applications, (J. Milton Harris (ed.), Plenum Press, New York, 1992), Poly(ethyleneglycol)Chemistry and Biological Applications, (J. Milton Harris and S. Zalipsky, eds., American Chemical Society, Washington DC, 1997), and Bioconjugation Protein Coupling Techniques for the Biomedical Sciences, (M. Aslam and A. Dent, eds., Grove Publishers, New York, 1998), and Chapman, 2002, Advanced Drug Delivery Reviews 54:531-545.

[0118] Pharmaceutical compositions and treatments Treatment of the disease includes the treatment of patients already diagnosed with having any form of the disease at any clinical stage or sign, delaying the onset, progression, worsening, or deterioration of the symptoms or signs of the disease, and / or preventing and / or reducing the severity of the disease.

[0119] The "subject" or "patient" to whom the anti-GPVI antibody or a functional fragment thereof is administered can be a mammal, such as a non-primate (e.g., a cattle, pig, horse, cat, dog, rat, etc.) or a primate (e.g., a monkey or human). Preferably, the "subject" or "patient" is human. In certain embodiments, the human is an adult patient. In other embodiments, the human is a pediatric patient.

[0120] Pharmaceutical compositions comprising an anti-GPVI antibody, an optionally pharmaceutically acceptable carrier and / or excipient, and an optionally one or more additional therapeutic agents are described herein. The composition is typically supplied as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any preferred form (depending on the desired method of administration to the patient).

[0121] Anti-GPVI antibodies and functional fragments can be administered to patients via various routes, including oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, subarachnoid, topical, or local. Typically, the anti-GPVI antibody or its functional fragment is administered intravenously to the patient. In a typical embodiment, the anti-GPVI antibody or functional fragment is present in the pharmaceutical composition at a concentration sufficient to enable intravenous administration at a dose of 0.5 mg / kg body weight to 20 mg / kg body weight. In some embodiments, suitable concentrations of antibodies or fragments for use in the compositions and methods described herein include, but are not limited to, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, or concentrations in a range between any of the aforementioned values, for example, 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, or 10 mg / kg to 18 mg / kg.

[0122] The effective dose of anti-GPVI antibody or functional fragment may range from approximately 0.001 to approximately 750 mg / kg per single (e.g., bolus), multiple, or consecutive dose, or any effective range or value within that range, depending on the treated condition, route of administration, and the age, weight, and condition of the subject. This can be used to achieve a serum concentration of 0.01 to 5000 μg / ml per single (e.g., bolus), multiple, or consecutive dose. In certain embodiments, each dose may range from approximately 0.5 mg to approximately 50 mg per kg of body weight, or from approximately 3 mg to approximately 30 mg per kg of body weight. The antibody can be formulated as an aqueous solution.

[0123] The pharmaceutical composition can be conveniently presented in unit dose form containing a predetermined amount of anti-GPVI antibody or functional fragment per dose. Such units may include 0.5 mg to 5 g, for example, but are not limited to, 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 750 mg, 1000 mg, 2000 mg, or any range between any two of the aforementioned values, for example, 10 mg to 1000 mg, 20 mg to 500 mg, or 30 mg to 300 mg. Pharmaceutically acceptable carriers can take a wide variety of forms, for example, depending on the condition being treated or the route of administration.

[0124] Determining the effective dosage, total number of doses, and duration of treatment of anti-GPVI antibodies or their functional fragments is within the scope of the skills of those skilled in the art and can be determined using standard dose-escalation studies.

[0125] Suitable therapeutic formulations of anti-GPVI antibodies and functional fragments according to the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an antibody or functional antibody fragment of the desired purity with any choice of pharmaceutically acceptable carriers, excipients, or stabilizers (all referred to herein as “carriers”) commonly used in the art, namely buffers, stabilizers, preservatives, isotonic agents, nonionic detergents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be nontoxic to the recipient at the dosage and concentration used.

[0126] Buffers help maintain a pH within a range close to physiological conditions. They can be present in concentrations ranging from approximately 2 mM to approximately 50 mM. Suitable buffers include citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinate-monosodium succinate mixture, succinate-sodium hydroxide mixture, succinate-disodium succinate mixture, etc.), tartaric acid buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), and fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate-disodium fumarate mixture, monosodium fumarate). This includes both organic and inorganic acids, as well as their salts, such as disodium fumarate mixtures, gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactic acid buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetic acid buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Furthermore, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, may be used.

[0127] Preservatives can be added to slow the growth of microorganisms and may be added in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, sometimes also known as “stabilizers,” can be added to ensure the isotonicity of liquid compositions and may include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients that can be functionally diverse, ranging from bulking agents to additives that help solubilize therapeutic agents or prevent denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, and threonine; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, and glycerol (including cyclitols such as inositol); polyethylene glycol; amino acid polymers; urea, glutathione, and thioctose. Potential stabilizers include acids, sulfur-containing reducing agents such as sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in amounts ranging from 0.1 to 10,000 parts by weight per 1 part by weight of active protein.

[0128] Nonionic surfactants or detergents (also known as "wetting agents") may be added to assist in the solubilization of therapeutic agents and to protect therapeutic proteins from agitation-induced aggregation, which also allows the formulation to be subjected to shear stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxomers (184, 188, etc.), Pluronic polyols, and polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Nonionic surfactants may be present in a range of about 0.05 mg / ml to about 1.0 mg / ml, or in a range of about 0.07 mg / ml to about 0.2 mg / ml.

[0129] Various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), protease inhibitors, and co-solvents.

[0130] The formulations described herein may also contain a second therapeutic agent in addition to an anti-GPVI antibody or a functional fragment thereof.

[0131] The medication schedule may vary from once a month to daily, depending on several clinical factors, including the type of disease, the severity of the disease, and the patient's sensitivity to the anti-GPVI antibody or functional fragment. In certain embodiments, the anti-GPVI antibody or its functional fragment may be administered daily, twice a week, three times a week, every other day, or every five days, etc.

[0132] The dosage of the anti-GPVI antibody or functional fragment administered will vary depending on the specific antibody or functional fragment, the subject, the nature and severity of the disease, the subject's physical condition, the treatment regimen (e.g., whether a second therapeutic agent is used), and the chosen route of administration. The appropriate dosage can be easily determined by those skilled in the art.

[0133] The optimal dose and interval of individual doses of anti-GPVI antibodies or their functional fragments will be determined by the nature and severity of the condition being treated, the form, route and site of administration, and the age and condition of the specific subject being treated, and it will be recognized by those skilled in the art that the physician ultimately determines the appropriate dose to be used. This medication can be repeated frequently as needed. If side effects occur, the dose and / or frequency of administration can be modified or reduced in accordance with the usual clinical practice.

[0134] Disorders that are treated The present invention further relates to a method for treating or preventing a GPVI-related condition in a subject, comprising administering an antibody or functional fragment as defined herein to the subject. Furthermore, the present invention also relates to an antibody or functional fragment as defined in any one of the above embodiments, or a pharmaceutical composition comprising such an antibody or functional fragment, for use in a method for treating or preventing a GPVI-related condition in a subject.

[0135] The terms “GPVI-related condition” or “GPVI-related disease,” as used herein, specifically and exclusively refer to the onset, progression, or persistence of one or more symptoms or disease states involving GPVI. Therefore, “GPVI-related condition” or “GPVI-related disease,” as used herein, is any condition / disease in which one or more symptoms or disease states can be treated or prevented by (preferably functional) inhibition / blockade of GPVI through the administration of antibodies capable of binding to GPVI. Exemplary GPVI-related conditions include, but are not limited to, thromboinflammatory diseases, cardiovascular diseases, cancer / cancer-related diseases, inflammation, and disorders related to the proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function of abnormal or ectopic megakaryocytes and / or platelets. As used herein, these categories of GPVI-related conditions are not necessarily mutually exclusive and may overlap. Similarly, as used herein, exemplary conditions, diseases, disorders, disease states, etc., in the different categories of GPVI-related conditions detailed below are not necessarily mutually exclusive and may, for example, overlap. According to one embodiment, GPVI-related conditions are thromboinflammatory diseases, cardiovascular diseases, inflammation, cancer / cancer-related diseases, and / or disorders related to the proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function of abnormal or ectopic megakaryocytes and / or platelets.

[0136] Exemplary cardiovascular diseases include thrombosis and thrombotic disorders (arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic diseases [e.g., diseases with leg swelling, pain and ulcers, pulmonary embolism, abdominal venous thrombosis], coronary artery thromboembolism, thrombotic microangiopathy, cancer-associated thrombosis (Trousseau syndrome), immunothrombosis and thrombosis associated with infections [e.g., cerebral malaria]), restenosis, ischemic cerebrovascular disease, cerebrovascular disease, vascular purpura, coronary artery disease [e.g., coronary artery disease (e.g., arterial occlusive disease), myocardial infarction, coronary artery revascularization, coronary artery This includes, but is not limited to, conditions resulting from any vascular injury that may cause platelet aggregation, including but not limited to: restenosis of the heart pulsate, acute coronary artery disease, acute coronary syndrome, cardiac ischemia (including complications associated with coronary artery surgery such as percutaneous coronary angioplasty (balloon angioplasty) and percutaneous coronary intervention), atherosclerosis, plaque formation, cerebral artery disease, ischemic events, unstable angina, acute cerebrovascular isovascular disease (stroke), ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches, peripheral artery disease, acute phlebitis, pulmonary embolism, and conditions resulting from any vascular injury that may cause platelet aggregation.

[0137] According to a particular embodiment, the GPVI-associated condition is preferably a cardiovascular disease selected from the cardiovascular diseases specified above. With respect to coronary artery surgery, such treatment can be achieved through the administration of the protein of the present invention before, during, or after surgery. In a preferred embodiment, such administration can be used to prevent acute cardiac ischemia after angioplasty.

[0138] In another embodiment, a GPVI-related condition is a thrombosis or thrombotic disorder, a disease exhibiting quantitative or qualitative platelet dysfunction, or a disease exhibiting endothelial dysfunction. These diseases include, but are not limited to, coronary artery disease and cerebral artery disease. Generally, thrombotic disorders as used herein may refer to disorders associated with the pathological formation of thrombi in veins (e.g., deep vein thrombosis [DVT]), arteries (e.g., myocardial infarction, ischemic stroke), or ventricles. According to a preferred embodiment, a GPVI-related condition is a thrombosis or thrombotic disorder (including, but not limited to, arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic disease, coronary artery thromboembolic occlusion, thrombotic microangiopathy, cancer-associated thrombosis, immunothrombosis, and thrombosis associated with infection [e.g., cerebral malaria]).

[0139] In another embodiment, a GPVI-associated condition is a disorder resulting from any vascular injury that may cause platelet aggregation. The term “vascular injury” as used herein includes, but is not limited to, vascular wall injuries resulting from a highly thrombotic surface otherwise exposed within an intact vessel, such as vascular wall injuries resulting from the release of ADP, thrombin and / or epinephrine, fluid shear stress occurring at sites of vascular stenosis, rupture and / or fracture at sites of atherosclerotic plaque, and injuries resulting from balloon angioplasty or atherectomy.

[0140] According to another embodiment, GPVI-related conditions include inflammation, i.e., inflammation or thromboinflammatory conditions, including but not limited to platelet-mediated regulating of cellular function, including but not limited to: inflammation, i.e., infection [e.g., cerebral malaria], arthritis, fibrosis, acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury (IRI) of various organs (e.g., liver, colon), peripheral vascular disease, antiphospholipid syndrome (APS), deep vein thrombosis, thrombophlebitis and vasculitis, transfusion-associated acute lung injury (TRALI), transplant rejection, pre-eclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, sickle cell disease, bacterial and viral infections, ischemic restenosis, sepsis, major trauma, autoimmune diseases, and cancer cell proliferation and / or dissemination. As used herein, the term “thromboinflammatory disease” preferably refers to a disease involving activating interactions of platelet / coagulation and components of the immune system. Disorders / diseases related to the term "GPVI-related condition is inflammatory or thromboinflammatory" may also be "thromboinflammatory disorders" or "thromboinflammatory diseases." Similarly, for example, cardiovascular disease may be a thromboinflammatory disease. Autoimmune diseases include, but are not limited to, celiac disease, post-infectious IBS, diabetes mellitus type 1, Henoch-Schönlein purpura (HSP), sarcoidosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), alopecia areata, and multiple sclerosis (MS).

[0141] According to another embodiment, the GPVI-associated condition is cancer (including, but not limited to, colon cancer, breast cancer, ovarian cancer, lung cancer, skin cancers such as malignant melanoma, and metastatic cancer).

[0142] In yet another embodiment, a GPVI-related condition is a disorder related to the proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function of abnormal or ectopic megakaryocytes and / or platelets.

[0143] In another embodiment, the antibody or functional fragment of the present invention or the pharmaceutical composition described above is preferably used to modulate platelet aggregation and degranulation. In another embodiment, the antibody or functional fragment of the present invention or the pharmaceutical composition described above is used to modulate the immunomodulatory function of platelets. In another embodiment, the antibody or functional fragment of the present invention or the pharmaceutical composition described above is used to treat disorders of the liver, bone marrow, and peripheral blood.

[0144] Preferably, the antibodies or functional fragments of the present invention are used to treat cardiovascular diseases selected from thrombosis and thrombotic disorders such as arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic disease, thrombotic microangiopathy, cancer-associated thrombosis [Trousseau syndrome], immunothrombosis and infection [e.g., thrombosis associated with cerebral malaria], restenosis, acute coronary syndrome, ischemic cerebrovascular disease, cerebrovascular disease, and vascular purpura, coronary artery disease and cerebral artery disease, ischemic events, acute coronary syndrome, myocardial infarction (heart attack), acute cerebrovascular ischemia (stroke), percutaneous coronary intervention, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches (e.g., aortic aneurysm, thrombosis), peripheral artery disease, acute phlebitis, and pulmonary embolism.

[0145] In certain preferred embodiments, the antibody or functional fragment of the present invention is used to treat cardiovascular diseases selected from arterial or venous thrombosis, restenosis, acute coronary syndrome, or atherosclerosis, preferably cerebrovascular events resulting from arterial or venous thrombosis. In any embodiment of this specification, the patient treated with the anti-GPVI antibody or its functional fragment may also be treated with another conventional pharmaceutical.

[0146] In a further embodiment, the present invention relates to an antibody or functional fragment as defined in any one of the above embodiments, or a pharmaceutical composition comprising such an antibody or functional fragment, for use in a method for treating or preventing thrombosis or thrombotic disorders. "Thrombosis or thrombotic disorders" is preferably as defined above. In another embodiment, the present invention relates to an antibody or functional fragment as defined in any one of the above embodiments, or a pharmaceutical composition comprising such an antibody or functional fragment, for use as an antithrombotic pharmaceutical.

[0147] Further aspects of the present invention are methods for treating GPVI-related conditions, comprising administering an effective amount of the anti-GPVI antibody or a functional fragment thereof as defined above to a patient in need. The GPVI-related condition is preferably one of the conditions described above. Further aspects of the present invention are methods for treating or preventing thrombosis or thrombotic disorders, comprising administering an effective amount of the anti-GPVI antibody or a functional fragment thereof as defined above to a patient in need. "Thrombosis or thrombotic disorders" is preferably as defined above. Further aspects of the present invention are methods for preventing GPVI-related conditions, comprising administering an effective amount of the anti-GPVI antibody or a functional fragment thereof as defined above to a patient in need. The GPVI-related condition is preferably one of the conditions described above. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Examples]

[0148] 1. Materials and Methods 1.1 Antibodies and Reagents Horm collagen was purchased from Takeda (Linz, Austria), collagen-related peptide (CRP) from Cambridge Research Biochemicals (Cambridge, UK), ADP, apirase, prostacyclin (PGI2), fibrinogen and hematoxylin from Sigma Aldrich (Steinheim, Germany), thrombin from Roche Diagnostic (Mannheim, Germany), conbulxin from Enzo Life Sciences (New York, NY, USA), rabbit anti-GAPDH and rat anti-mouse IgG-HRP antibodies from Sigma-Aldrich (Steinheim, Germany), U46619 from Alexis Biochemicals (Enzo Life Sciences, New York, NY, USA), and anti-rabbit IgG-HRP from Jackson The antibodies were purchased from Immuno (Suffolk, UK), and goat anti-rat IgG-HRP from Dianova (Hamburg, Germany). Microcuvettes for agglutination analysis were purchased from LABITec (Ahrensburg, Germany). S-monovettes 3.2% citrate and Safety-Fly-Needle 21G were purchased from Sarstedt (Numbrecht, Germany) for human blood collection. Heparin was purchased from Ratiopharm (Ulm, Germany), and 5 mL polystyrene round-bottom tubes for flow cytometry were purchased from Corning Inc. (New York, NY, USA). Emf6 was produced in parallel with other Emf antibodies. 25 After repeated subcloning, a highly productive monoclonal subcloning (Emf6.1) was isolated and further characterized (Emfret Analytics, unpublished). Emf1 32 Emf2 32 Emf3, Emf6.1, JON / A 34 and WUG 1.9 34 It was self-generated, purified, and derivatized.

[0149] The humanized anti-human GPVI drug Fab Glenzocimab (code: PX-TA1552-1000, also known as ACT017) was purchased from ProteoGenix (Schiltigheim, France).

[0150] 1.2 Blood donors and blood collection Blood samples were collected from healthy volunteers who had not received anticoagulant or antiplatelet therapy for at least four weeks. Blood samples were obtained after obtaining written informed consent in accordance with the Declaration of Helsinki and after approval by the Institutional Review Board of the University of Würzburg. Blood was collected by venous puncture using a butterfly needle and placed in a 9 mL tube containing 3.2% trisodium citrate. For all tests, the blood was kept at room temperature and used within four hours. All methods were performed in accordance with relevant guidelines and regulations. Blood was collected by venous puncture using a butterfly needle and placed in a 9 mL tube containing 3.2% trisodium citrate. For all tests, the blood was kept at room temperature and used within four hours.

[0151] 1.3 Animals The animal experiments were approved by the Lower Franconia (Regierung von Unterfranken) administrative district and conducted in accordance with the current Animal Research: reporting of in vivo experiments guidelines (https: / / arriveguidelines.org / ). The mice used were matched for age, sex, and genetic background. All animal experiments described in this study were conducted using the aforementioned humanized mouse strain (hGP6) against GPVI. tg / tg ) was implemented using 32 .

[0152] 1.4 Washed human and mouse platelets Human washed platelets were obtained as follows: Citrated blood was collected in a 10 ml S-monovette and rinsed with 2 ml of ACD pH 4.5. The sample was centrifuged at 300 g for 20 minutes at room temperature. Platelet-rich plasma (PRP) was collected in a new 15 ml Falcon tube and supplemented with 1 / 10 ACD, 2 μL of apirase / ml (0.02 U ml-1; A6410, Sigma-Aldrich) and 5 μL of PGI2 / μL (0.1 μg ml-1; P6188, Sigma-Aldrich). Platelets were pelletized by centrifugation at 500g for 10 minutes, washed twice with Tyrode's buffer containing 2 μL apirase / ml and 5 μL PGI2 / ml (N-2-hydroxyethyl-piperazine-NO2-ethanesulfonic acid; 134 mM NaCl, 0.34 mM NaH2PO4, 2.9 mM KCl, 12 mM NaHCO3, 5 mM HEPES, 5 mM glucose, 0.35% BSA, pH 7.4), and finally resuspended in Tyrode's buffer at a concentration of 500,000 / μL and held at 37°C for 30 minutes before use. For the experiment, platelets were remineralized using calcified Tyrode's buffer.

[0153] Mouse washed platelets were obtained as follows: Whole blood was collected in heparin via retroorbital hemorrhage after isoflurane anesthesia. The heparinized blood was centrifuged at 300 g for 6 minutes. For human platelets, the obtained PRP was supplemented with 2 μL of apirase and 5 μL of PGI2. Next, the platelets were pelletized by centrifuging at 800 g for 5 minutes and washed twice with Tyrode's buffer (using 2 μL of apirase and 5 μL of PGI2). Finally, for human platelets, the platelets were resuspended and kept at 37°C for 30 minutes before use.

[0154] 1.5 Fluid Adhesion Assay (Whole Blood Perfusion Assay) Evaluation of platelet adhesion and aggregate formation (thrombus formation) on Horm collagen under flow conditions in heparinized human blood treated with 1, 2, 5, or 10 μg / mL anti-GPVI antibody or Fab, or control antibody or Fab. 200 μg / mL Horm collagen was coated onto coverslips at 37°C for 1 hour and then blocked with BSA in 1% PBS 1x. Heparinized human or mouse blood was diluted 1:2 in Tyrode's buffer and treated with 2 mM Ca 2+ The following was added: Mouse platelets were labeled with an anti-GPIX-Dylight488 conjugate antibody, and human platelets were labeled with an anti-GPIbβ antibody p0p1 conjugate to Dylight488. 35 Labeled using [method]. Blood was placed on a coverslip for 1000 seconds. -1 The plates were perfused at a shear rate for 4 minutes, followed by washing with calcium-supplemented Tyrode's buffer for another 4 minutes. After the washing step, eight representative fields were imaged using a Leica DMI6000B microscope (Leica Biosystems Technologies, Frankfurt, Germany) with a 63x objective lens. Finally, images were analyzed using Fiji.17 for overall platelet surface coverage and relative thrombus volume (integrated fluorescence density).

[0155] 1.6 Coagulation Flow Chamber For example, evaluation of platelet adhesion, thrombus formation, phosphatidylserine exposure, and fibrin deposition to Horm collagen + tissue factor under flow conditions in remineralized human blood treated with 10 μg / mL anti-GPVI antibody or Fab, or a control antibody or Fab. To analyze thrombus formation under flow conditions in vitro, coverslips were newly coated with 50 μg / mL Horm collagen at 37°C for 1 hour, followed by a second incubation in a humidified chamber with 500 pM tissue factor for 1 hour. The slides were then blocked at room temperature for 30 minutes using PBS 1 × 1% BSA. For in-situ blood remineralization, citrated blood and remineralization buffer (32 mM MgCl2 and 63 mM CaCl2 in Hepes buffer pH 7.45) were pumped into the chamber containing the blocked coverslip using a Y-shaped silicone tube. The blood was then pumped onto the coverslip for 1000 seconds. -1 Perfusion was performed at the specified shear rate. Images were taken every 30 seconds using a LEICA DMI6000B microscope (Leica Biosystems Technologies, Frankfurt, Germany) equipped with a 63x objective lens. Anti-GPIbβ antibody p0p1-A647 35 Human platelets were labeled using [a specific method], PS exposure was observed using auto-generated annexin 5-A546, and fibrin deposition was observed by adding fibrinogen-A488.

[0156] 1.7 Light transmission aggregometry (nephelometric agglutination) Agglutination response of washed human platelets treated with 1, 2, 5, or 10 μg / mL of anti-GPVI antibody or Fab, or control antibody or Fab, in light transmission agglutination assay at 37°C. Washed human or mouse platelets were treated with 2 mM Ca 2+The samples were then diluted (remineralized) in Tyrode's buffer supplemented with 100 μg / ml human fibrinogen. When thrombin was used as an agonist, fibrinogen was not supplemented in Tyrode's buffer. After pre-incubation with the antibody and addition of a specific agonist (0.5 μg / mL CRP, 2, 5, 10, or 20 μg / mL collagen), light transmission agglutination assays were performed using a 4-channel APACT agglutinator (LABITec Ahrensburg, Germany) under 10 minutes of agitation conditions to track platelet aggregation over time.

[0157] 1.8 Diffusion assay Washed platelets were pre-incubated with Fab fragments (e.g., 10 μg / mL anti-GPVI Fab, or control Fab), then further diluted to 100,000 / μL, and pipetteed onto a 100 μg / mL fibrinogen-coated surface. The platelets were diffused at 37°C for 45 minutes. The coverslips were then fixed using 4% PF4 for 10 minutes. The diffused platelets were visualized using a ZEISS Axiovert (Zeiss group, Oberkochen, Germany) microscope with a 100x objective lens. Images were analyzed using the Fiji.17 cell counter tool, and phase abundances were determined by identifying platelets based on four stages of diffusion: Stage 1: Adhesion, Stage 2: Foot formation, Stage 3: Lamellipodia formation, Stage 4: Completely diffused platelets.

[0158] 1.9 Measurement of platelet count and size To determine platelet count and size, hGP6 tg / tgMice were intravenously administered either anti-GPVI Fab or control Fab at a dose of 4 mg / kg, and their peripheral platelets were monitored for 5 days for automated cell analysis. To assess platelet count and size, mice were bled into EDTA-coated tubes at specific time points after administration of anti-GPVI Fab or control Fab. Platelet parameters were measured using an automated cell counter (ScilVet, scil animal care company GmbH, Viernheim, Germany). Platelet count and size values ​​from anti-GPVI Fab-treated mice were normalized using values ​​from control Fab samples that were not specific to GPVI-treated mice at the same time point.

[0159] 1.10 Flow cytometry analysis of GPVI surface level on platelets, binding of the anti-GPVI antibody or Fab of the present invention to GPVI on platelets, and platelet activation To determine the in vivo GPVI surface level, anti-GPVI Fab binding, and platelet activation on circulating platelets, hGP6 tg / tg Mice were given an anti-GPVI drug at a dose of 4 mg / kg (Emf6.1 Fab ) or a control Fab was administered intravenously, and their peripheral platelets were monitored for 5 or 6 days by flow cytometry and automated cell analysis. Emf-2 FITC (Used to test GPVI exposure, i.e., GPVI surface levels on circulating platelets) and Emf-3 FITC For detection using (used to evaluate the epitope saturation of the anti-GPVI antibody or Fab of the present invention on circulating platelets), Ca 2+ Mouse blood diluted 1:20 in Tyrode's buffer (which does not contain hGP6) was pre-incubated with the antibody for 10 minutes. In a different experimental setup, hGP6 was used. tg / tg Mice were intravenously administered 4 mg / kg of EMA601 (humanized Fab of the present invention) or glenzosimab (ACT017), and their peripheral platelets were monitored for 24 hours by flow cytometry and automated cell analysis. Emf-2 FITC (Used to test GPVI surface expression) and Emf-3 FITC or JAQ1FITC For detection using (used to evaluate the epitope saturation of EMA601 and glenzosimab on circulating platelets, respectively), Ca 2 + Mouse blood diluted 1:20 in Tyrode's buffer (which does not contain ) was pre-incubated with the antibody for 10 minutes. Anti-human IgG (Fab-specific)-FITC antibody (Sigma Aldrich, F5512) was used to detect surface-bound EMA601 or glenzosimab. For platelet activation analysis, mouse blood was treated with 2 mM Ca25. 2+ The sample was diluted in Tyrode's buffer containing [component name missing]. Activated integrin αIIbβ3 was detected using JON / A-PE (Emfret Analytics, Eibelstadt, Germany), while P-selectin exposure was used as a marker for platelet degranulation and detected with the FITC conjugate-specific anti-mP-selectin antibody WUG 1.9. 34 Mouse blood was collected at a specific time point after administration of anti-GPVI Fab or control Fab (as described in Section 1.2), diluted, and then mixed with either CRP (0.5 μg / mL), thrombin (0.1 U / mL), or vehicle solution, along with JON / A-PE and anti-Psel. FITC The samples were incubated for 12 minutes (6 minutes at 37°C and 6 minutes at room temperature). Finally, the blood was further diluted in 500 μL of PBS so that MFI could be measured using FACSCelesta (BD Biosciences, Franklin Lakes, New Jersey, USA). To determine the GPVI surface level on circulating platelets / the binding of anti-GPVI antibody or the Fab of the present invention to GPVI on platelets / platelet activation (exposure to activated integrin αIIbβ3, P-selectin), the respective MFI values ​​of the samples from anti-GPVI Fab-treated mice were normalized using a control Fab sample that was not specific to GPVI-treated mice at the same time point.

[0160] 1.11 In vivo models of thrombosis The abdominal aorta of anesthetized mouse-treated mice (e.g., 1 hour after injection of 4 mg / kg anti-GPVI Fab or control Fab) was exposed via access to the abdominal cavity. The aorta was separated from the vena cava by removing the fat layer, and an ultrasound flow probe (0.5PSB699; Transonic Systems, USA) was placed around the abdominal aorta. Thrombosis was induced by compressing the aorta once using forceps (Ultra Fine Hemostats clams, Fine Science Tools, Vancouver, Canada) upstream of the flow probe for 5 seconds (buckle setting 1). Blood flow was monitored for 30 minutes or until vascular occlusion occurred (blood flow was interrupted for more than 5 minutes). The significance of occluded and unoccluded vessels was statistically assessed using Fisher's exact test.

[0161] 1.12 Tail hemorrhage assay After anesthetizing the animals, the 2mm tip of the tail was removed using a scalpel. Tail bleeding was monitored by gently absorbing the blood onto filter paper at 20-second intervals without direct contact with the wound site. If no blood was observed on the paper, it was assumed that bleeding had stopped. After 20 minutes, the experiment was manually stopped by cauterization. The difference between occluded and unoccluded wounds was statistically evaluated using Fisher's exact test of mean bleeding time by the Mann-U-Whitney U test.

[0162] 1.13 Automatic μSPOT synthesis The GPVI ectodomain (residues 24-267 of UniProtKB:Q9HCN6-1; corresponding to SEQ ID NO: 36) was represented in microarray format as a 15-mer duplicate peptide shifted by three residues. The peptide array was synthesized using a MultiPep RSi robot (CEM GmbH, Kamp-Lintford, Germany) on a cellulose disk containing a 9-fluorenylmethyloxycarbonyl-β-alanine (Fmoc-β-Ala) linker (average load: 130 nmol / disk - 4 mm diameter). 36The synthesis was carried out by deprotecting the Fmoc group using 20% ​​piperidine in dimethylformamide (DMF). The peptide chain was extended using a coupling solution consisting of an amino acid (0.5M) with an oxima (1M) and diisopropylmethandiamine (1M) in DMF (1:1:1). The coupling step was performed three times (30 minutes each), followed by capping (4% anhydride acetate in DMF). To ensure cleavage of the cellulose support, the cleavable peptide was coupled with an acid-unstable linker (Fmoc-link-amide).

[0163] The cellulose disc was transferred to a 96-deep-well plate for peptide workup. First, the side chain groups were deprotected at room temperature (RT) for 1 hour with 90% trifluoroacetic acid (TFA), 2% dichloromethane (DCM), 5% H2O, and 3% triisopropylsilane (150 μL / well). The deprotection solution was then discarded, and the disc was solubilized overnight at room temperature (O / N) using a solvation mixture containing 88.5% TFA, 4% trifluoromethanesulfonic acid (TFMSA), 5% H2O, and 2.5% TIPS (250 μL / well). The resulting peptide-cellulose conjugate (PCC) was precipitated in ice-cold ether (700 μL / well) and centrifuged at 2000 × g at 4°C for 10 minutes. The resulting pellet was then washed twice with ice-cold ether. The resulting pellet was dissolved in DMSO (250 μL / well). The PCC solution was mixed with physiological saline-sodium citrate buffer (150 mM NaCl, 15 mM trisodium citrate, pH 7.0) in a 2:1 ratio and transferred to a 384-well plate. A SlideSpotter (CEM GmbH) was used to transfer the PCC solution to white-coated CelluSpot blank slides (76 × 26 mm, Intavis AG Peptide Services GmbH and CO.KG). After the printing procedure was complete, the slides were allowed to dry for at least 3 hours. The cleavable peptides were purified from the supernatant of the cleavage mixture and precipitated in the same manner. The peptide pellet was resuspended in water.

[0164] 1.14 Microarray-conjugated assay Microarray slides were blocked for 60 minutes with 5% (w / v) skim milk powder (Carl Roth) in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4). After blocking, the slides were incubated with Emf6.1 (5 μg / mL) in block buffer for 30 minutes, followed by washing with PBS for 3 × 1 minute. Antibody binding was detected using goat anti-mouse IgG-HRP (Thermo Fisher catalog no. 31430, 1:5000). Chemiluminescence readout was detected using an Azure imaging system c400 (lowest sensitivity) with a SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific GmbH, Schwerte, Germany). Epitope neutralization was performed by pre-incubating Emf6.1 with a cleavable peptide for 30 minutes, and then applied to the blocked slides.

[0165] Microarray binding intensity was quantified in FIJI using the "Microarray Profile" plugin (OptiNav Inc, Bellevue, WA, USA). After background subtraction of the mean grayscale values ​​of the microarray surface surrounding the spot, the raw grayscale intensity for each position was obtained for both the left and right sides of the internal replicas on each microarray slide. The standard deviation (STDEV) between the two sides was calculated.

[0166] 1.15 Antibody Humanization The Emf6.1 mouse variable domain was sequenced, the complementary determination region (CDR) was identified, and it was transplanted into an appropriate human acceptor framework as described in Section 2.2.1 below. The DNA encoding the amino acid sequence of the variant was cloned into the mammalian transient expression plasmid pETE V2. The variant was expressed using a CHO-based transient expression system and purified by affinity chromatography using an AKTA chromatography instrument as described in Section 2.2.2 below.

[0167] 1.16 Kinetic analysis (KD) Dissociation equilibrium constant (K D The parameters were determined using biolayer interferometry (BLI). A series of tests were performed to first optimize the assay parameters, and the following parameters were obtained. [Table 2]

[0168] K D Measurements were performed by immobilizing human GPVI (1.2 μg) on ​​the biosensor tip using a suitable capture surface and monitoring the binding of Emf6.1 variants (i.e., including humanized Fab fragments and chimeric Fab fragments, HC0 LC0, Emf6.1 mouse variable domain and human Ig constant domain) by BLI using an Octet instrument. The resulting sensorograms were analyzed using the provided software (Fortebio). Association / dissociation time (seconds): 900 / 1200; antibody screening range (seven concentrations using 3-fold serial dilutions): 10–0.014 nM; interaction / fitting model: 1:1. All samples were diluted in freshly prepared electrophoresis buffer.

[0169] K D Measurements were performed using human GPVI protein antigen captured by a biosensor. The antigen-capturing biosensor (i.e., tip) was immersed in wells containing different concentrations of Emf6.1 variant (association step), followed by a dissociation step by flowing buffer. To allow for reference correction, the antigen-capturing sensor was immersed in a well containing only buffer (reference well). This reference provided a means to compensate for the spontaneous dissociation of the captured antigen. The steps were performed at 25°C and a constant orbital flow rate of 1000 rpm (generated by moving the sample in the wells of a multi-well plate by orbital motion relative to the biosensor). A new sensor was used for each sample. Overall, the results were fitted, and ka, kd, and K were obtained. DTo obtain the best value, a dilution series of the Emf6.1 variant was used in the association step. Dissociation equilibrium constant (K D The interaction was calculated using ForteBio data analysis software, and the response data for the coupling was fitted to a 1:1 interaction / coupling model. All consumables used were those recommended by ForteBio.

[0170] 2.Results 2.1 Example 1: Production and testing of mouse IgG antibodies against human GPVI 2.1.1 Emf6.1 binds to human GPVI and blocks its function. A series of monoclonal mouse IgG antibodies (Emf antibodies) against human GPVI were generated using standard hybridoma technology, and 16 clones were isolated based on their specific binding to the human GPVI ectodomain (Emf1-Emf16). Flow cytometry analysis showed that only nine of these also bound to human platelets, i.e., the membrane expressed GPVI, including Emf1, Emf2, Emf3, and Emf6.1. These clones were further characterized for their ability to inhibit GPVI function. In fact, three of these monoclonal antibodies (mAbs) inhibited the CRP and collagen-induced response in human platelets (Emf1). 25 Emf2 32 Emf6.1 (mouse IgG2aκ) consistently showed the most potent inhibitory effect. Emf6.1 IgG (10 μg / mL) eliminated CRP and collagen-induced aggregation of human platelets, but did not affect the response to other agonists, including thrombin and the stable thromboxane A2 analog U46619 (Figure 1A). Furthermore, Emf6.1, when used at 10, 5, 2, and 1 μg / mL, showed the same effect on flow (1000s). -1 Under these conditions, aggregation of human platelets in heparinized blood on collagen was strongly blocked (Figure 1B, C). This was done to avoid the possible Fc-mediated effects of Emf6.1 IgG mediated by platelet-expressed Fc gamma receptor IIA (FcγRIIA, CD32), and also to prevent Fc-dependent thrombocytopenia and / or GPVI depletion.19 To exclude it, we generated a Fab fragment of Emf6.1 and used it for all further research.

[0171] 2.1.2 Emf6.1 Fab It inhibits GPVI-dependent activation of human platelets. Emf6.1 in reducing GPVI-dependent platelet adhesion and aggregate formation. Fab To determine the in vitro efficacy, anticoagulant human blood was tested in a flow state on a collagen-coated surface. At 10 μg / mL, Emf 6.1 Fab is 1000s -1 At this shear rate, adhesion and aggregate formation of human platelets were almost completely inactivated (Figure 9A). In particular, at lower concentrations of Emf6.1 Fab Aggregate formation was significantly inhibited at (5, 2, and 1 μg / mL), and thrombus volume was reduced by 75% compared to the control Fab at 1 μg / mL (Figure 9A). Next, citrated human blood was subjected to Emf6.1 at 10 μg / mL. Fab Alternatively, pre-incubation was performed with a control Fab, and then thrombus formation was tested in a flow chamber under active coagulation conditions. Notably, under these conditions, Emf6.1 Fab This strongly inhibited platelet deposition and thrombus formation on the collagen / TF coated surface (Figure 1D-F), and completely eliminated phosphatidylserine (PS) exposure and fibrin deposition (Figure 1D, G-H).

[0172] Next, the washed human platelets were treated with 10 μg / mL Emf6.1 Fab The samples were treated with [method name] and then tested using a standard agglutination assay with different agonists. Emf6.1 Fab This eliminated CRP and collagen-induced aggregation, but the thrombin-induced response remained unchanged, supporting the GPVI-specific inhibitory effect of Fab (Figure 1I). Similar to the results in fluid adhesion, Emf6.1 at 5, 2, and 1 μg / mL was observed. Fab Significant inhibition was observed by agglutination assay (Figure 9B). Human GPVI has been shown to promote platelet activation and diffusion on fibrinogen. 40、41 Emf6.1 FabTo test whether it interferes with this GPVI function, human platelets were attached to immobilized fibrinogen in the presence or absence of Fab. In fact, Emf6.1 Fab (10 μg / mL) strongly inhibited complete platelet diffusion, as evidenced by a dramatic reduction in stage IV (fully diffused) platelets and a significant increase in stage II (filamentous platelets) (Figure 1 J-K).

[0173] In summary, this data is from Emf6.1 Fab However, it has been shown to potently inhibit GPVI-dependent activation, aggregation, and diffusion of human platelets in vitro.

[0174] 2.1.3 emf6.1 Fab is hGP6 tg / tg Inhibits GPVI-mediated activation of platelets In vivo EMF 6.1 Fab To study the effects of GPVI, a newly generated humanized mouse strain (hGP6) was used. tg / tg ) 32 I used hGP6. tg / tg Emf6.1 for platelet agonist-induced activation Fab The effect was tested by whole blood flow cytometry. Emf6.1 Fab (10 μg / mL) eliminated CRP-induced activation but did not affect responses to other agonists such as thrombin, ADP, and U46619 (Figure 10A-B). Similar to the results for human platelets, Emf6.1 Fab This refers to hGP6 on collagen in a whole blood perfusion assay. tg / tg It completely inhibits platelet aggregate formation (Figure 2A-C), and the Emf 6.1 at 2 μg / mL. Fab The same effect was observed at the concentration (Figure 10D). Furthermore, Emf6.1 Fab (10 or 2 μg / mL) is hGP6 tg / tg GPVI-mediated aggregation of platelets was almost completely inhibited (Figure 2D and Figure 10C). Previous studies have shown that mouse platelets expressing human GPVI, rather than wild-type platelets, can completely diffuse onto fibrinogen. 32、42Therefore, Emf6.1 Fab We also tested whether this would interfere with the response. In fact, hGP6 tg / tg Platelets diffuse well onto the fibrinogen-coated surface, and this process is similar to what is observed in human platelets, as in Emf6.1 Fab It was eliminated in the presence of (Figures 2E-F).

[0175] In summary, these results are for humans and hGP6. tg / tg Emf6.1 for mouse platelets Fab It showed equivalent in vitro effects, and the mouse strain was Emf6.1 Fab This demonstrates that it is a suitable model system for studying the in vivo effects.

[0176] 2.1.4 Emf6.1 Fab is hGP6 tg / tg Efficiently block GPVI functionality in ex vivo environments in mice. Emf6.1 Fab To study the in vivo effects of hGP6, tg / tg Mice were intravenously administered a dose of 4 mg / kg, and their peripheral platelets were monitored for 5 days by flow cytometry and automated cell analysis. Platelet count and size remained unchanged compared to the control at all time intervals (Figure 3A-B), and no changes at the GPVI surface level on circulating platelets were detected (Figure 3C). Next, Emf6.1 Fab The GPVI occupancy rate on circulating platelets was evaluated over time using a FITC-labeled anti-GPVI mAb that competes with the Emf6.1 binding site (Emf3). Emf6.1 occupancy rate was assessed at 4 mg / kg bw. Fab Significant GPVI occupancy was detected for up to 96 hours after a single dose injection (Figure 3D). Specifically, 1 hour after injection, approximately 90% of the binding epitopes on GPVI were Emf6.1 FabThe percentage was occupied by GPVI, and gradually decreased to approximately 76% at 24 hours, 50% at 48 hours, 36% at 72 hours, 19% at 96 hours, and 13% at 120 hours. This sustained GPVI blockade was confirmed by flow cytometry analysis of CRP-induced platelet activation in ex vivo diluted blood. Emf6.1 Fab In treated mice, Emf 6.1 at 4 mg / kg bw. Fab For up to 72 hours after injection, a significantly impaired response to CRP-, but not to thrombin, was detected compared to the control group (Figure 3E-H).

[0177] According to these results, Emf6.1 was obtained with 4 mg / kg bw, not with the control Fab. Fab No significant inhibition of CRP-induced platelet aggregation was observed with ex vivo for up to 72 hours after injection (Figure 4A). Furthermore, collagen-induced aggregation was observed with Emf6.1 Fab Treatment with [agent] strongly inhibited GPVI, showing a significant decrease at 1 and 24 hours post-treatment, but substantial GPVI inhibition was still observable at 48 hours and further decreased at 72 hours (Figure 4B). As expected, thrombin-induced aggregation was not altered at any of the test time points (Figure 4C). Next, Emf6.1 on GPVI-dependent aggregate formation on collagen-coated surfaces. Fab The effect of the treatment was evaluated using an ex vivo total blood flow adhesion assay. Additionally, control Fab-treated hGP6 was evaluated up to 48 hours after injection. tg / tg Compared to a mouse, Emf6.1 Fab In this case, a significant reduction in surface coverage and thrombus volume was observed (Figures 4D-E).

[0178] In conclusion, these data are from Emf6.1 Fab This study demonstrates that treatment of human GPVI-expressing mice with [specific agent] results in large-scale and sustained inhibition of GPVI-dependent platelet activation ex vivo.

[0179] 2.1.5 Emf6.1 Fab It is highly protective in vivo in arterial thrombosis models. Emf6.1 Fab To evaluate the antithrombotic capacity of GPVI blockade, a model of arterial thrombosis was used in which the abdominal aorta was mechanically damaged and blood flow / occlusive thrombus formation was monitored by an ultrasound flow probe. 43、44 Notably, Emf6.1 Fab Treated mice (1 hour after injection of 4 mg / kg bw) were strongly protected from thrombotic vascular occlusion (Figure 5A), with 91.7% (11 out of 12) not forming stable thrombi within a 30-minute observation period, compared to 100% of the control group which developed occlusion. Emf6.1 Fab Most treated mice experienced a transient decrease in blood flow, followed by the formation of initial aggregates, and then rapid separation or embolization of the formed thrombi (Figure 5B).

[0180] Finally, Emf6.1 for trauma-induced hemostasis in a tail hemorrhage model. Fab The effectiveness of the process was evaluated. Emf6.1 Fab Processing hGP6 tg / tg In mice, control Fab-treated hGP6 tg / tg Furthermore, no significant difference in tail bleeding time was detected compared to control Fab-treated WT mice (Figure 5C). Overall, these data suggest that Emf6.1 Fab Processing by hGP6 tg / tg This study demonstrates that the treatment strongly protects mice from arterial thrombosis while not significantly affecting tail bleeding time, i.e., normal hemostasis.

[0181] 2.1.6 Emf6.1 binds to the proposed GPVI dimerization site. To determine the epitope of Emf6.1 in human GPVI, a 15-mer duplicate peptide library covering the hGPVI ectodomain (residues 24-267 of UniProtKB Q9HCN6-1) was synthesized and printed in microarray format. GPVI peptide microarray binding of Emf6.1-IgG was detected using an HRP-labeled anti-mouse IgG antibody. Epitope mapping revealed a distinct discontinuous binding epitope located between D2 and the transmembrane helix, more precisely on residues Val201-Glu215 and Ser246-Pro260 (Figure 6A). The significant contributions of both binding surfaces to Emf6.1 binding were verified by neutralization of Emf6.1 using the corresponding soluble peptide (Figure 6B). In particular, the Emf6.1 binding epitope is located at the C-terminus of all previously reported GPVI epitopes (residues 58-187) and is therefore located at structurally degraded collagen binding sites (Trp76, Arg38, and Glu40) (Figure 6C). Thus, the mapping suggests that Emf6.1 inhibits GPVI function via a non-canonical mechanism likely related to GPVI dimerization, which is structurally degraded early and mapped to overlapping surfaces (Asp196-Thr203). 18 (Figure 6C) 17 .

[0182] Therefore, peptide microarray-based mapping and neutralization 49~51 This supports the non-canonical discontinuous binding site of Emf6.1 in GPVI (Val201-Glu215 and Ser246-Pro260) between D2 and the transmembrane helix that overlaps with the proposed GPVI dimerization residues (Val201-Thr203), although D2 does not overlap with the collagen binding interface of D117. 18 Based on this finding, Emf6.1 Fab / EMA601 is primarily like the other GPVI antagonists previously described. 21、52、53 It can be hypothesized that GPVI function can be blocked not by interfering with ligand binding, but perhaps by regulating its clustering / signaling ability.

[0183] 2.2 Example 2: Generation and Characterization of Humanized Emf6.1 Fab Variants 2.2.1 Fully Humanized Emf6.1 Fab Variant generation Emf6.1 Fab Considering its large-scale GPVI inhibitory activity, a set of humanized Fab fragments derived from the Emf6.1 sequence was designed with the aim of producing non-immunogenic monovalent antibody fragments suitable for injection into humans and capable of preserving specific GPVI binding activity. The Emf6.1 mouse variable domain was sequenced, and canonical and subclass complementary determination regions (CDRs) were identified. 37、38 Mouse V H and V L CDR residues within the domain were identified using a combination of IMGT and the Kabat numbering system. 37、38 .

[0184] Mouse Emf6.1 V L and V H The domain had the following sequence, which did not contain the mouse signal peptide sequence: >Original mouse Emf6.1 V L (VL0) [Sequence List 1] JPEG2026509897000011.jpg14170>Original Mouse EMF6.1 V H (VH0) [Sequence Listing 2] JPEG2026509897000012.jpg14170

[0185] The IMGT numbering system was used to identify the gray-highlighted CDR residues, and the Kabat numbering system was used to... Underlined The CDR residues were identified. The identified CDR sequences are summarized in Table 3 below. [Table 3]

[0186] The closest human germline gene V H The region was Homo sapiens IGHV4-4. A database of human IgG and human IgK sequences was searched using the BLAST search algorithm, and then mouse V H and V L Each domain was searched for comparison, and suitable candidates were selected based on a combination of framework homology, maintenance of key framework residues, and canonical loop structure. The humanized variant is mouse V H and V L This was created by porting the CDR to these acceptor frameworks. >VL1 (based on BAH04725) [Sequence Listing 3] JPEG2026509897000014.jpg12170>VL2 (based on AIT38570) [Sequence Listing 4] JPEG2026509897000015.jpg12170>VL3 (based on AMK70118) [Sequence Listing 5] JPEG2026509897000016.jpg11170>VL4 (based on APZ85375) [Sequence Listing 6] JPEG2026509897000017.jpg11170>VH1 (based on AAV40414) [Sequence Listing 7] JPEG2026509897000018.jpg10170>VH2 (based on AAY23326) [Sequence Listing 8] JPEG2026509897000019.jpg11170>VH3(>Based on AEX28400) [Sequence Listing 9] JPEG2026509897000020.jpg12170>VH4 (based on AAD31716) [Sequence Listing 10] JPEG2026509897000021.jpg12170>VH5 (based on IGHV4-4) [Sequence List 11] JPEG2026509897000022.jpg10170

[0187] In Figures 7A and 7B, mouse EMF 6.1 V L and V H The sequences (VL0 and VH0) are, respectively, humanized V L Variant V H The variants are aligned. In order of homology, the humanized variants are VL1>VL4>VL2>VL3. In order of homology, the humanized variants are VH5>VH1>VH2>VH4>VH3.

[0188] V H and V L Each domain was synthesized in frame along with the constant domain sequences of human IgG1 and human IgK isotypes and cloned into the mammalian transient expression plasmid pETE V2. The humanized variants were checked to determine whether they were humanized according to the WHO definition of humanized antibodies. The variable domains of the humanized chain, when analyzed as a whole, have a V-region amino acid sequence closer to that of humans than other species (evaluated using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool, Table 4). 39 Original mouse antibody V H and V LFurthermore, humanized variant sequences were screened for MHC class II-binding peptides, and it was determined that peptide sequences with high affinity for the humanization process were removed using an in silico algorithm. Additionally, Fab variants were tested for motifs prone to deamidation from asparagine to aspartic acid. Such motifs were not present in mouse or humanized Fab variants. The structure of the variable domain binding site was modeled using Schrödinger software. Based on RMSD analysis, the VH2:VL3 and VH1:VL3 combinations had the lowest scores compared to the mouse VH0 VL0 domain. This ranked them as having the closest structural similarity to the mouse variable domain. All humanized variable domain combinations had RMSD values ​​less than 2 and were therefore predicted to have good agreement with the mouse VH0 VL0 domain structure (see Table 5). [Table 4] [Table 5]

[0189] For higher levels of expression in CHO cells, N-terminal signal peptides were added to each heavy and light chain (heavy chain signal peptide: MGWTLVFLFLLSVTAGVH = SEQ ID NO: 63, light chain signal peptide: MVSSAQFLGLLLLCFQGTRC = SEQ ID NO: 62). H Each domain was synthesized in frame along with the C-terminal human IgG1 isotype constant domain sequence (allotype G1m17,1) corresponding to the N-terminal signal peptide (SEQ ID NO: 63) and SEQ ID NO: 61. LEach domain was synthesized in frame along with the N-terminal signal peptide (SEQ ID NO: 62) and the C-terminal human IgK isotype constant domain sequence (allotype Km3) corresponding to SEQ ID NO: 60. The sequences were codon-optimized (ATUM, USA), and each variant chain was validated by DNA sequencing analysis. The complete amino acid sequences of each heavy and light chain are shown in Table 1 below.

[0190] 2.2.2 Transient Expression and Purification of Humanized Fab Variants The next step was transient transfection and expression of each humanized Fab fragment. There was one chimeric Fab fragment expressed for use as a positive control, possessing both a mouse variable domain and a human Ig constant domain, as well as 20 humanized variants possessing both a humanized variable domain and a human Ig constant domain. The Emf6.1 humanized mutant is a 50 kilodalton (kDa) monovalent Fab fragment, with each fragment consisting of a heavy chain (with the entire Fc region missing) and a light chain, complexed together via disulfide bonds. Mammalian expression vectors encoding each variant (pETE V2) were transfected into Chinese hamster ovary (CHO) cells, and batch cultures of each variant were grown for up to 7 days.

[0191] The expressed Fab fragment was purified from the cell culture supernatant via affinity chromatography. For this purpose, the cell culture supernatant containing the Fab fragment was clarified by centrifugation and filtration. The Emf6.1 variant was purified from the cell culture supernatant via affinity chromatography (using state-of-the-art AKA chromatography equipment). The purified Fab fragment was dialyzed / buffered with phosphate-buffered saline. Quality control experiments, including concentration and purity measurements of the purified Fab product, were performed to ensure that specific criteria were met. The purity of the Fab fragment was determined to be greater than 95% by reducing and denaturing a sodium dodecyl sulfate polyacrylamide gel. The Fab concentration was determined by measuring the absorbance at 280 nm and calculated using a calculated extinction coefficient of 1.49 at 1.0 mg / ml = A280 (assuming MW = 50 kDa of the Fab fragment).

[0192] All Fab variants were successfully expressed and purified according to the above criteria. SDS-PAGE analysis revealed that all Fab fragments showed sufficient purity under reducing conditions. Under reducing conditions, two bands were observed with molecular weights of approximately 25 kDa and 30 kDa, respectively. Under non-reducing conditions, two bands were observed with molecular weights of approximately 20 kDa and 40 kDa. The additional bands (impurities) are likely the result of unpaired heavy and / or light chains.

[0193] 2.2.3 Dynamic analysis of humanized Fab variants For kinetic analysis, human GPVI-Fc fusion protein (1.2 μg) was immobilized on a biosensor using a suitable capture surface, and binding of the Emf6.1 variant was monitored by biolayer interferometry (BLI) using an Octet instrument. The resulting sensorogram was analyzed using the provided software (Fortebio). For experimental details, please refer to Section 1.16 above. [Table 6]

[0194] The kinetic (octet) analysis showed that, in most cases, the experimental data fit a 1:1 binding model. Under the experimental conditions used, numerous Fab fragments, including HC1 LC2 (KD: 195 pM), HC1 LC3 (KD: 284 pM), HC2 LC2 (KD: 175 pM), and HC2 LC3 (KD: 250 pM), showed higher affinity than that of the chimeric HC0 LC0 control antibody (KD: 427 pM) (see Table 6).

[0195] 2.2.3 Further Characterization of the HC1 LC2 Variant (EMA601) The HC1 LC2 variant (referred to as EMA601) was selected for further characterization due to its most potent GPVI blocking effect. Firstly, EMA601 at a concentration of 5 μg / mL strongly inhibited the formation of human platelet aggregates on collagen in a whole blood perfusion system (Figures 8A-C). Notably, EMA601 was also effective at lower concentrations (1 μg / mL) in this assay (Figure 11A).

[0196] Furthermore, EMA601 (5 μg / mL) eliminated CRP and collagen-induced aggregation of human platelets without affecting the thrombin-induced response (Figure 8D). This effect was also achieved at a low EMA601 concentration of 1 μg / mL (Figure 11B), and dose-response experiments further demonstrated that humanized EMA601 strongly inhibited activation / aggregation at high collagen concentrations (10 and 20 μg / mL). In addition, EMA601 (10 μg / mL) was found to strongly inhibit GPVI-dependent diffusion of human platelets on fibrinogen (Figures 8E-F). Finally, EMA601 also strongly inhibited platelet deposition and thrombus formation on collagen / TF-coated surfaces (Figures 8G-I), completely eliminating phosphatidylserine (PS) exposure and fibrin deposition (Figures 8G, J-K). Overall, these data establish EMA601 as a promising clue for targeting human GPVI in pathological conditions.

[0197] 2.3 Example 3: Face-to-face comparison of EMA601 and ACT017 (glenzosimab) 2.3.1 In vitro binding of GPVI on platelets In vitro and in vivo studies were conducted to directly compare the GPVI inhibitory activity of EMA601 and ACT017 (glenzosimab). In the first set of experiments, hGP6 tg / tg Diluted mouse blood was pre-incubated with EMA601 or ACT017 at different concentrations (ranging from 50 to 0.1 μg / mL), and epitope saturation on GPVI was examined by flow cytometry. Emf3 FITC and JAQ1 FITC (5 μg / mL each) was used to detect epitope occupancy by EMA601 and ACT017, respectively. Emf3 FITC Binding was strongly inhibited by EMA601 even at a concentration of 0.5 μg / mL, but ACT017 was strongly inhibited by EMF3 even at very high concentrations (n=4). FITC Without affecting binding (Figure 12A, B), EMA601 and ACT017 support binding to different epitopes on human GPVI. In contrast, JAQ1FITC binding was inhibited by ACT017 at high concentrations (50, 20, 10 μg / mL), but the effect gradually disappeared at concentrations below 5 μg / mL, suggesting a relatively low affinity of ACT017 for GPVI. Notably, EMA601 bound to JAQ1 at concentrations more than 10 times lower (below 0.5 μg / mL). FITC The binding was inhibited. Next, hGP6 tg / tgDiluted mouse blood was pre-incubated with EMA601 or ACT017 at different concentrations (ranging from 50 to 0.1 μg / mL), and bound Fab was detected using fluorescently labeled anti-human IgG-Fab antibodies (n=4) (Figure 12C). At all test concentrations, EMA601 produced a significantly higher signal than ACT017, demonstrating a higher binding affinity for EMA601 compared to ACT017. This was further supported by the rapid decrease in surface-bound ACT017 at concentrations below 2 μg / mL. Specifically, at the lowest concentrations of 0.2 μg / mL and 0.1 μg / mL, ACT017 was virtually absent from the platelet surface, while EMA601 still occupied 86.2% and 57.3% of the epitopes, respectively, compared to the highest test concentration (50 μg / mL) (Figure 12C).

[0198] 2.3.2 Standard light transmission agglutination assay and flow adhesion assay Next, washed human platelets were incubated with different concentrations of EMA601, ACT017, or control Fab, and their responses to collagen (10 μg / mL) and CRP (0.5 μg / mL) were tested by standard light transmission agglutination assays. EMA601 eliminated collagen-induced aggregation at 5 μg / mL and still reduced it by approximately 50% even at 1 μg / mL. In stark contrast, ACT017 at concentrations up to 10 μg / mL did not have a significant inhibitory effect under these conditions. Notably, even at the highest test concentration (50 μg / mL), ACT017 could not completely inhibit the response, demonstrating its overall limited GPVI blocking capacity (Figure 12D). Furthermore, CRP-induced aggregation was eliminated at all test concentrations of EMA601 (5, 1, and 0.5 μg / mL), while ACT017 showed only partial inhibition at 1 μg / mL and no inhibitory effect at 0.5 μg / mL (Figure 12D). Next, the in vitro efficacy of EMA601 and ACT017 in reducing GPVI-dependent adhesion and aggregate formation of human platelets on collagen was tested in a whole blood flow chamber under arterial shear stress (1000 s-1) using a flow adhesion assay. Notably, EMA601 significantly reduced both platelet adhesion and aggregate formation at all test concentrations, while ACT017 did not affect adhesion on collagen and only reduced thrombus volume. Interestingly, EMA601 showed a large and strong reduction in thrombus volume compared to ACT017 at all test concentrations (Figures 12F-H).

[0199] 2.3.3 Ex vivo study after intravenous administration Finally, to compare the efficacy of EMA601 or ACT017 in vivo for blocking hGPVI, hGP6 tg / tgMice were intravenously administered 4 mg / kg of humanized Fab, and either GPVI epitope occupancy or platelet activation was tested ex vivo 1 hour later. Notably, approximately 1.8 times higher levels of bound EMA601 were detected compared to ACT017 (Figure 12H). Consistent with this, CRP-induced platelet activation (JON / A-PE binding) was eliminated in the blood of EMA 601-treated mice, but no significant inhibition was detected in the blood of ACT017-treated mice (Figure 12I).

[0200] In summary, these results indicate that EMA601 is effective in human platelets in vitro, as well as in hGP6 in vitro and in vivo. tg / tg The study demonstrated that ACT017 exhibited significantly higher GPVI inhibitory effects on mouse platelets compared to ACT017. Furthermore, the data showed that ACT017 at concentrations up to 50 μg / mL could not completely block GPVI function in vitro, while high doses (4 mg / kg) of hGP6 tg / tg We revealed that the same thing can be observed in in vivo / ex vivo studies.

[0201] 2.4 Conclusion Monovalent antibody Emf6.1 Fab And its humanized variant EMA601 was tested in vitro in humans and hGP6 tg / tg It was shown to potently inhibit GPVI function in mouse platelets. (Emf6.1 transgenic mouse) Fab The treatment did not affect bleeding time but provided sustained GPVI blockade and significant protection from occlusive arterial thrombosis. On the one hand, it involved high affinity binding to GPVI and hGP6. tg / tg Good pharmacokinetics in mice, and on the other hand, unexpected binding sites on the receptor, Emf6.1 Fab We highlight / EMA601 as a novel clue to achieving potent yet safe GPVI inhibition in clinical settings.

[0202] This data is from 9O12 Fab and ACT017 (Glenzosimab) )23、29、30 Compared to what has been reported about hGP6 tg / tgEmf6.1 on circulating platelets in mice Fab It showed remarkably long persistence, with receptor occupancy rates of 50% and 35% at 48 and 72 hours after a single dose (4 mg / kg) injection, respectively, resulting in sustained inhibition of GPVI function ex vivo. Emf6.1 Fab One possible explanation for this remarkably extended in vivo activity could be its high affinity for human GPVI (kD: 0.46 nM), which is about 10 times higher than the reported affinity for ACT017 (kD: 4.1 nM). 26 Notably, Emf6.1 Fab The affinity for hGP6 increased by approximately 2.3 times during the humanization process, with EMA601 showing a kD of 0.195 nM (thus approximately 21 times higher than ACT017). Direct face-to-face comparison of EMA601 and ACT017 showed that EMA601 was superior to human hGP66 in vitro. tg / tg It showed more than 10 times higher efficacy in inhibiting GPVI function in platelets, hGP6 tg / tg EMA601 was also confirmed to efficiently block GPVI function in mice, but no significant inhibitory effect was observed under the same conditions as ACT017 (4 mg / kg bw) (Figure 12). Based on these results, EMA601 is expected to achieve the desired level of GPVI inhibition in humans at a relatively lower dose and / or longer duration compared to ACT017.

[0203] This data represents Emf 6.1 relative to the total recalcified blood. Fab In vitro application of this drug strongly inhibits stable thrombus formation, eliminating PS exposure, followed by a reduction in fibrin deposition on the collagen / TF coated surface. Emf6.1 in vivo. Fab This significantly inhibits the formation of stable thrombi at arterial injury sites, suggesting that its potent antithrombotic effect may be based on two main activities: namely, a reduction in platelet activation on the exposed extracellular matrix / thrombus surface, and indirectly, a potent inhibition of local platelet-dependent coagulation.

[0204] Peptide microarray-based mapping and neutralization49~51 This confirms the non-canonical discontinuous binding site of Emf6.1 in GPVI (Val201~Glu215 and Ser246~Pro260) between D2 and the transmembrane helix that overlaps with the GPVI dimerization residues (Val201-Thr203), which does not overlap with the collagen binding interface of D117. 18 Based on this finding, Emf6.1 Fab / EMA601 primarily interferes with ligand binding, rather than as previously described for other GPVI antagonists. 21、52、53 This may block GPVI function by modifying its clustering / signaling ability. This mechanism of action is described in Emf6.1. Fab However, this could explain how platelet GPVI-induced activation is inhibited on a large scale while preserving the receptor's initial adhesion function.

[0205] In conclusion, these data provide a preclinical characterization of the function of blocking the Fab fragment, which has very high affinity for human GPVI, and may enable sustained and safe inhibition of GPVI function in circulating platelets, possibly via a non-canonical mechanism of action.

[0206] Example 4: Emf6.1-fab treatment in experimental stroke Materials and methods Mice. The animals used in this study were matched for age, sex, and genetic background. The experiments were conducted in accordance with local government regulations and followed the current ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https: / / www.nc3rs.org.uk / arrive-guidelines).

[0207] Animal processing. To block human GPVI hGp6 tg / tgMice were intravenously administered 4 mg / kg bwEmf6.1-fab or a control fab one hour before tMCAO. Six hours after tMCAO, a second dose of the fab fragment (4 mg / kg bw) was administered subcutaneously.

[0208] Local ischemia model. As previously explained. 63 Localized cerebral ischemia was diagnosed by tMCAO in 10-14 week olds with hGp6 tg / tg mouse 32 Anesthesia was induced using 2% isoflurane. The duration of the surgical procedure per animal was kept to less than 10 minutes. A silicone rubber-coated 6.0 nylon monofilament (6021PK10, Doccol, Redlands, CA, USA) was advanced through the carotid artery to the origin of the MCA to induce MCA infarction. After 60 minutes of occlusion, the filament was removed and reperfusion was performed. Animals were sacrificed 23 hours after reperfusion and checked for intracerebral hemorrhage.

[0209] Infarct size measurement. The extent of infarct was quantitatively assessed 24 hours after reperfusion. Animals were sacrificed, and the brain was cut into three 2 mm thick coronal sections. The slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich; 2% wt / volume (w / v) solution) at 37°C for 20 minutes to visualize the infarct (Junge et al.). Edema-corrected infarct volume was calculated by area measurement (Image J software, National Institutes of Health) according to the following formula: V indirect (mm 3 )=V infarct ×(1-(VI-VC) / VC) (VI-VC) represents the volume difference between the ischemic hemisphere (VI) and the control hemisphere (VC), and (VI-VC) / VC) expresses this difference as a percentage of the control hemisphere.

[0210] Statistical analysis. All data from animal experiments were given as box plots including the 25th and 75th percentiles, and the median with minimum and maximum deviations. The data were tested for Gaussian distribution using, for example, the D'Agostino and Pearson omnibus normality test, and then analyzed, where applicable, by Student's t-test, one-way ANOVA, or Mann-Whitney U test. Scores dealing with functional outcomes were compared using the Mann-Whitney U test. P<0.05 was considered statistically significant. The GraphPad Prism 7.05 software package (GraphPad Software) was used for statistical analysis.

[0211] Results - Effect of hGPVI blockade in a focal cerebral ischemia (stroke) model To test the possible effects of hGPVI blockade in cerebral thrombotic inflammation, hGPVI tg / tg Mice were treated with Emf6.1-fab (to block hGPVI) or a control fab and subjected to 1 hour of transient midbrain ischemia (tMCAO) and 23 hours of reperfusion. Surprisingly, triphenyltetrazolium chloride (TTC) staining showed (Med.: 105.9 (25%: 88.5; 75%: 121.0) vs. 137.1 (25%: 119.6; 75%: 142.3) mm 3 As measured by p<0.05 (Figures 13A and 13B), 24 hours after tMCAO, infarct volume in Emf6.1-fab treated mice was significantly reduced compared to control-fab treated (Ctrl.) littermates. No signs of intracerebral hemorrhage were detected in any of the tested animals.

[0212] The result is hGp6 processed with Emf6.1-fab. tg / tg The study showed that mice were significantly protected from cerebral infarct proliferation after tMCAO, and that GPVI blockade did not increase the risk of intracerebral hemorrhage in this setting.

[0213] Example 5: Analysis of EMA601 (HC1 LC2) variant HC1LC2 contains free cysteine ​​at Kabat L55 in VLCDR2. Nineteen variants of HC1LC2 were generated and tested in which the cysteine ​​at Kabat L55 was replaced with a different amino acid.

[0214] Materials and methods The generation of DNA constructs for expressing Fab variants was outsourced to an external provider. The parental variable light chain and variable heavy chain sequences were generated via gene synthesis, followed by subcloning into expression vectors, a kappa light chain vector for the variable light chain, and an IgG1 Fab vector for the variable heavy chain. Light chain variants encoding alternative amino acids to replace unpaired cysteine ​​residues within CDRL2 were constructed by site-directed mutagenesis followed by subcloning.

[0215] Variant expression in the Fab format was performed by transient transfection of Expi293F cells. Proteins were purified using a HighTrap Protein L column, followed by preparative sorting using a HighLoad 16 / 600 Superdex 200 column.

[0216] The recombinant huGPVI-Fc fusion protein was used for kinetic analysis by BLI (Octet). The analysis was performed as described in Section 1.16 above.

[0217] The variants were functionally evaluated using agglutination assays with human platelets and GPVI-specific agonists. The evaluations were performed as described in Section 1.7 above.

[0218] result All Fab fragments were tested in BLI (octet) at the following concentrations: 10, 3.3, 1.1, 0.37, 0.12, 0.04, and 0.014 nM. Glenzosimab, a humanized anti-GPVI Fab from Acticor, was tested at concentrations of 100, 33, 11, 3.7, 1.23, 0.41, and 0.14 nM.

[0219] The determined dissociation constant (KD) for the variant was as follows: [Table 7]

[0220] All variants exhibited higher affinity for human glycoprotein VI than the antibody glenzosimab.

[0221] All variants were further tested for their ability to block GPVI-dependent human platelet aggregation at a concentration of 0.5 μg / mL, and, where indicated, 10 μg / mL. The agonists used were as follows: 1. Collagen (10 μg / mL) 2. Collagen-related peptide (CRP - 0.5 μg / mL) Agglutination tests were performed using washed human platelets with an aggregation time of 15 minutes. A control Fab or HC1LC2 variant was added 5 minutes before the agonist was introduced. As shown in Figure 14, all variants at a concentration of 0.5 μg / mL completely inhibited CRP-induced aggregation, and most of them also potently inhibited collagen-induced aggregation. Notably, some variants inhibited GPVI function even with higher potency than the parent version. Variants with isoleucine, leucine, glutamic acid, threonine, valine, or serine at the Kabat 55 position performed particularly well. 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Claims

1. An antibody or functional fragment thereof capable of binding to human glycoprotein VI (GPVI), comprising: (i) a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 1, a CDR2 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 9, or SEQ ID NO: 64, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 3 Ｌ (ii) a domain and a V having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 4, a CDR1 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 5, and a CDR3 region having an amino acid sequence according to the amino acid sequence shown in SEQ ID NO: 6 Ｈ An antibody or a functional fragment thereof containing a domain.

2. The antibody or functional fragment according to claim 1, which is monovalent.

3. The antibody or functional fragment has a dissociation equilibrium constant (K) of less than 700 pM, preferably less than 300 pM, and more preferably less than 200 pM. Ｄ The antibody or functional fragment according to claim 1 or 2, which binds to human GPVI.

4. The antibody or functional fragment contains an amino acid sequence that has at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, and 35. Ｌ V containing an amino acid sequence having at least 90% sequence identity with the domain and / or a sequence selected from the group consisting of SEQ ID NOs: 21, 22, 23, 24, and 25. Ｈ An antibody or functional fragment according to any one of the prior claims, comprising a domain.

5. The antibody or functional fragment contains the amino acid sequence shown in SEQ ID NOs: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. Ｌ V containing the domain and / or the amino acid sequence shown in SEQ ID NOs. 21, 22, 23, 24, or 25 Ｈ An antibody or functional fragment according to any one of the prior claims, comprising a domain.

6. A functional fragment according to any one of the prior claims, which is a fragment antigen-binding (Fab), F(ab'), Fv, monovalent IgG, disulfide-linked variable fragment (dsFv), or single-stranded variable fragment (scFv).

7. The antibody or functional fragment according to any one of the prior claims, wherein the antibody or functional fragment comprises a light chain including an N-terminal light chain signal peptide having an amino acid sequence according to the sequence shown in SEQ ID NO: 62, and / or a constant domain having an amino acid sequence shown in SEQ ID NO: 60, and / or a heavy chain including an N-terminal light chain signal peptide having an amino acid sequence according to the sequence shown in SEQ ID NO: 63, and / or a constant domain having an amino acid sequence shown in SEQ ID NO:

61.

8. The functional fragment according to claim 7, wherein the functional fragment comprises or is a Fab comprising a light chain having the amino acid sequence shown in one of sequence numbers 38, 39, 40, 41, 55, 56, 57, or 58, and a heavy chain having the amino acid sequence shown in one of sequence numbers 44, 45, 46, 47, or 48.

9. The functional fragment according to any one of the prior claims, wherein the functional fragment is a Fab consisting of a pair of light chains and heavy chains having the amino acid sequences shown in SEQ ID NOs: 39 and 44, 39 and 45, 40 and 45, 56 and 44, 56 and 45, or 57 and 45, preferably SEQ ID NOs: 39 and 44, or 56 and 44.

10. An antibody or functional fragment according to any one of the prior claims, capable of binding to human GPVI in a binding epitope corresponding to amino acids V178 to E192 and / or S223 to P237 of SEQ ID NO:

36.

11. A nucleic acid encoding an antibody or functional fragment as described in any one of the prior claims.

12. A cell containing the nucleic acid described in claim 11.

13. A method for preparing an antibody or functional fragment according to any one of claims 1 to 10, comprising: culturing the cells according to claim 12 in a culture medium under conditions that enable the expression of nucleic acids encoding the antibody or functional fragment; and recovering the antibody or functional fragment from the cells or the culture medium.

14. A pharmaceutical composition comprising an antibody or functional fragment according to any one of claims 1 to 10, and optionally a pharmaceutically acceptable carrier and / or excipient.

15. This invention is intended for use in methods of treating or preventing GPVI-related conditions in subjects, and optionally, the GPVI-related condition may be cardiovascular diseases preferably selected from thromboinflammatory diseases; including arterial thrombosis, venous thrombosis, atherothrombosis, stent thrombosis, venous thromboembolic disease, thrombotic microangiopathy, cancer-associated thrombosis, immunothrombosis and infection-related thrombosis, restenosis, acute coronary syndrome, ischemic cerebrovascular disease, cerebrovascular disease, vascular purpura, coronary artery disease and cerebral artery disease, thrombosis and thrombotic diseases, ischemic events, acute coronary syndrome, myocardial infarction, stroke, percutaneous coronary intervention, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches, peripheral artery disease, acute phlebitis, and pulmonary embolism; inflammation, preferably persistent or long-term inflammation related to infection, arthritis, fibrosis, acute respiratory distress syndrome (ARDS), and various organs (liver, colon, etc.); inflammation, preferably persistent or long-term inflammation related to infection; arthritis, fibrosis, acute respiratory distress syndrome (ARDS); and various organs (liver, colon, etc.). An antibody or functional fragment according to any one of claims 1 to 10, or the pharmaceutical composition according to claim 14, which is a disorder in which platelets regulate cellular function, including but not limited to ischemia-reperfusion injury (IRI), peripheral vascular disease, antiphospholipid syndrome (APS), deep vein thrombosis, thrombophlebitis and vasculitis, transfusion-related acute lung injury (TRALI), transplant rejection, pre-eclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, pre-eclampsia, sickle cell disease, bacterial and viral ischemic restenosis, sepsis, major trauma, autoimmune diseases, and cancer cell proliferation and / or dissemination; cancer, preferably skin cancer, colon cancer, breast cancer, ovarian cancer, lung cancer, or metastatic cancer; and / or a disorder related to the proliferation, differentiation, morphology, migration, aggregation, degranulation and / or function of abnormal or ectopic megakaryocytes and / or platelets.

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