Anti-GPVI antibodies and functional fragments thereof

Abstract

The present invention relates to antibody molecules and functional fragments thereof capable of binding to human glycoprotein VI (GPVI), methods for their production and therapeutic uses thereof.

Description

Technical Field

[0001] This invention relates to antibody molecules capable of binding to human glycoprotein VI (GPVI) and their functional fragments, methods of their production, and their therapeutic uses. Background Technology

[0002] Platelet adhesion and aggregation at the site of vascular injury are essential for normal hemostasis and maintaining vascular integrity. 1-3 The pathological deviation in hemostasis is thrombosis, which is associated with cardiovascular diseases such as myocardial infarction, stroke, lower limb ischemia, and venous thromboembolism, and is associated with many pathologies such as cancer, infection, or inflammatory diseases. It represents a leading cause of death and severe disability worldwide and remains a major global health burden.

[0003] Thrombosis is the uncontrolled formation of a blood clot within a blood vessel, such as at the site of a ruptured atherosclerotic plaque. This can lead to a blockage of the blood vessel and cause life-threatening cardiovascular conditions such as myocardial infarction or ischemic stroke. 4，5 Therefore, antiplatelet drugs such as acetylsalicylic acid and P2Y 12 ADP receptor blockers or glycoprotein (GP) IIb / IIIa inhibitors have become indispensable therapies for the effective prevention or treatment of arterial thrombosis. However, the mechanisms of action of these drugs are often aimed at hemostasis, and therefore their use leads to an inherent risk of anti-hemostatic effects and an increased risk of uncontrolled bleeding complications associated with their antithrombotic efficacy, which limits their use, most notably in multidisease patients who require dual platelet inhibition or combined anticoagulation effects. 2，6，7，26 Therefore, there is a need for new, safe, and effective treatments that do not have systemic side effects such as bleeding. However, developing new antithrombotic therapies that focus on reducing the risk of bleeding without sacrificing drug efficacy remains challenging.

[0004] Glycoprotein VI (GPVI) is an activating platelet receptor for collagen / fibrin(ogen) and an important antithrombotic target, as its deficiency and functional inhibition prevent experimental thrombosis and thrombotic inflammatory states without impairing hemostasis. In particular, both GPVI deficiency and functional inhibition provide protection against pathological thrombosis and thrombotic inflammatory mechanisms such as (hyper)acute ischemic stroke. 8 It does not cause significant hemorrhagic complications in several mammalian model organisms. 9-13 Therefore, functional inhibition of GPVI is a promising and appropriate strategy for treating thrombosis and related diseases.

[0005] GPVI is a transmembrane signaling receptor of approximately 65 kDa from collagen and fibrin, expressed only on platelets and megakaryocytes. It non-covalently binds to the common γ-subunit (FcRγ-chain) of the Fc receptor containing the immune receptor tyrosine activation motif (ITAM), which serves as the signal transduction subunit of the receptor complex. 14，15 The extracellular domain of GPVI consists of two Ig-like domains, D1 and D2, followed by a presumed intrinsic disordered region proximal to the transmembrane region. 16 Based on X-ray crystallography, collagen binding is attributed to D1. 17 GPVI dimerization is largely mediated by D2 and the subsequent disordered region. 18

[0006] Different pharmacological approaches targeting GPVI-mediated platelet activation have been reported, including antibody (IgG)-mediated GPVI immune attenuation. 9，19，20 The competitive inhibitor Revacept (dimeric GPVI-Fc fusion protein), 21，22 and blocking GPVI via monovalent IgG-derived Fab fragments 20，23，24，25，54 Furthermore, these studies have demonstrated the significant benefits of these different anti-GPVI strategies in experimental models of thrombosis and thrombotic inflammation.

[0007] To date, several antibodies and antibody fragments have been reported, and their GPVI binding sites have been located at various interfaces within residues 58 to 187 (but not beyond). 31 Few of these antibodies and antibody fragments are able to block GPVI-collagen interactions, some induce GPVI shedding, and they generally suffer from weak affinity for GPVI, making them unsuitable for clinical investigations. 26 The first reported functionally blocking anti-GPVI antibody was against JAQ1 (rat IgG) produced by GPVI in mice. 33 It also recognizes human GPVI, but does not block its function. 32，55，56 However, in vivo administration of JAQ1IgG to mice induces GPVI reduction via an Fc-dependent mechanism. 19 This leads to a sustained GPVI knockout-like phenotype and long-term antithrombotic protection. 9 Further research later showed that a similar GPVI reduction mechanism can also occur in humans. 14，48，20 GPVI reduction is undesirable because it cannot be controlled and is irreversible (i.e., it prolongs platelet lifespan, or even longer, due to GPVI reduction on megakaryocytes). Other anti-GPVI antibodies that induce a GPVI reduction phenotype are described in the art. 57，58，59Typically, intact bivalent IgG can potentially bridge platelet membrane GPVI to low-affinity FcγRIIA receptors, leading to platelet activation and potentially inducing GPVI deficiency through internalization or shedding. 26

[0008] One antibody fragment with slightly high but still moderate binding affinity for GPVI is a humanized monovalent GPVI-blocking Fab (ACT017, glenzocimab) with a KD of 4.1 nM, which is based on a KD of 17 nM. D Parental mouse antibody fragment 9O12 Fab . 26，60，61 Mapping of the ACT017 binding site to GPVI reveals a discontinuous epitope consisting of two extensions of hGPVI located in the D2 domain, 114 to 142 and 165 to 187 (numbered according to Q9HCN6). 31

[0009] In ACT017, 9O12 Fab In the initial study of the monovalent parental mouse antibody fragment, 26 It effectively inhibited GPVI in vitro in non-human primates 29 and in mouse lines expressing human GPVI but not mouse GPVI. 23 First data from a phase II clinical trial of humanized GPVI-blocking Fab (ACT017, glomcizumab) demonstrate that treatment with the anti-GPVI Fab fragment is effective as adjunctive therapy in the acute phase of ischemic stroke. 26，27 It also reduces the thromboinflammatory response that drives infarction progression. In contrast, another GPVI inhibitor, Revacept, which recently entered clinical trials, failed to reduce the incidence of myocardial injury in patients with stable ischemic heart disease. 22 Furthermore, different groups reported that this indirect approach was less effective than the direct functional inhibition of GPVI by antibodies in mouse and human platelets. 46，47

[0010] However, 9O12 Fab The in vivo half-life in mice (approximately 2.5 hours at an intravenous dose of 4 mg / kg) is [not specified]. 23，29 The in vivo half-life of ACT017 (glencimab) in humans is approximately 10 hours at an intravenous dose >16 mg / kg. 30 The length is relatively short, which can at least partially be achieved with 9O12. Fab The fairly moderate binding affinity of both ACT017 and GPVI can be explained by (K) D (The values ​​are 17.08 nM and 4.1 nM, respectively).26，29，30 The antibody fragments were completely cleared in mice 24 hours after injection. 23 and 9O12 in primates Fab The rapid decline in the binding assay results with circulating platelets parallels the moderate binding affinity and relatively short plasma half-life, where 9O12 on circulating platelets... Fab Reduced to 50% of peak levels, and 9O12 within 24 hours after injection. Fab The number of positive circulating platelets is reduced by more than half. 29

[0011] Other GPVI antibodies described in the prior art exist, including four mouse monoclonal anti-GPVI antibodies, OM1, OM2, OM3 and OM4, which have been found to inhibit GPVI binding to collagen, collagen-induced secretion and in vitro formation of thromboxane A2 (TxA2), in vitro collagen-induced platelet aggregation after intravenous injection into cynomolgus monkeys, and OM4 also appears to inhibit thrombosis in a rat thrombosis model. 62 These antibodies are reported to have a smaller Kc than, for example, ACT017. D Value, however, K of ACT017 D The value is greater than 0.7 nM. Furthermore, relevant patent literature reports the use of bivalent IgG antibodies for K... D Measurements show that monovalent fragments typically have slightly reduced affinity compared to their corresponding intact IgG. 62

[0012] Therefore, there is a persistent need for improved inhibitors of human GPVI, which, as antiplatelet agents, can effectively prevent or treat arterial or venous thrombosis and thromboinflammatory conditions in high-risk populations with a low risk of bleeding. This GPVI inhibitor should have at least a high affinity for human GPVI (e.g., at least K+). D < 700 pM), and preferably with a long plasma half-life. Ideally, such GPVI inhibitors are characterized by prolonged persistence on circulating platelets and efficacy in inhibiting thrombus formation as assessed in vitro and / or in vivo. Summary of the Invention

[0013] The inventors of this application have discovered certain anti-GPVI antibodies and their functional fragments with extremely high binding affinity for human GPVI, possessing a dissociation equilibrium constant (K0.1) of less than 700 pM and as low as 195 pM (thus approximately 21-fold lower than ACT017). DFurthermore, the novel anti-GPVI antibody and functional fragment (i) unexpectedly bind to a novel epitope on human GPVI, which includes the proposed GPVI dimerization residues; and (ii) exhibit a long plasma half-life and potently inhibit humanized GPVI-targeted mice (hGP6) in vitro. tg / tg (mice) and hGP6 tg / tg (iii) GPVI function in mouse platelets; and providing sustained GPVI blockade in vivo (e.g., where receptor occupancy is >50% at 48 h post-injection on circulating platelets and >35% at 72 h post-injection) and significant thrombotic protection (e.g., effective inhibition of thrombosis as assessed in vitro and / or in vivo), while providing protection against hGP6. tg / tg The tail hemorrhage time in the mouse model had no significant effect. Based on these results, it is reasonable to expect that, compared to, for example, ACT017, the anti-GPVI antibody and functional fragment of the present invention will achieve the desired level of GPVI inhibition in humans at a comparable lower dose and / or for a longer period of time. The monovalent form of the antibody and its functional fragment excludes Fc-dependent thrombocytopenia and / or GPVI reduction, and as a Fab fragment, circumvents the possible Fc-mediated effects of full-length IgG via platelet-expressed Fc γ receptor IIA (FcγRIIA, CD32). Therefore, the present invention provides an anti-GPVI antibody and functional fragment with very high affinity for human GPVI, which allows for sustained and safe inhibition of human GPVI function on circulating platelets (presumably through a non-canonical mechanism of action) with a very low risk of bleeding.

[0014] This invention provides antibody molecules and their functional fragments capable of binding to human GPVI with very high affinity and blocking its function in vitro and in vivo with low bleeding risk. Therefore, this invention relates to the subject matter defined in the following items (1) to (133):

[0015] (1) An antibody or a functional fragment thereof capable of binding to human glycoprotein VI (GPVI), wherein the antibody or functional fragment comprises (i) V L The domain comprises 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, and (ii) V HThe domain comprises 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.

[0016] (2) An antibody or a functional fragment thereof capable of binding to human glycoprotein VI (GPVI), wherein the antibody or functional fragment comprises (i) V L The domain comprises 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, and (ii) V H The domain comprises 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.

[0017] (3) The antibody or functional fragment thereof of item (1) or (2), wherein in the amino acid sequence SEQ 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.

[0018] (4) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is A in the amino acid sequence SEQ ID NO: 9.

[0019] (5) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is R.

[0020] (6) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is N.

[0021] (7) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is D in the amino acid sequence SEQ ID NO: 9.

[0022] (8) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is Q.

[0023] (9) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is E.

[0024] (10) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is G in the amino acid sequence SEQ ID NO: 9.

[0025] (11) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is H.

[0026] (12) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is I in the amino acid sequence SEQ ID NO: 9.

[0027] (13) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is L.

[0028] (14) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is K.

[0029] (15) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is M in the amino acid sequence SEQ ID NO: 9.

[0030] (16) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is F in the amino acid sequence SEQ ID NO: 9.

[0031] (17) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is S.

[0032] (18) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is T.

[0033] (19) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is W.

[0034] (20) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein in the amino acid sequence SEQ ID NO: 9, residue X is Y.

[0035] (21) An antibody or a functional fragment thereof of any one of items (1)-(3), wherein residue X is V in the amino acid sequence SEQ ID NO: 9.

[0036] (22) The antibody or functional fragment thereof of item (1), wherein the antibody or functional fragment comprises (i) V L The domain comprises 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, and (ii) V H The domain comprises 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.

[0037] (23) The antibody of item (1) or a functional fragment thereof, wherein in the amino acid sequence SEQ ID NO: 13, residue X (X5) at position 5 is D or E; and / or residue X (X6) at position 6 is G or A.

[0038] (24) The antibody or its functional fragment in any of the aforementioned items is monovalent.

[0039] (25) The functional fragment of any of the preceding items is an antigen-binding fragment (Fab), F(ab'), Fv, disulfide-linked variable fragment (dsFv), monovalent IgG, or single-chain variable fragment (scFv).

[0040] (26) The functional fragment of any of the aforementioned items is an antigen-binding fragment (Fab).

[0041] (27) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment thereof specifically binds to human GPVI.

[0042] (28) Any antibody or functional fragment of the preceding items, wherein the antibody or functional fragment thereof does not bind significantly to a protein that is structurally closely related to human GPVI.

[0043] (29) An antibody or functional fragment, particularly any of the preceding items, that is capable of binding human GPVI at the binding epitopes corresponding to amino acids V178 to E192 and / or S223 to P237 of SEQ ID NO: 36.

[0044] (30) The antibody of item (29), wherein the binding epitope is a discontinuous epitope comprising or consisting of amino acids V178 to E192 and S223 to P237 of SEQ ID NO: 36.

[0045] (31) Any antibody or functional fragment of the aforementioned projects is capable of binding human GPVI at the GPVI residues proposed to participate in dimerization.

[0046] (32) An antibody or functional fragment of any of the preceding items, wherein the binding of the antibody or functional fragment to human GPVI is interrupted or interferes with GPVI dimerization.

[0047] (33) An antibody or a functional fragment thereof that binds to a binding epitope substantially the same as that of any of the antibodies or functional fragments in the preceding items.

[0048] (34) The antibody or functional fragment of any of the foregoing items, wherein 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, even more preferably about 175 pM or less. D It combines with human GPVI.

[0049] (35) An antibody or functional fragment of any of the aforementioned items, with a dissociation equilibrium constant (K0) of 195 pM or less. D It combines with human GPVI.

[0050] (36) Antibodies or functional fragments of items (34) or (35), wherein the dissociation equilibrium constant (K) D Biolayer interferometry (BLI), surface plasmon resonance (SPR) techniques (e.g., in a BIACORE instrument), or ELISA is used; BLI is preferred (e.g., using an Octet instrument and Fortebio software) for determination.

[0051] (37) An antibody or functional fragment of any of items (34) to (36) wherein the dissociation equilibrium constant (K) is determined using biolayer interferometry (BLI) (e.g., using an Octet instrument). D ), preferably where K DThe determination is carried out as in the example in Section 1.16.

[0052] (38) The antibody or functional fragment of any of the aforementioned items, wherein the dissociation equilibrium constant (K) D The following criteria were used to determine the use of biolayer interferometry (BLI) at 25°C, preferably with an Octet instrument and preferably at a constant orbital flow rate, such as 1000 rpm; an antigen loading concentration of 1.2 µg / ml (human GPVI) for immobilizing the antigen on the biosensor; an association time of 900 s and a dissociation time of 1200 s; a screening range of 7 antibody concentrations ranging from 10 to 0.014 nM using 3-fold serial dilutions; and a 1:1 fitting model.

[0053] (39) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V L A domain comprising or consisting of the following: 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 with the sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35, preferably 16, 17, 18, 19, 32, 33, 34 and 35, even more preferably at least 16, 17, 18, 32, 33 and 34.

[0054] (40) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V L A domain comprising or consisting of the following: 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 with the sequence selected from the group consisting of SEQ ID NO: 26, 27, 28, 32, 33, 34 and 35, preferably 32, 33 and 34.

[0055] (41) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V LA domain comprising or consisting of the following: 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 with the sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 29, 30 and 31, preferably 16, 17, 18 and 19, more preferably 16, 17 and 18.

[0056] (42) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V H A domain comprising or consisting of the following: 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 with the sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 and 25, preferably 21, 22 and 23.

[0057] (43) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V L The domain comprises or consists of the following SEQ ID NO: 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 the amino acid sequence shown in 16, 17, 18, 32, 33 or 34.

[0058] (44) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains V L The domain comprises or consists of the following: SEQ ID NO: 26, 27, 28, 32, 33, 34 or 35, preferably 32, 33, 34 or 35, more preferably the amino acid sequence shown in 32, 33 or 34.

[0059] (45) An antibody or functional fragment of any one of items (1)-(43), wherein the antibody or functional fragment contains V L The domain comprises or consists of the following: SEQ ID NO: 16, 17, 18, 19, 29, 30 or 31, preferably 16, 17, 18 or 19, more preferably the amino acid sequence shown in 16, 17 or 18.

[0060] (46) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment contains VH The domain contains the amino acid sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25, preferably 21, 22 or 23.

[0061] (47) An antibody or functional fragment of any of the aforementioned items, wherein V L Domain and V H The domain contains or consists of the following: SEQ ID NO: 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 NO: 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 2 4 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, 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 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; even 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; even 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; even more preferably 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.

[0062] (48) An antibody or functional fragment of any of the aforementioned items, wherein V L Domain and V HThe domain contains or consists of the following: SEQ ID NO: 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, 2 6 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 22, 34 and 23, 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 22, 27 and 21 or 27 and 22; even more preferably a pair of amino acid sequences shown in 33 and 21, 33 and 22, 34 and 22 or 27 and 22.

[0063] (49) An antibody or functional fragment of any of the aforementioned items, wherein V L Domain and V HThe field contains or consists of the following: SEQ ID NO: 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 23. 33 and 24, 33 and 25, 34 and 21, 34 and 22 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, 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 a pair of amino acid sequences shown in 33 and 21, 33 and 22 or 34 and 22.

[0064] (50) An antibody or functional fragment of any one of items (1)-(47), wherein V L Domain and V HThe domain contains or consists of the following: SEQ ID NO: 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, 2 9 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; 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 22, 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 and 23, 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, 30 and 21 or 30 and 22; even more preferably a pair of amino acid sequences shown in 17 and 21, 17 and 22, 18 and 22 or 30 and 22.

[0065] (51) An antibody or functional fragment of any one of items (1)-(47), wherein V L Domain and V HThe field contains or consists of the following: SEQ ID NO: 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 23. 17 and 24, 17 and 25, 18 and 21, 18 and 22 or 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 a pair of amino acid sequences shown in 17 and 21, 17 and 22 or 18 and 22.

[0066] (52) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment comprises: V L 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%, even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; and V H 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%, even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.

[0067] (53) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment comprises: V L The domain 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% sequence identity with the amino acid sequence shown in SEQ ID NO: 33; and V HThe 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%, even more preferably at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.

[0068] (54) An antibody or functional fragment of any one of items (1)-(47) and (50)-(52), wherein the antibody or functional fragment contains V L Domain and V H Domain, the V L The domain contains or consists of an amino acid sequence as shown in SEQ ID NO: 17; the V H Contains or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0069] (55) An antibody or functional fragment of any one of items (1)-(49), wherein the antibody or functional fragment contains V L Domain and V H Domain, the V L The domain contains or consists of an amino acid sequence as shown in SEQ ID NO: 33; the V H Contains or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0070] (56) An antibody or functional fragment of any one of items (1) to (42), wherein the antibody or functional fragment contains V L Domain and / or V H Domain, the V L The domain contains or consists of an amino acid sequence as shown in SEQ ID NO: 15; the V H Contains or consists of the amino acid sequence shown in SEQ ID NO: 20; optionally, wherein the V L Domain and / or V H The domains contain 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions, respectively, relative to the sequences shown in SEQ ID NO:15 and SEQ ID NO:20.

[0071] (57) The antibody or functional fragment of any of the preceding items is immunoglobulin G (IgG) or a functional fragment thereof, preferably IgG1 or a functional fragment thereof.

[0072] (58) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment comprises or is composed of the following: containing the V L Light chains of the domain and containing the VH Relinks of domains.

[0073] (59) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment comprises or is composed of a light chain and a heavy chain, wherein the light chain, in addition to the V L Beyond the domain, it also includes the preferred option in the aforementioned V. L The constant field at the C-terminus of the domain (C L ), this heavy chain, besides the V H It is also included in the V outside the domain. H The constant field at the C-terminus of the domain (C H ).

[0074] (60) An antibody or functional fragment of item (58) or (59), wherein the antibody or functional fragment comprises a light chain comprising a constant domain derived from a human IgK light chain constant domain (allotype Km3), and preferably wherein the sum of the number of amino acids differing from, for example, the amino acid sequence of the human IgK light chain constant domain (allotype Km3) shown in SEQ ID NO: 60 is less than 10, 9, 8, 7, 6, 5, 4, 3 or 2; and / or wherein the antibody or functional fragment comprises a heavy chain comprising a human IgG1 heavy chain C H 1. A constant domain (allotype G1m17,1), and preferably wherein it is associated with, for example, the human IgG1 heavy chain C shown in SEQ ID NO: 61. H The total number of different amino acids in the amino acid sequence of the constant domain (allotype G1m17,1) is less than 10, 9, 8, 7, 6, 5, 4, 3 or 2.

[0075] (61) An antibody or functional fragment of any one of items (59) to (60), wherein C L 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%, even more preferably at least 99% sequence identity with the amino acid shown in SEQ ID NO: 60.

[0076] (62) An antibody or functional fragment of any one of items (59) to (61), wherein C H 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%, even more preferably at least 99% sequence identity with the amino acid shown in SEQ ID NO: 61.

[0077] (63) An antibody or functional fragment of any one of items (58) to (62), wherein the light chain comprises an N-terminal light chain signal peptide and / or a light chain constant domain (C L The N-terminal light chain signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 62, and the light chain constant domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 60, and preferably is located in the V... L The C-terminus of the domain.

[0078] (64) An antibody or functional fragment of any one of items (58) to (63), wherein the heavy chain comprises an N-terminal heavy chain signal peptide and / or a heavy chain constant domain (C H The N-terminal heavy chain signal peptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 63, and the heavy chain constant domain comprises or is composed of the amino acid sequence shown in SEQ ID NO: 61, and preferably is located in the V... H The C-terminus of the domain.

[0079] (65) An antibody or functional fragment of any of the foregoing items, which is monovalent, preferably Fab, comprising or composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following: 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%, even more preferably at least 99.5% sequence identity with the sequence selected from SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58; the heavy chain comprises or is composed of the following: 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%, even more preferably at least 99.5% sequence identity with the sequence selected from SEQ ID NO: The sequences of the group consisting of 44, 45, 46, 47 and 48 have amino acid sequences with 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%, even more preferably at least 99.5% sequence identity.

[0080] (66) An antibody or functional fragment of any of the foregoing items, which is monovalent, preferably Fab, comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed 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%, even more preferably at least 99.5% sequence identity with the sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, and 48.

[0081] (67) An antibody or functional fragment of any of the preceding items comprising or composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequence shown in one of SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, and the heavy chain comprises or is composed of the following amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0082] (68) An antibody or functional fragment of any of the preceding items comprising or composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 55, 56, 57 and 58 (preferably SEQ ID NO: 55, 56 and 57); and the heavy chain comprises or is composed of the following amino acid sequence as shown in SEQ ID NO: 44, 45, 46, 47 and 48 (preferably SEQ ID NO: 44, 45 and 46).

[0083] (69) An antibody or functional fragment of any of items (1)-(67) comprising or composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequence as shown in one of SEQ ID NO: 38, 39, 40 and 41 (preferably SEQ ID NO: 38, 39 and 40); and the heavy chain comprises or is composed of the following amino acid sequence as shown in one of SEQ ID NO: 44, 45, 46, 47 and 48 (preferably SEQ ID NO: 44, 45 and 46).

[0084] (70) The antibody or functional fragment of any of the preceding items is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of the following light and heavy chains, wherein the light chain comprises or consists of the following amino acid sequence shown in one of SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, and the heavy chain comprises or consists of the following amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0085] (71) The antibody or functional fragment of any of the preceding items is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of the following light and heavy chains, the light chain comprising or consisting of the following amino acid sequence as shown in one of SEQ ID NO: 52, 53, 54, 55, 56, 57 and 58, and the heavy chain comprising or consisting of the following amino acid sequence as shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0086] (72) The antibody or functional fragment of any of the preceding items is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of the following light and heavy chains, wherein the light chain comprises or consists of the following amino acid sequence as shown in one of SEQ ID NO: 55, 56, 57 and 58; and the heavy chain comprises or consists of the following amino acid sequence as shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0087] (73) The antibody or functional fragment of any of the preceding items is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of the following light and heavy chains, the light chain comprising or consisting of the following amino acid sequence as shown in one of SEQ ID NO: 55, 56 and 57, and the heavy chain comprising or consisting of the following amino acid sequence as shown in one of SEQ ID NO: 44, 45 and 46.

[0088] (74) An antibody or functional fragment of any one of items (1)-(70), which is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of a light chain and a heavy chain, wherein the light chain comprises or consists of the amino acid sequence shown in one of SEQ ID NO: 38, 39, 40, 41, 49, 50 and 51, and the heavy chain comprises or consists of the amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0089] (75) An antibody or functional fragment of any one of items (1)-(70), which is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of a light chain and a heavy chain, wherein the light chain comprises or consists of an amino acid sequence shown in one of SEQ ID NO: 38, 39, 40 and 41; and the heavy chain comprises or consists of an amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.

[0090] (76) An antibody or functional fragment of any one of items (1)-(70), which is monovalent, preferably an antigen-binding fragment (Fab), comprising or consisting of a light chain and a heavy chain, wherein the light chain comprises or consists of an amino acid sequence shown in one of SEQ ID NO: 38, 39 and 40, and the heavy chain comprises or consists of an amino acid sequence shown in one of SEQ ID NO: 44, 45 and 46.

[0091] (77) An antibody or functional fragment of any one of items (1)-(70), which (preferably monovalent, more preferably Fab, and) comprises or consists of: having SEQ ID NO: 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 5 4 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 4 4, 57 and 45, 57 and 46, 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, 53 and 44 or 53 and 45; even more preferably 56 and 44, 56 and 45, 57 and 45 or 53 and 45; even more preferably a pair of light and heavy chains of the amino acid sequences shown in 56 and 44.

[0092] (78) An antibody or functional fragment of any one of items (1)-(70), which (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains, the pair of light and heavy chains comprising or consisting of: SEQ ID NO: 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 and 44, 57 and 45 or 57 and 46; even more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45 or 57 and 46; even more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45 or 57 and 46; even more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44 or 57 and 45; even more preferably 56 and 44, 56 and 45, 56 and 46 or 57 and 45; even more preferably 56 and 44, 56 and 45 or 57 and 45, even more preferably the amino acid sequence shown in 56 and 44.

[0093] (79) An antibody or functional fragment of any one of items (1)-(70), which (preferably monovalent, more preferably Fab, and) comprises or consists of: having SEQ ID NO: 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 5 1 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 4 4, 40 and 45, 40 and 46, 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, 50 and 44 or 50 and 45; even more preferably 39 and 44, 39 and 45, 40 and 45 or 50 and 45; even more preferably a pair of light and heavy chains of the amino acid sequences shown in 39 and 44.

[0094] (80) An antibody or functional fragment of any one of items (1)-(70), which (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains, the pair of light and heavy chains comprising or consisting of: SEQ ID NO: 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; more 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; even more preferably 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45 or 40 and 46; even more preferably 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45 or 40 and 46; even more preferably 39 and 44, 39 and 45, 39 and 46, 40 and 44 or 40 and 45; even more preferably 39 and 44, 39 and 45, 39 and 46 or 40 and 45; even more preferably 39 and 44, 39 and 45 or 40 and 45, even more preferably the amino acid sequence shown in 39 and 44.

[0095] (81) An antibody or functional fragment of any one of items (1)-(70), which is monovalent, preferably Fab, and consists of a pair of light and heavy chains having SEQ ID NO: 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, 4 1 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.

[0096] (82) An antibody or functional fragment of any one of items (1)-(70), which is monovalent, preferably Fab, consisting of a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NO: 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 46, 49 and 48, 50 and 44, 50 and 45 or 50 and 46.

[0097] (83) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, consisting of a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NO: 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.

[0098] (84) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, consisting of a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NO: 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45 or 40 and 46.

[0099] (85) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, consisting of a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NO: 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45 or 40 and 46.

[0100] (86) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, consisting of a pair of light and heavy chains having the amino acid sequence shown in SEQ ID NO: 39 and 44, 39 and 45, 39 and 46, 40 and 44 or 40 and 45.

[0101] (87) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, and consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45 or 40 and 45.

[0102] (88) An antibody or functional fragment of any of items (1)-(70), which is monovalent, preferably Fab, and consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44.

[0103] (89) An antibody or functional fragment of any one of items (1) to (66), which is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, the pair of light and heavy chains comprising or consisting of the amino acid sequences shown in SEQ ID NO: 37 and 43.

[0104] (90) The antibody or functional fragment of any of the preceding items is capable of continuously inhibiting 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, even more preferably at least 96 hours.

[0105] (91) The antibody or functional fragment of any of the foregoing items, which (preferably in monovalent form, e.g., as Fab) is capable of prolonging binding to circulating platelets after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment, preferably having a GPVI receptor epitope occupancy of >50% when normalized with a negative control (e.g., a GPVI-nonspecific control Fab), 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, for example, as determined by flow cytometry analysis in ex vivo diluted blood, for example using 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, as described in the examples in Section 1.10.

[0106] (92) The antibody or functional fragment of any of the foregoing items, which (preferably in monovalent form, e.g., as Fab) is capable of prolonging binding to circulating platelets after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment, preferably having at least 20% GPVI receptor epitope occupancy when normalized with a negative control (e.g., a GPVI-nonspecific control Fab), for at least 36 hours, 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, even more preferably at least 96 hours, for example, determined by flow cytometry analysis in ex vivo diluted blood, for example using 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, as described in the examples in Section 1.10.

[0107] (93) Any antibody or functional fragment of the preceding items that, at a concentration of ≤ 10 μg / ml, preferably ≤ 5 μg / ml, more preferably ≤ 2 μg / ml, or even more preferably ≤ 1 μg / ml, can completely inhibit CRP-induced and / or collagen-induced aggregation of washed human platelets, as determined by standard light-transmission aggregometry, for example as described in the examples in Section 1.7.

[0108] (94) An antibody or functional fragment of any of the aforementioned items, administered intravenously at a dose of 4 mg / kg to a humanized mouse model (hGP6) targeting GPVI. tg / tg In mice, at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 60 hours after administration, the washed-out mouse hGP6 was completely inhibited. tg / tg CRP-induced and / or collagen-induced aggregation of platelets, as determined using standard light transmission aggregation assays, such as those described in the examples in Section 1.7.

[0109] (95) An antibody or functional fragment of any of the foregoing items, at a concentration of 5 μg / ml, in heparinized human blood under flow (preferably with a shear rate of 1000 s⁻¹), is capable of completely inhibiting human platelet adhesion (platelet surface coverage) and thrombus formation (relative thrombus volume) on collagen-coated surfaces, as determined by a flow adhesion assay, for example as described in the examples in Section 1.5; optionally, wherein complete inhibition is defined as a reduction of platelet surface coverage by at least 60%, preferably at least 65%, more preferably at least 70%, and / or a reduction of relative thrombus volume by at least 80%, preferably at least 85%, more preferably at least 87%, and even more preferably at least 90%, compared to a negative control.

[0110] (96) The antibody or functional fragment of any of the foregoing items, at a concentration of ≤ 10 μg / ml, preferably ≤ 5 μg / ml, more preferably ≤ 2 μg / ml, or even more preferably ≤ 1 μg / ml, is capable of reducing the relative thrombus volume on a collagen-coated surface (preferably coated at 200 μg / ml) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹) in a flow adhesion assay (e.g., as described in the examples in Section 1.5).

[0111] (97) The antibody or functional fragment of any of the aforementioned items, compared with the negative control, when administered intravenously at a dose of 4 mg / kg to a humanized mouse model (hGP6) targeting GPVI. tg / tg After at least 12 hours, preferably at least 24 hours, more preferably at least 48 hours, and even more preferably at least 60 hours in mice, the mice were able to maintain the condition under flow conditions (preferably using 1000 s). -1 In heparinized human blood at a shear rate of at least 50%, preferably at least 60%, relative thrombus volume on collagen-coated surfaces (preferably coated at 200 μg / ml) is reduced by at least 50% in a flow adhesion assay (e.g., as described in the examples in Section 1.5).

[0112] (98) An antibody or functional fragment of any of the foregoing items, at a concentration of 10 μg / ml, is capable of completely inhibiting thrombus formation (relative thrombus volume), phosphatidylserine (PS) exposure, and fibrin deposition on surfaces coated with collagen and tissue factor (preferably 200 μg / ml and 500 pM for collagen and tissue factor, respectively) in flowing (preferably using a shear rate of 1000 s⁻¹) human blood, as determined in a flow adhesion assay suitable for coagulation (e.g., as described in the examples in section 1.6); optionally, wherein complete inhibition is defined as a reduction of at least 90%, preferably at least 95%, in relative thrombus volume / phosphatidylserine (PS) exposure / fibrin deposition compared to a negative control.

[0113] (99) An antibody or functional fragment of any of the foregoing items, when used at a concentration of 10 μg / ml in a spreading assay on washed human platelets that allows binding to the fibrinogen-coated surface (e.g., as described in the example in Section 1.8), is capable of reducing the portion of stage 4 (fully spread) platelets by at least 40%, preferably at least 50%, and increasing the similarity of the portion of stage 2 (platelets forming filamentous pseudopodia).

[0114] (100) The antibody or functional fragment of any of the foregoing items, at a concentration of 10 μg / ml, is capable of flowing under conditions (preferably 1000 s) -1 Complete inhibition of human platelet adhesion (platelet surface coverage) and thrombus formation (relative thrombus volume) on collagen-coated surfaces (preferably coated at 200 μg / ml) in heparinized human blood using a flow adhesion assay, as determined by the shear rate, for example as described in the examples in Section 1.5; optionally, where complete inhibition means a reduction of at least 80%, preferably at least 85%, in platelet surface coverage and / or a reduction of at least 90%, preferably at least 95%, in relative thrombus volume compared to a negative control.

[0115] (101) The antibody or functional fragment of any of the foregoing items, preferably in monovalent form, preferably Fab, is administered intravenously at a dose of 4 mg / kg to, for example, a humanized mouse model (hGP6) targeting GPVI. tg / tgFollowing administration to mice, significantly impaired CRP-induced circulating platelet activation was achieved for at least 12, 24, 36, 48, 60, 72, 84, or 96 hours after administration, as determined by flow cytometry analysis in ex vivo diluted blood. The use of fluorescently labeled antibodies capable of specifically binding to activated integrin αIIbβ3 and / or specifically binding to P-selectin is preferred, as illustrated in the examples in Section 1.10. "Significantly impaired" preferably means a reduction in fluorescence signal of >50% when using circulating platelets sampled at least 12, 24, 36, 48, 60, 72, 84, or 96 hours after administration, compared to a negative control.

[0116] (102) The antibody or functional fragment of any of the preceding items is preferably monovalent, preferably Fab, and is capable of inhibiting the dimerization of GPVI, optionally without abolishing or partially abolishing ligand binding to GPVI.

[0117] (103) The antibody or functional fragment of any of the preceding items is monovalent, preferably Fab, and is capable of inhibiting GPVI-induced platelet activation, optionally while maintaining the initial adhesion function of the GPVI receptor.

[0118] (104) An antibody or functional fragment of any of the foregoing items that provides sustained protection against occlusive thrombosis, such as arterial thrombosis, preferably using a humanized mouse model of occlusive arterial thrombosis (hGP6) targeting GPVI. tg / tg The results were determined by mice, as illustrated in the examples in Section 1.11, for instance.

[0119] (105) An antibody or functional fragment of any of the foregoing items that can inhibit the formation of stable thrombi at the site of arterial injury, preferably by reducing platelet activation at exposed extracellular matrix sites within the blood vessel and / or by effectively inhibiting local platelet-dependent coagulation, optionally such as using a humanized mouse model of occlusive arterial thrombosis targeting GPVI (hGP6). tg / tg The results were determined by mice, as illustrated in the examples in Section 1.11, for example.

[0120] (106) An antibody or functional fragment of any of the preceding items, wherein the antibody or functional fragment has significantly greater potency in inhibiting GPVI function compared to ACT017 (glenciemab) i) as determined by an aggregation assay using a standard light transmission aggregation assay, for example as described in the examples in Section 1.7; ii) as determined by a flow adhesion assay (for example as described in the examples in Section 1.5) using heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹); and / or iii) as determined 1 hour after intravenous administration based on CRP-induced activation of circulating platelets in ex vivo diluted blood by flow cytometry using a fluorescently labeled antibody capable of specifically binding to activated integrin αIIbβ3.

[0121] (107) The antibody or functional fragment of any of the foregoing items, in monovalent form, preferably as Fab, when normalized with a negative control (e.g., a Fab control nonspecific to GPVI), can increase the binding to circulating platelets (preferably with an increase of ≥ 1.5-fold, more preferably ≥ 1.7-fold, ≥ 1.8-fold, or ≥ 1.9-fold) compared to ACT017 (grencimab), as measured 1 hour after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment or ACT017 (grencimab), as determined by flow cytometry analysis in ex vivo diluted blood, for example with a fluorescently labeled Fab-specific antibody and using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, as described in the examples in Section 1.10.

[0122] (108) The antibody or functional fragment of any of the foregoing items, in monovalent form, preferably Fab, when normalized with a negative control (e.g., a Fab control nonspecific to GPVI), is capable of increasing binding to circulating platelets (preferably with an increase of ≥ 2-fold, more preferably ≥ 2.5-fold, ≥ 3-fold, ≥ 3.5-fold, or ≥ 3.75-fold in GPVI receptor epitope binding), as measured 3 hours after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment or ACT017, as determined by flow cytometry analysis in ex vivo diluted blood, for example, using a fluorescently labeled Fab-specific antibody and using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, as described in the examples in Section 1.10.

[0123] (109) The antibody or functional fragment of any of the foregoing items, preferably in monovalent form, more preferably in Fab form, is administered intravenously at a dose of 4 mg / kg to, for example, a humanized mouse model (hGP6) targeting GPVI. tg / tg One hour after administration to mice, significantly impaired CRP-induced activation of circulating platelets, as determined, for example, by flow cytometry analysis in diluted blood in vitro, is preferably achieved using a fluorescently labeled antibody capable of (specifically) binding to activated integrin αIIbβ3, such as as described in the examples in Section 1.10 (while ACT017 (glencimab) does not cause significantly impaired CRP-induced activation); optionally, "significantly impaired" means a reduction in fluorescence signal of >85%, preferably >90%, >95%, >97%, >98%, >99%, or >99.5%, when using circulating platelets sampled 24 hours after administration compared to a negative control.

[0124] (110) The antibody or functional fragment of any of the foregoing items, wherein, compared with ACT017 (glencimab), the antibody or functional fragment, preferably at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, has a greater in vitro efficacy in inhibiting GPVI function in human platelets by >2 times, preferably >4 times, >6 times, >8 times or >10 times, as determined using a standard light transmission aggregation assay, for example as described in the examples in Section 1.7, optionally wherein the fold power is determined as the ratio of the maximum aggregation percentage of the sample treated with the antibody or functional fragment to the maximum aggregation percentage of the sample treated with ACT017.

[0125] (111) The antibody or functional fragment of any of the preceding items, wherein, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, the antibody or functional fragment has, in vitro, a greater than 2-fold, preferably greater than > 4-fold, > 6-fold, > 8-fold or > 10-fold greater efficacy than ACT017 (glencimab) in inhibiting collagen-induced and / or CRP-induced aggregation of washed human platelets, as determined using a standard light transmission aggregation assay, such as as described in the examples in Section 1.7, optionally wherein the fold power is determined as the ratio of the maximum aggregation percentage of the sample treated with the antibody or functional fragment to the maximum aggregation percentage of the sample treated with ACT017.

[0126] (112) Any antibody or functional fragment of the preceding items, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, compared with the negative control, is able to significantly reduce (e.g., more than 1.5 times, preferably more than 2 times, 2.5 times or 3 times) platelet surface coverage on collagen-coated surfaces (preferably coated at 200 μg / ml) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹) in flow adhesion assays (e.g., as described in the examples in Section 1.5) (while ACT017 (glencimab) treatment does not result in (significant) reduction).

[0127] (113) The antibody or functional fragment of any of the preceding items, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, is able to reduce the platelet surface coverage on the collagen-coated surface (preferably coated at 200 μg / ml) by more than 1.5 times, preferably more than 2 times, 2.5 times or 3 times, in a flow adhesion assay (e.g., as described in the examples in Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹).

[0128] (114) The antibody or functional fragment of any of the foregoing items, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, is able to reduce the relative thrombus volume on the collagen-coated surface (preferably coated at 200 μg / ml) by more than 2 times, preferably more than 4 times, 6 times, 8 times or 10 times, in a flow adhesion assay (e.g., as described in the examples in Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹).

[0129] (115) An antibody or a functional fragment thereof that binds to a human GPVI and is substantially the same epitope as an antibody exhibiting one or more of the features mentioned in items (1) to (114) above.

[0130] (116) Nucleic acid, which encodes an antibody or functional fragment of any of the aforementioned items.

[0131] (117) A vector or plasmid containing the nucleic acid of item (116).

[0132] (118) A cell containing the nucleic acid of item (116) or the vector or plasmid of item (117).

[0133] (119) A method for preparing an antibody or functional fragment of any one of items (1) to (115), comprising culturing the cells of item (118) in a culture medium under conditions that allow expression of nucleic acids encoding the antibody or functional fragment, and recovering the antibody or functional fragment from the cells or the culture medium.

[0134] (120) The method of Project (119) wherein the cells are Chinese hamster ovary (CHO) cells, and the codon-optimized nucleic acid for expression in CHO cells is transiently expressed from a vector or plasmid, and wherein recovery involves affinity chromatography.

[0135] (121) A pharmaceutical composition comprising an antibody or functional fragment of any one of items (1) to (115), and optionally a pharmaceutically acceptable carrier and / or excipient.

[0136] (122) An antibody or functional fragment as defined in any of items (1) to (115) is used in a method of treating or preventing GPVI-related conditions in a subject.

[0137] (123) Based on the antibody or functional fragment used in project (122), the GPVI-related condition is a thrombotic inflammatory disease.

[0138] (124) Based on the antibody or functional fragment used in project (122), GPVI-related conditions are cardiovascular diseases.

[0139] (125) The antibody or functional fragment used in item (123) or (124) is selected from thrombosis and thrombotic conditions (such as arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic disease, thrombotic coronary occlusion, thrombotic microangiopathy, cancer-related thrombosis (Trousseau syndrome), immune thrombosis and thrombosis associated with infection [e.g., cerebral malaria]), restenosis, acute coronary syndrome, ischemic stroke, cerebrovascular disease and vascular purpura, coronary artery disease and cerebral artery disease, ischemic events, acute coronary syndrome, myocardial infarction (heart attack), acute cerebral ischemia (stroke), percutaneous coronary intervention, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches (such as aortic aneurysm, thrombosis), peripheral artery disease, acute phlebitis and pulmonary embolism.

[0140] (126) An antibody or functional fragment for use according to item (123) or (124), wherein the cardiovascular disease is thrombosis or a thrombotic condition, preferably selected from arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic disease, thrombotic coronary occlusion, thrombotic microangiopathy, cancer-related thrombosis (Trusso syndrome), immune thrombosis and thrombosis associated with infection [e.g., cerebral malaria].

[0141] (127) The antibody or functional fragment used in the project (124) wherein the cardiovascular disease is selected from arterial or venous thrombosis, restenosis, acute coronary syndrome, or cerebrovascular accident caused by atherosclerosis, preferably arterial or venous thrombosis.

[0142] (128) The antibody or functional fragment used in item (122) or (123) wherein the GPVI-related condition is inflammatory or thrombotic inflammation, preferably selected from persistent or prolonged inflammation associated with infection [e.g., cerebral malaria], arthritis, autoimmune diseases [such as celiac disease, post-infectious IBS, type 1 diabetes, allergic purpura (HSP), sarcoidosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, eosinophilic granulomatous polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis (RA), ankylosing spondylitis, polymyositis (PM), dermatomyositis (DM), alopecia areata and polymyositis]. [Multiple sclerosis (MS)], fibrosis, acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury (IRI) of various organs (liver, colon, etc.), 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, sickle cell disease, ischemic restenosis due to bacterial and viral infections, sepsis, major trauma, and diseases involving platelet-regulating cell function, including but not limited to cancer cell proliferation and / or dissemination.

[0143] (129) Based on the antibody or functional fragment used in the project (122), the GPVI-related condition is cancer, preferably skin cancer (especially malignant melanoma), colon cancer, breast cancer, ovarian cancer, lung cancer or metastatic cancer.

[0144] (130) Based on the antibody or functional fragment used in item (122), GPVI-related conditions are conditions associated with abnormal or abnormal megakaryocyte and / or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation and / or function.

[0145] (131) The antibody or functional fragment used in any of the items (122)-(130) is a human subject.

[0146] (132) The antibody or functional fragment used in any of items (122) to (131), wherein the method includes intravenous administration of the antibody or functional fragment to a human subject.

[0147] (133) A method for treating or preventing GPVI-related conditions, comprising administering to a patient in need an effective amount of an anti-GPVI antibody or a functional fragment thereof as defined in any one of items (1) to (115) or a pharmaceutical composition of item (121), preferably wherein the GPVI-related condition is one specified in items (123) to (130). Attached Figure Description

[0148] Figure 1 Emf6.1 Fab Inhibition of GPVI-induced human platelet aggregation, thrombus formation, and spreading. (A) Aggregation response of washed human platelets treated with 10 μg / mL Emf6.1 IgG or control IgG in a turbidity aggregation assay. Gray arrows indicate the addition of agonists. (BC) Aggregation response of heparinized human blood treated with 10 μg / mL Emf6.1 IgG or control IgG in a flow (1000 s) assay. -1 Evaluation of platelet adhesion (B) and aggregate formation (C) of Horm collagen (200 μg / mL) under flow conditions. (DH) In recalcified human blood treated with 10 μg / mL Emf6.1Fab or control Fab, platelet adhesion was evaluated under flow conditions (1000 s). -1 Assessment of platelet adhesion (E), thrombosis (F), phosphatidylserine exposure (G), and fibrin deposition (H) with Horm collagen (200 μg / mL) plus tissue factor (500 pM). Values ​​are mean ± SD (n = 4). Unpaired, Mann-U-Whitney test. **P < 0.01, ***P < 0.001. (D) Representative images are shown, scale bar 50 µm. (I) Assay using 10 μg / mL Emf6.1 FabAggregation response of washed human platelets treated with Fab (n = 4). (JK) Washed human platelets were spread on fibrinogen (100 μg / mL) at 37°C for 45 min. DIC images (100x objective) were taken (J) and the abundance of stages was determined. Stage 1: adhesion; Stage 2: filopodia formation; Stage 3: lamellar podia formation; Stage 4: complete platelet spreading (K). Values ​​are mean ± SD (n = 8); unpaired, two-tailed Student's t-test *** P < 0.001.

[0149] Figure 2 Emf6.1 Fab Inhibition of GPVI-induced hGP6 tg / tg Platelet aggregation, thrombus formation, and spreading. (AC) with 5 μg / mL EMF 6.1 Fab Or control Fab-treated heparinized hGP6 tg / tg In the blood, during flow (1000 s) -1 Evaluation of platelet adhesion (B) and aggregate formation (C) with Horm collagen (200 μg / mL) at the following parameters. Values ​​are mean ± SD (n = 5). Unpaired, Mann-U-Whitney test. **P < 0.01, ***P < 0.001. (A) shows representative images, scale bar 50 µm. (D) Platelet adhesion (B) and aggregate formation (C) with 10 μg / mL Emf6.1 at the following parameters in the light transmission aggregation assay. Fab Or compare with the cleaned hGP6 treated with Fab. tg / tg Platelet aggregation response (n=5); gray arrows indicate the addition of agonists. (EF) Washed hGP6 at 37°C tg / tg Platelets spread on fibrinogen (100 μg / mL) for 45 min. DIC images (100x objective, scale bar 30 µm) were taken (E) and the abundance of each stage was determined. Stage 1: adhesion; Stage 2: filopodia formation; Stage 3: lamellar podia formation; Stage 4: complete platelet spreading (F). Values ​​are mean ± SD (n = 3); unpaired, Mann-U-Whitney test. *P<0.05, **P<0.01.

[0150] Figure 3 Emf6.1 Fab Highly effective blocking of hGP6 tg / tg GPVI function in mice. At 4 mg / kg bw emf 6.1 Fab Or compare with Fab treatment hGP6 tg / tgAnimals (n=5). (AB) Platelet counts (A) and size (B) determined using an automated cell counter at specified time points post-treatment. (CD) GPVI exposure was assessed using Emf2 IgG-FITC (C), while epitope saturation of Emf6.1 was assessed using Emf3 IgG-FITC (D). (EH) Degranulation (α-P-selectin-FITC) in response to CRP (E, G) or thrombin (F, H) (EF) and activation of platelet αIIbβ3 integrin (JON / A-PE) (G, H) were determined by flow cytometry at specified time points post-treatment. Data are expressed as mean ± SD; significance is expressed as *p < 0.05, ***p < 0.001, relative to the indicator group (two-way Anova) (n = 5).

[0151] Figure 4 Emf6.1 Fab Sustained GPVI inhibition in vivo. (AC) In the phototransmission aggregation assay, from 4 mg / kg bw Emf 6.1 Fab Or control Fab intravenous treatment of hGP6 tg / tg Aggregation response of washed platelets in mice to CRP (A), collagen (B), or thrombin (C) (n = 6); arrows indicate the addition of an agonist. (DF) from patients receiving or receiving 4 mg / kg bw emf 6.1 Fab Or refer to Fab's hGP6 tg / tg In the heparinized blood of mice, the flow rate (1000 s) was observed at different time points after injection. -1 The effects of Horm collagen (200 g / mL) on platelet adhesion (E) and aggregate formation (F) were assessed. Values ​​are mean ± SD. Two-way Anova *P<0.05, ***P<0.001.

[0152] Figure 5 Emf6.1 Fab Treatment (4 mg / kg bw) was highly protective in the arterial thrombosis model without affecting bleeding time. (AB). In the mechanical injury of the aortic thrombosis model, compared with control Fab or Emf 6.1 Fab Arterial thrombosis was assessed at the longest timeframe of 30 min post-treatment. (A) The time to vascular occlusion is depicted, with each circle representing one animal. ***P < 0.001, Fisher's exact test was used. (B) Data from controls Fab and Emf6.1 are shown. Fab Representative blood flow traces of the treated mice. (C) Hemostasis was assessed using tail bleeding time, where each circle represents a mouse.

[0153] Figure 6 Emf6.1 binds to a novel GPVI epitope. Mapping of the Emf6.1 binding epitope in the extracellular domain of human GPVI. (A) 15-mer overlapping peptides covering the extracellular domain of human GPVI (residues 24 to 267) are displayed as a peptide microarray. Emf6.1 IgG binding is visualized by HRP-labeled anti-mouse IgG secondary antibody. Two distinct epitopes, 201V-E215 and 246S-P260, were identified by mapping. (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 not with the ligand binding site (displayed against collagen, PDB-ID 5ou9).

[0154] Figure 7 Alignment of humanized variants. (AB) The Emf6.1 VL sequence (VL0) has been aligned with four humanized VL variants (A), and the Emf6.1 VH sequence (VH0) has been aligned with five humanized VH variants (B). VL0 is a mouse sequence, VL1-4 are humanized variants; VH0 is a mouse sequence, VH1-5 are humanized variants. CDRs are underlined. Key residues important for the VH / VL interface and canonical loop structure have been maintained as much as possible in the humanized variants using the CDRx platform. (C) Alignment with mouse VL / VH ... L (i.e., a humanized variant homologous to VL0). (D) and mouse V H (i.e., a humanized variant that is homologous to VH0).

[0155] Figure 8 EMA601 inhibits GPVI function. (AC) Heparinized hGPVI treated with 5 μg / mL EMA601 or control Fab... tg / tg In the blood, during flow (1000 s) -1 Evaluation of platelet adhesion (B) and aggregate formation (C) with Horm collagen (200 μg / mL) under light transmission aggregation assay. Values ​​are mean ± SD (n = 3). Unpaired, Mann-U-Whitney test *P<0.05, **P<0.01. (A) shows representative images, scale bar 50 µm. (D) Washed hGP6 treated with 5 μg / mL EMA601 or control Fab in light transmission aggregation assay. tg / tg Platelet aggregation response (n=3), arrows indicate the addition of agonists. (EF) Washed hGP6 at 37°C tg / tgPlatelets spread on fibrinogen (100 μg / mL) for 45 min. DIC images (100x objective, scale bar 30 µM) were taken (E) and the abundance of each stage was determined. Stage 1: adhesion; Stage 2: filopodia formation; Stage 3: lamellar podia formation; Stage 4: complete platelet spreading (F). Values ​​are mean ± SD (n = 3); unpaired, Mann-U-Whitney test *P<0.05, **P<0.01. (GK) In recalcified human blood treated with 10 μg / mL EMA601 (grey) or control Fab (black), platelets were observed in flow (1000 s) -1 Assessment of platelet adhesion (H), thrombosis (I), phosphatidylserine exposure (J), and fibrin deposition (K) with Horm collagen (200 μg / mL) plus tissue factor (500 pM). Values ​​are mean ± SD (n = 4). Unpaired, Mann-U-Whitney test. ***P < 0.001. (G) shows representative images, scale bar: 50 µm.

[0156] Figure 9 Emf6.1 Fab Inhibits GPVI-induced activation and aggregation of human platelets. (A) Administered with 10 μg / mL (row 2), 5 μg / mL (row 3), 2 μg / mL (row 4), or 1 μg / mL (bottom row) Emf6.1 Fab Or, in control Fab (top row) treated heparinized human blood, during flow (1000 s) -1 Evaluation of platelet adhesion (bar graph next to the image) and aggregate formation (bar graph on the right-hand side) with Horm collagen (200 μg / mL) was performed. Values ​​are mean ± SD (n = 4). Unpaired, Mann-U-Whitney test. *P<0.05, **P<0.001. Representative images are shown on the left-hand side, scale bar 50 µm. (B) Evaluation of platelet adhesion (platelet adhesion) with 5, 2, or 1 μg / mL Emf6.1 using light transmission aggregation assay. Fab Or compare the aggregation response of washed human platelets treated with Fab (n = 4).

[0157] Figure 10 Emf6.1 Fab Inhibition of GPVI-induced hGP6 tg / tg Platelet activation and aggregate formation. (AB) After activation with a specified agonist, platelet activity was determined by flow cytometry using 10 μg / mL Emf6.1. Fab Or compare Fab hGP6 processing tg / tgPlatelet degranulation (α-P-selectin-FITC) (A) and activation of platelet αIIbβ3 integrin (JON / A-PE) (B) (n=5). Unpaired, Mann-U-Whitney test. ***P<0.001. (C) In light transmission aggregation assay with 2 μg / mLEmf6.1 Fab Or compare Fab pre-warmed and cleaned hGP6 tg / tg Platelet aggregation response (n = 4). Arrows indicate the addition of agonists. (D) With 2 μg / mL EMF 6.1 Fab Or control Fab-treated heparinized hGP6 tg / tg In the blood, flowing (1000s) -1 Platelet adhesion and aggregate formation under the influence of Horm collagen (200 μg / mL). Left: Representative images shown, scale bar 50 µM. Right: Surface area coverage and thrombus volume data at the end of perfusion are expressed as mean ± SD (n = 4). Unpaired, Mann-U-Whitney test *** P < 0.001.

[0158] Figure 11 EMA601 inhibits GPVI function in human platelets. (A) In control group Fab In heparinized human blood pre-incubated with (10 µg / mL) or EMA601 (20, 10, or 1 µg / mL), under flowing conditions (1000 s) -1 Evaluation of platelet adhesion and aggregate formation with Horm collagen (200 μg / mL) at the end of the experiment. Left: Representative fluorescence and bright-field images at the end of the experiment, scale bar 50 µM. Right: Data on surface area coverage and thrombus volume at the end of perfusion are expressed as mean ± SD (n = 3). Unpaired, Mann-U-Whitney test. *P<0.05, **P<0.01. (B) In the light transmission aggregation assay, compared with 10 μg / mL (left) or 1 μg / mL (right) EMA601 or control. Fab Aggregation response of washed human platelets pre-incubated for 10 min (n=4).

[0159] Figure 12 Head-to-head comparison of EMA601 and ACT017 (glencimab) in vitro and in vivo. (AB) hGP6 tg / tg Diluted heparinized blood from mice was pre-incubated with specified concentrations of EMA601 or ACT017. Epitope saturation was assessed by flow cytometry using (A)Emf3. FITC For use in EMA601 and (B) JAQ1 FITCUsed for ACT017 (n=4). (C) hGP6 tg / tg Diluted heparinized blood from mice was pre-incubated with specified concentrations of EMA601 or ACT017, and the binding of Fab was detected using fluorescently labeled anti-human IgG-Fab antibody (n=4). (DE) Aggregation traces and quantification of washed human platelets pretreated with specified concentrations of EMA601 or ACT017 and stimulated with collagen or CRP. (EG) In heparinized human blood treated with specified concentrations of EMA601, ACT017, or control Fab, the binding was detected in flowing (1000 s) -1 Evaluation of platelet adhesion (F) and aggregate formation (G) of Horm collagen (200 μg / mL) at a scale bar of 50 μm. (HI) hGP6 tg / tg Mice (n=3) were intravenously treated with 4 mg / kg bw EMA601, ACT017, or control Fab (n=3). One hour after treatment, Fab binding (H) was detected in vitro by flow cytometry using fluorescently labeled anti-human IgG-Fab antibody, while platelet αIIbβ3 integrin activation (I) in response to 0.5 μg / mL CRP was measured using JON / A-PE. Data are expressed as mean ± SD; significance was defined as * p < 0.05, ** p < 0.01, *** p < 0.001 relative to the specified group (ordinary one-way ANOVA).

[0160] Figure 13: Emf6.1 Fab Treatment of mice in a tMCAO model protecting against ischemic stroke. A: 24 hours after tMCAO, hGP6 mice treated with either 4 mg / kg bw control-fab (control) or Emf6.1-fab. tg / tg Infarct volume in the mouse brain (shown in cross-section), as measured by triphenyltetrazolium chloride (TTC) staining. Day 1 after tMCAO, control (top row) and Emf6.1-fab treatment (bottom row) hGP6 tg / tg Representative TTC-stained 2 mm brain sections from mice. Grid areas represent unstained TTC areas, indicating tissue involvement due to infarction. Non-grid areas represent stained TTC areas, and therefore unaffected by infarction. B: 24 hours after tMCAO, hGP6 treated with either 4 mg / kg bw control-fab (Ctrl.) or Emf6.1-fab. tg / tg Infarct volume in the brain of mice (n = 9 mice per group). The x-axis represents the two groups of samples (left: control - fab-treated hGp6).tg / tg Mice, right: hGp6 treated with Emf6.1-fab tg / tg (Mice), with the y-axis representing infarct volume in mm³. Each point represents a data point, i.e., data from one mouse. Further markers are the median and the 25th and 75th percentiles forming the bottom and top lines of the rectangle. Finally, the lowest and highest values ​​for each sample type are also represented by lines. Statistical significance was analyzed by Student's t-test, *p<0.05.

[0161] Figure 14 All EMA601 (HC1LC2) variants, in which the cysteine ​​at Kabat L55 is replaced with different amino acids, inhibit GPVI function in human platelets. Aggregation response was measured in washed human platelets pre-incubated for 5 min with 0.5 µg / mL of the corresponding HC1LC2 variant or, if indicated, 10 µg / mL of the control Fab (n = 4) in a light transmission aggregation assay. Platelets were stimulated with 10 μg / mL collagen (left) or 0.5 μg / mL CRP. All variants inhibited GPVI more efficiently than glomsimab (last inset). Detailed Implementation

[0162] This invention relates to antibodies or functional fragments thereof capable of binding 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 the IgG, IgM, IgE, IgA, or IgD class (or any subclass thereof), and includes all conventionally known antibodies and their functional fragments. In the context of this invention, a "functional fragment" of an antibody / immunoglobulin, also referred to as a "functional antibody fragment," is defined as an antigen-binding fragment or other derivative of a parent antibody that substantially retains one or more of the properties of such parent antibodies mentioned in items (1) to (114) above.

[0163] An "antigen-binding fragment" of an antibody / immunoglobulin is defined as a fragment that retains 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, namely CDR-1, CDR-2, and / or CDR-3 regions. The "antigen-binding fragments" of this invention include Fab fragments, F(ab')2 fragments, and F(ab') fragments. The "functional fragments" of this invention include scFv, Fv, dsFv, monovalent IgG, biantibodies, triantibodies, tetraantibodies, and Fc fusion proteins. The antibodies or functional fragments of this invention may be part of a bifunctional or multifunctional construct.

[0164] As used in this article, "Fab," also known as "F(ab)," "antigen-binding fragment," or "Fab fragment," refers to a monovalent antigen-binding fragment containing both a heavy chain and a light chain, each containing a variable domain and a constant domain (i.e., a variable heavy domain ("V")). H ")+constant weight 1 domain ("C H 1”) and variable light domain (“V”) L ") + constant light domain ("C L ), but lacks a fragment crystallizable region (Fc region). Typically, the light and heavy chains in a Fab are linked by disulfide bonds. Fab may contain additional N-terminal and / or C-terminal sequences, such as the C-terminal portion of the N-terminal signal peptide and / or hinge region; however, it does not include such residues in the hinge region that participate in the formation of disulfide bonds.

[0165] "F(ab')2", also known as "F(ab')2 fragment", is bivalent and contains two antigen-binding regions (i.e., two antigen-binding fragment regions) and retains a portion of the hinge region that they are 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 yields two monovalent Fab' fragments, also known as "F(ab') fragments". 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 can be engineered to minimize or completely remove C. H 1 and C L Intermolecular disulfide interactions exist between the domains.

[0166] The “Fv” segment contains two chains, one of which contains V. H The area, and a line containing V L This region constitutes the antigen-binding site, but it lacks the constant region of Fab (C). H 1 and C L ), and without disulfide bonds connecting the two chains. "Monovalent IgG" preferably refers to a functional fragment containing only one full-length light chain and one full-length heavy chain, and therefore includes an antigen-binding fragment region. "dsFv" is a disulfide-linked variable fragment containing V linked by constructed interchain disulfide bonds. H Chain and V L Chain. “scFv” is a single-chain Fv segment, where the variable light (“V”) is a variable light (“V”). L ”) and variable weight (“V”) H The domains are connected by peptide bridges.

[0167] A "biantibody" is a dimer composed of two fragments, each having a variable region (hereinafter referred to as a biantibody-forming fragment) linked together via a linker or similar means, and typically containing two V... L and two VH Biantibody-forming fragments include those composed of V L and V H V L and V L V H and V H etc., preferred V H and V L The fragments that form the biantibody. In biantibody-forming fragments, the linker connecting the variable regions is not particularly limited, but is preferably short enough to avoid non-covalent bonds between variable regions within the same fragment. The length of such linkers can be suitably determined by those skilled in the art, but is typically 2-14 amino acids, preferably 3-9 amino acids, and especially 4-6 amino acids. In this case, V encoded on the same fragment L and V H Connected via a sufficiently short joint to avoid V on the same chain. L and V H Non-covalent bonds between the fragments prevent the formation of single-chain variable regions, thus allowing the fragment to form a dimer. This dimer can be formed via covalent or non-covalent bonds, or both, between the biantibody-forming fragments.

[0168] Furthermore, biantibody-forming fragments can be linked via linkers to form single-chain biantibodies (sc(Fv)2). By using long linkers of approximately 15-20 amino acids to link biantibody-forming fragments, non-covalent bonds can be formed between biantibody-forming fragments existing on the same chain to form dimers. Based on the same principle as in the preparation of biantibodies, polymeric antibodies such as trimers or tetramers can also be prepared by linking three or more biantibody-forming fragments.

[0169] Antibodies or functional fragments can be monovalent or multivalent, such as bivalent, like full-length IgG molecules. According to a preferred embodiment of the invention, the antibody or its functional fragment is monovalent. According to another preferred embodiment of the invention, the antibody or its functional fragment is a monovalent functional fragment of an antibody. Monovalent fragments of antibodies include antigen-binding fragments (Fab), F(ab'), Fv, disulfide-linked variable fragments (dsFv), monovalent IgG, and single-chain variable fragments (scFv). Preferred functional fragments of the invention are Fab, Fv, dsFv, scFv, and biantibodies. Particularly preferred functional antibody fragments of the invention are antigen-binding fragments (Fab). According to another preferred embodiment of the invention, the antibody or functional fragment, preferably a functional antibody fragment, is multivalent, such as bivalent; however, it contains only one GPVI antigen-binding region.

[0170] The terms "human glycoprotein VI" or "human GPVI" are known to those skilled in the art and refer to platelet membrane glycoproteins involved in platelet-collagen interactions. GPVI is a transmembrane collagen receptor expressed on the surface of platelets. In one embodiment, human GPVI refers to a protein comprising or composed of the following: amino acids having the UniProt accession number Q9HCN6 (preferably Q9HCN6-1 having the NCBI accession number NP_057447), or any amino acid sequence exhibiting at least about 90% identity with the amino acid sequence of Q9HCN6-1, Q9HCN6-2, or Q9HCN6-3, preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of Q9HCN6-1, Q9HCN6-2, or Q9HCN6-3, or higher identity with Q9HCN6-1, Q9HCN6-2, or Q9HCN6-3.

[0171] Preferably, the antibody or functional fragment of the present invention specifically binds to GPVI. As used herein, the antibody or functional fragment thereof "specifically recognizes" or "specifically binds" to human GPVI when it is able to distinguish between human GPVI and one or more reference molecules. Preferably, the IC50 binding time to each reference molecule is [not specified]. 50 ICs combining value ratio and GPVI 50 The value is at least 1000 times greater. In its most general form (and when no reference to the definition is mentioned), “specific binding” refers to the ability of an antibody or functional fragment to distinguish between human GPVI and unrelated biomolecules, as determined, for example, by specificity assays known in the art. Such methods include, but are not limited to, biolayer interferometry (BLI), surface plasmon resonance (SPR), ELISA assays, Western blotting, and immunofluorescence-based assays such as immunohistochemistry (IHC). For example, standard BLI, SPR, or ELISA assays can be performed. Typically, binding specificity is determined not by using a single reference biomolecule, but by using a group of approximately three to five unrelated biomolecules, such as milk powder, BSA, transferrin, etc.

[0172] There are no particular limitations on the binding epitopes on GPVI to which the antibody or functional fragment of the present invention binds. According to one embodiment of the invention, the antibody or functional fragment binds to human GPVI near or at the proposed GPVI dimerizing residues. According to a preferred embodiment of the invention, the antibody or functional fragment binds to human GPVI at a binding epitope that at least partially overlaps with the proposed GPVI dimerizing residues. The proposed GPVI dimerizing residues can be found in the extracellular domain of GPVI, and preferably refer to residues D173 to T180 of SEQ ID NO: 36 (corresponding to a portion of the extracellular domain of a GPVI isoform defined as isoform 1 by UniProt and isoform 2 by NCBI), which correspond to residues D196 to T203 of GPVI isoforms 1 to 3 (i.e., Q9HCN6-1, Q9HCN6-2, and Q9HCN6-3) in UniProt. According to another embodiment of the invention, the binding of the antibody or functional fragment to human GPVI is interrupted or interferes with GPVI dimerization. According to a preferred embodiment of the invention, the antibody or functional fragment binds to human GPVI at a binding epitope comprising or consisting of amino acids V178 to E192 and / or S223 to P237 of SEQ ID NO: 36. According to another preferred embodiment of the invention, the antibody or functional fragment binds to a discontinuous binding epitope comprising or consisting of amino acids V178 to E192 and S223 to P237 of SEQ ID NO: 36.

[0173] The antibody of the present invention or the functional fragment of the present invention comprises V L Domain and V H Domain. V L The domain includes the CDR1 area (CDRL1), CDR2 area (CDRL2), CDR3 area (CDRL3), and the frame area. H The domain includes CDR1 area (CDRH1), CDR2 area (CDRH2), CDR3 area (CDRH3) and frame area.

[0174] The term “CDR” or “complementarity-determining region” refers to one of the six hypervariable regions within an antibody’s variable domain that primarily contribute to antigen binding. One of the most commonly used definitions of 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 sequence of an antibody or functional fragment is identified using the antibody numbering system derived from IMGT and Kabat (i.e., as a combination of IMGT / Kabat CDR sequences).

[0175] V of the antibody or functional fragment of the present invention L The domain comprises 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 comprises 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 embodiment, the V of the antibody or functional fragment of the present invention L The domain comprises 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 embodiment, the V of the antibody or functional fragment of the present invention LThe domain comprises 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 comprises 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.

[0176] In the amino acid sequence SEQ ID NO: 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 specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is A. In another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is R. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is N. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is D. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is Q. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is E. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is G. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is H. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is I. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is L. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is K. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is M. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is F. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is S. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is T. In yet another specific embodiment of the invention, residue X in amino acid sequence SEQ ID NO:9 is W. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is Y. In yet another specific embodiment of the invention, residue X in the amino acid sequence SEQ ID NO: 9 is V.

[0177] V of the antibody or functional fragment of the present invention HThe domain comprises 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 substantially corresponds to the amino acid sequence of SEQ ID NO: 5, provided that 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 invention, the V of the antibody or functional fragment of the invention... H The domain comprises 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.

[0178] In alternative embodiments (Kabat only), the antibody of the present invention or the functional fragment of the present invention comprises (i)V L The domain comprises 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, and (ii) V H The domain comprises 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.

[0179] In another alternative embodiment, the antibody of the present invention or the functional fragment of the present invention comprises (i) V L The domain comprises 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, and (ii) V H The region comprises 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.

[0180] In another embodiment, the antibody or functional fragment of the present invention comprises V L 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%, even more preferably at least 99% sequence identity with the sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35, preferably 16, 17, 18, 19, 32, 33, 34 and 35, even more preferably at least 16, 17, 18, 32, 33 and 34. In another embodiment, the antibody or functional fragment of the present invention comprises V L A domain comprising or consisting of the following: 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 with the sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35, preferably 16, 17, 18, 19, 32, 33, 34 and 35, even more preferably at least 16, 17, 18, 32, 33 and 34.

[0181] In yet another embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of 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. In another embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of 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 NO: 16, 17, 18, and 19, preferably SEQ ID NO: 16, 17, and 18. In a further embodiment, the antibody or functional fragment of the present invention comprises V HThe domain comprises or consists of an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 and 25, preferably SEQ ID NO: 21, 22 and 23.

[0182] According to a preferred embodiment, the antibody or functional fragment of the present invention comprises V L Domain and V H Domain, the V L The domain comprises or consists of the following: an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; the V H The domain contains or consists of an amino acid sequence having at least 80%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21.

[0183] According to one embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the following SEQ ID NO: 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 the amino acid sequence shown in 16, 17, 18, 32, 33 or 34. According to another embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the following: SEQ ID NO: 26, 27, 28, 32, 33, 34 or 35, preferably 32, 33, 34 or 35, more preferably the amino acid sequence shown in 32, 33 or 34.

[0184] In another embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the following amino acid sequences shown in SEQ ID NO: 16, 17, 18, 19, 29, 30, or 31. In another embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17, 18, or 19. In another embodiment, the antibody or functional fragment of the present invention comprises V LThe domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 29, 17, 30, or 31. In a preferred embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the following amino acid sequences: SEQ ID NO: 16, 17, 18, 32, 33, or 34. In another preferred embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 32, 33, or 34. In yet another preferred embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 16, 17, or 18. In a particularly preferred embodiment, the antibody or functional fragment of the present invention comprises V L The domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17. In another particularly preferred embodiment, the antibody or functional fragment of the present invention comprises V L The domain contains or consists of the amino acid sequence shown in SEQ ID NO: 33.

[0185] In a further embodiment, the antibody or functional fragment of the present invention comprises V H The domain comprises or consists of the following amino acid sequences shown in SEQ ID NO: 21, 22, 23, 24, or 25. In a preferred embodiment, the antibody or functional fragment of the present invention comprises V H The domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 21, 22, or 23. In a particularly preferred embodiment, the antibody or functional fragment of the present invention comprises V H The domain contains or consists of the amino acid sequence shown in SEQ ID NO: 21.

[0186] According to a specific preferred embodiment of the present invention, V L Domain and V HThe domain contains or consists of the following: SEQ ID NO: 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 2 4, 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, representing a pair of amino acid sequences (i.e., V) L Contains or consists of the sequence shown in SEQ ID NO: 32, and V H Contains or consists of the sequence shown in SEQ ID NO: 21, or V L Contains or consists of the sequence shown in SEQ ID NO: 32, and V H Contains or consists of the sequence shown in SEQ ID NO: 22, etc.; preferably a pair of amino acid sequences shown in SEQ ID NO: 33 and 21, 33 and 22, 34 and 22, 17 and 21, 17 and 22 or 18 and 22.

[0187] According to another specific preferred embodiment of the invention, V L Domain and V H The domain contains or consists of the following: SEQ ID NO: 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, 2 The amino acid sequences shown are 6 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, pairs of amino acid sequences shown are 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 specific preferred embodiment of the invention, VL Domain and V H The domain contains or consists of the following: SEQ ID NO: 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 a pair of amino acid sequences shown in 32 and 22, 33 and 21, 33 and 22, 33 and 23, 34 and 21, 34 and 22 or 34 and 23.

[0188] According to yet another specific preferred embodiment of the invention, V L Domain and V H The domain contains or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 Contains or consists of the sequence shown in SEQ ID NO: 16, and V H Contains or consists of the sequence shown in SEQ ID NO: 21, or V L Contains or consists of the sequence shown in SEQ ID NO:16, and V H Contains or consists of the sequence shown in SEQ ID NO: 22, etc.). According to another specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences as shown in SEQ ID NO: 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 specific preferred embodiment of the invention, V L Domain and V HThe domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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. According to yet another specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, V L Domain and V H The domain contains or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 17 and 21, 17 and 22, 18 and 22 or 30 and 22.

[0189] According to a further specific preferred embodiment of the invention, V L Domain and V H The domain contains or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 Contains or consists of the sequence shown in SEQ ID NO: 16, and V H Contains or consists of the sequence shown in SEQ IDNO: 21, or V L Contains or consists of the sequence shown in SEQ ID NO: 16, and V H Contains or consists of the sequence shown in SEQ ID NO: 22, etc.). According to another specific preferred embodiment of the invention, V L Domain and V HThe domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 17 and 21, 17 and 22, 17 and 23, 18 and 21, 18 and 22, or 18 and 23. According to yet another specific preferred embodiment of the invention, V L Domain and V H The domain comprises or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 17 and 21, 17 and 22, 17 and 23, 18 and 21, or 18 and 22. According to yet another specific preferred embodiment of the invention, V L Domain and V H The domain contains or consists of the following: a pair of amino acid sequences shown in SEQ ID NO: 17 and 21, 17 and 22 or 18 and 22.

[0190] According to a particularly preferred embodiment of the invention, the VL and VH domains comprise or consist of the pair of amino acid sequences shown in SEQ ID NO: 33 and 21. According to another particularly preferred embodiment of the invention, the VL and VH domains comprise or consist of the pair of amino acid sequences shown in SEQ ID NO: 17 and 21.

[0191] In alternative embodiments, the VL domain of the antibody or functional fragment of the present invention comprises or consists of the following amino acid sequences as 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 above embodiments.

[0192] In a further embodiment, the antibody or functional fragment of the present invention comprises V L Domain and / or V H Domain, the V LThe domain contains or consists of an amino acid sequence as shown in SEQ ID NO: 15; the V H Contains or consists of the amino acid sequence shown in SEQ ID NO: 20.

[0193] 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. IgG subclasses suitable for the present invention include, but are not limited to, IgG1, IgG2, IgG3, and IgG4. Preferably, IgG is subclass 1, i.e., it is an IgG1 molecule.

[0194] Typically, the antibodies or functional fragments of the present invention comprise V L Light chains of the domain and including V H Heavy chain containing the VL domain. According to certain embodiments of the invention, the antibody or functional fragment consists of a light chain containing the VL domain and a heavy chain containing the VH domain. According to a preferred embodiment of the invention, the antibody or functional fragment is monovalent, such as Fab, having a VH domain. L Light chains of domains and having V H The heavy chain of the domain, the V L The domain includes the above-mentioned V L The amino acid sequence specified in or composed of the domain in any implementation thereof, the V L The domain includes the above-mentioned V H The amino acid sequence specified in or composed of the domain in any implementation thereof.

[0195] According to a preferred embodiment of the invention, except for V L In addition to the domain, light chains also contain the light chain constant domain (C). L ), preferably located in V L The C-terminus of the domain. According to a further preferred embodiment of the invention, besides V... H In addition to the domain, heavy chains also contain heavy chain constant domains (C). H ), preferably located in V H The C-terminus of the domain.

[0196] According to a specific embodiment, the antibody or functional fragment of the present invention comprises a light chain derived from the human immunoglobulin κ (IgK) light chain (preferably allotype Km3). According to another specific embodiment, the antibody or functional fragment of the present invention comprises a light chain containing a constant domain derived from a human IgK light chain constant domain (preferably allotype Km3). According to yet another specific embodiment, the antibody or functional fragment of the present invention comprises a light chain constant domain containing or composed of a human IgK light chain constant domain (preferably allotype Km3). According to yet another specific embodiment, the antibody or functional fragment of the present invention comprises a heavy chain containing a constant domain derived from a human IgG1 heavy chain CH1 constant domain (preferably allotype G1m17,1). According to yet another specific embodiment, the antibody or functional fragment of the present invention comprises a heavy chain containing a constant domain containing or composed of a human IgG1 heavy chain CH1 constant domain (preferably allotype G1m17,1).

[0197] According to yet another specific embodiment, the antibody or functional fragment of the present invention comprises a light chain and / or a heavy chain, the light chain comprising a constant domain derived from a human IgK light chain constant domain (allotype Km3), preferably wherein the sum of the number of amino acids differing from the amino acid sequence of, for example, the human IgK light chain constant domain (allotype Km3) shown in SEQ ID NO: 60 is less than 10, 9, 8, 7, 6, 5, 4, 3 or 2; the heavy chain comprises a constant domain derived from a human IgG1 heavy chain CH1 constant domain (allotype G1m17,1), preferably wherein the sum of the number of amino acids differing from the amino acid sequence of, for example, the human IgG1 heavy chain CH1 constant domain (allotype G1m17,1) shown in SEQ ID NO: 61 is less than 10, 9, 8, 7, 6, 5, 4, 3 or 2.

[0198] According to a preferred embodiment of the present invention, the light chain constant domain (C L The amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid shown in SEQ ID NO: 60. According to another preferred embodiment of the invention, the light chain constant domain (C... L It consists of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid shown in SEQ ID NO: 60.

[0199] According to a preferred embodiment of the present invention, the heavy chain constant domain (C HThe amino acid sequence comprises an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid shown in SEQ ID NO: 61. According to another preferred embodiment of the invention, the light chain constant domain (C... H It consists of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid shown in SEQ ID NO: 61.

[0200] According to a further preferred embodiment of the invention, the light chain comprises an N-terminal light chain signal peptide and / or a light chain constant domain (CL), the N-terminal light chain signal peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 62, and the light chain constant domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 60, and preferably in the V L The C-terminus of the domain. According to another preferred embodiment of the invention, the heavy chain comprises an N-terminal heavy chain signal peptide and / or a heavy chain constant domain (CH), the N-terminal heavy chain signal peptide comprising or consisting of the amino acid sequence shown in SEQ ID NO: 63, and the heavy chain constant domain comprising or consisting of the amino acid sequence shown in SEQ ID NO: 61, and preferably at the C-terminus of the VH domain. The N-terminal signal peptide and / or constant region may be arranged on the light chain and / or heavy chain such that they are directly linked to the VH domain. L Domain and / or V H The domain, or can be linked via a linker peptide. Preferably, the N-terminal signal peptide and the constant region are arranged on the light and heavy chains, such that they are directly linked to the V domain. L Domain and V H domain.

[0201] According to a preferred embodiment of the invention, the antibody or functional fragment is preferably monovalent, more preferably Fab, and comprises (a) a light chain containing V L Domain (which includes the above for V) L (Amino acids specified in any implementation of the domain or composed of them), C L Domain (which includes the above for C) L (a) the amino acids specified in any embodiment of the domain or composed thereof, and optionally the N-terminal light chain signal peptide as described above, or composed thereof; and (b) the heavy chain, which contains V H Domain (which includes, as described above, for V) H The amino acids specified in any implementation of the domain or that constitute it), C H Domain (which includes, as described above, for C) HThe amino acids specified in any embodiment of the domain (or those that constitute it) and optionally the N-terminal heavy chain signal peptide as described above, or those that constitute it.

[0202] Light chains and heavy chains can be included in V L and V H Further amino acid sequences at the N-terminus or C-terminus of the domain, such as other signal peptides, binding peptides, adaptor peptides, other functional protein domains, etc. In addition, light and heavy chains may be contained within further molecules covalently attached to or otherwise bound to the N-terminus or C-terminus, such as other signal molecules, binding moieties, (cross-linked) adaptor molecules, other functional moieties, etc.

[0203] In a preferred embodiment of the invention, the antibody or its functional fragment comprises or is composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, preferably SEQ ID NO: 38, 39, 40, 41, 55, 56, 57 and 58, more preferably the amino acid sequences shown in one of SEQ ID NO: 38, 39, 40, 55, 56 and 57; the heavy chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 44, 45, 46, 47 and 48, preferably the amino acid sequences shown in one of SEQ ID NO: 44, 45 and 46. In another preferred embodiment of the invention, the antibody or its functional fragment comprises or is composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 52, 53, 54, 55, 56, 57 and 58, preferably the amino acid sequence shown in one of SEQ ID NO: 52, 53 and 54; and the heavy chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 44, 45, 46, 47 and 48, preferably the amino acid sequence shown in one of SEQ ID NO: 44, 45 and 46. In yet another preferred embodiment of the invention, the antibody or its functional fragment comprises or is composed of the following light and heavy chains, wherein the light chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 38, 39, 40, 41, 49, 50 and 51, preferably the amino acid sequence shown in one of SEQ ID NO: 38, 39 and 40; and the heavy chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 44, 45, 46, 47 and 48, preferably the amino acid sequence shown in one of SEQ ID NO: 44, 45 and 46.

[0204] According to a preferred embodiment of the present invention, the antibody or its functional fragment is a monovalent functional antibody fragment, preferably an antigen-binding fragment (Fab), F(ab'), Fv, a disulfide-linked variable fragment (dsFv), monovalent IgG, or a single-chain variable fragment (scFv). A particularly preferred functional antibody fragment of the present invention is a monovalent Fab. In a preferred embodiment of the invention, the functional fragment is a monovalent functional antibody fragment comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence selected from SEQ ID NO: 38, 39, 40, or 41; and the heavy chain comprises or is composed of an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with the sequence selected from SEQ ID NO: 44, 45, 46, 47, or 48.

[0205] In another preferred embodiment of the invention, the functional fragment is a monovalent functional antibody fragment having a light chain and a heavy chain, wherein the light chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, preferably SEQ ID NO: 38, 39, 40, 41, 55, 56, 57 and 58, more preferably the amino acid sequences shown in one of SEQ ID NO: 38, 39, 40, 55, 56 and 57; and the heavy chain comprises or is composed of the following amino acid sequences: SEQ ID NO: 44, 45, 46, 47 and 48, preferably the amino acid sequences shown in one of SEQ ID NO: 44, 45 and 46. In another preferred embodiment of the invention, a monovalent functional antibody fragment is functionally comprising a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NOs: 52, 53, 54, 55, 56, 57, and 58; and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48. In another preferred embodiment of the invention, a monovalent functional antibody fragment is functionally comprising a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NOs: 38, 39, 40, 41, 49, 50, and 51; and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NOs: 44, 45, 46, 47, and 48.

[0206] In a further preferred embodiment of the invention, the functional fragment is monovalent, such as an antigen-binding fragment (Fab), comprising or consisting of: (a) a light chain comprising, or consisting of, an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 38, 39, 40, or 41; and (b) a heavy chain comprising, or consisting of, an amino acid sequence having at least 80%, 85%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 44, 45, 46, 47, or 48. In another preferred embodiment of the invention, the functional fragment is monovalent, preferably Fab, comprising or consisting of: (a) a light chain comprising, or consisting of, the amino acid sequence shown in one of SEQ ID NO: 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 NO: 44, 45, 46, 47, and 48. In another preferred embodiment of the invention, the functional fragment is monovalent, preferably Fab, comprising or consisting of: (a) a light chain comprising, or consisting of, the amino acid sequence shown in one of SEQ ID NO: 38, 39, 40, and 41, and (b) a heavy chain comprising, or consisting of, the amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47, and 48. According to another preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 38, 39, 40, 41, 49, 50, 51, 52, 53, 54, 55, 56, 57 and 58, and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48. According to another preferred embodiment, the antibody or its functional fragment is monovalent, preferably Fab, comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 52, 53, 54, 55, 56, 57 and 58, and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48.According to yet another preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 55, 56, 57 and 58, and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 44, 45, 46, 47 and 48. According to yet another preferred embodiment, the antibody or its functional fragment is monovalent, preferably Fab, comprising or composed of a light chain and a heavy chain, wherein the light chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 55, 56 and 57, and the heavy chain comprises or is composed of the amino acid sequence shown in one of SEQ ID NO: 44, 45 and 46.

[0207] In a specific preferred embodiment of the invention, the antibody or its functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains, wherein the pair of heavy and light chains comprises or consists of the following: SEQ ID NO: 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 Amino acid sequences shown in SEQ ID NO: 38 and 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: 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.).

[0208] In another specific preferred embodiment of the invention, the antibody or its functional fragment (preferably monovalent, more preferably Fab, and) comprises or is composed of the following: having SEQ ID NO: 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 4 7 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 53 and 46; even 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 a pair of light and heavy chains of the amino acid sequences shown in 56 and 44, 56 and 45, 57 and 45 or 53 and 45.

[0209] In another specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, wherein the pair of light and heavy chains comprises or consists of the following: SEQ ID NO: 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 and 44, 57 and 45 or 57 and 46; even more preferably 55 and 45, 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45 or 57 and 46; even more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44, 57 and 45 or 57 and 46; even more preferably 56 and 44, 56 and 45, 56 and 46, 57 and 44 or 57 and 45; even more preferably 56 and 44, 56 and 45, 56 and 46 or 57 and 45; even more preferably 56 and 44, 56 and 45 or 57 and 45, even more preferably 56 and 44.

[0210] In yet another preferred embodiment of the invention, the antibody or its functional fragment (preferably monovalent, more preferably Fab) comprises or consists of a pair of light and heavy chains, wherein the pair of heavy and light chains comprises or consists of the following: SEQ ID NO: The amino acid sequences shown in SEQ ID NO: 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 (i.e., the light chain contains or is composed of the sequence shown in SEQ ID NO: 38, and the heavy chain contains SEQ ID NO: 38). (The sequence shown in SEQ ID NO: 44 or composed of it, 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 specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or composed of a pair of light and heavy chains, which comprises or is composed of the following amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains comprising or consisting of the amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains comprising or consisting of the amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, the pair of light and heavy chains comprising or consisting of the amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains, the pair of light and heavy chains comprising or consisting of the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45, 40 and 45, or 50 and 45.

[0211] In a further preferred embodiment of the invention, the antibody or functional fragment (preferably monovalent, more preferably Fab, and) comprises or is composed of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 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 comprises or is composed 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 specific preferred embodiment of the invention, the functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 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 specific preferred embodiment of the invention, the functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 38 and 45, 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46. In yet another specific preferred embodiment of the invention, the functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45, 39 and 46, 40 and 44, 40 and 45, or 40 and 46.

[0212] In yet another specific preferred embodiment of the invention, the antibody or functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45, 39 and 46, 40 and 44, or 40 and 45. In yet another specific preferred embodiment of the invention, the antibody or functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45, 39 and 46, or 40 and 45. In yet another specific preferred embodiment of the invention, the antibody or functional fragment (preferably monovalent, more preferably Fab, and) comprises or consists of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44, 39 and 45, or 40 and 45. In another embodiment of the invention, the functional fragment (preferably monovalent, more preferably Fab) consists of a pair of light and heavy chains, which respectively contain or consist of the amino acid sequences shown in SEQ ID NO: 37 and 43.

[0213] In a particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 45. In another particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 46. In yet another particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 40 and 44. In yet another particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 40 and 45. In a particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 39 and 44. In another particular preferred embodiment of the invention, the antibody or its functional fragment is monovalent, preferably Fab, comprising or consisting of a pair of light and heavy chains having the amino acid sequences shown in SEQ ID NO: 56 and 44.

[0214] According to a further embodiment of the invention, in the amino acid sequences shown in SEQ ID NO: 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 specific embodiment of the invention, in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58, residue X is A. In another specific embodiment of the invention, in the amino acid sequences shown in SEQ ID NO: 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 invention, residue X is N in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is D in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is Q in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is E in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is G in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is H in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is I in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57 and 58. In yet another specific embodiment of the invention, residue X is L in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57 and 58.In yet another specific embodiment of the invention, residue X is K in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is M in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is F in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is S in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is T in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, residue X is W in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57, and 58. In yet another specific embodiment of the invention, in the amino acid sequences shown in SEQ ID NO: 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 invention, in the amino acid sequences shown in SEQ ID NO: 26, 27, 28, 32, 33, 34, 35, 52, 53, 54, 55, 56, 57 and 58, residue X is V.

[0215] According to a specific embodiment of the invention, in cases involving a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) or V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is A. In another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) or V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is R. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) LIn any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is N. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) or a V chain having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is D. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) or V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is Q. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is E. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is G. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) or V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is H. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is I. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is L. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is K. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V LIn any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is M. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is F. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is S. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is T. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is W. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) V L In any of the foregoing embodiments of an antibody or a functional fragment thereof, residue X is Y. In yet another specific embodiment of the invention, in the case of an antibody having a light chain (which comprises or consists of the amino acid sequences shown in SEQ ID NO: 32, 33, 34 and 35) L In any of the foregoing embodiments of the antibody or its functional fragment, residue X is V.

[0216] According to a specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58), residue X is A. In another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58), residue X is R. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed of the amino acid sequence shown in SEQ ID NO: 55, 56, 57, or 58), residue X is N. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is Q. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is E. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is G. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is H. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody or a functional fragment thereof having a light chain (which comprises or is composed 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 invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is W. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is Y. In yet another specific embodiment of the invention, in any of the foregoing embodiments relating to an antibody having a light chain (which comprises or is composed of the amino acid sequence shown in or composed of SEQ ID NO: 55, 56, 57 or 58), residue X is V.

[0217] In a further embodiment, the present invention relates to an antibody or a functional fragment thereof that binds substantially the same binding epitopes as defined in any of the above embodiments, but includes different CDR sequences and / or V as defined in any of the above embodiments. L and V H Sequences and / or light and heavy chain sequences.

[0218] Affinity

[0219] The antibody or functional fragment of this invention has a very high affinity for human GPVI. The term "K" is used in this context. D"KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction. Typically, the antibodies or functional fragments of the present invention bind to human GPVI with a dissociation equilibrium constant (KD) 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, about 195 pM or less, or about 175 pM or less. According to a particularly preferred embodiment, the antibodies or functional fragments of the present invention bind to human GPVI with a dissociation equilibrium constant (KD) of 195 pM or less. D It combines with human GPVI.

[0220] K D The dissociation equilibrium constant (Ki) can be readily determined using conventional techniques such as biolayer interferometry (BLI), surface plasmon resonance (SPR), or ELISA. Depending on the specific implementation, the dissociation equilibrium constant (Ki) can be determined. D Determined using BLI, SPR technology (e.g., in BIACORE instruments), or ELISA.

[0221] Preferably, the dissociation equilibrium constant (K) D Using BLI, for example in Octet instruments and with Fortebio software, to determine. Specifically, K... D The determination is preferably performed as described in Section 1.16 of the embodiments. According to another specific embodiment, the dissociation equilibrium constant (K...) is... D The antigen was determined by BLI at 25°C, for example using an Octet instrument, preferably at a constant orbital flow rate of, for example, 1000 rpm; the antigen was immobilized on the biosensor using an antigen (human GPVI) loading concentration of 1.2 µg / ml, with an association time of 900 s and a dissociation time of 1200 s; a screening range of 7 antibody concentrations ranging from 10 to 0.014 nM was used with 3-fold serial dilutions; and a 1:1 interaction / fitting model was applied.

[0222] Functional properties of antibodies or functional antibody fragments

[0223] Due to their very high affinity for human GPVI and high stability in plasma, the antibodies or functional fragments of the present invention exhibit high potency in inhibiting human GPVI on plasma platelets. In certain embodiments, the antibodies or functional fragments of the present invention are capable of sustained 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, 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 as specified in any embodiment herein preferably have one or more of the following further functional properties.

[0224] Typically, the antibodies or functional fragments of the present invention (preferably in monovalent form, e.g., as Fab) are capable of prolonging binding to GPVI on circulating platelets without affecting 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. Platelet count and size can be determined, for example, as described in the examples in Section 1.9.

[0225] In one embodiment of the invention, the antibody or functional fragment of the invention (preferably in monovalent form, e.g., as Fab) can be administered intravenously at a dose of 4 mg / kg (e.g., administered to a humanized mouse model of GPVI (hGP6)). tg / tg In mice, prolonged binding to circulating platelets, when normalized with a negative control (e.g., a GPVI-nonspecific control Fab), exhibits a GPVI receptor epitope occupancy greater than 50% for at least 12, 24, 36, 48, or 60 hours, as determined, for example, by flow cytometry analysis in ex vivo diluted blood, using competitive binding to different fluorescently labeled anti-GPVI antibodies or functional fragments and employing mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy, as illustrated in the examples in Section 1.10.

[0226] In another embodiment of the invention, the antibody or functional fragment of the invention (preferably in monovalent form, e.g., as Fab) can be administered intravenously at a dose of 2 mg / kg (e.g., administered to a humanized mouse model of GPVI (hGP6)). tg / tgIn mice, prolonged binding to circulating platelets, when normalized with a negative control (e.g., a GPVI-nonspecific control Fab), exhibits a GPVI receptor epitope occupancy greater than 50% for at least 8, 12, 16, 20, 28, or 28 hours, as determined, for example, by flow cytometry analysis in ex vivo diluted blood, using competitive binding to different fluorescently labeled anti-GPVI antibodies or functional fragments and employing mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy, as illustrated in the examples in Section 1.10.

[0227] In another embodiment of the invention, the antibody or functional fragment of the invention (preferably in monovalent form, e.g., as Fab) can be administered intravenously at a dose of 4 mg / kg (e.g., administered to a humanized mouse model of GPVI (hGP6)). tg / tg In mice, prolonged binding to circulating platelets, when normalized with a negative control (e.g., a GPVI-nonspecific control Fab), exhibits at least 20% GPVI receptor epitope occupancy for at least 36, 48, 60, 72, 84, or 96 hours, as determined, for example, by flow cytometry analysis in ex vivo diluted blood, using competitive binding to different fluorescently labeled anti-GPVI antibodies or functional fragments and employing mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope occupancy, as illustrated in the examples in Section 1.10.

[0228] In another embodiment of the invention, the antibody or functional fragment of the invention (preferably in monovalent form, e.g., as Fab) is administered intravenously at a dose of 4 mg / kg (e.g., administered to a humanized mouse model of GPVI (hGP6)). tg / tg In mice, the surface level of GPVI on circulating platelets was not affected, as assessed at 12, 24, 36, 48, 60, 72, 84, 96, 120, or 144 hours, and was determined, for example, by flow cytometry analysis in ex vivo diluted blood, as illustrated in the examples in Section 1.10.

[0229] In another embodiment of the invention, the antibody or functional fragment of the invention (preferably in monovalent form, such as Fab) is administered intravenously at a dose of 4 mg / kg (e.g., to a humanized mouse model of GPVI (hGP6)). tg / tg After mouse, the in vivo platelet count and size on circulating platelets, as assessed at 12, 24, 36, 48, 60, 72, 84, 96, 120, or 144 hours, are not affected, and the in vitro test is performed in the sampled blood, for example, as described in the examples in Section 1.9.

[0230] Typically, the antibodies or functional fragments of the present invention are capable of completely inhibiting CRP (collagen-related peptide)-induced and collagen-induced aggregation of washed human platelets. CRP-induced or collagen-induced aggregation of washed human platelets can be determined using a standard optical transmission aggregation assay, as described in the examples in Section 1.7. According to specific embodiments, the antibodies or functional fragments of the present invention, at concentrations ≤ 10 μg / ml, preferably ≤ 5 μg / ml, more preferably ≤ 2 μg / ml, and even more preferably ≤ 1 μg / ml, are capable of completely inhibiting CRP-induced and collagen-induced aggregation of washed human platelets, as determined using a standard optical transmission aggregation assay, as described in the examples in Section 1.7.

[0231] According to another specific embodiment, the antibody or functional fragment of the present invention is administered intravenously at a dose of 4 mg / kg to, for example, a humanized mouse model (hGP6) targeting GPVI. tg / tg (in mice) at least 12, 24, 48, or 60 hours after administration, it completely inhibited hGP6 in washed mice. tg / tg CRP-induced and / or collagen-induced aggregation of platelets, as determined using standard light transmission aggregation assays, such as those described in the examples in Section 1.7.

[0232] According to another specific embodiment, compared with ACT017 (glencimab), the antibody or functional fragment of the present invention, preferably at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, has > 2 times, preferably > 4 times, > 6 times, > 8 times or > 10 times greater potency in inhibiting GPVI function in human platelets in vitro, as determined using a standard light transmission aggregation assay, for example as described in the examples in Section 1.7. According to another specific embodiment, compared to ACT017 (glenciemab), the antibody or functional fragment of the present invention, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, has, in vitro, a >2-fold, preferably >4-fold, >6-fold, >8-fold, or >10-fold greater potency in inhibiting collagen-induced and / or CRP-induced aggregation of washed human platelets, as determined using a standard light transmission aggregation assay, for example, as described in the examples in Section 1.7. Optionally, the fold change in potency is determined as the ratio of the maximum aggregation percentage of a sample treated with the antibody or functional fragment to the maximum aggregation percentage of a sample treated with glenciemab.

[0233] Typically, the antibodies or functional fragments of the present invention are able to completely inhibit human platelet adhesion (defined as platelet surface coverage) and thrombus formation (defined as relative thrombus volume) on collagen-coated surfaces (preferably coated at 200 g / ml) in heparinized human blood under flow.

[0234] According to a specific embodiment, at a concentration of 5 μg / ml, the antibody or functional fragment of the present invention can be delivered under flow conditions (preferably 1000 s). -1 The heparinized human blood at a shear rate that completely inhibits human platelet adhesion (platelet surface coverage) and / or thrombus formation (relative thrombus volume) on collagen-coated surfaces (preferably coated at 200 μg / ml), as determined using a flow adhesion assay, for example as described in the examples in Section 1.5. According to a specific preferred embodiment of the invention, "complete inhibition" means a reduction of at least 60%, 65%, or 70% in platelet surface coverage and / or a reduction of at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, etc., in relative thrombus volume compared to a negative control (e.g., a control Fab if a functional fragment in the form of anti-GPVI Fab is used), for example as described in the examples in Section 1.5.

[0235] According to another specific embodiment, the antibody or functional fragment of the present invention, at concentrations of ≤ 10 μg / ml, preferably ≤ 5 μg / ml, more preferably ≤ 2 μg / ml, and even more preferably ≤ 1 μg / ml, is capable of reducing the relative thrombus volume on a collagen-coated surface (preferably coated at 200 μg / ml) in heparinized human blood under flow conditions (preferably using a shear rate of 1000 s⁻¹) in a flow adhesion assay (e.g., as described in the examples in Section 1.5). According to another preferred embodiment, the antibody or functional fragment of the present invention, when administered intravenously at a dose of 4 mg / kg to a humanized mouse model (hGP6) against GPVI, reduces the relative thrombus volume on a collagen-coated surface (preferably coated at 200 μg / ml) by at a flow adhesion assay (e.g., as described in the examples in Section 1.5). tg / tg After at least 12, 24, 48, or 60 hours, or more preferably at least 60 hours, in mice, compared with the negative control, it was able to be used in flow (preferably using 1000 s) -1 In heparinized human blood at a shear rate of at least 50%, 55%, 60%, or 60%, the relative thrombus volume on the collagen-coated surface (preferably coated at 200 μg / ml) is reduced by at least 50%, 55%, 60%, or 60% in a flow adhesion assay (e.g., as described in the examples in Section 1.5).

[0236] According to another specific embodiment, the antibody or functional fragment of the present invention, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, is able to significantly reduce (e.g., greater than 1.5 times, preferably greater than 2 times, 2.5 times, or 3 times) platelet surface coverage on collagen-coated surfaces (preferably coated at 200 μg / ml) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹) in flow adhesion assays (e.g., as described in the examples in Section 1.5), while ACT017 (glencimab) treatment does not result in a significant reduction.

[0237] According to another specific embodiment, the antibody or functional fragment of the present invention, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, is able to reduce platelet surface coverage on collagen-coated surfaces (preferably coated at 200 μg / ml) by more than 1.5 times, preferably more than 2 times, 2.5 times, or 3 times in a flow adhesion assay (e.g., as described in the examples in 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, at a concentration of 10 μg / ml, preferably 5 μg / ml, more preferably 1 μg / ml, is able to reduce the relative thrombus volume on a collagen-coated surface (preferably coated at 200 μg / ml) by more than 2, preferably more than 4, 6, 8, or 10 times in a flow adhesion assay (e.g., as described in the examples in Section 1.5) in heparinized human blood under flow (preferably using a shear rate of 1000 s⁻¹).

[0238] Typically, the antibodies or functional fragments of the present invention, in citrated and recalcified human blood under flow (preferably using a shear rate of 1000 s⁻¹), are able to completely inhibit platelet deposition (thrombus volume), phosphatidylserine (PS) exposure, and / or fibrin deposition on surfaces coated with collagen and tissue factor (preferably 200 μg / ml and 500 pM for collagen and tissue factor, respectively), as determined using a flow adhesion assay suitable for coagulation (e.g., as described in the examples in Section 1.6).

[0239] According to a specific embodiment, the antibody or functional fragment of the present invention, at a concentration of 10 μg / ml, is subjected to flow under conditions of 1000 s⁻¹. -1In citric acid- and recalcified human blood at a shear rate of [specific value], it can completely inhibit thrombus formation (defined as relative thrombus volume), phosphatidylserine (PS) exposure, and fibrin deposition on surfaces coated with collagen and tissue factor (preferably 200 μg / ml and 500 pM, respectively), as determined in a flow adhesion assay suitable for coagulation, for example as described in the examples in Section 1.6. According to a specific embodiment of the invention, "complete inhibition" means a reduction of at least 85%, 90%, 93%, 95%, 97%, or 99% in relative thrombus volume / phosphatidylserine (PS) exposure / fibrin deposition compared to a negative control, for example as described in the examples in Section 1.6.

[0240] Typically, the antibodies or functional fragments of the present invention, when used on washed human platelets that are allowed to bind to the fibrinogen-coated surface, can significantly reduce the portion of stage 4 (fully spread) platelets in a spreading assay (as described in the examples in Section 1.8), while increasing the portion of stage 2 (platelets forming filamentous pseudopodia) to a similar degree.

[0241] According to a specific embodiment, when the antibody or functional fragment of the present invention is used at a concentration of 10 μg / ml in a spreading assay on washed human platelets that are allowed to bind to the fibrinogen-coated surface (e.g., as described in the example in Section 1.8), it is able to reduce the portion of stage 4 (fully spread) platelets by at least 30%, 35%, 40%, 45%, 50%, or 55% compared to a negative control, and increase the similarity of the portion of stage 2 (platelets forming filopodia).

[0242] Typically, the antibody or functional fragment of the present invention (preferably in monovalent form, such as Fab) is administered intravenously at a dose of 4 mg / kg to, for example, a humanized mouse model (hGP6) targeting GPVI. tg / tg Following administration to mice, significantly impaired CRP-induced circulating platelet activation was induced for at least 12, 24, 36, 48, 60, 72, 84, or 96 hours, as determined by flow cytometry analysis in ex vivo diluted blood. Preferably, fluorescently labeled antibodies capable of specifically binding to activated integrin αIIbβ3 and / or specifically binding to P-selectin were used, as illustrated in the examples in Section 1.10. In this context, “significantly impaired” preferably means a reduction in fluorescence signal of >50% 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 examples in Section 1.10.

[0243] According to a specific embodiment, the antibody or functional fragment of the present invention (in monovalent form, preferably Fab), when normalized with a negative control (e.g., a Fab control nonspecific to GPVI), is able to increase the binding to circulating platelets (preferably with an increase of ≥ 1.5-fold, more preferably ≥ 1.7-fold, ≥ 1.8-fold, or ≥ 1.9-fold) as measured 1 hour after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment or glomsimab, compared to ACT017 (grencizumab), for example determined by flow cytometry analysis in ex vivo diluted blood, for example with fluorescently labeled Fab-specific antibodies and using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, as described in the examples in Section 1.10.

[0244] According to another specific embodiment, the antibody or functional fragment of the present invention (in monovalent form, preferably Fab), when normalized with a negative control (e.g., a Fab control nonspecific to GPVI), is able to increase the binding to circulating platelets measured 3 hours after intravenous administration of, for example, 4 mg / kg of anti-GPVI antibody or functional fragment or glomenciumab (preferably with an increase of ≥ 2-fold, more preferably ≥ 2.5-fold, ≥ 3-fold, ≥ 3.5-fold, or ≥ 3.75-fold in GPVI receptor epitope binding), for example, by flow cytometry analysis in ex vivo diluted blood, for example with fluorescently labeled Fab-specific antibodies and using mean fluorescence intensity (MFI) as a measure of GPVI receptor epitope binding, as described in the examples in Section 1.10.

[0245] According to another specific embodiment, the antibody or functional fragment of the present invention (preferably in monovalent form, more preferably Fab) is administered intravenously at a dose of 4 mg / kg to, for example, a humanized mouse model (hGP6) targeting GPVI. tg / tg One hour after administration to mice, significantly impaired CRP-induced activation of circulating platelets was induced, as determined, for example, by flow cytometry analysis in diluted blood in vitro. Preferably, a fluorescently labeled antibody capable of specifically binding to activated integrin αIIbβ3 was used, such as as described in the examples in Section 1.10, while ACT017 (glencimab) did not induce significantly impaired CRP-induced activation. Optionally, “significantly impaired” means a reduction in fluorescence signal of >85% compared to the negative control, preferably >90%, >95%, >97%, >98%, >99%, or >99.5%.

[0246] According to a specific embodiment, the antibody or functional fragment of the present invention (preferably in monovalent form (e.g., Fab)) is capable of inhibiting GPVI dimerization, optionally without disabling or partially disabling ligand binding to GPVI. Generally, the antibody or functional fragment of the present invention (preferably in monovalent form (e.g., Fab)) is capable of inhibiting GPVI dimerization, optionally without disabling or partially disabling ligand binding to GPVI.

[0247] Typically, the antibodies or functional fragments of this invention provide sustained protection against occlusive thrombosis, such as arterial thrombosis, as exemplified by a humanized mouse model of occlusive arterial thrombosis (hGP6) targeting GPVI. tg / tg The results were determined by mice, as illustrated in the examples in Section 1.11, for instance.

[0248] According to a specific embodiment, the antibody or functional fragment of the present invention can inhibit the formation of stable thrombi at the site of arterial injury, preferably by reducing platelet activation at exposed extracellular matrix sites within the blood vessel and / or by effectively inhibiting local platelet-dependent coagulation, optionally as in a humanized mouse model of occlusive arterial thrombosis targeting GPVI (hGP6). tg / tg The results were determined by mice, as illustrated in the examples in Section 1.11, for example.

[0249] Typically, in vivo use of the antibodies or functional fragments (preferably Fab) of the present invention is associated with a low risk of bleeding, for example by not significantly increasing bleeding time, as determined by tail bleeding assays as described in the examples in Section 1.12.

[0250] In some embodiments, the antibody or functional fragment of the present invention has high target selectivity, i.e., it can distinguish between GPVI and structurally closely related proteins. Preferably, the IC50 of structurally closely related proteins... 50 Value ratio of GPVI IC 50 The value is at least 1,000 times greater, preferably at least 5,000 times greater, and more preferably at least 10,000 times greater, as determined in competitive BLI, ELISA, or SPR assays.

[0251] Typically, the antibodies or functional fragments (preferably Fab) of this invention exhibit high stability. Stability can be assessed using various methods. The "melting temperature" T of Fab is also relevant. mThis can be determined by differential scanning fluorometry (DSF). Stability can also be assessed by prolonged binding to GPVI on circulating platelets, as determined, for example, in the examples in Section 1.10. In one embodiment, the antibody or functional fragment of the present invention is capable of prolonged binding to circulating platelets, having a greater than 50% GPVI receptor epitope occupancy for at least 12, 24, 36, 48, 60, 72, 84, or 96 hours, when administered in vivo (e.g., at a dose ranging from 0.01 to 500 mg, preferably to a human subject), when normalized with a negative control (e.g., a control antibody or functional fragment nonspecific to GPVI), as determined, for example, in the examples in Section 1.10.

[0252] In one embodiment, the antibody or functional fragment of the present invention, when administered in vivo (e.g., at a dose ranging from 0.01 to 500 mg, preferably to a human subject), does not induce a decrease in platelet count, i.e., thrombocytopenia. In another embodiment, the antibody or functional fragment of the present invention, when administered in vivo (e.g., at a dose ranging from 0.01 to 500 mg, preferably to a human subject), does not induce a decrease in GPVI surface level on platelets.

[0253] Antibodies and functional fragments

[0254] 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, biantibodies, triantibodies, and tetraantibodies. Preferably, the functional fragment is monovalent, including but not limited to Fab, F(ab'), Fv, monovalent IgG, dsFv, and scFv. In a particularly preferred embodiment, the functional fragment is an antigen-binding fragment (Fab). More preferably, the non-CDR sequence of Fab is a human sequence.

[0255] 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 eukaryotic clone, prokaryotic clone, or phage clone), rather than the method of producing it. A variety of techniques known in the art can be used to prepare monoclonal antibodies, including hybridoma, recombinant, and phage display technologies, or combinations thereof. (Harlow and Lane, "Antibodies, A Laboratory Manual," CSH Press 1988, Cold SpringHarbor NY).

[0256] In other embodiments (including those involving the in vivo use of anti-GPVI antibodies or functional fragments thereof in humans), chimeric, primate-derived, humanized, or human monovalent antibodies and antibody fragments may be used. In a preferred embodiment, the antibody or functional fragment thereof is a human antibody or functional fragment thereof, or a humanized antibody or functional fragment thereof, more preferably a monoclonal human antibody or humanized antibody or functional fragment thereof, and even more preferably a monovalent monoclonal human or humanized antibody or functional fragment thereof.

[0257] As used herein, the term "chimeric" antibody or antibody fragment refers 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; US Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.

[0258] In some embodiments, the anti-GPVI antibody or a functional fragment thereof is a humanized antibody or a functional fragment thereof. Various recombinant methods are readily 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 (such as Fv, Fab, F(ab'), F(ab')2, or other target-binding sequences of the antibody), containing minimal sequences derived from such non-human immunoglobulins. Typically, the resulting recombinant antibody will contain substantially all, and usually both, variable domains, wherein all or substantially all of the CDR regions correspond to those of non-human immunoglobulins, and all or substantially all of the FR regions are human immunoglobulin sequences, particularly those common human immunoglobulin sequences. CDR-transplanted antibodies are antibody molecules having one or more complementarity-determining regions (CDRs) from antibodies originally generated in non-human species, which bind to the desired antigen and frame (FR) region from human immunoglobulin molecules (EP239400; PCT Publication WO 91 / 09967; US Patent Nos. 5,225,539; 5,530,101 and 5,585,089). Typically, in a process known as “humanization,” frame residues in the human frame region are additionally replaced with corresponding residues from the CDR donor antibody to alter, preferably, improve, antigen binding. These frame substitutions are identified by methods well known in the art, such as by mimicking the interaction between CDRs and frame residues to identify frame residues important for antigen binding and by sequence comparison to identify unusual frame residues at specific locations. See, for example, Riechmann et al., Nature 332:323-7 and Queen et al., US Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762 (each incorporated herein by reference in its entirety). A variety of additional techniques known in the art can be used to make antibodies more human, 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 tampering (US Patent No. 5,565,332), all of which are incorporated herein by reference in their entirety. CDR-transplanted or humanized antibodies may also contain at least a portion of the immunoglobulin constant region (Fc), typically at least a portion of a selected human immunoglobulin template.

[0259] In some embodiments, the humanized antibody or its functional fragment is prepared as described in Queen et al., US Patent Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370.

[0260] In some embodiments, the anti-GPVI antibody or a functional fragment thereof is a human antibody or functional fragment. A fully "human" anti-GPVI antibody may be desirable for therapeutic treatment of human patients. As used herein, "human antibody or functional fragment" includes antibodies having the amino acid sequence of human immunoglobulins, and includes antibodies isolated from a human immunoglobulin library or from animals genetically modified against one or more human immunoglobulins and not expressing endogenous immunoglobulins. Human antibodies can be prepared by a variety of methods known in the art, including the phage display method described above using an antibody library derived from a human immunoglobulin sequence. See US Patents 4,444,887 and 4,716,111; and PCT Publications WO 98 / 46645; WO 98 / 50433; WO 98 / 24893; WO 98 / 16654; WO 96 / 34096; WO 96 / 33735; and WO91 / 10741, each of which is incorporated herein by reference in its entirety. Human antibodies can also be generated using transgenic mice that do not express functional endogenous immunoglobulins but do express human immunoglobulin genes. See, for example, PCT Publications WO 98 / 24893; WO 92 / 01047; WO 96 / 34096; WO 96 / 33735; US Patent Nos. 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. A technique known as “guided selection” can be used to generate fully human antibodies that recognize selected epitopes. In this method, a non-human monoclonal antibody, such as a mouse antibody, is selected to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., 1988, Biotechnology 12:899-903).

[0261] In some embodiments, the anti-GPVI antibody or a functional fragment thereof is derivatized. For example, but not by any means limited, the derivatized antibody or functional fragment may be modified, such as through glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage with cellular ligands or other proteins (see the discussion of antibody conjugates below). Any of a variety of chemical modifications can be performed using known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, the derivative may contain one or more non-classical amino acids.

[0262] In other respects, anti-GPVI antibodies have one or more amino acids inserted into one or more hypervariable regions therein, such as those described in US 2007 / 0280931.

[0263] Antibody conjugates

[0264] In some embodiments, the anti-GPVI antibody or functional fragment is a modified antibody / antibody fragment conjugate, for example, by covalently attaching any type of molecule (e.g., an effector molecule) to the antibody such that the covalent attachment does not interfere with binding to GPVI. Techniques for conjugating the effector moiety to the antibody are well known in the art (see, for example, Hellstrom et al., Controlled Drag Delivery, 2nd ed., pp. 623-53 (Robinson et al., ed., 1987)); Thorpe et al., 1982, Immunol. Rev. 62: 119-58; and Dubowchik et al., 1999, Pharmacology and Therapeutics 83: 67-123).

[0265] In one example, an antibody or a functional fragment thereof (optionally at the N-terminus or C-terminus) is fused via a covalent bond (e.g., a peptide bond) to the amino acid sequence of another protein (or a portion thereof; preferably a portion of at least 10, 20, or 50 amino acids of a protein). In one embodiment, the antibody or a functional fragment thereof is linked to another protein at the C-terminus of a constant domain of the antibody or functional fragment. Recombinant DNA procedures can be used to generate such fusions, as described, for example, in WO 86 / 01533 and EP0392745. In another example, an effector molecule can increase the in vivo half-life. Examples of suitable effector molecules of this type include polymers, albumins, albumin-binding proteins, or albumin-binding compounds, such as those described in WO 2005 / 117984.

[0266] In some embodiments, the anti-GPVI antibody or a functional fragment thereof may be attached to the poly(ethylene glycol) (PEG) moiety. For example, if the antibody is an antibody fragment, the PEG moiety can be attached via any available amino acid side chain or terminal amino acid functional group (e.g., any free amino, imino, thiol, hydroxyl, or carboxyl group) located on the antibody fragment. Such amino acids may be naturally present in the antibody fragment or engineered into the fragment using recombinant DNA methods. See, for example, US Patent No. 5,219,996. Multiple sites may be used to attach two or more PEG molecules. Preferably, the PEG moiety is covalently linked via a thiol group located on at least one cysteine ​​residue in the antibody fragment. When the thiol group is used as an attachment site, a suitably activated effector moiety may be used, such as thiol-selective derivatives like maleimide and cysteine ​​derivatives.

[0267] In another example, the anti-GPVI antibody conjugate is a modified Fab or F(ab') fragment that is polyethylene glycolated, i.e., has a PEG (poly(ethylene glycol)) covalently linked to it, for example, according to the method disclosed 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 D. C, 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.

[0268] Pharmaceutical Compositions and Treatments

[0269] Treatment of disease encompasses the treatment of patients diagnosed with any form of disease at any clinical stage or presentation; the onset, evolution, aggravation, or deterioration of the symptoms or signs of disease; and / or prevention and / or reduction of the severity of disease.

[0270] The “subject” or “patient” to which the anti-GPVI antibody or a functional fragment thereof is administered can be a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys or humans). Preferably, the “subject” or “patient” is a human. In some respects, the human is an adult patient. In other respects, the human is a pediatric patient.

[0271] This article describes a pharmaceutical composition comprising an anti-GPVI antibody and optional pharmaceutically acceptable carriers and / or excipients, as well as optional one or more additional therapeutic agents. The composition is typically provided as part of a sterile pharmaceutical composition comprising a pharmaceutically acceptable carrier. The composition may be in any suitable form (depending on the desired method of administration to the patient).

[0272] Anti-GPVI antibodies and functional fragments can be administered to patients via various routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intrathecally, topically, or locally. Typically, the anti-GPVI antibody or its functional fragment will be administered intravenously to the patient. In a typical implementation, the anti-GPVI antibody or functional fragment is present in the pharmaceutical composition at a concentration sufficient to allow intravenous administration from 0.5 mg / kg body weight to 20 mg / kg body weight. In some embodiments, suitable concentrations for use in the antibodies or fragments of 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 ranging from any of the foregoing values, such as 1 mg / kg to 10 mg / kg, 5 mg / kg to 15 mg / kg, or 10 mg / kg to 18 mg / kg.

[0273] The effective dose of the anti-GPVI antibody or functional fragment can range from about 0.001 to about 750 mg / kg per single (e.g., bolus) administration, multiple administrations, or continuous administration, or to achieve a serum concentration of 0.01-5000 μg / ml per single (e.g., bolus) administration, multiple administrations, or continuous administration, or any effective range or value thereof, depending on the condition being treated, the route of administration, and the age, weight, and condition of the subject. In some embodiments, each dose can range from about 0.5 mg to about 50 mg per kilogram of body weight or from about 3 mg to about 30 mg per kilogram of body weight. The antibody can be formulated as an aqueous solution.

[0274] The pharmaceutical composition can conveniently be presented in unit doses containing a predetermined amount of anti-GPVI antibody or functional fragment per dose. Such units can comprise 0.5 mg to 5 g, for example, but 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 foregoing values, such as 10 mg to 1000 mg, 20 mg to 500 mg, or 30 mg to 300 mg. Pharmaceutically acceptable loads can take various forms, depending on, for example, the route of inclusion or administration to be treated.

[0275] The determination of the effective dose, total number of doses, and treatment length of the anti-GPVI antibody or its functional fragment is within the capabilities of those skilled in the art and can be determined using standard dose escalation studies.

[0276] Therapeutic formulations of anti-GPVI antibodies and functional fragments suitable for the methods described herein can be prepared for storage as lyophilized formulations or aqueous solutions by mixing antibodies or functional antibody fragments of desired purity with optional pharmaceutically acceptable loaders, excipients, or stabilizers (all of which are referred to herein as “loaders”), namely buffers, stabilizers, preservatives, isotonifiers, nonionic detergents, antioxidants, and a wide variety of other additives. See Remington’s Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives must be non-toxic to the recipient at the dosage and concentration used.

[0277] Buffers help maintain pH levels within a range close to physiological conditions. They can be present at concentrations from about 2 mM to about 50 mM. Buffers include organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate mixtures, trisodium citrate mixtures, monosodium citrate-citrate mixtures, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixtures, succinic acid-sodium hydroxide mixtures, disodium succinate-monosodium succinate mixtures, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixtures, tartaric acid-potassium tartrate mixtures, tartaric acid-sodium hydroxide mixtures, etc.), and fumarate buffers (e.g., fumaric acid-monosodium fumarate mixtures, fumaric acid-...). Examples of buffer solutions include: disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc.; gluconate buffers (e.g., gluconate-sodium gluconate mixtures, gluconate-sodium hydroxide mixtures, gluconate-potassium gluconate mixtures, etc.); oxalate buffers (e.g., oxalate-sodium oxalate mixtures, oxalate-sodium hydroxide mixtures, oxalate-potassium oxalate mixtures, etc.); lactate buffers (e.g., lactate-sodium lactate mixtures, lactate-sodium hydroxide mixtures, lactate-potassium lactate mixtures, etc.); and acetate buffers (e.g., acetate-sodium acetate mixtures, acetate-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts, such as Tris, can be used.

[0278] Preservatives can be added to slow microbial growth, and can be added in amounts ranging from 0.2% to 1% (w / v). Suitable preservatives include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyl dimethyl benzyl ammonium chloride, benzalkonium halides (e.g., chlorides, bromides, and iodides), hexamethylammonium chloride, and alkylparaben esters (such as methylparaben or propylparaben), catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonics, sometimes called "stabilizers," can be added to ensure the isotonicity of the liquid composition, and include polyols, preferably ternary or higher sugar alcohols, such as glycerol, erythritol, arabinol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients, ranging in function from fillers to additives that dissolve therapeutic agents or help prevent denaturation or adhesion to container walls. Typical stabilizers can be polyols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, inositol, galactitol, glycerol, etc., including cyclic alcohols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents. Stabilizers include urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight peptides (e.g., peptides with 10 or fewer residues); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone monosaccharides such as xylose, mannose, fructose, and glucose; disaccharides such as lactose, maltose, and sucrose; and trisaccharides such as raffinose; and polysaccharides such as dextran. Stabilizers may be present in quantities ranging from 0.1 to 10,000 units by weight per part of active protein.

[0279] Nonionic surfactants or detergents (also known as "wetting agents") can be added to help dissolve therapeutic agents and protect them from aggregation caused by stirring. This also allows the formulation to be exposed to shear surface stress without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), poloxamer (184, 188, etc.), Pluronic polyols, and polyoxyethylene sorbitol monoethers (Tween®-20, Tween®-80, etc.). Nonionic surfactants can be present in the range of about 0.05 mg / ml to about 1.0 mg / ml, or in the range of about 0.07 mg / ml to about 0.2 mg / ml.

[0280] Additional excipients include fillers (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), protease inhibitors, and cosolvents.

[0281] In addition to anti-GPVI antibodies or their functional fragments, the formulations described herein may also contain a second therapeutic agent.

[0282] Dosing regimens can vary from once a month to once daily, depending on a number of clinical factors, including disease type, disease severity, and patient sensitivity to anti-GPVI antibodies or functional fragments. In specific implementations, anti-GPVI antibodies or functional fragments thereof may be administered once daily, twice weekly, three times weekly, every other day, or every five days.

[0283] The dosage of the anti-GPVI antibody or functional fragment to be administered will vary depending on the specific antibody or functional fragment, the subject and the nature and severity of the disease, the subject's physical condition, the therapeutic regimen (e.g., whether a second therapeutic agent is used) and the chosen route of administration; the appropriate dosage can be readily determined by those skilled in the art.

[0284] Those skilled in the art will recognize that the optimal amount and interval of an individual dose of anti-GPVI antibody or a functional fragment thereof will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the age and condition of the specific subject being treated, and the physician will ultimately determine the appropriate dose to be used. This dose may be repeated frequently as needed. If side effects develop, the amount and / or frequency of the dose may be modified or reduced in accordance with normal clinical practice.

[0285] Diseases awaiting treatment

[0286] The present invention further relates to a method of treating or preventing GPVI-related conditions in a subject, comprising administering to the subject an antibody or functional fragment as defined herein. Furthermore, the present invention relates to an antibody or functional fragment as defined in any of the above embodiments, or a pharmaceutical composition comprising such an antibody or functional fragment, for use in a method of treating or preventing GPVI-related conditions in a subject.

[0287] As used herein, the terms “GPVI-related condition” or “GPVI-related disease” specifically and exclusively refer to such conditions: the onset, progression, or persistence of one or more symptoms or disease states involving the involvement of GPVI. Therefore, as used herein, “GPVI-related condition” or “GPVI-related disease” is any condition / disease whose one or more symptoms or disease states can be treated or prevented by (preferably functionally) inhibiting / blocking GPVI through the administration of an antibody capable of binding to GPVI. Exemplary GPVI-related conditions include, but are not limited to, thrombotic inflammatory diseases, cardiovascular diseases, cancer / cancer-related diseases, inflammation, and conditions associated with abnormal or aberrant megakaryocyte and / or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function. As used herein, these categories of GPVI-related conditions are not necessarily mutually exclusive and may overlap. Similarly, as used herein, exemplary conditions, diseases, symptoms, 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 implementation plan, GPVI-related conditions are thrombotic inflammatory diseases, cardiovascular diseases, inflammation, cancer / cancer-related diseases, and / or conditions associated with abnormal or abnormal megakaryocytes and / or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function.

[0288] Exemplary cardiovascular diseases include, but are not limited to, thrombosis and thrombotic conditions (such as arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic diseases [such as diseases involving leg swelling, pain, and ulcers, pulmonary embolism, abdominal venous thrombosis], thrombotic coronary artery occlusion, thrombotic microangiopathy, cancer-related thrombosis (Trusso syndrome), immune thrombosis and thrombosis associated with infection [e.g., cerebral malaria], restenosis, ischemic stroke, cerebrovascular disease, vascular purpura, and coronary artery disease [such as coronary artery disease (e.g., occlusive artery disease)]. Diseases, myocardial infarction, coronary revascularization, coronary restenosis, acute coronary syndrome, acute coronary syndrome, cardiac ischemia (including complications associated with coronary procedures such as percutaneous coronary angioplasty (balloon angioplasty), percutaneous coronary intervention), atherosclerosis, plaque formation, cerebral artery disease, ischemic events, unstable angina, acute cerebral ischemia (stroke), ischemic restenosis, acute ischemia, chronic ischemia, aortic and its branches disease, peripheral artery disease, acute phlebitis, pulmonary embolism, and any condition caused by vascular damage that can cause platelet aggregation.

[0289] According to a specific embodiment, GPVI-related conditions are cardiovascular diseases, preferably selected from the cardiovascular diseases described above. In the context of coronary artery surgery, such treatment can be achieved by administering the protein of the present invention before, during, or after the surgery. In a preferred embodiment, such administration can be used to prevent acute myocardial ischemia after angioplasty.

[0290] In another embodiment, GPVI-related conditions are thrombotic or thrombotic disorders, diseases exhibiting quantitative or qualitative platelet dysfunction, or diseases exhibiting endothelial dysfunction. These diseases include, but are not limited to, coronary and cerebral artery diseases. Generally, as used herein, thrombotic disorders can refer to conditions associated with the pathological formation of thrombi in veins (e.g., deep vein thrombosis [DVT]), arteries (e.g., myocardial infarction, ischemic stroke), or cardiac chambers. According to a preferred embodiment, GPVI-related conditions are thrombotic or thrombotic disorders (including, but not limited to, arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic disease, thrombotic coronary occlusion, thrombotic microangiopathy, cancer-related thrombosis, immune thrombosis, and thrombosis associated with infection (e.g., cerebral malaria)).

[0291] In another embodiment, GPVI-related conditions are conditions caused by any vascular injury that can cause platelet aggregation. As used herein, the term "vascular injury" includes, but is not limited to, vascular wall injuries, such as vascular injuries resulting in exposure to highly thrombosed surfaces within otherwise intact blood vessels, vascular wall injuries leading to the release of ADP, thrombin, and / or adrenaline, fluid shear stress occurring at sites of vascular stenosis, rupture and / or tearing of atherosclerotic plaque sites, and injuries resulting from balloon angioplasty or atherectomy.

[0292] According to another implementation, GPVI-related conditions are inflammation, i.e., inflammation or thrombotic inflammation, including but not limited to persistent or prolonged inflammation associated with infection [e.g., cerebral malaria], arthritis, fibrosis, acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury (IRI) of various organs (liver, colon, etc.), 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, sickle cell disease, ischemic restenosis due to bacterial and viral infections, sepsis, major trauma, autoimmune diseases, and conditions in which platelet-regulating cell function is involved, including but not limited to cancer cell proliferation and / or dissemination. As used herein, the term "thrombotic inflammatory disease" preferably refers to a disease involving the activating interactions of platelet / coagulation and immune system components. Conditions / diseases associated with the term "GPVI-related condition is inflammation or thrombotic inflammation" may also be "thrombotic inflammatory condition" or "thrombotic inflammatory disease." Similarly, cardiovascular diseases, for example, may be thrombotic inflammatory diseases. Autoimmune diseases include, but are not limited to, celiac disease, post-infectious IBS, type 1 diabetes, Henoch-Schönlein purpura (HSP), sarcoidosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, eosinophilic granulomatous 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).

[0293] According to another implementation plan, GPVI-related conditions are cancers (including but not limited to colon cancer, breast cancer, ovarian cancer, lung cancer, skin cancer such as malignant melanoma, metastatic cancer, etc.).

[0294] According to another implementation scheme, GPVI-related conditions are conditions associated with abnormal or abnormal megakaryocyte and / or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation and / or function.

[0295] In another embodiment, the antibody or functional fragment of the present invention, or the pharmaceutical composition as described above, is used to regulate, preferably prevent, platelet aggregation and degranulation. In another embodiment, the antibody or functional fragment of the present invention, or the pharmaceutical composition as described above, is used to regulate the immunomodulatory function of platelets. In another embodiment, the antibody or functional fragment of the present invention, or the pharmaceutical composition as described above, is used to treat conditions of the liver, bone marrow, and peripheral blood.

[0296] Preferably, the antibodies or functional fragments of the present invention are used to treat cardiovascular diseases selected from thrombosis and thrombotic conditions (such as arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic disease, thrombotic microangiopathy, cancer-related thrombosis [Trousso syndrome], immune thrombosis and thrombosis associated with infection [e.g., cerebral malaria]), restenosis, acute coronary syndrome, ischemic stroke, cerebrovascular disease and vascular purpura, coronary artery disease and cerebral artery disease, ischemic events, acute coronary syndrome, myocardial infarction (heart attack), acute cerebral ischemia (stroke), percutaneous coronary intervention, ischemic restenosis, acute ischemia, chronic ischemia, diseases of the aorta and its branches (such as aortic aneurysm, thrombosis), peripheral artery disease, acute phlebitis, and pulmonary embolism.

[0297] In certain preferred embodiments, the antibodies or functional fragments of the present invention are used for cardiovascular diseases selected from arterial or venous thrombosis, restenosis, acute coronary syndrome, or cerebrovascular accidents caused by atherosclerosis, preferably arterial or venous thrombosis. In any embodiment herein, patients being treated with anti-GPVI antibodies or their functional fragments may also be treated with another conventional medicine.

[0298] In a further aspect, the present invention relates to antibodies or functional fragments as defined in any of the above embodiments, or pharmaceutical compositions comprising such antibodies or functional fragments, for use in methods of treating or preventing thrombosis or thrombotic disorders in a subject. "Thrombosis or thrombotic disorders" is preferably one as defined above. In a further aspect, the present invention relates to antibodies or functional fragments as defined in any of the above embodiments, or pharmaceutical compositions comprising such antibodies or functional fragments, for use as antithrombotic drugs.

[0299] A further aspect of the invention is a method for treating GPVI-related conditions, comprising administering to a patient in need an effective amount of an anti-GPVI antibody or a functional fragment thereof as defined above. GPVI-related conditions are preferably one of the conditions described above. A further aspect of the invention is a method for treating or preventing thrombosis or thrombotic disorders, comprising administering to a patient in need an effective amount of an anti-GPVI antibody or a functional fragment thereof as defined above. "Thrombosis or thrombotic disorders" are preferably one as defined above. A further aspect of the invention is a method for preventing GPVI-related conditions, comprising administering to a patient in need an effective amount of an anti-GPVI antibody or a functional fragment thereof as defined above. GPVI-related conditions are preferably one of the conditions described above.

[0300] Table 1. Summary of amino acid sequences

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] * Amino acids designated as “X” have the meanings defined in the attached sequence listing. The underlined and grey residues in SEQ ID NO: 15-35 correspond to Kabat- and IMGT-based CDR residues, respectively. The bold residues in SEQ ID NO: 36 are binding residues on human GPVI. The underlined residues in SEQ ID NO: 37-58 are V... L or V H Region. Bold residues in SEQ ID NO: 26-35 and 49-58 indicate amino acid substitutions.

[0310] Example

[0311] 1. Materials and Methods

[0312] 1.1 Antibodies and Reagents

[0313] Horm collagen was purchased from Takeda (Linz, Austria); collagen-related peptide (CRP) was purchased from Cambridge Research Biochemicals (Cambridge, UK); ADP, adenosine triphosphate bisphosphatase (apyrase), prostacyclin (PGI2), fibrinogen, and hematoxylin were from Sigma Aldrich (Steinheim, Germany); thrombin was purchased from Roche Diagnostic (Mannheim, Germany); convulxin was purchased from Enzo Life Sciences (New York, NY, USA); rabbit anti-GAPDH and rat anti-mouse IgG-HRP antibodies were purchased from Sigma-Aldrich (Steinheim, Germany); U46619 was purchased from Alexis Biochemicals (Enzo Life Sciences, New York, NY, USA); anti-rabbit IgG-HRP was purchased from Jackson Immuno (Suffolk, UK); and goat anti-rat IgG-HRP was purchased from Dianova (Hamburg, Germany). Microcuvettes used for aggregation assays were purchased from LABITec (Ahrensburg, Germany). S-monovettes 3.2% citrate and Safety-Fly-Needle 21G were purchased from Sarstedt (Nümbrecht, Germany) for human blood collection. Heparin was purchased from Ratiopharm (Ulm, Germany); 5 mL polystyrene round-bottom tubes for flow cytometry were purchased from Corning Inc. (New York, NY, USA). Emf6 was generated in parallel with other Emf antibodies. 25 Following repeated subcloning, a high-yield single-clone subclone (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 is generated, purified, and derivatized internally.

[0314] Humanized anti-human GPVI Fab Glenzocimab (code: PX-TA1552-1000, also known as ACT017) was purchased from ProteoGenix (Schiltigheim, France).

[0315] 1.2 Blood Donors and Blood Collection

[0316] Blood was collected from healthy volunteers who had not received anticoagulant or antiplatelet therapy for at least 4 weeks. Blood samples were obtained with written informed consent in accordance with the Declaration of Helsinki and approved by the Institutional Review Committee of the University of Würzburg. Blood was drawn via venipuncture using a butterfly needle and collected in 9 mL tubes containing 3.2% trisodium citrate. For all studies, the blood was kept at room temperature and used within 4 hours. All methods were performed in accordance with relevant guidelines and regulations. Blood was drawn via venipuncture using a butterfly needle and collected in 9 mL tubes containing 3.2% trisodium citrate. For all studies, the blood was kept at room temperature and used within 4 hours.

[0317] 1.3 Animals

[0318] Animal experiments were approved by the district government of Lower Franconia (Regierung von Unterfranken) and conducted in accordance with the current Animal Research: Reporting of In Vivo Experiments guidelines (https: / / arriveguidelines.org / ). The age, sex, and genetic background of the mice used were matched. All animal experiments described in this study used the previously described humanized GPVI mouse strain (hGP6). tg / tg (This was done) 32

[0319] 1.4 Cleansed human and mouse platelets

[0320] Human washed platelets were obtained as follows: Citrated blood was collected in 10 ml S-monovette and administered with 2 ml ACD pH 4.5. The sample was centrifuged at 300 g for 20 min at room temperature. Platelet-rich plasma (PRP) was collected in a fresh 15 ml falcon and supplemented with 1 / 10 ACD, 2 μL adenosine triphosphate bisphosphatase / ml (0.02 U ml−1; A6410, Sigma-Aldrich), and 5 μl PGI2 / µL (0.1 μg ml−1; P6188, Sigma-Aldrich). Platelets were centrifuged at 500g for 10 min to precipitate them. They were washed twice with Tyrode buffer (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) containing 2 μL adenosine triphosphate bisphosphonate (APT) and 5 μL PGI2. Finally, the platelets were resuspended in Tyrode buffer at a concentration of 500,000 / μL and incubated at 37°C for 30 min before use. For experiments, platelets were recalcified using calcified Tyrode buffer.

[0321] Cleansed platelets from rodents were obtained as follows: Whole blood was collected via retroorbital hemorrhage in heparin after isoflurane anesthesia. The heparinized blood was centrifuged at 300 g for 6 min. The obtained PRP was supplemented with 2 μL of adenosine triphosphate bisphosphatase and 5 μL of PGI2, similar to human platelets. Next, the platelets were precipitated by centrifugation at 800 g for 5 min and washed twice with Tyrode buffer (using 2 μL of adenosine triphosphate bisphosphatase and 5 μL of PGI2). Finally, the platelets were resuspended as human platelets and incubated at 37°C for 30 min before use.

[0322] 1.5 Flow Adhesion Assay (Whole Blood Perfusion Assay)

[0323] Evaluation of platelet adhesion and aggregate formation (thrombosis) of Horm collagen in heparinized human blood treated with 1, 2, 5, or 10 µg / mL anti-GPVI antibody or Fab, or control antibody or Fab, under flow conditions. 200 μg / mL Horm collagen was coated onto coverslips at 37°C for 1 hour and then blocked with 1% BSA in PBS 1x. Heparinized human or mouse blood was diluted 1:2 in Tyrode buffer and treated with 2 mM Ca2+. 2+Additional information: Mouse platelets were labeled with an antibody conjugated to anti-GPIX-Dylight 488, while anti-GPIX-Dylight 488 antibody p0p1 conjugated to Dylight 488 was used. 35 Label human platelets. Blood was pumped at 1000 s... -1 The shear rate was controlled by perfusion on coverslips for 4 min, followed by washing with calcium-supplemented Tyrode buffer for another 4 min. After the washing step, eight representative fields of view were imaged using a Leica DMI 6000B microscope (Leica Biosystems Technologies, Frankfurt, Germany) with 63x objectives. Finally, the overall platelet surface coverage and relative thrombus volume of the images were analyzed using Fiji.17 (integral fluorescence density).

[0324] 1.6 Coagulation Flow Chamber

[0325] Horm collagen + tissue factor was used to assess platelet adhesion, thrombosis, phosphatidylserine exposure, and fibrin deposition in recalcified human blood treated with, for example, 10 µg / mL anti-GPVI antibody or Fab, or control antibody or Fab, under flow conditions. To analyze thrombus formation under coagulation conditions in vitro under flow conditions, coverslips were freshly coated with 50 μg / mL Horm collagen at 37°C for 1 hour, followed by a second incubation with 500 pM tissue factor in a humidified chamber for 1 hour. The coverslips were then blocked with PBS (1x 1% BSA) at RT for 30 min. For in situ blood recalcification, citrated blood and recalcification buffer (Hepes buffer solution of 32 mM MgCl2 and 63 mM CaCl2, pH 7.45) were pumped into a chamber containing blocked coverslips using Y-tubes. Blood was pumped at 1000 s⁻¹. -1 The shear rate was measured and infused onto a coverslip. Images were taken every 30 seconds using a LEICA DMI6000B microscope with a 63x objective lens (Leica Biosystems Technologies, Frankfurt, Germany). Anti-GPIbβ antibody p0p1-A647 was used. 35 Human platelets were labeled, and PS exposure was observed using internally generated annexin 5-A546, while fibrin deposition was observed by adding fibrinogen-A488.

[0326] 1.7 Light transmission aggregation method (turbidity aggregation method)

[0327] Aggregation response of washed human platelets treated with 1, 2, 5, or 10 µg / mL anti-GPVI antibody or Fab, or control antibody or Fab, at 37°C. Washed human or mouse platelets were then supplemented with 2 mM Ca.2+ Diluted with 100 μg / mL human fibrinogen in Tyrode buffer (recalcified). When thrombin was used as an agonist, the Tyrode buffer was not supplemented with fibrinogen. A light transmission aggregation assay was performed to track platelet aggregation over time for 10 min under stirring conditions using a 4-channel APACT agglutinometer (LABITec Ahrensburg, Germany) 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), and the transmission was recorded.

[0328] 1.8 Spreading Measurement

[0329] Washed platelets were pre-incubated with Fab fragments (e.g., with 10 μg / mL anti-GPVI Fab or control Fab) and then further diluted to 100,000 / μL, and then pipetted onto a 100 μg / mL fibrinogen-coated surface. Platelets were allowed to spread at 37°C for 45 min. Next, coverslips were fixed with 4% PF4 for 10 min. Spreading platelets were observed using a ZEISS Axiovert microscope (Zeiss group, Oberkochen, Germany) with 100x objectives. Images were analyzed using the Fiji.17 cell counter tool, and platelet abundance was determined by differentiating platelets into four stages based on diffusion: Stage 1: Adhesion; Stage 2: Filamentous pseudopodia formation; Stage 3: Lamellar pseudopodia formation; Stage 4: Fully spread platelets.

[0330] 1.9 Platelet count and size measurement

[0331] To determine platelet count and size, hGP6 tg / tg Mice were intravenously administered 4 mg / kg of anti-GPVI Fab or control Fab, and their peripheral blood platelets were monitored for 5 days using automated cell analysis. To assess platelet count and size, mice were exsanguinated into EDTA-coated tubes at specific time points following 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 samples from mice treated with control Fab, which is non-GPVI specific, at the same time points.

[0332] 1.10 Platelet GPVI surface level, binding of the anti-GPVI antibody or Fab of the present invention to GPVI on platelets. Flow cytometry analysis of platelet activation

[0333] To determine in vivo GPVI surface levels, anti-GPVI Fab binding, and platelet activation on circulating platelets, hGP6 was used. tg / tg Mice were administered anti-GPVI Fab (Emf6.1) intravenously at a dose of 4 mg / kg. Fab (or control Fab) and monitor peripheral blood platelet counts for 5 or 6 days using flow cytometry and automated cell analysis. For use with Emf-2 FITC (Used to test GPVI exposure, i.e., the GPVI surface level on circulating platelets) and Emf-3 FITC (For assessing epitope saturation of the anti-GPVI antibody or Fab of the present invention on circulating platelets) will be detected, in the absence of Ca 2+ Mouse blood diluted 1:20 in Tyrode buffer was incubated with antibody for 10 min.

[0334] In another set of experiments, hGP6 tg / tg Mice were administered 4 mg / kg EMA601 (the humanized Fab of this invention) or glomsimab (ACT017) intravenously, and their peripheral blood platelet counts were monitored for 24 hours by flow cytometry and automated cell analysis. This was to facilitate the use of Emf-2... FITC (used to test GPVI surface expression) and Emf-3 FITC Or JAQ1 FITC (To assess epitope saturation of EMA601 and glomcizumab on circulating platelets, respectively) will be tested in Ca-free... 2+ Mouse blood diluted 1:20 in Tyrode buffer was incubated with the antibody for 10 min. To detect surface-bound EMA601 or glomsizumab, an anti-human IgG (Fab-specific)-FITC antibody (Sigma Aldrich, F5512) was used.

[0335] To perform platelet activation analysis, mouse blood was incubated with 2 mM Ca2+. 2+ Diluted in Tyrode buffer. JON / A-PE (Emfret Analytics, Eibelstadt, Germany) was used to detect activated integrin αIIbβ3, while P-selectin exposure was used as a marker of platelet degranulation and detected with FITC-conjugated specific anti-mPselectin antibody WUG1.9. 34 Mouse blood was drawn at specific time points following administration of anti-GPVI Fab or control Fab (as described in Section 1.2), diluted, and then mixed with CRP (0.5 μg / mL), thrombin (0.1 U / mL), or a mediator solution, as well as JON / A-PE and anti-Psel. FITCIncubate together for 12 min (6 min at 37°C and 6 min at RT). Finally, further dilute the blood in 500 μL PBS to allow for MFI measurement using FACSCelesta (BD Biosciences, Franklin Lakes, New Jersey, USA). To determine the GPVI surface level on circulating platelets / binding of the anti-GPVI antibody or Fab of the present invention to GPVI on platelets / platelet activation (activated integrin αIIbβ3, P-selectin exposure), the corresponding MFI values ​​from samples from mice treated with anti-GPVI Fab were normalized using samples from control Fab-treated mice at the same time points that are not specific to GPVI.

[0336] 1.11 In vivo model of thrombosis

[0337] Mice were anesthetized via intraperitoneal exposure, and their abdominal aortas were treated (e.g., 1 hour after injection of 4 mg / kg anti-GPVI Fab or control Fab). The aorta was separated from the vena cava by removing the fat layer, and an ultrasonic flow probe (0.5 PSB699; Transonic Systems, USA) was placed around the abdominal aorta. Thrombosis was induced by compressing the aorta for 5 seconds (buck setting 1) with forceps (Ultra Fine Hemostats clams, Fine Science Tools, Vancouver, Canada) upstream of the flow probe. Blood flow was monitored for 30 min or until vascular occlusion occurred (blood flow interruption > 5 min). The significance of occluded and non-occluded vessels was statistically assessed using Fisher's exact test.

[0338] 1.12 Tail hemorrhage measurement

[0339] After anesthetizing the animals, a 2 mm section of the tail tip was removed using a scalpel. Tail bleeding was monitored by gently absorbing blood with filter paper at 20-second intervals without direct contact with the wound site. Bleeding was considered to have stopped when no blood was observed on the paper. The experiment was manually stopped by cauterization after 20 minutes. Fisher's exact test was used to statistically assess the difference between occluded and non-occluded wounds, and the mean bleeding time was assessed using the Mann-U-Whitney test.

[0340] 1.13 Automated μSPOT Synthesis

[0341] The extracellular domain of GPVI (residues 24-267 of UniProtKB: Q9HCN6-1; corresponding to SEQ ID NO: 36) is displayed as a 15-mer overlapping peptide with a 3-residue shift in the form of a microarray. The peptide array was synthesized on a cellulose disk containing a 9-fluorenylmethoxycarbonyl-β-alanine (Fmoc-β-Ala) linker (average loading: 130 nmol / disk—4 mm diameter) using a MultiPep RSi robot (CEM GmbH, Kamp-Lintford, Germany). 36 Synthesis was performed by deprotecting the Fmoc group using a 20% piperidine solution in dimethylformamide (DMF). The peptide chain was extended using a coupling solution consisting of amino acids (0.5 M) in DMF with an oxime (1 M) and diisopropylmethanediimide (1 M) (1:1:1). The coupling step was performed three times (30 min each time), followed by end-capping (4% acetic anhydride in DMF). The cleavable peptide was coupled with an acid-unstable linker (Fmoc-rink-amide) to ensure cleavage from the cellulose support.

[0342] Cellulose discs were transferred to 96-well plates for peptide post-processing. First, the protected side chains were removed for 1 h at room temperature (RT) using a solution of 90% trifluoroacetic acid (TFA), 2% dichloromethane (DCM), 5% H₂O, and 3% triisopropylsilane (150 μL / well). Then, the deprotection solution was discarded, and the discs were dissolved overnight at RT (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 diethyl ether (700 μL / well) and rotated at 2000 × g for 10 min at 4°C, followed by two additional washes with ice-cold diethyl ether. The resulting precipitate was dissolved in DMSO (250 μL / well). The PCC solution was mixed with saline-sodium citrate buffer (150 mM NaCl, 15 mM trisodium citrate, pH 7.0) at a 2:1 ratio and transferred to a 384-well plate. A SlideSpotter (CEMGmbH) was used to transfer the PCC solution onto white-coated CelluSpot blank slides (76 × 26 mm, Intavis AG Peptide Services GmbH and CO. KG). After the printing process was complete, the slides were allowed to dry for at least 3 h. The cleavable peptides were post-processed from the supernatant of the cleavage mixture and precipitated in the same manner. The peptide precipitate was resuspended in water.

[0343] 1.14 Microarray Binding Measurement

[0344] Microarray slides were blocked for 60 min 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) blocking buffer for 30 min, followed by washing three times with PBS for 1 min each. Antibody binding was detected using goat anti-mouse IgG-HRP (Thermo Fisher Cat. No. 31430, 1:5000). Chemiluminescence readings were detected using the Azure imaging system c400 (lowest sensitivity) with the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific GmbH, Schwerte, Germany). Epitope neutralization was achieved by pre-incubating Emf6.1 with cleavable peptides for 30 min and then applying it to the blocked slides.

[0345] The binding intensity of microarrays was quantified using FIJI with the "microarray profile" plugin (OptiNav Inc, Bellevue, WA, USA). After subtracting the background average grayscale value surrounding the microarray surface, the original grayscale intensity at each location on the left and right sides of the internal duplicate on each microarray slide was obtained. The standard deviation (STDEV) between the two sides was calculated.

[0346] 1.15 Antibody Humanization

[0347] The variable domains of Emf6.1 mice were sequenced to identify the complementarity-determining region (CDR) and transplanted into the appropriate human receptor framework, as described in Section 2.2.1 below. 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 apparatus, as described in Section 2.2.2 below.

[0348] 1.16 Kinetic Analysis (KD)

[0349] Determining the dissociation equilibrium constant (K) using biolayer interference (BLI). D Initially, a series of tests were conducted to optimize the measurement parameters, resulting in the following parameters:

[0350] Table 2: Summary of optimized kinetic screening parameters for characterizing IgG:antigen interactions.

[0351]

[0352] Human GPVI (1.2 μg) was immobilized on the tip of a biosensor using a suitable capture surface, and K-cell binding was monitored via BLI using the Octet instrument and the binding of Emf6.1 variants (i.e., humanized Fab fragments and chimeric Fab fragments HC0 LC0, which contain the Emf6.1 mouse variable domain and the human Ig constant domain) was performed. D Measurements. Analyze the resulting sensor maps using the provided software (Fortebio). Association / dissociation time (s): 900 / 1200; Antibody screening range (using seven concentrations with 3-fold serial dilutions): 10⁻⁴ - 0.014 nM; Interaction / fit model: 1:1. Dilute all samples in freshly prepared run buffer.

[0353] K-cell antigens captured on a biosensor were used for K-cell assay. D Measurements. The antigen-captured biosensor (i.e., the tip) was immersed in wells containing different concentrations of the Emf6.1 variant (association phase), followed by a dissociation step in running buffer. To allow for reference calibration, the antigen-captured sensor was immersed in wells containing only buffer (reference wells). This reference provides a means of compensating for the natural dissociation of the captured antigen. The steps were performed at a constant orbital flow rate of 1000 rpm at 25°C (generated by moving the sample in the well of the multi-well plate relative to the biosensor in orbital motion). A fresh sensor was used for each sample. A series of dilutions of the Emf6.1 variant was used in the association step to globally fit the results and obtain ka, kd, and K. D The optimal value was determined using ForteBio data analysis software. The dissociation equilibrium constant (K0) was calculated. D The combined response data was fitted to a 1:1 interaction / binding model. All consumables used were those recommended by ForteBio.

[0354] 2. Results

[0355] 2.1 Example 1: Generation and testing of mouse IgG antibodies against human GPVI

[0356] 2.1.1 Emf6.1 binds to human GPVI and blocks its function.

[0357] A series of monoclonal mouse IgG antibodies (Emf antibodies) against human GPVI were generated using standard hybridoma technology, and 16 clones (Emf1-Emf16) were isolated based on their specific binding to the extracellular domain of human GPVI. Flow cytometry analysis showed that only 9 of these also bound to human platelets, i.e., membrane-expressing GPVI, including Emf1, Emf2, Emf3, and Emf6.1. The ability of these clones to inhibit GPVI function was further characterized. In fact, three of these monoclonal antibodies (mAbs) inhibited both CRP-induced and collagen-induced human platelet responses (Emf1...). 25 Emf2 32 Among them, Emf6.1 (mouse IgG2aκ) consistently showed the most potent inhibitory effect. Emf6.1 IgG (10 µg / mL) eliminated CRP-induced and collagen-induced human platelet aggregation, while showing no effect on responses to other agonists, including thrombin and the stable thromboxane A2 analog U46619. Figure 1 A). Furthermore, when used at concentrations of 10, 5, 2, and 1 µg / mL, Emf6.1 effectively blocked flow (1000 s). -1 The formation of heparinized human platelets aggregated on collagen in the blood. Figure 1 (B, C) To avoid the possible Fc-mediated effects of Emf6.1 IgG via platelet-expressed Fc γ receptor IIA (FcγRIIA, CD32), and to exclude Fc-dependent thrombocytopenia and / or GPVI reduction. 19 The Emf6.1 Fab fragment was generated and used in all further studies.

[0358] 2.1.2 Emf6.1 Fab Inhibition of GPVI-dependent activation of human platelets

[0359] To determine Emf6.1 Fab The in vitro efficacy in reducing GPVI-dependent platelet adhesion and aggregate formation was tested on anticoagulated human blood on a collagen-coated surface under flow conditions. At 10 μg / mL, Emf6.1 Fab In 1000 s -1 At shear rates of [specific value], human platelet adhesion and aggregate formation were almost completely eliminated. Figure 9 A). It is worth noting that at lower concentrations of Emf6.1 Fab Aggregate formation was also significantly inhibited at (5, 2, and 1 μg / mL), with a 75% reduction in thrombus volume compared to the control Fab at 1 μg / mL. Figure 9A). Next, citric acid-modified human blood was mixed with 10 μg / mL Emf6.1 Fab Alternatively, pre-incubate at Fab and then test for thrombosis in a flow chamber under active coagulation conditions. It is noteworthy that under these conditions, Emf6.1 Fab It effectively inhibited platelet deposition and thrombus formation on collagen / TF-coated surfaces. Figure 1 DF), and completely eliminated phosphatidylserine (PS) exposure and fibrin deposition ( Figure 1 D, GH).

[0360] Next, use 10 μg / mL Emf6.1 Fab Cleaned human platelets were processed and then tested using standard aggregation assays with different agonists. Emf6.1 Fab The elimination of CRP-induced and collagen-induced aggregation, while the thrombin-induced response remained unchanged, confirms the GPVI-specific inhibitory effect of Fab. Figure 1 I). Similar to the results of flow adhesion, at 5, 2, and 1 μg / mL Emf6.1 Fab Significant inhibition was observed in the aggregation assay ( Figure 9 B). Human GPVI has been shown to contribute to platelet activation and spreading on fibrinogen. 40, 41 To test Emf 6.1 Fab Whether this GPVI function is interfered with allows human platelets to adhere to immobilized fibrinogen in the presence or absence of Fab. In fact, Emf6.1 Fab (10 μg / mL) potently inhibits complete platelet spreading, as revealed by the sharp decrease in stage 4 (complete spreading) platelets and the significant increase in stage 2 (platelets forming filamentous pseudopodia). Figure 1 JK).

[0361] In summary, these data show that Emf6.1 Fab It effectively inhibits GPVI-dependent activation, aggregation, and diffusion of human platelets in vitro.

[0362] 2.1.3 Emf6.1 Fab Inhibition of GPVI-mediated hGP6 tg / tg Platelet activation

[0363] In order to study Emf6.1 Fab The in vivo effects were assessed using the recently developed GPVI humanized mouse line (hGP6). tg / tg ) 32 First, Emf6.1 was tested in whole blood flow cytometry. FabFor hGP6 tg / tg The agonist-induced activation effect on platelets. Emf6.1 Fab (10 μg / mL) eliminated CRP-induced activation, but had no effect on responses to other agonists such as thrombin, ADP, and U46619. Figure 10 (AB). Similar to the results for human platelets, Emf6.1 Fab hGP6 was completely inhibited in whole blood perfusion assays. tg / tg Platelet aggregation on collagen ( Figure 2 AC), and at 2 μg / mL Emf6.1 Fab The same effect was observed at the same concentration. Figure 10 D). In addition, Emf6.1 Fab (10 or 2 μg / mL) almost completely inhibited GPVI-mediated hGP6. tg / tg Platelet aggregation ( Figure 2 D and Figure 10 C). It has been previously shown that mouse platelets expressing human GPVI, rather than wild-type platelets, can spread completely on fibrinogen. 32, 42 Therefore, Emf6.1 was also tested. Fab Does it interfere with this response? Actually, hGP6 tg / tg Platelets successfully spread on the fibrinogen-coated surface, and the observation was similar to that of human platelets, a process observed in Emf 6.1. Fab Eliminated under the condition of existence ( Figure 2 EF).

[0364] In summary, these results show that Emf6.1 Fab For people and hGP6 tg / tg The in vitro effects of mouse platelets were comparable, indicating that the mouse strain was suitable for studying Emf6.1. Fab A suitable model system for in vivo effects.

[0365] 2.1.4 Emf6.1 Fab Highly efficient ex vivo blocking hGP6 tg / tg GPVI function in mice

[0366] In order to study Emf6.1 Fab The in vivo effects of hGP6 tg / tg Mice were administered a dose of 4 mg / kg intravenously, and their peripheral blood platelets were monitored for 5 days by flow cytometry and automated cell analysis. At all test intervals, platelet count and size remained unchanged compared to the control group. Figure 3 AB), and no change in the GPVI surface level on circulating platelets could be detected ( Figure 3C). Next, the anti-GPVI mAb (Emf3) with a FITC-tagged anti-Emf6.1 binding site was evaluated over time. Fab GPVI occupancy in circulating platelets. After a single injection of 4 mg / kg bwEmf6.1 Fab A significant GPVI occupancy rate was detected after 96 hours. Figure 3 D). Specifically, one hour after injection, approximately 90% of the binding epitopes on GPVI were converted to Emf6.1. Fab The percentage of platelet activation was approximately 76% at 24 hours, approximately 50% at 48 hours, approximately 36% at 72 hours, approximately 19% at 96 hours, and approximately 13% at 120 hours. This sustained GPVI blockade was confirmed by in vitro flow cytometry analysis of CRP-induced platelet activation in diluted blood. Compared with the control group, after injection of 4 mg / kg bw emf 6.1 Fab Emf6.1 was detected after 72 hours. Fab Significantly impaired response to CRP, but not thrombin, in treated mice ( Figure 3 EH).

[0367] Based on these results, after injection of 4 mg / kg bw Emf 6.1 Fab In contrast to the non-control Fab, a significant inhibitory effect on CRP-induced platelet aggregation was observed in vitro for up to 72 hours. Figure 4 A). Additionally, using Emf6.1 Fab Treatment effectively inhibited collagen-induced aggregation, showing a significant reduction at 1 and 24 hours post-treatment. Although significant GPVI inhibition was still observed at 48 h, it further decreased after 72 h. Figure 4 B). As expected, thrombin-induced aggregation remained unchanged at all test times. Figure 4 C). Next, Emf6.1 was evaluated in an isolated whole blood flow adhesion assay. Fab The effect of treatment on GPVI-dependent aggregate formation on collagen-coated surfaces. Also here, up to 48 hours post-injection, compared to the control Fab treatment, at Emf 6.1... Fab hGP6 processing tg / tg A significant reduction in surface coverage and thrombus volume was observed in mice. Figure 4 DE).

[0368] In summary, these data indicate that using Emf 6.1... Fab Treatment of mice expressing human GPVI resulted in a significant and sustained inhibition of GPVI-dependent platelet activation in vitro.

[0369] 2.1.5 Emf6.1 Fab It exhibits high in vivo protective activity in arterial thrombosis models.

[0370] To evaluate the results of Emf6.1 Fab The antithrombotic potential of GPVI blockade was demonstrated using an arterial thrombosis model in which the abdominal aorta was mechanically injured and blood flow / occlusive thrombosis was monitored using an ultrasound flow probe. 43, 44 It is worth noting that Emf 6.1 Fab The treated mice (1 hour after injection of 4 mg / kg bw) were strongly protected from thromboembolic occlusion. Figure 5 A) Of these, 91.7% (11 out of 12) did not develop stable thrombi within the 30-minute observation period, while 100% of the control group showed occlusion. In most cases of Emf6.1 Fab In the treated mice, a transient reduction in blood flow occurred, indicating the formation of initial aggregates, followed by rapid disaggregation or embolization of the formed thrombus. Figure 5 B).

[0371] Finally, the Emf6.1 model of tail hemorrhage was evaluated. Fab The effect of treatment on trauma-induced hemostasis. hGP6 compared to the control Fab-treated hGP6. tg / tg Compared to WT mice treated with Fab, in Emf6.1 Fab hGP6 processing tg / tg No significant difference in tail hemorrhage time was detected in mice. Figure 5 C). In summary, these data demonstrate the effectiveness of Emf 6.1. Fab Treatment effectively protects hGP6 tg / tg Mice were spared from arterial thrombosis without significantly affecting tail bleeding time, i.e., normal hemostasis.

[0372] 2.1.6 Emf6.1 binds to the proposed GPVI dimerization site

[0373] To determine the epitope of Emf6.1 in human GPVI, a 15-mer overlapping peptide library covering the extracellular domain of hGPVI (residues 24 to 267 of UniProtKB Q9HCN6-1) was synthesized and printed as a microarray. GPVI peptide microarray binding to Emf6.1-IgG was detected using an HRP-labeled anti-mouse IgG antibody. Epitope mapping revealed a clear, discontinuous binding epitope located between D2 and the transmembrane helix. Figure 6 A), more precisely, on residues Val201 to Glu215 and Ser246 to Pro260 ( Figure 6A). The key contributions of the two binding surfaces to Emf6.1 binding were verified by neutralizing Emf6.1 with the corresponding soluble peptides. Figure 6 B). Notably, the Emf6.1 binding epitope is located at the C-terminus of all GPVI epitopes reported to date (residues 58 to 187), and therefore also at the C-terminus of structurally resolved collagen binding sites (Trp76, Arg38, and Glu40). Figure 6 C). Therefore, this plot shows that Emf6.1 suppresses GPVI function via a non-canonical mechanism associated with GPVI dimerization, which was structurally resolved early and located at the overlapping surface (Asp196-Thr203). 18 ( Figure 6 C). 17

[0374] Therefore, mapping and neutralization based on peptide microarrays 49-51 The non-canonical discontinuous binding sites of Emf6.1 in GPVI (Val201 to Glu215 and Ser246 to Pro260) were confirmed to be located between D2 and the transmembrane helix. They do not overlap with the collagen binding interface in D117, but rather with the proposed GPVI dimerized residues (Val201-Thr203). 18 Based on this discovery, it can be inferred that Emf6.1... Fab / EMA601 may not be as previously described for other GPVI antagonists. 21, 52, 53 It primarily blocks GPVI function by interfering with ligand binding, and possibly by modulating its aggregation / signal transduction capabilities.

[0375] 2.2 Example 2: Generation and Characterization of Humanized Emf6.1 Fab Variants

[0376] 2.2.1 Generation of a fully humanized Emf6.1Fab variant

[0377] Given Emf6.1 Fab To investigate the significant GPVI-inhibiting activity of Emf6.1, a set of humanized Fab fragments derived from the Emf6.1 sequence was designed. The aim was to create non-immunogenic monovalent antibody fragments suitable for injection into humans and capable of retaining specific GPVI-binding activity. The Emf6.1 mouse variable domain was sequenced, and canonical and subclass complementarity-determining regions (CDRs) were identified. 37, 38 The mouse V was identified using a combination of the IMGT and Kabat numbering systems. H and V L CDR residues in the domain. 37, 38

[0378] Mouse Emf6.1 V L and V HThe domain has the following sequence, which does not include the mouse signal peptide sequence:

[0379] >Primitive mouse Emf6.1 V L (VL0)

[0380]

[0381] >Primitive mouse EMF 6.1 V H (VH0)

[0382]

[0383] Identification using the IMGT numbering system The highlighted CDR residues were identified using the Kabat numbering system. Down Draw lines The CDR residues identified are summarized in Table 3 below.

[0384] Table 3: The following sequence data for Emf6.1 CDR were obtained:

[0385]

[0386] 1 The amino acid designated as “X” has the meaning as defined in the attached sequence listing.

[0387] The closest human genotype V H The region is Homo sapiens IGHV4-4. The BLAST search algorithm was used to search databases of human IgG and human IgK sequences to compare them with mouse V... H and V L The domains were compared, and suitable candidates were selected based on combinations of framework homology, maintenance of key framework residues, and canonical loop structures. This was achieved by comparing mouse V... H and V L The CDRs are transplanted into these receptor frameworks to generate humanized variants.

[0388] VL1 (based on BAH04725)

[0389]

[0390] VL2 (based on AIT38570)

[0391]

[0392] VL3 (based on AMK70118)

[0393]

[0394] VL4 (based on APZ85375)

[0395]

[0396] VH1 (based on AAV40414)

[0397]

[0398] VH2 (based on AAY23326)

[0399]

[0400] VH3 (based on AEX28400)

[0401]

[0402] VH4 (based on AAD31716)

[0403]

[0404] VH5 (based on IGHV4-4)

[0405]

[0406] exist Figure 7 In A and B, the mouse EMF was 6.1 V. L and V H The sequences (VL0 and VH0) have been associated with humanized V L Variant V H Variant alignment. According to the hierarchy of homology, the humanized variants are VL1>VL4>VL2>VL3. According to the hierarchy of homology, the humanized variants are VH5>VH1>VH2>VH4>VH3.

[0407] V H and V L Each of the domains was synthesized within the constant domain sequence frames of human IgG1 and human IgK isotypes, respectively, and cloned into the mammalian transient expression plasmid pETE V2. Humanized variants were examined to determine if they had been humanized according to the WHO definition of humanized antibodies: the variable domains of the humanized chains had the V region amino acid sequence, which, as a whole, was more closely similar to humans than other species (using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool, Table 4). 39 Using computer algorithms to analyze the original mouse antibody V H and V LMHC class II binding peptides were screened using humanized variant sequences to determine if the humanization process had removed high-affinity peptide sequences. Furthermore, motifs that readily deamidate asparagine to aspartic acid in the Fab variants were tested. Such motifs were absent in either the mouse or humanized Fab variants. The structures of the variable domain binding sites were modeled using Schrodinger software. Based on RMSD analysis, the combinations of VH2:VL3 and VH1:VL3 had the lowest scores compared to the mouse VH0 VL0 domain. These were classified as having the closest structural similarity to the mouse variable domain. All humanized variable domain combinations had RMSD values ​​less than 2 Å and were therefore predicted to have a good structural match with the mouse VH0 VL0 domain (see Table 5).

[0408] Table 4: WHO-assigned antibodies INN for mouse and humanized variants

[0409]

[0410] Table 5: Structural similarity predicted using Schrodinger software.

[0411]

[0412] To achieve higher expression levels 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). Each V was synthesized within the box. H The VL domain and N-terminal signal peptide (SEQ ID NO: 63) and the C-terminal human IgG1 isotype constant domain sequence corresponding to SEQ ID NO: 61 (allotype G1m17,1) were synthesized within the box. Each VL domain and N-terminal signal peptide (SEQ ID NO: 62) and the C-terminal human IgK isotype constant domain sequence corresponding to SEQ ID NO: 60 (allotype Km3) were synthesized within the box. Codon optimization of the sequences (ATUM, USA) was performed, 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.

[0413] 2.2.2 Transient expression and purification of humanized Fab variants

[0414] The next stage involved transient transfection and expression of each humanized Fab fragment. One chimeric Fab fragment expression was used as a positive control, containing a mouse variable domain and a human Ig constant domain, along with 20 humanized variants containing both a humanized variable domain and a human Ig constant domain. The Emf6.1 humanized variant was a 50 kDa monovalent Fab fragment, each consisting of a heavy chain (with the entire Fc region missing) and a light chain linked together via disulfide bonds. The mammalian expression vector (pETE V2) encoding each variant was transfected into Chinese hamster ovary (CHO) cells, and batch cultures of each variant were grown for up to seven days.

[0415] The expressed Fab fragment was purified from cell culture supernatant via affinity chromatography. For cell cultures containing the Fab fragment, the supernatant 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 / buffer-exchanged into phosphate-buffered saline solution. Quality control experiments were performed, including determining the concentration and purity of the purified Fab product to ensure compliance with specified standards. The purity of the Fab fragment was determined to be >95%, as determined by reduction and denaturation of sodium dodecyl sulfate polyacrylamide gel electrophoresis. The Fab concentration was determined by measuring the absorbance at 280 nm and calculated using the calculated extinction coefficient, where 1.0 mg / ml = 1.49 A280 (assuming MW = 50 kDa for the Fab fragment).

[0416] All Fab variants were successfully expressed and purified according to the above criteria. SDS-PAGE analysis showed that all Fab fragments exhibited sufficient purity levels under reducing conditions. Under reducing conditions, two bands were observed at molecular weights of approximately 25 and 30 kDa, respectively. Under non-reducing conditions, two bands were observed at molecular weights of approximately 20 and 40 kDa. The additional bands (impurities) may be a result of unpaired heavy chains and / or light chains.

[0417] 2.2.3 Kinetic Analysis of Humanized Fab Variants

[0418] For kinetic analysis, the human GPVI-Fc fusion protein (1.2 μg) was immobilized on the biosensor using a suitable trapping surface, and binding to the Emf6.1 variant was monitored using the Octet instrument via biolayer interferometry (BLI). The resulting sensor maps were analyzed using the provided software (Fortebio). Experimental details are described in Section 1.16 above.

[0419] Table 6: Kinetic parameters of antibody variant-antigen interaction

[0420]

[0421] The R² value indicates the degree of correlation between the fit and the experimental data, and a value above 0.95 is considered a good fit; X² is the sum of squares of the deviations, which should generally be below 3. X² is a measure of the error between the experimental data and the fitted line. Italicized text: chimeric control antibody, HC0 LC0.

[0422] Octet analysis showed that, in most cases, the experimental data conformed to a 1:1 binding model. Under the experimental conditions used, many Fab fragments, including HC1 LC2 (KD: 195 pM), HC1 LC3 (KD: 284 pM), HC2 LC2 (KD: 175 pM), and HC2 LC3 (KD: 250 pM), exhibited higher affinity than the chimeric HC0LC0 control antibody (KD: 427 pM) (see Table 6).

[0423] 2.2.3 Further characterization of the HC1 LC2 variant (EMA601)

[0424] The HC1 LC2 variant (designated EMA601) was chosen for further characterization because it exhibited the most potent GPVI blocking effect. Firstly, EMA601 at a concentration of 5 μg / mL potently inhibited the formation of human platelet aggregates on collagen in a whole blood perfusion system. Figure 8 AC). It is worth noting that EMA601 is also effective in this assay at lower concentrations (1 μg / mL). Figure 11 A).

[0425] Furthermore, EMA601 (5 µg / mL) eliminated CRP-induced and collagen-induced human platelet aggregation, but had no effect on thrombin-induced responses. Figure 8 D). This effect was also achieved at concentrations as low as 1 μg / mL of EMA601. Figure 11 B), and dose-response experiments further revealed that humanized EMA601 effectively inhibited activation / aggregation at high collagen concentrations (10 and 20 μg / mL). Furthermore, EMA601 (10 μg / mL) was found to strongly inhibit GPVI-dependent spreading of human platelets on fibrinogen. Figure 8 EF). Finally, EMA601 also strongly inhibited platelet deposition and thrombus formation on collagen / TF-coated surfaces (EF). Figure 8 GI), and completely eliminated phosphatidylserine (PS) exposure and fibrin deposition ( Figure 8 (G, JK). Overall, these data establish EMA601 as a promising lead for targeting human GPVI under pathological conditions.

[0426] 2.3 Example 3: Head-to-head comparison of EMA601 and ACT017 (Glenciemab)

[0427] 2.3.1 In vitro binding of GPVI to platelets

[0428] To directly compare the GPVI inhibitory efficacy of EMA601 and ACT017 (glenciemab), in vitro and in vivo studies were conducted. In the first set of experiments, hGP6... tg / tg Diluted mouse blood was pre-incubated with different concentrations (ranging from 50 to 0.1 μg / mL) of EMA601 or ACT017, and epitope saturation on GPVI was detected by flow cytometry. Emf3 FITC and JAQ1 FITC (Both 5 µg / mL) were used to detect the epitope occupancy of EMA601 and ACT017, respectively. Although Emf3 FITC Binding was strongly inhibited by EMA601, but even at a concentration of 0.5 μg / mL EMA601, ACT017 also inhibited Emf3. FITC The binding has no effect, even at very high concentrations (n=4). Figure 12 (A, B) This confirms that EMA601 and ACT017 bind to different epitopes on human GPVI. In contrast, ACT017 inhibits JAQ1 at high concentrations (50, 20, 10 µg / mL). FITC The binding of ACT017 to GPVI is observed, but the effect gradually diminishes at concentrations ≤ 5 µg / mL, indicating a relatively low affinity of ACT017 for GPVI. Notably, EMA601 inhibits JAQ1 at concentrations >10-fold lower (≤ 0.5 µg / mL). FITC Combine. Next, hGP6 tg / tg Diluted mouse blood was pre-incubated with different concentrations (range 50 to 0.1 μg / mL) of EMA601 or ACT017, and the binding of Fab (anti-human IgG-Fab antibody) was detected using fluorescently labeled anti-human IgG-Fab antibody (n=4). Figure 12 C). At all tested concentrations, EMA601 produced a significantly higher signal than ACT017, indicating a higher binding affinity of EMA601 compared to ACT017. This was further confirmed by the rapid decrease in surface-bound ACT017 at concentrations ≤ 2 µg / mL. Specifically, at the lowest concentrations of 0.2 and 0.1 μg / mL, almost no ACT017 was present from the platelet surface, while EMA601 still occupied 86.2% and 57.3% of the epitopes, respectively, compared to the highest tested concentration (50 μg / mL). Figure 12 C).

[0429] 2.3.2 Standard light transmission aggregation measurement method and flow adhesion measurement

[0430] 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 using a standard phototransfer aggregation assay. EMA601 eliminated collagen-induced aggregation at 5 µg / mL and still reduced it by approximately 50% at 1 µg / mL. In stark contrast, ACT017 at concentrations up to 10 µg / mL did not show significant inhibitory effects under these conditions. Notably, even at the highest tested concentration (50 μg / mL), ACT017 could not completely inhibit the response, indicating that ACT017 has limited overall GPVI blocking efficacy. Figure 12 D). Furthermore, CRP-induced aggregation was eliminated at all tested 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 12 D). 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-flow chamber under arterial shear stress (1000 s⁻¹) using a flow adhesion assay. Notably, at all tested concentrations, EMA601 significantly reduced platelet adhesion and aggregate formation, while ACT017 did not affect adhesion on collagen but only reduced thrombus volume. Interestingly, at all tested concentrations, EMA601 showed a significantly stronger reduction in thrombus volume compared to ACT017. Figure 12 FH).

[0431] 2.3.3 In vitro testing after intravenous administration

[0432] Finally, to compare the efficacy of EMA601 or ACT017 in blocking hGPVI in vivo, hGP6 tg / tg Mice were intravenously administered one of two humanized Fab formulations at 4 mg / kg, and GPVI epitope occupancy and platelet activation were measured in vitro 1 hour later. Notably, approximately 1.8 times higher levels of EMA601 binding were detected compared to ACT017. Figure 12 H). Consistent with this, CRP-induced platelet activation (JON / A-PE binding) was eliminated in the blood of EMA601-treated mice, while no significant inhibition was detected in the blood of ACT017-treated mice. Figure 12 I).

[0433] In summary, these results indicate that, compared to ACT017, EMA601 has better efficacy against human platelets in vitro and against hGP6 both in vitro and in vivo. tg / tgMouse platelets exhibited a significantly higher inhibitory effect on GPVI. Furthermore, data revealed that ACT017 at concentrations up to 50 µg / mL could not completely block GPVI function in vitro, and that high doses (4 mg / kg) of hGP6... tg / tg The same situation was observed in vivo and in vitro.

[0434] 2.4 Conclusion

[0435] The monovalent antibody Emf6.1 has been shown to be effective. Fab Its humanized variant EMA601 potently inhibits human and hGP6 in vitro. tg / tg GPVI function in mouse platelets. Emf6.1 in transgenic mice. Fab Treatment resulted in sustained GPVI blockade and significant protection against occlusive arterial thrombosis without affecting bleeding time. On one hand, it exhibits high affinity binding to GPVI and at hGP6 tg / tg The favorable pharmacokinetics in mice, and, on the other hand, its unexpected binding site on the receptor, highlight Emf6.1. Fab / EMA601 is a novel lead inhibitor for achieving potent yet safe GPVI inhibition in a clinical setting.

[0436] The data in this application show that, compared with 9O12 Fab And ACT017 (glenciemab) reported 23, 29, 30 In comparison, Emf 6.1 Fab In hGP6 tg / tg The persistence of GPVI on mouse circulating platelets was significantly prolonged, with receptor occupancy >50% and >35% at 48 h and 72 h after a single dose (4 mg / kg), respectively, leading to sustained inhibition of GPVI function in vitro. This significantly prolonged Emf6.1 Fab One possible explanation for its in vivo activity is 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 It is worth noting that Emf 6.1 Fab The affinity for EMA601 increased by approximately 2.3-fold during humanization, with a kD of 0.195 nM (thus approximately 21-fold higher than ACT017). Direct head-to-head comparisons of EMA601 and ACT017 confirmed that EMA601 exhibits inhibitory effects on human and hGP6 in vitro. tg / tg It has >10 times the potency of GPVI in platelets and also effectively blocks hGP6. tg / tgGPVI function in mice was inhibited, while no significant inhibitory effect was observed with ACT017 (4 mg / kg bw) under the same conditions. Figure 12 Based on these results, it is expected that EMA601 will achieve the desired level of GPVI inhibition in humans at a relatively lower dose and / or for a longer period of time compared to ACT017.

[0437] The data in this application shows that Emf6.1 Fab In vitro application on whole recalcified blood strongly inhibits stable thrombus formation, characterized by the elimination of PS exposure, followed by a reduction in fibrin deposition on collagen / TF-coated surfaces. Emf6.1 Fab It significantly inhibited the formation of stable thrombi at the site of arterial injury in vivo, suggesting that its strong antithrombotic effect may be based on two main activities: reducing platelet activation at the exposed extracellular matrix / thrombus surface and indirectly through potent inhibition of local platelet-dependent coagulation.

[0438] Peptide microarray-based mapping and neutralization 49-51 The non-canonical discontinuous binding sites of Emf6.1 in GPVI (Val201 to Glu215 and Ser246 to Pro260) were confirmed to overlap with the collagen binding interface in D117 between D2 and the transmembrane helix, rather than with the dimerized residues of GPVI (Val201-Thr203). 18 Based on this finding, Emf6.1 is a likely candidate. Fab / EMA601 is not as previously described for other GPVI antagonists. 21, 52, 53 Primarily by interfering with ligand binding, it may block GPVI function by modulating its aggregation / signal transduction capabilities. This mechanism of action could explain Emf6.1. Fab How to significantly inhibit GPVI-induced platelet activation while preserving the initial adhesion function of the receptor.

[0439] In summary, the data from this invention provide preclinical characterization of a functionally blocking Fab fragment with very high affinity for human GPVI, which allows for sustained and safe inhibition of GPVI function in circulating platelets, presumably through a non-canonical mechanism of action.

[0440] Example 4: Emf6.1-fab treatment in experimental stroke

[0441] Materials and methods

[0442] Mice.The animals used in this study were matched for age, sex, and genetic background. The experiments were conducted in accordance with the regulations of the local authorities and the current ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines (https: / / www.nc3rs.org.uk / arrive-guidelines).

[0443] Animal processing. In order to block human GPVI, hGp6 tg / tg Mice received 4 mg / kg bw Emf6.1-fab or control fab intravenously 1 hour before tMCAO. Six hours after tMCAO, a second dose of the fab fragment (4 mg / kg bw) was administered subcutaneously.

[0444] Focal ischemia model. As previously mentioned, hGP6 at 10 to 14 weeks of age was monitored using tMCAO. tg / tg mice 32 Induced focal cerebral ischemia. 63 Inhalation anesthesia was induced by 2% isoflurane. Surgical duration for each animal was kept under 10 minutes. A silicone-coated 6.0mm nylon monofilament (6021PK10, Doccol, Redlands, CA, USA) was passed through the carotid artery to the origin of the MCA, causing MCA infarction. After a 60-minute occlusion period, the filament was removed, allowing reperfusion. Animals were sacrificed 23 hours after reperfusion, and the brain was examined for intracranial hemorrhage.

[0445] Infarct size measurement. The extent of infarction was quantitatively assessed 24 h after reperfusion. Animals were euthanized and the brain was divided into three 2 mm thick coronal sections. Sections were stained at 37°C for 20 min with 2% 2,3,5-triphenyltetrazolium chloride (Sigma-Aldrich; 2% w / v solution) to visualize the infarction (Junge et al.). Edema-corrected infarct volume was calculated using planimetry (Image J software, National Institutes of Health) according to the following equation: V 间接 (mm 3 ) = V 梗死 × (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.

[0446] Statistical analysis. All data from animal experiments are presented as box plots, including the median and 25th percentile, 75th percentile, minimum bias, and maximum bias. Gaussian distribution tests were performed on the data, such as the D'Agostino and Pearson combined normality test, followed by analysis using Student's t-test, one-way ANOVA, or Mann-Whitney U test (if applicable). The Mann-Whitney U test was used to compare scores for functional outcomes. A p-value < 0.05 was considered statistically significant. Statistical analysis was performed using the GraphPad Prism 7.05 software package (GraphPadSoftware).

[0447] Results - Effects of hGPVI blockade in a focal cerebral ischemia (stroke) model

[0448] To test the potential effects of hGPVI blockade in cerebral thrombosis inflammation, hGP6 tg / tg Mice were treated with Emf6.1-fab (to block hGPVI) or control fab and subjected to one hour of transient midbrain ischemia (tMCAO) and 23 h of reperfusion. Notably, the infarct volume in Emf6.1-fab-treated mice was significantly reduced at 24 h after tMCAO compared with control-fab-treated (Ctrl.) litters, as measured by triphenyltetrazolium chloride (TTC) staining (Med.: 105.9 (25%: 88.5; 75%: 121.0) vs. 137.1 (25%: 119.6; 75%: 142.3) mm). 3 p<0.05; Figure 13A , Figure 13B No signs of intracranial hemorrhage were detected in any of the test animals.

[0449] The results show that the hGP6 processed by Emf6.1-fab tg / tg Mice were significantly protected from cerebral infarction growth after tMCAO, and GPVI blockade in this context did not increase the risk of intracerebral hemorrhage.

[0450] Example 5: Analysis of the EMA601 (HC1 LC2) variant

[0451] HC1LC2 contains a free cysteine ​​residue at the Kabat L55 position in VLCDR2. Nineteen variants of HC1LC2 were generated and tested, with the cysteine ​​residue at Kabat L55 replaced by different amino acids.

[0452] Materials and methods

[0453] The generation of DNA constructs expressing Fab variants was outsourced to an external provider. Parental variable light chain and variable heavy chain sequences were generated via gene synthesis and then subcloned into expression vectors: a κ 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 in CDRL2 were generated through site-directed mutagenesis followed by subcloning.

[0454] Expression of the Fab variant was achieved by transient transfection of Expi293F cells. The protein was purified using a HighTrap ProteinL column and then preparatively sized on a HighLoad 16 / 600 Superdex 200 column.

[0455] Kinetic analysis was performed using the recombinant huGPVI-Fc fusion protein via BLI (Octet). The analysis was performed as described in Section 1.16 above.

[0456] Functional assessment of the variants was performed using a human platelet and GPVI-specific agonist assay. The assessment was conducted as described in Section 1.7 above.

[0457] result

[0458] All Fab fragments were tested in BLI (Octet) at 10, 3.3, 1.1, 0.37, 0.12, 0.04, and 0.014 nM.

[0459] Glencimab (humanized anti-GPVI Fab from Acticor) was tested at 100, 33, 11, 3.7, 1.23, 0.41, and 0.14 nM.

[0460] The definite dissociation constant (KD) of the variant is as follows.

[0461] Table 7.

[0462]

[0463] All variants showed higher affinity for human glycoprotein VI than the antibody glenzamab.

[0464] The ability of all variants to block GPVI-dependent human platelet aggregation was further tested at concentrations of 0.5 μg / mL and 10 μg / mL at the indicated concentration. The agonists used were:

[0465] 1. Collagen (10 μg / mL)

[0466] 2. Collagen-related peptide (CRP - 0.5 μg / mL)

[0467] Aggregation assay was performed using washed human platelets, with an aggregation time of 15 min. A control Fab or HC1LC2 variant was added 5 min prior to the agonist.

[0468] like Figure 14 As shown, 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, several variants even inhibited GPVI function with greater potency than the parental versions. Variants containing isoleucine, leucine, glutamic acid, threonine, valine, or serine at the Kabat 55 position performed particularly well.

[0469] All variants that showed only minor inhibition of collagen-induced aggregation at a concentration of 0.5 µg / mL exhibited potent inhibition when added at a concentration of 10 µg / mL.

[0470] In contrast, even at 10 µg / mL, glomcitabine showed little to no inhibition of collagen-induced aggregation and only partial inhibition of CRP-induced aggregation.

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Claims

1. An antibody or a functional fragment thereof capable of binding to human glycoprotein VI (GPVI), wherein said antibody or functional fragment comprises (i) V L The domain comprises 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, and (ii) V H The domain comprises 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.

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

3. The antibody or functional fragment according to claim 1 or 2, wherein the antibody or functional fragment has a dissociation equilibrium constant (K0) of less than 700 pM, preferably less than 300 pM, more preferably less than 200 pM. D It combines with human GPVI.

4. The antibody or functional fragment according to any one of the preceding claims, wherein the antibody or functional fragment comprises V L Domain and / or V H Domain, the V L The domain comprises an amino acid sequence having at least 90% sequence identity with the sequence selected from the group consisting of SEQ ID NO: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34 and 35; the V H The domain contains an amino acid sequence that has at least 90% sequence identity with the sequence selected from the group consisting of SEQ ID NO: 21, 22, 23, 24 and 25.

5. The antibody or functional fragment according to any one of the preceding claims, wherein the antibody or functional fragment comprises V L Domain and / or V H Domain, the V L The field contains the amino acid sequence shown in SEQ ID NO: 16, 17, 18, 19, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35, wherein the V H The domain contains the amino acid sequence shown in SEQ ID NO: 21, 22, 23, 24 or 25.

6. The functional fragment according to any one of the preceding claims is an antigen-binding fragment (Fab), F(ab'), Fv, monovalent IgG, disulfide-linked variable fragment (dsFv), or single-chain variable fragment (scFv).

7. An antibody or functional fragment according to any one of the preceding claims, wherein the antibody or functional fragment comprises a light chain and / or a heavy chain, the light chain comprising 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 the heavy chain comprising 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 segment of claim 7, wherein the functional segment is a Fab, comprising or consisting of the following: A light chain having an amino acid sequence as shown in one of SEQ ID NO: 38, 39, 40, 41, 55, 56, 57 or 58, and a heavy chain having an amino acid sequence as shown in one of SEQ ID NO: 44, 45, 46, 47 or 48.

9. The functional fragment according to any one of the preceding claims, wherein the functional fragment is a Fab consisting of a pair of light and heavy chains having an amino acid sequence as shown in SEQ ID NO: 39 and 44, 39 and 45, 40 and 45, 56 and 44, 56 and 45 or 57 and 45, preferably SEQ ID NO: 39 and 44 or 56 and 44.

10. An antibody or functional fragment according to any one of the preceding claims, which is capable of binding human GPVI at the binding epitopes corresponding to amino acids V178 to E192 and / or S223 to P237 of SEQ ID NO:

36.

11. A nucleic acid that encodes an antibody or functional fragment according to any one of the preceding claims.

12. A cell comprising the nucleic acid of 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 of claim 12 in a culture medium under conditions that allow expression of a nucleic acid 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. An antibody or functional fragment as defined in any one of claims 1 to 10, or a pharmaceutical composition according to claim 14, used in a method of treating or preventing GPVI-related conditions in a subject; optionally, wherein said GPVI-related conditions are thrombotic inflammatory diseases; cardiovascular diseases, preferably selected from thrombosis and thrombotic disorders, including arterial thrombosis, venous thrombosis, atherosclerotic thrombosis, stent thrombosis, venous thromboembolic disease, thrombotic microangiopathy, cancer-related thrombosis, immune thrombosis and infection-related thrombosis, restenosis, acute coronary syndrome, ischemic stroke, cerebrovascular accident, cerebrovascular disease, vascular purpura, coronary artery disease and cerebral artery disease, 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. Pulmonary embolism; inflammation, preferably persistent or prolonged inflammation related to infection, arthritis, fibrosis, acute respiratory distress syndrome (ARDS), ischemia-reperfusion injury (IRI) of various organs (liver, colon, etc.), peripheral vascular disease, antiphospholipid syndrome (APS), deep vein thrombosis, thrombophlebitis and vasculitis, transfusion-related acute lung injury (TRALI), transplant rejection, preeclampsia, severe burns, atherosclerosis, hypertension, antiphospholipid syndrome, preeclampsia, sickle cell disease, bacterial and viral infections, ischemic restenosis, sepsis, major trauma, autoimmune diseases, and conditions involving platelet-modulating cell function, including but not limited to cancer cell proliferation and / or dissemination; cancer, preferably skin cancer, colon cancer, breast cancer, ovarian cancer, lung cancer, or metastatic cancer; and / or conditions associated with abnormal or abnormal megakaryocytes and / or platelet proliferation, differentiation, morphology, migration, aggregation, degranulation, and / or function.

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