Platelet-binding protein and pharmaceutical use thereof

Abstract

Provided are a platelet-binding protein and a pharmaceutical use thereof. Specifically, provided are a platelet-binding protein, e.g., a TLT-1-binding protein, and an encoding nucleic acid, vector, cell, pharmaceutical composition and use thereof.

Description

Platelet-binding proteins and their medicinal uses

[0001] This disclosure claims priority to Chinese patent application CN202510052109.8, filed on January 13, 2025. Technical Field

[0002] This disclosure relates to platelet-binding proteins encoding polynucleotides, carriers, cells, compositions, preparation methods, and pharmaceutical uses, particularly for the preparation of drugs for thrombolysis. Background Technology

[0003] Urokinase-type plasminogen activator (uPA, urokinase) is a serine protease that promotes the fibrinolytic process. The main function of uPA is to promote the conversion of plasminogen into plasmin, which degrades fibrin, dissolving the thrombus and achieving a thrombolytic effect.

[0004] uPA is expressed in a single-chain, inactive (or very low-activity) proenzyme form, pro-urokinase (pro-UK). The peptide bond between Lys158 and Lys159 is hydrolyzed by plasmin or other proteases, forming two peptide chains, A and B, still linked by interchain disulfide bonds; this is called high molecular weight (HMW) uPA. The peptide bond between Lys136 and Lys137 can be further hydrolyzed, releasing the N-terminal fragment. The remaining protease domain is linked to a small polypeptide segment of chain A via disulfide bonds; this is called low molecular weight (LMW) uPA. The protease activity of LMW uPA is comparable to that of HMW uPA, and it also possesses thrombolytic function. Both double-chain forms of uPA are active and can activate the conversion of plasminogen to plasmin. Prourokinase (pro-UK; also known as single-chain urokinase plasminogen activator (scu-PA)) or low molecular weight single-chain uPA (LMW-scuPA) have particularly low basic enzyme activity, and therefore pose a lower risk of causing systemic bleeding than urokinase, making them superior recombinant thrombolytic drugs.

[0005] Ischemic stroke refers to cerebral circulatory disturbances leading to cerebral vascular occlusion or severe stenosis, resulting in decreased cerebral blood flow perfusion, and consequently ischemia, hypoxia, and brain tissue damage and death. The key to treating ischemic stroke is to open the blocked blood vessel as early as possible, restore cerebral blood flow, and salvage the ischemic penumbra. Intravenous thrombolysis is an effective and important treatment for early vascular recanalization. Currently used intravenous thrombolytic drugs have the following limitations: 1) Narrow therapeutic window, requiring treatment to be initiated within 3-4.5 hours after stroke onset; statistics show that less than 3% of ischemic stroke patients currently receive intravenous thrombolysis; 2) Short half-life, requiring intravenous bolus and infusion administration, resulting in a long administration time; 3) High risk of hemorrhagic transformation, damage to the blood-brain barrier, neurotoxicity, and other side effects, and may exacerbate immunosuppression.

[0006] Based on the above, there is an urgent clinical need for innovative drugs that improve thrombolytic efficiency and safety, extend the treatment time window, reduce drug dosage, and shorten administration time. Ischemic stroke accounts for approximately 62.4% of all stroke cases, and in ischemic stroke caused by embolism, arterial emboli account for 60%-70% of the emboli origin. Arterial thrombi have a higher platelet composition than other types of thrombi; platelet targeting can specifically target active drugs to the pathological site, resulting in faster and more effective action.

[0007] In view of this, this disclosure provides a platelet-targeting protein that innovatively fuses uPA with a TLT-1 (Trem-like transcript 1 protein) targeting antibody. TLT-1 is specifically expressed and distributed on the surface of activated platelets, enabling uPA to be rapidly delivered to the pathological thrombus site for thrombolysis. It exhibits better thrombolytic efficiency against platelet-rich thrombi, prolonging the therapeutic window and reducing bleeding risk and dosage. Low molecular weight single-chain uPA (LMW-scuPA) is inactive in the circulatory system and has little effect on plasma endogenous plasminogen. It only exerts its thrombolytic effect when activated on the thrombus surface by plasmin or kininase, becoming an active low molecular weight double-chain uPA, thereby reducing the risk of systemic bleeding. Simultaneously, LMW-scuPA does not bind to UPAR or binds weakly, reducing its receptor-mediated clearance and contributing to a suitable extension of its half-life. The platelet-targeting protein disclosed herein can avoid the damage to nerve cells and the impact on the blood-brain barrier caused by tPA drugs, thereby improving drug safety and the reliability and convenience of clinical use, and providing an innovative drug with clinical application potential for the treatment of ischemic stroke. Summary of the Invention

[0008] This disclosure provides TLT-1 binding protein, platelet-binding protein, polynucleotides encoding TLT-1 binding protein or platelet-binding protein, carriers, pharmaceutical compositions and their pharmaceutical uses.

[0009] TLT-1 binding protein

[0010] This disclosure provides a TLT-1 binding protein comprising at least one immunoglobulin single variable domain that specifically binds to TLT-1.

[0011] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 can bind to the TLT-1 37mer peptide.

[0012] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 does not bind to the TLT-1 extracellular region (TLT-1_ECD) other than TLT-1 37mer.

[0013] In some embodiments, the amino acid sequence of the TLT-1 37mer polypeptide is shown in SEQ ID NO: 5.

[0014] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes CDR3 in the amino acid sequence shown in Formula (I):

[0015] Formula (I) (SEQ ID NO: 80),

[0016] in,

[0017] X1-X 18 Each group was independently selected from any amino acid;

[0018] The CDR3 is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR3 is defined according to the Kabat numbering system.

[0019] In some specific implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes CDR3 as shown in SEQ ID NO: 37 (FRQAGGSWYISATYKY).

[0020] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 further includes CDR1 and CDR2 selected from the following group:

[0021] The CDR1 shown in X1YSMX2 (SEQ ID NO: 81)

[0022] The CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82)

[0023] Among them, each of groups X1-X4 is independently selected from any amino acid; or,

[0024] X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 X1YSMX2WFRQAPGKX11REGVX 12 AISX3X4GERTYYADSVKGRFTISRDNX 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 The amino acid sequence YYCAGFRQAGGSWYISATYKYWGQGTLVTVSS shows CDR1 and CDR2, where X1-X 18 Each amino acid is independently selected from any amino acid;

[0025] In some implementations, the aforementioned CDR1 and CDR2 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR1 and CDR2 are defined according to the Kabat numbering system.

[0026] In some specific implementations, CDR1 shown in X1YSMX2 (SEQ ID NO: 81) and CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82) are as follows:

[0027] X1 is selected from A, S, T, G, D, E;

[0028] X2 is selected from A, S, G;

[0029] X3 is selected from A, S, T, G; and / or

[0030] X4 is selected from G, K, H.

[0031] In some specific implementations, CDR1 shown in X1YSMX2 (SEQ ID NO: 81) and CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82) are as follows:

[0032] X1 is selected from A, S, T, G;

[0033] X2 is selected from A, S, G;

[0034] X3 is selected from A, S, T, G; and / or

[0035] X4 is selected from G, K, H.

[0036] In some embodiments, the CDR1 and CDR2 of the TLT-1 binding protein are selected from:

[0037] CDR1 of amino acid sequences such as AYSMA, DYSMA (SEQ ID No. 83), EYSMA (SEQ ID No. 84), SYSMA, TYSMA (SEQ ID No. 85), AYSMG, GYSMA, or AYSMS; and / or,

[0038] CDR2 of amino acid sequences such as AISAGGERTYYADSVKG, AISSGGERTYYADSVKG, AISGGGERTYYADSVKG, AISAKGERTYYADSVKG, AISAHGERTYYADSVKG, or AISTGGERTYYADSVKG;

[0039] In some implementations, the aforementioned CDR1-3, which specifically binds to the TLT-1 immunoglobulin single variable domain, is selected from:

[0040] (1-1) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 36 and 37 respectively;

[0041] (1-2) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 65, 36 and 37 respectively;

[0042] (1-3) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 36 and 37 respectively;

[0043] (1-4) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 67, 36 and 37 respectively;

[0044] (1-5) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 68, 36 and 37 respectively;

[0045] (1-6) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 69 and 37 respectively;

[0046] (1-7) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 70 and 37 respectively;

[0047] (1-8) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 71 and 37 respectively;

[0048] (1-9) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 72 and 37 respectively;

[0049] (1-10) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 73 and 37 respectively;

[0050] (1-11) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 70 and 37 respectively;

[0051] (1-12) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 73 and 37 respectively;

[0052] or,

[0053] (1-13) CDR1, CDR2 and CDR3, respectively, as shown in the amino acid sequences of SEQ ID NO: 67, 70 and 37.

[0054] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 further includes a framework region FR selected from any of the following groups (4-1)-(4-2).

[0055] (4-1)X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 FR1,WFRQAPGKX shown in (SEQ ID No. 86) 11 REGVX 12 FR2,RFTISRDNX shown in (SEQ ID No. 87) 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 YYCAG (SEQ ID No. 88) represents FR3, and WGQGTLVTVSS (SEQ ID No. 89) represents FR4.

[0056] Among them, X5-X 18 Each group was independently selected from any amino acid;

[0057] (4-2)

[0058] X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 X1YSMX2WFRQAPGKX 11 REGVX 12 AISX3X4GERTYYADSVKGRFTISRDNX 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 The amino acid sequence YYCAGFRQAGGSWYISATYKYWGQGTLVTVSS shows FR1, FR2, FR3, and FR4.

[0059] Among them, X1-X 18 Each amino acid is independently selected from any amino acid;

[0060] In the aforementioned groups (4-1) and (4-2), FR1, FR2, FR3, and FR4 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, FR1, FR2, FR3, and FR4 are defined according to the Kabat numbering system.

[0061] In some implementations, in the aforementioned groups (4-1)-(4-2):

[0062] X5 is selected from D and E;

[0063] X6 is selected from L,V;

[0064] X7 is selected from S,L;

[0065] X8 is selected from A, P;

[0066] X9 is selected from G and F;

[0067] X 10 Selected from G, E, N;

[0068] X1 is selected from A, D, E, S, T, G;

[0069] X2 is selected from A, S, G;

[0070] X 11 Selected from Q, D, E;

[0071] X 12 Selected from A, S;

[0072] X3 is selected from A,S,T,G;

[0073] X4 is selected from G, K, H;

[0074] X 13 Selected from A, S;

[0075] X 14 Selected from L, V;

[0076] X 15 Selected from L, M;

[0077] X 16 Selected from K,R;

[0078] X 17 Selected from P, A;

[0079] X 18 Selected from M,V.

[0080] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes the amino acid sequence shown in formula (I).

[0081] Formula (I) (SEQ ID NO: 80),

[0082] in,

[0083] X1-X 18 Each amino acid is independently selected from any amino acid;

[0084] In some embodiments, the amino acid sequence represented by formula (I) contains:

[0085] X1 is selected from A, S, T, G;

[0086] X2 is selected from A, S, G;

[0087] X3 is selected from A,S,T,G;

[0088] X4 is selected from G, K, H;

[0089] X5 is selected from D and E;

[0090] X6 is selected from L,V;

[0091] X7 is selected from S,L;

[0092] X8 is selected from A, P;

[0093] X9 is selected from G and F;

[0094] X 10 Selected from G, E, N;

[0095] X 11 Selected from Q, D, E;

[0096] X 12Selected from A, S;

[0097] X3 is selected from A,S,T,G;

[0098] X4 is selected from G, K, H;

[0099] X 13 Selected from A, S;

[0100] X 14 Selected from L, V;

[0101] X 15 Selected from L, M;

[0102] X 16 Selected from K,R;

[0103] X 17 Selected from P, A;

[0104] X 18 Selected from M,V.

[0105] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the following (5-1)-(5-25), or an amino acid sequence having at least 80% sequence identity with it:

[0106] (5-1)

[0107] (5-2)

[0108] (5-3)

[0109] (5-4)

[0110] (5-5)

[0111] (5-6)

[0112] (5-7)

[0113] (5-8)

[0114] (5-9)

[0115] (5-10)

[0116] (5-11)

[0117] (5-12)

[0118] (5-13)

[0119] (5-14)

[0120] (5-15)

[0121] (5-16)

[0122] (5-17)

[0123] (5-18)

[0124] (5-19)

[0125] (5-20)

[0126] (5-21)

[0127] (5-22)

[0128] In this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity, as well as the range between any two of the foregoing values, including integers and decimals.

[0129] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the above (7-1)-(7-25), or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0130] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the amino acid sequences shown in (7-1)-(7-25) above.

[0131] In some implementation schemes, the aforementioned single variable domain of the immunoglobulin is of camel origin.

[0132] In some implementations, the aforementioned immunoglobulin single variable domain is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0133] In some implementation schemes, the single variable domain of the immunoglobulin is humanized.

[0134] In some embodiments, the CDR1 of the immunoglobulin single variable domain that specifically binds to TLT-1 has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the CDR2 of the immunoglobulin single variable domain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the CDR3 of the immunoglobulin single variable domain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid mutations compared to any of the aforementioned CDR3s.

[0135] In some implementations, the aforementioned amino acid mutations are conserved substitutions, replacements, or modifications, and / or deletions and / or additions that do not affect function.

[0136] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any one or any combination of the aforementioned CDR1, CDR2 and CDR3 (e.g., any combination of any two or any three).

[0137] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes three complementarity-determining regions CDR1, CDR2, and CDR3 and four FRs.

[0138] In some embodiments, CDR1-3 and FR1-4 in the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 are arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0139] In some embodiments, the aforementioned TLT-1 binding protein is an antibody that binds to TLT-1 or its antigen-binding fragment, or a conjugate or fusion protein containing the antibody or its antigen-binding fragment.

[0140] In some implementations, the aforementioned TLT-1 binding protein is a fusion protein comprising an antibody that binds to TLT-1 or an antigen-binding fragment thereof.

[0141] In some implementations, the aforementioned TLT-1 binding protein is a single variable domain of an immunoglobulin that specifically binds to TLT-1.

[0142] In some implementation schemes, the aforementioned antibodies are camel antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0143] In some implementations, the aforementioned antigen-binding fragment is an sdAb or a bispecific antibody or a multispecific antibody.

[0144] In some implementations, the aforementioned single variable domain of the immunoglobulin is VHH, Fab, or scFv. In some specific implementations, the aforementioned single variable domain of the immunoglobulin is VHH.

[0145] In some implementations, the aforementioned VHH is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0146] In some implementations, the aforementioned VHH is a humanized VHH.

[0147] In some implementations, the aforementioned VHH is a VHH with reduced immunogenicity.

[0148] In some implementations, the aforementioned VHH is a VHH that removes T-cell epitopes. In some specific implementations, the aforementioned VHH is a VHH that removes T-cell epitopes while maintaining a non-significant reduction in affinity.

[0149] In some implementations, a TLT-1 binding protein is provided, which includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different, and any two immunoglobulin single variable domains may be directly linked or linked by a linker.

[0150] In some embodiments, a TLT-1 binding protein is provided that binds to or competes with the same epitope as the aforementioned immunoglobulin single variable domain of this disclosure.

[0151] In some embodiments, the aforementioned TLT-1 binding protein includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different.

[0152] In some implementations, the aforementioned TLT-1 binding protein does not contain the human immunoglobulin Fc region.

[0153] In some embodiments, the aforementioned TLT-1 binding protein may also include a human immunoglobulin Fc region; for example, the Fc region is derived from human IgG1, IgG2, or IgG4. The Fc region may or may not contain mutations.

[0154] In some implementations, the aforementioned Fc region is derived from the Fc region of human IgG4.

[0155] In some implementations, the aforementioned Fc region includes SEQ ID NO: 38.

[0156] In some implementations, the immunoglobulin single variable domain in the aforementioned TLT-1 binding protein is directly or via a linker connected to the Fc region.

[0157] In some specific implementations, the connector includes, but is not limited to, (G) m S n ) h Or (GGNGT) h (SEQ ID NO: 90) or (YGNGT) h (SEQ ID NO: 91) or (EPKSS) h The amino acid sequence shown in SEQ ID NO: 92, wherein m and n are each independently selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker may be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures. In some embodiments, the linker is a flexible linker. In some specific implementations, the connector is selected from G4S (SEQ ID NO: 77), GS, GAP, (G4S)2 (SEQ ID NO: 93), (G4S)3 (SEQ ID NO: 78), (G4S)4 (SEQ ID NO: 94), (G4S)5 (SEQ ID NO: 95), ASGS (SEQ ID NO: 96), for example (G4S)2 and (G4S)3.

[0158] In some embodiments, the TLT-1 binding protein of this disclosure may comprise any of the complete immunoglobulin single variable domains described above; it may also comprise a functional portion of any of the immunoglobulin single variable domains described above or a variant thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0159] In some embodiments, the variant of the functional portion of the single variable domain of the immunoglobulin may be a polypeptide that retains the TLT-1 binding function of CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3 and has at least 80% or at least 90% sequence identity with them. For example, it may be a polypeptide that retains the TLT-1 binding function of CDR3 and has some sequence identity with it, such as a polypeptide that has at least 80% or at least 90% sequence identity with any of the above CDR3s.

[0160] In some embodiments, the TLT-1 binding protein of this disclosure comprises one or more therapeutic or diagnostic agents, wherein the therapeutic or diagnostic agent is covalently or non-covalently linked to a single variable domain of the immunoglobulin.

[0161] In some embodiments, the therapeutic or diagnostic agent is selected from therapeutic or diagnostic proteins, nucleic acids, and small molecule compounds.

[0162] In some embodiments, the TLT-1 binding protein of this disclosure, or an immunoglobulin single variable domain therein, has a K value of about 1.68E-06 to 5.00E-06. D The value is combined with TLT-1.

[0163] In some embodiments, the TLT-1 binding protein of this disclosure, or the immunoglobulin single variable domain therein, binds to TLT-1 in a steady-state mode that is both fast-binding and fast-dissociating.

[0164] In some embodiments, the Ka of the TLT-1 binding protein of this disclosure or the immunoglobulin single variable domain therein that binds to TLT-1 is relatively large, and the Kd is also large.

[0165] In some implementations, the TLT-1 binding protein of this disclosure or the immunoglobulin single variable domain thereof enters the body and quickly binds to TLT-1 on activated platelets, enabling the therapeutic molecule to exert a rapid thrombolytic effect, and then quickly dissociates from TLT-1 and is metabolized and cleared.

[0166] Platelet-binding protein

[0167] This disclosure provides platelet-targeting molecules, which include molecules that specifically bind to platelets and anticoagulants. In some specific embodiments, the platelet-targeting molecule is a platelet-targeting protein.

[0168] This disclosure provides platelet-targeting proteins, which include antibodies or antigen-binding fragments that specifically bind to platelets and anticoagulants.

[0169] This disclosure provides a platelet-targeting protein comprising at least one immunoglobulin single variable domain that specifically binds to TLT-1, and at least one anticoagulant.

[0170] In some embodiments, the anticoagulant is a plasminogen activator. In some specific embodiments, the plasminogen activator is selected from urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), tenecteplase (TNK, TNK-tPA), reteplase (r-PA), alteplase, monteplase, lateplase, saruplase (r-scu-PA), stethokinase (SK), or plasminogen-streptokinase activator complexes.

[0171] In some embodiments, the plasminogen activator is a urokinase-type plasminogen activator or a functional variant thereof. In some embodiments, the plasminogen activator is a tissue-type plasminogen activator or a functional variant thereof. In some specific embodiments, the plasminogen activator is uPA (urokinase-type plasminogen activator; urokinase). In some specific embodiments, the uPA is selected from LMW-scuPA (low molecular weight single-chain uPA), HMW-UK, LMW-UK, or Pro-UK, and functional variants thereof.

[0172] In some embodiments, the uPA comprises amino acid sequences as shown in SEQ ID NO: 74, 75.

[0173] In some embodiments, the uPA may or may not contain a signal peptide. In some specific embodiments, the uPA does not contain a signal peptide.

[0174] In some embodiments, the TNK comprises an amino acid sequence as shown in SEQ ID NO: 76.

[0175] In some embodiments, the immunoglobulin single variable domain in the platelet-targeting protein of this disclosure is as described in any of the places above.

[0176] In some specific embodiments, the TLT-1 binding protein includes at least one immunoglobulin single variable domain that specifically binds to TLT-1.

[0177] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 can bind to the TLT-1 37mer peptide.

[0178] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 does not bind to the TLT-1 extracellular region (TLT-1_ECD) other than TLT-1 37mer.

[0179] In some embodiments, the amino acid sequence of the TLT-1 37mer polypeptide is shown in SEQ ID NO: 5.

[0180] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes CDR3 in the amino acid sequence shown in Formula (I):

[0181] Formula (I) (SEQ ID NO: 80),

[0182] in,

[0183] X1-X 18 Each group was independently selected from any amino acid;

[0184] The CDR3 is defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR3 is defined according to the Kabat numbering system.

[0185] In some specific implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes CDR3 as shown in SEQ ID NO: 37 (FRQAGGSWYISATYKY).

[0186] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 further includes CDR1 and CDR2 selected from the following group:

[0187] The CDR1 shown in X1YSMX2 (SEQ ID NO: 81)

[0188] The CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82)

[0189] Among them, each of groups X1-X4 is independently selected from any amino acid; or,

[0190] X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 X1YSMX2WFRQAPGKX11REGVX 12 AISX3X4GERTYYADSVKGRFTISRDNX 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 The amino acid sequence YYCAGFRQAGGSWYISATYKYWGQGTLVTVSS shows CDR1 and CDR2, where X1-X 18 Each amino acid is independently selected from any amino acid;

[0191] In some implementations, the aforementioned CDR1 and CDR2 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific implementations, the CDR1 and CDR2 are defined according to the Kabat numbering system.

[0192] In some specific implementations, CDR1 shown in X1YSMX2 (SEQ ID NO: 81) and CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82) are as follows:

[0193] X1 is selected from A, S, T, G, D, E;

[0194] X2 is selected from A, S, G;

[0195] X3 is selected from A, S, T, G; and / or

[0196] X4 is selected from G, K, H.

[0197] In some specific implementations, CDR1 shown in X1YSMX2 (SEQ ID NO: 81) and CDR2 shown in AISX3X4GERTYYADSVKG (SEQ ID NO: 82) are as follows:

[0198] X1 is selected from A, S, T, G;

[0199] X2 is selected from A, S, G;

[0200] X3 is selected from A, S, T, G; and / or

[0201] X4 is selected from G, K, H.

[0202] In some embodiments, the CDR1 and CDR2 of the TLT-1 binding protein are selected from:

[0203] CDR1 of amino acid sequences such as AYSMA, DYSMA, EYSMA, SYSMA, TYSMA, AYSMG, GYSMA, or AYSMS; and / or,

[0204] CDR2 of amino acid sequences such as AISAGGERTYYADSVKG, AISSGGERTYYADSVKG, AISGGGERTYYADSVKG, AISAKGERTYYADSVKG, AISAHGERTYYADSVKG, or AISTGGERTYYADSVKG;

[0205] In some implementations, the aforementioned CDR1-3, which specifically binds to the TLT-1 immunoglobulin single variable domain, is selected from:

[0206] (1-1) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 36 and 37 respectively;

[0207] (1-2) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 65, 36 and 37 respectively;

[0208] (1-3) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 36 and 37 respectively;

[0209] (1-4) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 67, 36 and 37 respectively;

[0210] (1-5) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 68, 36 and 37 respectively;

[0211] (1-6) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 69 and 37 respectively;

[0212] (1-7) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 70 and 37 respectively;

[0213] (1-8) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 71 and 37 respectively;

[0214] (1-9) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 72 and 37 respectively;

[0215] (1-10) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 73 and 37 respectively;

[0216] (1-11) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 70 and 37 respectively;

[0217] (1-12) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 73 and 37 respectively;

[0218] or,

[0219] (1-13) CDR1, CDR2 and CDR3, respectively, as shown in the amino acid sequences of SEQ ID NO: 67, 70 and 37.

[0220] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 further includes a framework region FR selected from any of the following groups (4-1)-(4-2).

[0221] (4-1)X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 The FR1 shown is WFRQAPGKX 11 REGVX 12 FR2,RFTISRDNX shown 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 YYCAG shows FR3, WGQGTLVTVSS shows FR4.

[0222] Among them, X5-X 18 Each group was independently selected from any amino acid;

[0223] (4-2)

[0224] X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 X1YSMX2WFRQAPGKX11 REGVX 12 AISX3X4GERTYYADSVKGRFTISRDNX 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 The amino acid sequence YYCAGFRQAGGSWYISATYKYWGQGTLVTVSS shows FR1, FR2, FR3, and FR4.

[0225] Among them, X1-X 18 Each amino acid is independently selected from any amino acid;

[0226] In the aforementioned groups (4-1) and (4-2), FR1, FR2, FR3, and FR4 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering systems. In some specific embodiments, FR1, FR2, FR3, and FR4 are defined according to the Kabat numbering system.

[0227] In some implementations, in the aforementioned groups (4-1)-(4-2):

[0228] X5 is selected from D and E;

[0229] X6 is selected from L,V;

[0230] X7 is selected from S,L;

[0231] X8 is selected from A, P;

[0232] X9 is selected from G and F;

[0233] X 10 Selected from G, E, N;

[0234] X1 is selected from A, DE, ST, G;

[0235] X2 is selected from A, S, G;

[0236] X 11 Selected from Q, D, E;

[0237] X 12 Selected from A, S;

[0238] X3 is selected from A,S,T,G;

[0239] X4 is selected from G, K, H;

[0240] X 13 Selected from A, S;

[0241] X14 Selected from L, V;

[0242] X 15 Selected from L, M;

[0243] X 16 Selected from K,R;

[0244] X 17 Selected from P, A;

[0245] X 18 Selected from M,V.

[0246] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes the amino acid sequence shown in formula (I).

[0247] X5VQLX6ESGGGX7VQX8GGSLRLSCAASGX9TFX 10 X1YSMX2WFRQAPGKX11REGVX 12 AISX3X4GERTYYADSVKGRFTISRDNX 13 KNTX 14 YLQX 15 NSLX 16 X 17 EDTAX 18 YYCAGFRQAGGSWYISATYKYWGQGTLVTVSS

[0248] Formula (I) (SEQ ID NO: 80),

[0249] in,

[0250] X1-X 18 Each amino acid is independently selected from any amino acid;

[0251] In some embodiments, the amino acid sequence represented by formula (I) contains:

[0252] X1 is selected from A, S, T, G;

[0253] X2 is selected from A, S, G;

[0254] X3 is selected from A,S,T,G;

[0255] X4 is selected from G, K, H;

[0256] X5 is selected from D and E;

[0257] X6 is selected from L,V;

[0258] X7 is selected from S,L;

[0259] X8 is selected from A, P;

[0260] X9 is selected from G and F;

[0261] X 10 Selected from G, E, N;

[0262] X 11 Selected from Q, D, E;

[0263] X 12 Selected from A, S;

[0264] X3 is selected from A,S,T,G;

[0265] X4 is selected from G, K, H;

[0266] X 13 Selected from A, S;

[0267] X 14 Selected from L, V;

[0268] X 15 Selected from L, M;

[0269] X 16 Selected from K,R;

[0270] X 17 Selected from P, A;

[0271] X 18 Selected from M,V.

[0272] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the following (5-1)-(5-25), or an amino acid sequence having at least 80% sequence identity with it:

[0273] (5-1)

[0274] (5-2)

[0275] (5-3)

[0276] (5-4)

[0277] (5-5)

[0278] (5-6)

[0279] (5-7)

[0280] (5-8)

[0281] (5-9)

[0282] (5-10)

[0283] (5-11)

[0284] (5-12)

[0285] (5-13)

[0286] (5-14)

[0287] (5-15)

[0288] (5-16)

[0289] (5-17)

[0290] (5-18)

[0291] (5-19)

[0292] (5-20)

[0293] (5-21)

[0294] (5-22)

[0295] In this disclosure, "at least 80% (sequence) identity" encompasses at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% (sequence) identity, as well as the range between any two of the foregoing values, including integers and decimals.

[0296] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the above (7-1)-(7-25), or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with it.

[0297] In some embodiments, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any of the amino acid sequences shown in (7-1)-(7-25) above.

[0298] In some implementation schemes, the aforementioned single variable domain of the immunoglobulin is of camel origin.

[0299] In some implementations, the aforementioned immunoglobulin single variable domain is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0300] In some implementation schemes, the single variable domain of the immunoglobulin is humanized.

[0301] In some embodiments, the CDR1 of the immunoglobulin single variable domain that specifically binds to TLT-1 has 0, 1, 2, 3, 4, or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the CDR2 of the immunoglobulin single variable domain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the CDR3 of the immunoglobulin single variable domain has 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid mutations compared to any of the aforementioned CDR3s.

[0302] In some implementations, the aforementioned amino acid mutations are conserved substitutions, replacements, or modifications, and / or deletions and / or additions that do not affect function.

[0303] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes any one or any combination of the aforementioned CDR1, CDR2 and CDR3 (e.g., any combination of any two or any three).

[0304] In some implementations, the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 includes three complementarity-determining regions CDR1, CDR2, and CDR3 and four FRs.

[0305] In some embodiments, CDR1-3 and FR1-4 in the aforementioned immunoglobulin single variable domain that specifically binds to TLT-1 are arranged in the following order from the amino terminus to the carboxyl terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0306] In some embodiments, the aforementioned TLT-1 binding protein is an antibody that binds to TLT-1 or its antigen-binding fragment, or a conjugate or fusion protein containing the antibody or its antigen-binding fragment.

[0307] In some implementations, the aforementioned TLT-1 binding protein is a fusion protein comprising an antibody that binds to TLT-1 or an antigen-binding fragment thereof.

[0308] In some implementations, the aforementioned TLT-1 binding protein is a single variable domain of an immunoglobulin that specifically binds to TLT-1.

[0309] In some implementation schemes, the aforementioned antibodies are camel antibodies, chimeric antibodies, humanized antibodies, or fully human antibodies.

[0310] In some implementations, the aforementioned antigen-binding fragment is an sdAb or a bispecific antibody or a multispecific antibody.

[0311] In some implementations, the aforementioned single variable domain of the immunoglobulin is VHH, Fab, or scFv. In some specific implementations, the aforementioned single variable domain of the immunoglobulin is VHH.

[0312] In some implementations, the aforementioned VHH is modified by any of the following: humanization, affinity maturation, removal of T cell epitopes, reduction of antibody deamidation, reduction of antibody aggregation, reduction of antibody isomerization, or a combination thereof.

[0313] In some implementations, the aforementioned VHH is a humanized VHH.

[0314] In some implementations, the aforementioned VHH is a VHH with reduced immunogenicity.

[0315] In some implementations, the aforementioned VHH is a VHH that removes T-cell epitopes. In some specific implementations, the aforementioned VHH is a VHH that removes T-cell epitopes while maintaining a non-significant reduction in affinity.

[0316] In some implementations, a TLT-1 binding protein is provided, which includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different, and any two immunoglobulin single variable domains may be directly linked or linked by a linker.

[0317] In some embodiments, a TLT-1 binding protein is provided that binds to or competes with the same epitope as the aforementioned immunoglobulin single variable domain of this disclosure.

[0318] In some embodiments, the aforementioned TLT-1 binding protein includes one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different.

[0319] In some implementations, the aforementioned TLT-1 binding protein does not contain the human immunoglobulin Fc region.

[0320] In some embodiments, the aforementioned TLT-1 binding protein may also include a human immunoglobulin Fc region; for example, the Fc region is derived from human IgG1, IgG2, or IgG4. The Fc region may or may not contain mutations.

[0321] In some implementations, the aforementioned Fc region is derived from the Fc region of human IgG4.

[0322] In some implementations, the aforementioned Fc region includes SEQ ID NO: 38.

[0323] In some implementations, the immunoglobulin single variable domain in the aforementioned TLT-1 binding protein is directly or via a linker connected to the Fc region.

[0324] In some specific implementations, the connector includes, but is not limited to, (G) m S n ) h Or (GGNGT) h Or (YGNGT) h Or (EPKSS) h The amino acid sequence shown, wherein m and n are each independently selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and h is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). In some embodiments, the linker may be a non-functional amino acid sequence of 1-20 or more amino acids without secondary or higher structures. In some embodiments, the linker is a flexible linker. In some embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, for example (G4S)2 or (G4S)3.

[0325] In some embodiments, the TLT-1 binding protein of this disclosure may comprise any of the complete immunoglobulin single variable domains described above; it may also comprise a functional portion of any of the immunoglobulin single variable domains described above or a variant thereof, such as CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR1-CDR1-FR2-CDR2-FR3-CDR3, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0326] In some embodiments, the variant of the functional portion of the single variable domain of the immunoglobulin may be a polypeptide that retains the TLT-1 binding function of CDR3, CDR3-FR4, CDR2-FR3-CDR3, CDR2-FR3-CDR3-FR4, FR2-CDR2-FR3-CDR3-FR4, CDR1-FR2-CDR2-FR3-CDR3-FR4, or FR1-CDR1-FR2-CDR2-FR3-CDR3, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and has at least 80% or at least 90% sequence identity with them. For example, it may be a polypeptide that retains the TLT-1 binding function of CDR3 and has some sequence identity with it, such as a polypeptide that has at least 80% or at least 90% sequence identity with any of the above CDR3s.

[0327] In some embodiments, the TLT-1 binding protein of this disclosure comprises one or more therapeutic or diagnostic agents, wherein the therapeutic or diagnostic agent is covalently or non-covalently linked to a single variable domain of the immunoglobulin.

[0328] In some embodiments, the therapeutic or diagnostic agent is selected from therapeutic or diagnostic proteins, nucleic acids, and small molecule compounds. In some embodiments, the platelet-targeting protein is a fusion protein.

[0329] In some embodiments, the fusion protein includes at least one immunoglobulin single variable domain that specifically binds to TLT-1, and at least one uPA (urokinase plasminogen activator; urokinase).

[0330] In some embodiments, the fusion protein comprises a single variable immunoglobulin domain that specifically binds to TLT-1 and a uPA (urokinase-type plasminogen activator; urokinase). In some specific embodiments, the fusion protein comprises a single variable immunoglobulin domain that specifically binds to TLT-1 and an LMW-scuPA.

[0331] In some embodiments, the fusion protein further includes a linker L1. In some specific embodiments, L1 comprises (G m S n ) h , where m and n are each independently selected from integers 1-8, and h is independently selected from integers 1-20.

[0332] In some implementations, the fusion protein is a single-chain protein.

[0333] This disclosure provides platelet-targeting proteins including a single variable immunoglobulin domain that specifically binds to TLT-1, as shown in any of SEQ ID NO: 15, 40-43, 48-64.

[0334] In some embodiments, the fusion protein comprises a single variable immunoglobulin domain that specifically binds to TLT-1, as shown in any of SEQ ID NO: 15, 40-43, and 48-64, and an LMW-scuPA, as shown in any of SEQ ID NO: 75-76. In some specific embodiments, the fusion protein comprises SEQ ID NO: 15 and SEQ ID NO: 74. In some specific embodiments, the fusion protein comprises SEQ ID NO: 42 and SEQ ID NO: 74. In some specific embodiments, the fusion protein comprises SEQ ID NO: 62 and SEQ ID NO: 74.

[0335] In some embodiments, the platelet-binding protein of this disclosure or the immunoglobulin single variable domain therein has a K value of about 1.68E-06 to 5.00E-06. D The value is combined with TLT-1.

[0336] In some embodiments, the platelet-binding protein of this disclosure or the immunoglobulin single variable domain therein binds to TLT-1 in a steady-state mode that binds and dissociates rapidly.

[0337] In some embodiments, the platelet-binding protein of this disclosure or the immunoglobulin single variable domain therein binds to TLT-1 with a relatively large Ka and a large Kd.

[0338] In some implementations, the platelet-binding protein of this disclosure or the immunoglobulin single variable domain thereof enters the body and quickly binds to TLT-1 on activated platelets, enabling the therapeutic molecule to exert a rapid thrombolytic effect, and then quickly dissociates from TLT-1 and is metabolized and cleared.

[0339] In some embodiments, the platelet-binding protein of this disclosure, or a single variable immunoglobulin domain therein, exerts thrombolytic activity after entering the body.

[0340] A conjugate containing a TLT-1 binding domain and an anticoagulant is provided.

[0341] In some embodiments, the TLT-1 binding domain is as described in any of the TLT-1 binding domains in this disclosure; the anticoagulant is as described in any of the anticoagulants in this disclosure.

[0342] Polynucleotides

[0343] A polynucleotide is provided that encodes the TLT-1 binding protein disclosed herein.

[0344] Provides a polynucleotide that encodes the platelet-targeting protein disclosed herein.

[0345] The aforementioned polynucleotides may be RNA, DNA, or cDNA. According to some embodiments of this disclosure, the polynucleotides disclosed are isolated polynucleotides.

[0346] The polynucleotides disclosed herein may also be in vector form, may exist in a vector and / or may be part of a vector, such as a plasmid, sticky-terminal plasmid, YAC, or viral vector. The vector may be particularly an expression vector, i.e., capable of providing…

[0347] Vectors for the in vitro and / or in vivo (i.e., in suitable host cells, host organisms, and / or expression systems) expression of TLT-1 binding proteins or platelet-targeting proteins. These expression vectors typically contain at least one polynucleotide of this disclosure operably linked to one or more suitable expression regulatory elements (e.g., promoters, enhancers, terminators, etc.). Selection of these elements and their sequences for expression in a specific host is common knowledge to those skilled in the art. Regulatory elements and other elements useful or necessary for the expression of the TLT-1 binding proteins or platelet-targeting proteins of this disclosure include, for example, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.

[0348] The polynucleotides disclosed herein can be prepared or obtained by known means (e.g., by automated DNA synthesis and / or recombinant DNA technology) based on information about the amino acid sequence of the polypeptides / proteins disclosed herein, and / or can be isolated from suitable natural sources.

[0349] host cells

[0350] In some embodiments, a host cell is provided that expresses or is capable of expressing one or more of the TLT-1 binding proteins or platelet-targeting proteins disclosed herein, and / or a recombinant host cell containing polynucleotides or vectors disclosed herein.

[0351] In some implementations, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0352] Bacterial cells include, for example, cells of Gram-negative bacterial strains (such as Escherichia coli, Proteus, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus, Streptomyces, Staphylococcus, and Lactococcus strains).

[0353] Fungal cells include, for example, cells of species from the genera *Trichoderma*, *Neurospora*, and *Aspergillus*; or cells of species from the genera *Saccharomyces* (e.g., *Saccharomyces cerevisiae*), *Schizosaccharomyces* (e.g., *Schizosaccharomyces pombe*), *Pichia* (e.g., *Pichia pastoris* and *Pichia methanolica*), and *Hansenula*.

[0354] For example, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, etc.

[0355] This disclosure may also be used with amphibian cells, insect cells, plant cells, and any other cells in the art used for expressing heterologous proteins.

[0356] The cells disclosed herein cannot develop into complete plant or animal individuals.

[0357] Production or preparation method

[0358] This disclosure provides a method for preparing the TLT-1 binding protein or platelet-targeting protein of this disclosure, comprising:

[0359] - Culture the host cells of this disclosure under conditions that allow expression of the TLT-1 binding protein or platelet-targeting protein of this disclosure; and

[0360] -Recover TLT-1 binding protein or platelet-targeting protein expressed by said host cells from the culture; and

[0361] -Optionally, this may include further purification and / or modification of the TLT-1 binding protein or platelet-targeting protein disclosed herein.

[0362] The TLT-1 binding protein or platelet-targeting protein disclosed herein may be produced in cells as described above in an intracellular manner (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), followed by isolation from the host cell and optionally further purification; or may be produced in an extracellular manner (e.g., in a culture medium for culturing host cells), followed by isolation from the culture medium and optionally further purification.

[0363] Methods and reagents for recombinantly generating peptides or proteins, such as specific expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, and culture conditions, are known in the art. Similarly, isolation and purification techniques suitable for producing target proteins such as binding molecules or antibodies of this disclosure are well known to those skilled in the art. Methods for producing and purifying antibodies are well known and available in the prior art, such as Cold Spring Harbor's Guide to Laboratory Antibody Techniques (Chapters 5-8 and 15). The engineered antibodies of this disclosure can also be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect cells. Mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves or ion exchange. The resulting product must be frozen immediately, such as at -70°C, or freeze-dried.

[0364] However, the TLT-1 binding protein or platelet-targeting protein disclosed herein can also be obtained by other protein-generating methods known in the art, such as chemical synthesis, including solid-phase or liquid-phase synthesis.

[0365] Pharmaceutical Composition

[0366] This disclosure provides pharmaceutical compositions comprising, in a preventive or therapeutically effective amount of the TLT-1 binding protein or platelet-targeting protein of this disclosure as described above, and / or a polynucleotide encoding the TLT-1 binding protein or platelet-targeting protein, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

[0367] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of TLT-1 binding protein or platelet-targeting protein per unit dose. In other specific embodiments, the amount of TLT-1 binding protein or platelet-targeting protein per unit dose is 0.1-20000 mg. In some specific embodiments, it is 1-10000 mg.

[0368] Methods of treating and preventing diseases and pharmaceutical uses

[0369] A method for improving platelet targeting of a therapeutic or diagnostic agent is provided, comprising covalently or non-covalently linking the TLT-1 binding protein or platelet-targeting protein of this disclosure to the therapeutic or diagnostic agent. The method used for linking is known in the art, or may be any future method, such as fusion by chemical coupling, fusion at the DNA level, fusion at the mRNA level, or fusion at the protein level, etc.

[0370] In some implementations, the aforementioned therapeutic or diagnostic molecules can be therapeutic or diagnostic targets. Targets can be any protein, peptide, nucleic acid, oligonucleotide, sugar, polysaccharide, or glycoprotein for therapeutic or diagnostic purposes. Examples include, but are not limited to, receptors, receptor ligands, viral capsid proteins, immune system proteins, hormones, enzymes, antigens, cell signaling proteins, or fragments thereof.

[0371] In some implementations, the therapeutic molecule is a thrombolytic molecule. In some specific implementations, the thrombolytic molecule is uPA as described above.

[0372] This disclosure provides methods for treating and / or preventing diseases, comprising administering to a subject in need a therapeutically and / or preventively effective amount of the disclosed TLT-1 binding protein or platelet-targeting protein.

[0373] This disclosure also provides a method for treating and / or preventing disease using TLT-1 binding protein or platelet-targeting protein, the method comprising administering a therapeutically and / or preventively effective amount of the TLT-1 binding protein or platelet-targeting protein of this disclosure to a subject in need.

[0374] This disclosure also provides the use of TLT-1 binding protein or platelet-targeting protein in the preparation of medicaments for the treatment and / or prevention of diseases.

[0375] In some implementation schemes, the aforementioned diseases are indications related to abnormal platelet activation, adhesion, and / or aggregation.

[0376] In some specific implementation plans, the subjects have the following conditions: thrombosis or thrombosis-related conditions.

[0377] In some specific implementation plans, the subjects have the following conditions: diseases related to the fibrinolytic system, thrombolysis, or thrombus regression.

[0378] In some specific implementations, the aforementioned thrombosis includes, but is not limited to: venous, arterial or capillary thrombosis, cardiac thrombosis, thromboembolism, and thrombosis during and / or after contact of human or animal subject blood with an artificial surface.

[0379] In some embodiments, the TLT-1 binding protein, platelet-targeting protein, polynucleotide, carrier, and / or pharmaceutical composition disclosed herein can be administered by any suitable method known in the art, and the administration can be systemic or local. Attached Figure Description

[0380] Figure 1 shows the binding of TLT-1 single-domain antibody 183a-1 (Fc) to human, mouse and rat TLT-1 on the cell surface.

[0381] Figure 2 shows the sensorgram diagram of the binding of 183a-1_hu3 to human TLT-1ECD.

[0382] Figure 3 shows the binding of the antibody to human TLT-1 on the cell surface as detected by flow cytometry.

[0383] Figure 4 shows the sensorgram diagram of 183a-1_hu3-15 binding to human TLT-1ECD.

[0384] Figure 5 shows a schematic diagram of the structure of the platelet-targeted thrombolytic molecule. In Figure 5, A represents the molecular structure of VHH at the N-terminus, and in Figure 5, B represents the molecular structure of VHH at the C-terminus.

[0385] Figure 6 shows the sensorgram diagram of the binding of 9004 to human TLT-1ECD.

[0386] Figure 7 shows the sensorgram diagram of the binding of 9008 with human TLT-1ECD.

[0387] Figure 8 shows the sensorgram diagram of 9018 binding to human TLT-1ECD.

[0388] Figure 9 shows the in vitro urokinase activity of non-targeted and targeted uPA molecules detected by urokinase substrates.

[0389] Figure 10 illustrates the plasmin substrate detection activity of non-targeted and targeted uPA molecules in in vitro conversion of plasminogen to plasmin. Detailed Implementation

[0390] definition

[0391] To facilitate understanding of this disclosure, certain techniques and scientific methods are specifically defined below. Unless otherwise expressly defined in this disclosure, all other techniques and scientific methods used in this disclosure have the meaning commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0392] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0393] The terms "anticoagulant" and "anticoagulant" are interchangeable and refer to agents that inhibit the activity of coagulation factors or stimulate thrombolytic activity. They can be coagulation factor inhibitors or thrombolytic agents. For example, an anticoagulant may contain a thrombolytic peptide corresponding to urokinase-type plasminogen activator (uPA).

[0394] The terms "plasminogen activator," "plasminogen pathway activator," or "plasminogen pathway activator" are general terms that encompass components of the plasminogen activation pathway, compounds that can directly activate plasminogen or indirectly activate plasminogen by activating upstream components of the plasminogen activation pathway, compounds that mimic the activity of plasminogen or plasmin, compounds that can upregulate the expression of plasminogen or plasminogen activator, plasminogen analogs, plasmin analogs, tPA or uPA analogs, and antagonists of plasminogen inhibitors. The term "components of the plasminogen activation pathway" or "components of the plasminogen activation pathway" encompasses: plasminogen, Lys-plasminogen, Glu-plasminogen, micro-plasminogen, delta-plasminogen; variants or analogs thereof; plasminogen and its variants or analogs; and plasminogen activators, such as tPA and uPA, and tPA or uPA variants and analogs containing one or more domains of tPA or uPA (such as one or more kringle domains and proteolytic domains). The term "antagonists of fibrinolysis inhibitors" encompasses antagonists of PAI-1, complement C1 inhibitors, α2 anti-plasmin, or α2 macroglobulin, such as antibodies against PAI-1, complement C1 inhibitors, α2 anti-plasmin, or α2 macroglobulin. The plasminogen disclosed herein may be a human plasminogen ortholog from primates or rodents, or a variant thereof that still retains plasminogen activity and / or lysine-binding activity. Non-limiting examples include functional variants of uPA, t-PA, glucosamine, and streptokinase.

[0395] The terms "u-PA," "uPA," or "u-PA polypeptide" used in this disclosure are generic and should be understood in the broadest sense within the scope of this disclosure, referring to any u-PA polypeptide, including but not limited to recombinant polypeptides, synthetic polypeptides, and u-PA polypeptides extracted or isolated from cells or tissues, and their functional variants, including but not limited to liver and blood. Interchangeable alternative names for u-PA include, but are not limited to, urokinase, urokinase plasminogen activator, urokinase plasminogen activator, urinary plasminogen activator, and urokinase-type plasminogen activator. u-PA includes related polypeptides from different species, including but not limited to human and non-human sources. Human u-PA includes u-PA, allele variants, isotypes, synthetic molecules derived from nucleic acids, proteins isolated from human tissues and cells, and their modified forms. Exemplary unmodified human u-PA peptides include, but are not limited to, unmodified and wild-type prourokinase (containing 431 amino acid residues) including a propeptide and / or a signal peptide, cell-secreted prourokinase after the removal of the signal peptide (pro-uPA, i.e., mature u-PA peptide; exemplarily, such as SEQ ID NO: 75), and protease domains (exemplarily, such as positions 159-411 of SEQ ID NO: 75).

[0396] The u-PA disclosed herein encompasses u-PA peptides in activated or double-chain form. Double-chain or high-molecular-weight u-PA (HMW-uPA) is formed from single-chain forms, such as prourokinase (pro-uPA), via protein cleavage. For example, protein cleavage can be accomplished by plasminogen lyase, kallikrein, cathepsin B, proteolytic enzymes, and nerve growth factor-γ. The u-PA peptides provided herein can be further modified, such as chemically or post-translationally. Such modifications include, but are not limited to, glycosylation, polyethylene glycolation, albuminization, farnesylation, carboxylation, hydroxylation, phosphorylation, and other peptide modifications known in the art.

[0397] The u-PA disclosed herein includes a precursor polypeptide in single-chain or double-chain form and a mature u-PA polypeptide having an active truncated form (such as its low molecular weight form), a separated protease domain, including allelic variants and species variants, variants encoded by splicing variants, and other variants, comprising at least or at least about 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the precursor polypeptide, its mature form, or its protease domain. Combinations of the above different forms are also included, such as single-chain and truncated strategies forming a low molecular weight single-chain uPA (LMW-scuPA). u-PA polypeptides include, but are not limited to, tissue-specific isotypes and their allelic variants, synthetic or recombinant molecules prepared by transcription-translation of nucleic acids, proteins produced by chemical synthesis including the synthesis of shorter polypeptides linked by recombinant methods, proteins isolated from human and non-human tissues and cells, chimeric u-PA polypeptides and their modified forms. u-PA peptides also include u-PA peptide fragments or portions having sufficient length or including suitable regions that retain the activity of at least one full-length mature peptide (if desired, after activation). In one example, a portion of u-PA is a protease domain corresponding to amino acids 159-411 of the u-PA sequence shown in SEQ ID NO: 75. u-PA peptides also include those containing chemical or post-translational modifications and those not containing chemical or post-translational modifications. Such modifications include, but are not limited to, PEGylation, albuminization, glycosylation, farnesylation, carboxylation, hydroxylation, phosphorylation, HES-ization (extending half-life by conjugating a drug molecule to biodegradable hydroxyethyl starch (HES), PAS-ization (genetic fusion or chemical conjugation of a drug-active compound such as a protein, peptide, or low molecular weight drug with a naturally disordered biosynthetic polymer consisting of small L-amino acids Pro, Ala, and / or Ser), and other peptide modifications known in the art.

[0398] The u-PA disclosed herein includes u-PA from any species, including human and non-human species. Non-human u-PA peptides include, but are not limited to, u-PA peptides from mice, dogs, rabbits, birds, cattle, sheep, pigs, and other primates. Exemplary non-human u-PA peptides include, for example, mice (Mus musculus), rats (Rattus norvegicus), cattle (Bos taurus), pigs (Sus scrofa), rabbits (Oryctolagus cuniculus), chickens (Gallus gallus), yellow baboons (Papio cynocephalus), Sumatran orangutans (Pongo abelii), dogs (Canis lupus), sheep (Ovis aries), marmosets (Callithrix jacchus), rhesus monkeys (Macaca mulatta), northern white-cheeked gibbons (Nomascus leucogenys), and chimpanzees (Pan troglodytes).

[0399] "TLT-1" refers to TREM-like transcription factor-1, or TREM (myeloid trigger receptor)-like transcript-1, a membrane protein found only in the α-granules of platelets and megakaryocytes. TLT-1 contains an extracellular type V Ig-like domain, a proline-rich region, and a cytoplasmic tail region containing an immunoreceptor tyrosine repressive motif. Upon platelet activation, TLT-1 is rapidly released onto the platelet surface, thereby enhancing calcium absorption. 2+ Influx and promotion of platelet aggregation. A non-restrictive example of a full-length human TLT-1 sequence is NCBI Reference Sequence: NP_835468.1.

[0400] "TLT-1 binding molecule" refers to any molecule capable of specifically binding to TLT-1 or its epitopes, including but not limited to proteins and peptides. TLT-1 binding molecules may include antibodies or antigen-binding fragments thereof targeting TLT-1 or its epitopes as defined in this disclosure, or conjugates or fusion proteins containing said antibodies, their antigen-binding fragments. Antigen-binding fragments may be, for example, sdAbs or bispecific or multispecific antibodies. The TLT-1 binding molecules of this disclosure may contain at least one (e.g., 2, 3, 4, or more) immunoglobulin single variable domain (such as VHH) that binds to TLT-1. In addition to the immunoglobulin single variable domain, the TLT-1 binding molecules of this disclosure may also contain linkers and / or portions having effector molecule functions, including but not limited to diagnostic or therapeutic agents, such as antitumor agents, immunomodulators, chromophores, fluorophores, chemiluminescent compounds, enzymes, metal ions, and any combinations thereof.

[0401] "Affinity-matured" TLT-1 antibodies (such as VHH) have one or more variations in one or more CDRs that result in an increased affinity for TLT-1 compared to their parent anti-TLT-1 antibodies. Affinity-matured anti-TLT-1 antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10: 779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996.

[0402] "Reverse mutation" refers to mutating the amino acid residues in the FR region of the human antibody to the corresponding amino acid residues in the original antibody. This is usually done to avoid the decrease in immunogenicity and activity caused by humanized antibodies. Minimal reverse mutations can be performed on the variable region of the humanized antibody to maintain the antibody's activity.

[0403] Whether referring to heavy chain antibodies or conventional tetrapeptide chains composed of two heavy chains and two light chains linked by interchain disulfide bonds, the term "antibody" or "immunoglobulin" is used as a general term to include full-length antibodies, their individual chains, and all their parts, domains, or fragments (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains).

[0404] The term “sequence” (as used in terms such as “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the sequence, unless further definition is required by this disclosure.

[0405] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0406] "Immunoglobulin domain" refers to the spherical region of an antibody chain.

[0407] An "immunoglobulin variable domain" is a domain essentially composed of "framework region 1" (FR1), "framework region 2" (FR2), "framework region 3" (FR3), and "framework region 4" (FR4), as well as "complementation-determining region 1" (CDR1), "complementation-determining region 2" (CDR2), and "complementation-determining region 3" (CDR3). Therefore, the general structure (or sequence) of a variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Variable domains confer antigen specificity due to the presence of antigen-binding sites.

[0408] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0409] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the required VH / VL interaction between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNARs, domains, (single-domain) antibodies (such as dAbsTM) that are VH domains or derived from VH domains, and (single-domain) antibodies (such as dAbsTM) that are VL domains or derived from VL domains. Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of a single variable domain of an immunoglobulin is the “VHH domain” (or simply “VHH”) as defined below.

[0410] The “VHH domain,” also known as a heavy-chain single-domain antibody, VHH, VHH domain, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy-chain antibody” (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish this variable domain from the VH and VL domains present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to epitopes without the need for other antigen-binding domains (for the VH or VL domains in conventional tetrapeptide chain antibody structures, the epitope is recognized by the VL domain along with the VH domain). The VHH domain is a small, stable, and highly efficient antigen recognition unit formed by a single immunoglobulin domain. Terms include "heavy chain single-domain antibody," "VHH domain," "VHH," "VHH domain," "VHH antibody fragment," and "VHH antibody." “ The term "VHH domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) is used interchangeably. "VHH domain" includes, but is not limited to, naturally occurring antibodies produced by camelids, humanized antibodies produced by camelids, or those obtained through phage display technology. The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure. Methods for obtaining VHHs that bind to specific antigens or epitopes have previously been disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., J Biol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17 June, 2009 and WO94 / 04678.

[0411] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0412] "Humanized antibody," also known as CDR-grafted antibody, refers to an antibody generated by grafting a non-human CDR sequence into the variable region framework of a human antibody. This overcomes the strong immune response induced by chimeric antibodies due to their carrying of numerous non-human protein components. To avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse mutations can be performed on the fully human antibody variable region to maintain activity. Examples of "humanization" include the substitution of one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding position in the VH domain of a conventional human tetrapeptide antibody by a VHH domain derived from the Camelidae family. The humanized VHH domain may contain one or more fully human framework region sequences, and in some specific embodiments, may contain the human framework region sequence of IGHV3. Humanization methods include protein surface amino acid resurfacing and antibody humanization using a universal framework grafting method (CDR grafting to a universal framework), which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes, for example, can be found in the VBase human germline sequence database and in Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0413] Those skilled in the art can use any of the many known numbering schemes to determine the amino acid sequence boundaries of antibody CDRs. Known numbering schemes include those described in the following literature: Kabat et al., above (“Kabat” numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (“Chothia” numbering scheme); MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (“Contact” numbering scheme); Lefran et al., Dev. Comp. Immunol., 2003, 27:55-77 (“IMGT” numbering scheme); and Honegge and Plückthun, J. Mol. Biol., 2001, 309:657-70 (“AHo” numbering scheme); each of which is incorporated herein by reference in its entirety. Unless otherwise specified, Kabat numbering is used in the embodiments of this disclosure.

[0414] An epitope, or the interchangeable term "antigenic determinant," refers to the site on an antigen where an immunoglobulin or antibody specifically binds. Antigenic determinants typically contain chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and / or specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a distinctive spatial conformation; it can be a "linear" epitope or a "conformal" epitope. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996).

[0415] In linear epitopes, all points of interaction between the antigen and the interacting molecule (e.g., antibody) are linearly located along the primary amino acid sequence of the antigen. In conformational epitopes, the points of interaction are located across amino acid residues that are separated from each other. Epitopes of a given antigen can be identified using many epitope localization techniques well known in the art. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996). For example, linear epitopes can be determined by, for instance, by simultaneously synthesizing a large number of peptides on a solid support, wherein these peptides correspond to portions of a protein molecule, and by reacting these peptides with an antibody while still attached to the support. These techniques are known in the art and described, for example, in U.S. Patent No. 4,708,871; Geysen et al. (1984) Proc. Natl. Acad. Sci. USA 81:3998-4002; and Geysen et al. (1986) Molec. Immunol. 23:709-715. Conformational epitopes can also be identified by determining the spatial configuration of amino acids, for example, through X-ray crystallography and two-dimensional nuclear magnetic resonance. Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen. A high-throughput method for obtaining antibodies binding to the same epitope based on their cross-competition is described in International Patent Application WO03 / 48731. Therefore, antibodies and their antigen-binding fragments that compete with the antibody molecules of this disclosure for binding to the same epitope on TLT-1 can be obtained using conventional techniques known to those skilled in the art.

[0416] Generally, the term "specificity" refers to the number of different types of antigens or epitopes that a particular antigen-binding molecule or antigen-binding protein (such as the TLT-1 binding molecule of this disclosure) can bind. Specificity can be determined based on the affinity and / or avidity of the antigen-binding protein. It is determined by the dissociation equilibrium constant (K0) between the antigen and the antigen-binding protein. D The affinity expressed by K is a measure of the strength of binding between an epitope and the antigen-binding site on an antigen-binding protein: K D The smaller the value, the stronger the binding strength between the epitope and the antigen-binding protein (or, affinity can also be expressed as the binding constant (K)). a ), which is 1 / K DAs those skilled in the art will understand, depending on the specific antigen of interest, affinity can be determined in a known manner. Affinity is a measure of the strength of binding between an antigen-binding protein (e.g., an immunoglobulin, antibody, a single variable domain of an immunoglobulin, or a polypeptide containing such a domain) and the associated antigen. Affinity relates to both the affinity between the protein and the antigen-binding site on the antigen-binding protein, and the number of associated binding sites present on the antigen-binding protein.

[0417] "Functional variants" include, but are not limited to, homologues, fragments, truncated forms, mutants, and modifications of wild-type proteins / peptides. These functional variants of proteins / peptides exhibit enhanced, reduced, or maintained protein / peptide activity compared to the wild-type protein / peptide. Non-limiting examples include alteplase (rt-PA), tenecteplase (TNK, TNK-tPA), reteplase (r-PA), monteplase, lateplase, and sarublase (r-scu-PA, RESCUPASE), all of which can be considered functional variants of tPA.

[0418] The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (ELISA), and sandwich competitive assays) as described in this disclosure.

[0419] In this disclosure, "homology" and "identity" refer to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position in two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of compared positions × 100. For example, at optimal sequence alignment, if 6 out of 10 positions in two sequences match or are homologous, then the two sequences are 60% homologous; if 95 out of 100 positions in two sequences match or are homologous, then the two sequences are 95% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0420] "Conservative amino acid mutation" or "conservative (amino acid) substitution" refers to the substitution of one or more amino acid residues in a protein or polypeptide with a conserved amino acid structure. The original amino acid residues are chemically similar to the substituted amino acid residues, and the substitution has little or no impact on the function, activity, or other biological properties of the protein or polypeptide. Such conserved amino acid substitutions are well-known in the art; for example, a preferred conserved amino acid substitution is the substitution of one amino acid residue from group (i)-(v) by another amino acid residue from the same group:

[0421] (i) Smaller aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly;

[0422] (ii) Polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;

[0423] (iii) Polar positively charged residues: His, Arg, and Lys; (iv) Larger aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and

[0424] (v) Aromatic residues: Phe, Tyr and Trp.

[0425] Particularly preferred conserved amino acid substitutions are as follows: Ala is substituted with Gly or Ser; Arg is substituted with Lys; Asn is substituted with Gln or His; Asp is substituted with Glu; Cys is substituted with Ser; Gln is substituted with Asn; Glu is substituted with Asp; Gly is substituted with Ala or Pro; His is substituted with Asn or Gln; Ile is substituted with Leu or Val; Leu is substituted with Ile or Val; Lys is substituted with Arg, Gln, or Glu; Met is substituted with Leu, Tyr, or Ile; Phe is substituted with Met, Leu, or Tyr; Ser is substituted with Thr; Thr is substituted with Ser; Trp is substituted with Tyr; Tyr is substituted with Trp or Phe; Val is substituted with Ile or Leu.

[0426] The terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are used interchangeably in this disclosure and refer to any single-stranded or double-stranded DNA or RNA molecule, and in the case of a single-stranded molecule, its complementary sequence, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is “effectively linked.” For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to said coding sequence.

[0427] The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is attached. In one embodiment, the vector is a "plasmid," which refers to a circular double-stranded DNA loop into which an additional DNA segment can be attached. In another embodiment, the vector is a viral vector into which an additional DNA segment can be attached to a viral genome. The vectors of this disclosure are capable of autonomous replication in host cells that have been introduced into them (e.g., bacterial vectors with bacterial origins of replication and episodic mammalian vectors) or can be integrated into the host cell's genome after introduction into the host cell, thereby replicating along with the host genome (e.g., non-episodic mammalian vectors).

[0428] The terms “cell,” “cell line,” and “cell culture” used in this disclosure are used interchangeably, and all such names include progeny. Therefore, “transformant” and “transformed cell” include primary test cells and cultures derived therefrom, regardless of passage number. It should also be understood that, due to intentional or unintentional mutations, all progeny cannot be exactly identical in terms of DNA content. This includes mutant progeny with the same function or biological activity as those screened from the original transformed cells. Where different names are intended, this is clearly apparent from the context.

[0429] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors into which nucleic acid inserts are incorporated. Host cell includes progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cell includes cells transfected and / or transformed in vivo with nucleic acids of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

[0430] "Pharmaceutical composition" refers to a mixture containing one or more of the TLT-1 binding molecules, platelet-targeting molecules, polynucleotides, and other components, such as physiologically / pharmaceutically acceptable carriers, diluents, buffers, or excipients described herein. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertion of its biological activity.

[0431] "Giving," "administering," and "treatment," when applied to animals, humans, subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Giving," "administering," and "treatment" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and cells, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cells. "Giving," "administering," and "treatment" also mean the treatment of cells in vitro and in vitro by means of a reagent, diagnostic agent, conjugate composition, or another cell. "Treatment," when applied to humans, veterinary subjects, or research subjects, refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0432] "Treatment" means administering an oral or topical therapeutic agent (such as the platelet-targeting protein of this disclosure) to a subject who has symptoms of one or more diseases, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, a therapeutic agent is administered in a treated patient or population in an amount that effectively relieves one or more symptoms of the disease, inducing the regression of such symptoms or inhibiting their progression to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the patient's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been relieved can be evaluated using any clinical test method commonly used by a physician or other healthcare professional to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating symptoms of each target disease, they should reduce symptoms of the target disease in a statistically significant number of patients, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0433] "Optional" or "optionally" means that the event or circumstance described below may, but does not necessarily, occur, and the description includes the possibility that the event or circumstance may or may not occur. "And / or" should be interpreted as specifically disclosing that each of the two specified features or components has or does not have the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" in this disclosure includes "A and B," "A or B," "A" (alone), and "B" (alone). Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," etc., should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; that is, the meaning of "including but not limited to."

[0434] In this disclosure, "subject" and "patient" refer to mammals, especially primates, and particularly humans.

[0435] The ordinal numbers “first,” “second,” “L1,” “L2,” etc., used in this disclosure are not intended to limit quantity, level, grade, or order, but are intended only to distinguish different elements, steps, or technical features.

[0436] Example

[0437] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure. Experimental methods in the embodiments of this disclosure that do not specify specific conditions are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual or Molecular Cloning Manual; or under conditions recommended by the raw material or commercial manufacturer. Reagents that do not specify a specific source are commercially available, conventional reagents.

[0438] Example 1. Preparation of targeted antigens and detection proteins and peptides

[0439] Using human TLT-1 (SEQ ID NO: 1) as a template, we designed related proteins for immunization, screening, and detection.

[0440] Human TLT-1 extracellular protein (human TLT-1_ECD, SEQ ID NO: 2) was fused with a His tag or an Avi-His tag, expressed and purified in HEK293 cells, and used for screening and detection of camel-derived single-domain antibody phage libraries.

[0441] A 37-amino acid polypeptide fragment (SEQ ID NO: 5) from the C-terminus of the extracellular region of human TLT-1 protein was chemically synthesized and KLH-conjugated. Simultaneously, this polypeptide fragment was fused with camel IgG1aFc for expression.

[0442] Partial sequences are as follows:

[0443] Human TLT-1 full length (Note: Italicized parts represent signal peptides; underlined parts represent extracellular domains; bolded italicized parts represent transmembrane domains; wavy underlined parts represent intracellular domains.)

[0444] Human TLT-1 extracellular region (human TLT-1_ECD)

[0445]

[0446] >Human TLT-1_ECD-His

[0447] >TLT-1_ECD-avi-His (Note: The underlined part is the extracellular region of human TLT-1 protein (human TLT-1_ECD); the underlined part is the G4S linker; the underlined part is the Avi tag; and the italic part is the His tag.)

[0448] Human TLT-1 37mer peptide

[0449]

[0450] Human TLT-1 37mer peptide-camel IgG1a Fc fusion protein (Note: The double-underlined part represents camel IgG1a Fc)

[0451] The above protein sequences can be expressed, purified, and isolated using conventional methods in this field. The polypeptide sequences are KLH-conjugated or biotin-labeled during synthesis.

[0452] Example 2. Screening for single-domain antibodies (VHH) that specifically bind to human TLT-1

[0453] 1. Camel immunity and phage library establishment

[0454] A native Bactrian camel from Inner Mongolia was immunized with a synthetically produced human TLT-1 37mer polypeptide fragment conjugated with KLH and a 37mer polypeptide-camel IgG1a Fc fusion protein. Freund's complete or incomplete adjuvant was mixed with the immunogen at a 1:1 volume ratio, and the camel was immunized subcutaneously at multiple sites. The initial immunization dose was 200 μg of protein, with booster immunizations every two weeks at a dose of 100 μg of protein each time.

[0455] Titer was determined after 4-5 immunizations. 2 μg / mL human TLT-1_ECD-His coated plates (Costar, Cat. #9018), 100 μL / well, were incubated overnight at 4°C. The next day, the plates were washed three times with PBST (0.05% Tween 20), 300 μL / well, followed by blocking with 4% skim milk powder and incubation at 37°C for 2 hours. After washing, serially diluted immunized camel serum was added, and the plates were incubated at 37°C for 1 hour. Negative controls included serially diluted pre-immunization serum and blank PBS solution. After incubation, the plates were washed three times with PBST, and horseradish peroxidase-labeled goat anti-camel Fc polyclonal antibody (Thermo, Cat No. A16060) was added, followed by incubation at 37°C for 1 hour. After washing three more times with PBST, TMB was added for color development, and the reaction was terminated with 1M sulfuric acid. The absorbance was read using a SpectraMax M5 microplate reader at OD 450 nm. After the titer was qualified, peripheral blood was collected from camels, peripheral blood lymphocytes (PBMCs) were separated using the Ficoll method, cells were lysed with Trizol, RNA was extracted, cDNA was obtained by reverse transcription, and phage libraries were constructed.

[0456] 2. Screening of phage libraries

[0457] Phage libraries were screened using biotinylated human TLT-1 37mer peptide and human TLT-1_ECD-Avi-His protein to obtain single-domain antibodies with affinity for human TLT-1.

[0458] 2 μg of biotinylated human TLT-1 37mer peptide was bound to 1 mg of Dynabeads M-280 streptavidin (Invitrogen, Cat. #11206D). After incubation at room temperature for 30 minutes, the sample was washed three times with 1×PBS, blocked with 2% skim milk at room temperature for 2 hours, and then the aforementioned camel-derived single-domain antibody phage display library was added and incubated at room temperature for 1 hour. The sample was washed nine times with PBST (0.05% Tween-20) to remove unbound phages. Phages specifically bound to the human TLT-1 37mer peptide were eluted with 1 mg / mL trypsin and used to infect *E. coli* TG1 bacteria in the logarithmic growth phase. Phages were produced and purified for the next round of screening.

[0459] The same screening process was repeated twice. In the second round, 10 μg of biotinylated human TLT-1_ECD-Avi-His protein was used as the screening antigen. In the third round, biotinylated human TLT-1 37mer peptide was used as the screening antigen. Positive clones were enriched after three rounds of screening.

[0460] 3. Phage ELISA identification and NGS sequence analysis

[0461] Single colonies were selected from the enriched clones and packaged into phage single-domain antibodies for phage ELISA testing. ELISA plates were coated overnight with 2 μg / mL of human, dog, rat, mouse, and porcine TLT-1 peptides and human TLT-1_ECD-His, respectively. After blocking with 4% skim milk at 37°C for 1 hour, phage supernatant diluted with 2% skim milk was added, and the plates were incubated at room temperature for 1 hour. The plates were washed three times with PBST (0.05% Tween 20) and detected with horseradish peroxidase-labeled anti-M13 secondary antibody. Clones with OD450 absorbance values ​​greater than 0.5 for both human TLT-1_ECD-His protein and human, rat, and mouse TLT-1 37mer peptides in the ELISA binding assay were sequenced. NGS sequencing analysis was also performed on the phage library before and after the second round of screening, yielding further sequences. Some VHH sequences are shown below:

[0462] >30a(VHH)

[0463] >31a(VHH)

[0464] >61a(VHH)

[0465] >70a(VHH)

[0466] >121a(VHH)

[0467] >154a(VHH)

[0468] >155a(VHH)

[0469] >170a-2(VHH)

[0470] >183a-1(VHH)

[0471] The anti-TLT-1 antibodies disclosed herein are summarized in Table 1 according to the CDR sequence encoded by Kabat.

[0472] Table 1. CDR sequence of VHH

[0473] 4. Preparation of intact antibodies

[0474] Taking 183a-1(VHH) as an example: the sequence of 183a-1(VHH) is fused with the following human IgG4-Fc (Hinge-CH2-CH3, containing S228P mutation) fragment to obtain 183a-1(Fc).

[0475] hIgG4-Fc(S228P)

[0476] >183a-1(Fc) (Note: The underlined part is human IgG4-Fc)

[0477] The 183a-1(Fc) sequence was cloned into the mammalian expression vector pTT5, transfected into HEK293E or ExpiCHOs (ThermoFisher, Cat.#A29127) cells for expression, and then isolated and purified to obtain the target antibody.

[0478] 293T cells were seeded in 6-well plates, with 6 × 10⁶ cells per well. 5 Cells were transfected with 3 μg of human TLT-1 plasmid (SinoBio, Cat.#HG11934-UT), rat TLT-1 plasmid (internal construction, NCBI reference number: NM_001419518.1), and mouse TLT-1 plasmid (SinoBio, Cat.#MG5A2386-UT) into cells using Lipofectamine 2000 transfection reagent and Opti-MEM medium. Cells were allowed to express the protein for 48 hours for binding assays.

[0479] Flow cytometry was used to detect the binding of a TLT-1 single-domain antibody (a fusion protein with human IgG4 Fc) to TLT-1 overexpressed cells. First, the antibody was diluted to the desired concentration with staining buffer. TLT-1-overexpressing cells were digested and resuspended in staining buffer. 50 μL of cell suspension and 50 μL of diluted antibody were mixed in a 96-well plate and incubated at 4°C for 30 min. After washing 1-2 times with PBS, 50 μL of the corresponding secondary antibody was added. The plate was incubated at 4°C for 30 min. After washing 1-2 times with PBS, flow cytometry was performed, and data analysis was conducted using Flowjo and GraphPad Prism 9.

[0480] As shown in Figure 1 and Table 2, the binding experiment with HEK293T cells overexpressing TLT-1 revealed that only 183a-1 cells had good human TLT-1 binding function, and also bound to rat and mouse TLT-1.

[0481] Table 2. Binding of TLT-1 single-domain antibody 183a-1 (Fc) to TLT-1 on cell surface

[0482] Example 3. Humanization of anti-human TLT-1 single-domain antibody

[0483] Three-dimensional structural homology modeling was performed on the specific anti-TLT-1 single-domain antibody 183a-1. Based on the alignment results with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the germline gene IGHV3-23, which has high sequence homology with 183a-1, was selected as the template for FR1, FR2, and FR3. IGJH4 was used as the template for FR4. The CDR of the camel-derived single-domain antibody was transplanted into the corresponding human template, forming a variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The transplanted single-domain antibody underwent another three-dimensional structural simulation and analysis. Reversion mutations were performed on specific sites in the FR region that affect the structural morphology of the CDR region. The humanized VHH framework region contains at least one of the following amino acid reversion mutations: 27G; 30G; 37F; 44Q; 45R; 47G; 49A; 74A; 78V; 82L; 94G. The modified antibody exhibits higher stability. The design of the reversion mutation sites and mutation modes is shown in Table 3.

[0484] Table 3. Reversal mutation sites and mutation modes

[0485] The obtained humanized sequence is as follows, with the CDR region represented by an underscore (Kabat encoding rule).

[0486] >183a-1_hu1

[0487] >183a-1_hu2

[0488] >183a-1_hu3

[0489] >183a-1_hu4

[0490] The above humanized sequence was cloned into the mammalian expression vector pTT5, and the plasmid was transiently transfected into HEK293E or ExpiCHOs cells. After expression, the target antibody was obtained by separation and purification.

[0491] Simultaneously, a fusion protein of a humanized single-domain antibody and the Fc region of hIgG4 was constructed using the method described in Example 2. The obtained complete humanized antibody sequence is shown below, with the underlined portion representing the CDR region and the dashed portion representing the Fc region of hIgG4 (S228P):

[0492] >183a-1_hu1-hIgG4Fc(S228P)

[0493] >183a-1_hu2-hIgG4Fc(S228P)

[0494] >183a-1_hu3-hIgG4Fc(S228P)

[0495] >183a-1_hu4-hIgG4Fc(S228P)

[0496] Example 4. Assay of binding of anti-human TLT-1 single-domain antibody to TLT-1

[0497] 1. The affinity of the single-domain antibody VHH to human TLT-1 was determined using a Biacore T200 (GE Healthcare).

[0498] Anti-His-tagged antibodies were conjugated to the CM5 sensor chip (GE, Cat.#29-1049-88) using an amino-conjugation kit (GE, Cat.#BR-1000-50) and a His capture kit (GE, Cat.#28-9950-56) according to the kit instructions. A certain amount of human TLT-1_ECD-His (SEQ ID NO: 3) was affinity-captured using this conjugated chip. Then, a series of serially diluted test antibody 183a-1_hu3 was flowed onto the chip surface at a flow rate of 50 μL / min. After each cycle, the chip was regenerated using glycine-hydrochloric acid (GE, Cat.#BR-1003-54) at pH 1.5. The reaction buffer was 1× HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P2O, pH 7.4).

[0499] The reaction signal was detected in real time using a Biacore T200 to obtain binding and dissociation curves. The data were fitted using BIAevaluation version 4.1 and GE software in a steady-state affinity mode to obtain affinity values. The results are shown in Table 4, and the corresponding sensorgrams are shown in Figure 2. It is evident that 183a-1_hu3 and human TLT-1 ECD exhibit a rapid binding and dissociation steady-state mode. After entering the body, it quickly binds to TLT-1 on activated platelets, enriching the thrombolytic molecule uPA at the pathological thrombus site, thus rapidly exerting a thrombolytic effect. The fusion molecule then quickly dissociates from TLT-1 and is metabolically cleared. This rapid binding and dissociation mode of the TLT-1 antibody, compared to the prolonged action time caused by continuous binding to TLT-1, is expected to reduce the risk of bleeding.

[0500] Table 4. Affinity data for 183a-1_hu3

[0501] 2. The binding of the single-domain antibody VHH to human TLT-1 was determined using an overexpression cell line.

[0502] 293T cells were seeded in 6-well plates, with 6 × 10⁶ cells per well. 5 3 μg of human TLT-1 plasmid (SinoBio, Cat.#HG11934-UT) was transfected into cells using Lipofectamine 2000 transfection reagent and Opti-MEM medium. Cells were allowed to express the protein for 48 hours for binding assays.

[0503] Flow cytometry was used to detect the binding of TLT-1 single-domain antibody (a fusion protein with human IgG4 Fc) to TLT-1 overexpressed cells. First, the antibody was diluted to the desired concentration with staining buffer. Overexpressing cells were digested and resuspended with staining buffer. 50 μL of cell suspension and 50 μL of diluted antibody were mixed in a 96-well plate and incubated at 4°C for 30 min. After washing 1-2 times with PBS, 50 μL of the corresponding secondary antibody was added. The plate was incubated at 4°C for 30 min. After washing 1-2 times with PBS, flow cytometry was performed, and data analysis was conducted using Flowjo and GraphPad Prism9.

[0504] The experimental results are shown in Figure 3 and Table 5. The single-domain antibody 183a-1 and its humanized antibody both showed good binding to human TLT-1.

[0505] Table 5. Binding of TLT-1 single-domain antibody (Fc) to TLT-1 on cell surface

[0506] Example 5. Genetic engineering of anti-human TLT-1 single-domain antibody

[0507] To reduce potential immunogenicity, we performed site-directed mutagenesis on 183a-1_hu3. The first round of modification yielded molecules 183a-1_hu3-1 to 183a-1_hu3-14, based on a combination of immunogenicity and affinity assays. In the second round of modification, selected mutation sites were used to construct multi-site combined mutant molecules 183a-1_hu3-15 to 183a-1_hu3-17. The specific sequences of the obtained modified single-domain antibodies are shown below, with CDR regions underlined (Kabat coding rules).

[0508] >183a-1_hu3-1

[0509] >183a-1_hu3-2

[0510] >183a-1_hu3-3

[0511] >183a-1_hu3-4

[0512] >183a-1_hu3-5

[0513] >183a-1_hu3-6

[0514] >183a-1_hu3-7

[0515] >183a-1_hu3-8

[0516] >183a-1_hu3-9

[0517] >183a-1_hu3-10

[0518] >183a-1_hu3-11

[0519] >183a-1_hu3-12

[0520] >183a-1_hu3-13

[0521] >183a-1_hu3-14

[0522] >183a-1_hu3-15

[0523] >183a-1_hu3-16

[0524] >183a-1_hu3-17

[0525] The modified anti-TLT-1 antibody molecules disclosed herein are summarized in Table 6 according to the CDR sequence encoded by Kabat.

[0526] Table 6. CDR sequence of modified VHH

[0527] The above single-domain antibody sequence was cloned into the mammalian expression vector pTT5, and the plasmid was transiently transfected into HEK293E cells. After expression, the target antibody was obtained by separation and purification.

[0528] The affinity of the single-domain antibody VHH for human TLT-1 was determined using a Biacore T200 (GE Healthcare). The method was the same as in Example 4. The data were fitted using steady-state affinity to obtain the affinity values, and the results are shown in Tables 7 and 8. The binding sensorgram of 183a-1_hu3-15 to human TLT-1 ECD is shown in Figure 4. Other modified molecules also showed similar fast binding and fast dissociation steady-state patterns.

[0529] Table 7. Affinity data of the first-round modified single-domain antibody with human TLT-1

[0530] Table 8. Affinity data of the second-round modified single-domain antibody with human TLT-1

[0531] Example 6. Design and preparation of platelet-targeting fusion protein

[0532] Pro-UK (uniprot, P00749, Ser21-Leu431), LMW-scuPA (low molecular weight single-chain uPA, Lys156-Leu431), and tenecteplase (TNK, WO9324635) are fused to the N-terminus or C-terminus of an anti-TLT-1 single-domain antibody via linkers, as shown in Figure 5. The molecular structures of each fusion protein are shown in Table 9 below, with examples as follows: 9001 is a non-targeted LMW-scuPA (low molecular weight single-chain uPA) with linkers G4S and His tags.

[0533] Table 9. Structure of platelet-targeting fusion molecules

[0534] The relevant sequences are as follows:

[0535] >LMW-scuPA

[0536] Pro-UK

[0537] TNK

[0538] G4S

[0539] >(G4S)×3

[0540] His tag

[0541] The DNA sequence of the fusion protein was cloned into the mammalian cell expression vector pTT5, and the expression plasmid was transiently transfected into HEK293 or expiCHOs cells. The cells were cultured and the culture supernatant was collected. The purified fusion protein was then obtained through separation and purification.

[0542] Example 7. Affinity determination of platelet-targeting uPA fusion protein with human TLT-1

[0543] The affinity of platelet-targeting uPA fusion molecules for human TLT-1 was determined using a Biacore T200. Using an amino-coupling kit (GE, Cat.#BR-1000-50), the FC2 channel of a CM5 sensor chip (GE, Cat.#29-1049-88) was conjugated with the antigen protein TLT-1_ECD-his (SEQ ID NO: 3) according to the kit instructions until the surface response value reached the predetermined RU value. The channel was then blocked with ethanolamine and set aside for later use. The FC1 channel, after activation by EDC / NHS and subsequent blocking with ethanolamine, was used as the reference channel. The coupled chip was used for target molecule affinity assays. A series of serially diluted antibody / fusion protein samples were flowed onto the chip surface at a flow rate of 50 μL / min, with a binding time of 30 seconds and a dissociation time of 60 seconds. After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (GE, Cat.#BR-1003-54). The reaction buffer was 1× HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P2O, pH 7.4). The reaction signal was monitored in real-time using a Biacore T200 (GE Healthcare) to obtain binding and dissociation curves. The data were fitted using BIAevaluation version 4.1 GE software to determine the steady-state affinity, and the results are shown in Table 10. The sensorgrams of binding of 9004, 9008, and 9018 to human TLT-1 ECD are shown in Figures 6 to 8, respectively.

[0544] Table 10. Affinity data of fusion molecules

[0545] Example 8. In vitro urokinase activity assay of platelet-targeted uPA fusion protein

[0546] uPA (urokinase-type plasminogen activator) is a serine protease. To verify the urokinase activity of the fusion protein disclosed in this invention, its substrate can be used for direct enzyme activity detection. For the in vitro urokinase activity assay of the fusion protein, the non-targeted uPA protein (9001) and the targeted fusion protein (9004, 9018) used in this embodiment were pre-digested with 0.1 U / mL plasmin (Sigma, cat.#P1867-150UG) at room temperature for 30 minutes. The experimental reaction system was a buffer solution at pH 8.8 containing 50 mM Tris–HCl and 38 mM NaCl. The plasmin-activated reaction mixture was added to a clear 96-well plate, and 0.5 mM urokinase substrate S-2444 (Asnail, cat.#AS00-0110) was added and mixed. The experimental reaction system was the aforementioned buffer solution at pH 8.8. Place the 96-well plate in a SpectraMax M5 microplate reader and read the absorbance every minute at 405 nm wavelength and room temperature to reflect the enzyme activity rate.

[0547] As shown in Figure 9, the enzyme activity of platelet-targeted uPA molecules and non-targeted uPA molecules in this study is basically the same, confirming that the tandem targeting fragment does not affect the urokinase activity of the activated molecule.

[0548] Example 9. In vitro conversion plasminogen activity assay of platelet-targeted uPA fusion molecule

[0549] To better simulate the in vivo enzymatic activity of the fusion protein, specifically its activity in converting plasminogen to plasmin, an in vitro indirect enzyme activity assay was performed using plasmin substrates. For the in vitro plasminogen-to-plasmin activity assay of the fusion protein, the non-targeted uPA protein and the targeted fusion protein used in this embodiment were co-incubated with 400 nM human glu-plasminogen (Enzyme Research, cat.#HPG 2001) and 1 mM plasmin substrate S-2251 (Asnail, cat.#AS00-0106) in a clear 96-well plate. The experimental reaction system was a pH 7.4 buffer containing 50 mM Tris–HCl and 0.1 M NaCl. The 96-well plate was placed in a SpectraMax M5 microplate reader, and absorbance was read every 30 seconds at 405 nm at room temperature to reflect the enzyme activity reaction rate. The plasminogen activation rate of different molecules was calculated using the method described in the reference (Res Pract Thromb Haemost. 2022; 6:e12771.).

[0550] As shown in Figure 10 and Table 11 below, the platelet-targeted uPA molecules and non-targeted uPA molecules exhibited essentially equivalent in vitro plasminogen activating activity, confirming that the tandem targeting of the target fragments did not affect the activity of the molecules.

[0551] Table 11. Platelet-targeted uPA fusion molecule conversion plasminogen conversion rate

[0552] Example 10. In vitro thrombolytic activity of platelet-targeting uPA fusion protein

[0553] For the in vitro thrombolytic activity of the fusion protein, this example used sodium citrate-anticoagulated whole blood (human blood or SD rat whole blood, C57 / BL6J mouse whole blood, beagle dog whole blood, volume ratio 1:9) to compare the in vitro thrombolytic effects of positive molecules TNK-tPA (TNK; Guangzhou Mingkang Biotechnology, Mingfule) and 9001, 9004, and 9018 at equimolar concentrations. A coagulant was prepared by mixing 134 mM CaCl2 solution and HEPES buffer (50 mM HEPES, 274 mM NaCl) in equal proportions. 5 μL of the coagulant was added to the bottom edge of a clear 96-well plate, 5 μL of ddH2O was added to the positive control well, and 25 μL of anticoagulated whole blood was aspirated. The pipette tip was slowly drawn in a circular motion along the edge of the well to evenly disperse the coagulant, while simultaneously slowly expelling the blood from the pipette tip, creating a hollow circular thrombus at the bottom of the well. Sealed well plates were incubated at 37°C for 15 minutes to promote thrombus formation and stabilization. Different concentrations of the test molecule were diluted in ddH2O. 70 μL of the diluted test molecule (70 μL of ddH2O was added to the negative and positive control wells) was added to the thrombus-forming wells. The 96-well plate was placed in a SpectraMax M5 microplate reader, and absorbance was read every minute at 510 nm wavelength and 37°C, with the plate shaken for 5 seconds at each time point. The in vitro thrombolytic activity of the test molecule was demonstrated by using absorbance values ​​read at different time points under different concentration conditions. The data were fitted to a four-parameter logarithmic equation using nonlinear regression analysis, and the corresponding EC5 values ​​were derived. 50 value.

[0554] This disclosure shows that the thrombolytic activity of targeting uPA molecules 9004 and 9018 in simulated thrombi prepared from whole blood samples of humans, rats, mice, and beagle dogs was superior to that of the non-targeting molecule 9001. Compared with positive molecules, under high concentration conditions, the thrombolytic activity of 9018≈9004>TNK was... The results of human blood sample testing are shown in Table 12.

[0555] Table 12. In vitro thrombus dissolution time (EC) of the analyte in humans 50 Note: *180 min is the experimental endpoint.

[0556] Example 11. Evaluation of the in vivo efficacy of platelet-targeting uPA molecules in a rat thromboembolic stroke model.

[0557] To assess the in vivo efficacy of fusion proteins in improving stroke, this embodiment uses a rat thromboembolic stroke model (embolic MCAo, eMCAo) to test the efficacy of 9004 or 9018 in improving infarction, neurological function, and restoring cerebral blood flow. Five- to six-week-old male SD rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased, and the model was established after the animals had acclimatized for a few days. Immediately after blood collection from the rats, it was aspirated into a PE50 tube (inner diameter 0.58 mm, outer diameter 0.96 mm) containing thrombin (Solarbio, cat.#T8021), incubated at room temperature for 2 hours, and then stored at 4°C for 22 hours. The thrombus was then pushed out into 30 mL of physiological saline and washed three times. An 8 mm long embolus was cut and aspirated into a PE50 tube for later use.

[0558] Before thromboembolization, baseline cerebral blood flow was measured using a SIM BFI HR PRO laser speckle flowmeter. Rats were anesthetized with isoflurane gas and fixed in a supine position on the operating table. The skin was incised along the midline of the neck, and the right common carotid artery and external carotid artery were freed. The pterygopalatine artery, a branch of the internal carotid artery, was isolated and clamped. A small incision was made at the external carotid artery, and the embolus from the catheter was pushed into the cranium with 0.4 mL of normal saline. The catheter was carefully withdrawn, and the skin was sutured. Fifty-five minutes after thromboembolization, a blinded modified neurological severity score (mNSS) was performed. Rats with a relatively uniform injury score of 9-10 and a ≥50% decrease in cerebral blood flow compared to the ipsilateral baseline, meeting both criteria, were eligible for enrollment. If cerebral blood flow was not monitored, rats with a neurological severity score of 9-11 were enrolled. Drug administration began 60 minutes after thromboembolization.

[0559] In this experiment, rats were randomly divided into groups of 10 each. A blinded method was used to administer a single intravenous injection of vehicle (model control group), 9004 (1.3 mg / kg), or 9018 (1.3 / 3.9 / 11.7 mg / kg), with a dosage volume of 2 mL / kg. Two hours after administration, cerebral blood flow was measured using a laser speckle imaging system. Neurological function was assessed at 6 and 24 hours post-administration. After the 24-hour assessment, the heart was perfused with saline, the rats were decapitated, and the forebrain was harvested for photographic recording of overall cerebral hemorrhage. The brain tissue was frozen at -20°C and then sliced ​​from front to back, with each slice being 2 mm thick. The cerebral hemorrhage was scored on the slices. The brain tissue slices were incubated in 1% red tetrazolium (TTC) solution at 37°C for 5 minutes; infarcted tissue appeared white, while non-infarcted tissue appeared red. ImageJ software was used to measure the infarct area and calculate the percentage of the total brain area covered by the infarct. The experimental data were analyzed using Graphpad statistical software. The results are shown in Tables 13 and 14. Compared to the model group, both 9004 and 9018 significantly improved the symptoms of ischemic stroke in rats, including increased cerebral blood flow, reduced infarct area, and improved neurological function. Furthermore, the multiple-dose 9018 group also showed a clear dose-dependent effect. Simultaneously, no significant bleeding risk was observed in either the 9004 or 9018 treatment groups.

[0560] Table 13. Improvement results of various indicators after drug administration

[0561] Table 14. Improvement results of various indicators after drug administration

[0562] Example 12. Evaluation of the in vivo safety of platelet-targeting uPA molecules using a mouse tail hemorrhage model.

[0563] Regarding the in vivo safety and safety window size of the fusion protein, this embodiment uses a mouse tail hemorrhage model to compare the effects of rt-PA (Boehringer Ingelheim) and 9004, 9018 on tail hemorrhage time in mice at clinically equivalent doses and twice the clinically equivalent doses; or the effects of positive molecules TNK (Guangzhou Mingkang Biotechnology, Mingfule) and 9018 (equimolar dose with TNK) on tail hemorrhage time in mice at ten times the clinically equivalent dose. Eight to nine-week-old C57BL / 6J mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were purchased, and experiments began after the animals had acclimatized for a few days. Mice were intraperitoneally injected with an anesthetic. After anesthesia was confirmed, they were then injected intravenously via the tail vein with PBS, rt-PA (9 mg / kg, 18 mg / kg) and 9004 (2 mg / kg, 4 mg / kg), or PBS, rt-PA (11 mg / kg, 22 mg / kg) and 9018 (2.4 mg / kg, 4.8 mg / kg), or PBS, TNK-tPA (30 mg / kg) and 9018 (24 mg / kg). The administration volume was 5 mL / kg. One minute after administration, the tip of the mouse tail (diameter = 1.3 mm) was vertically severed with the back of a blade, and timing was started. The mice were then transferred to a 37°C heating pad with their tails laid flat, the tail tip placed in a 50 mL centrifuge tube filled with physiological saline, and the centrifuge tube was placed in a 37°C water bath. Tail bleeding was observed, and the time of cessation of the first bleeding was recorded. The experimental data were analyzed using Graphpad statistical software. The results are shown in Tables 15-17. At equimolar doses, the bleeding time of the platelet-targeting uPA fusion molecules 9004 and 9018 disclosed in this paper was significantly shorter than that of the control molecule rt-PA, and the dosing safety window of 9018 was significantly better than that of TNK.

[0564] Table 15. Time to initial hemostasis in each group after drug administration

[0565] Table 16. Time to initial hemostasis in each group after drug administration

[0566] Table 17. Time to initial hemostasis in each group after drug administration

[0567] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. A fusion protein containing a TLT-1 binding domain and an anticoagulant.

2. The fusion protein of claim 1, wherein, The TLT-1 binding domain includes an immunoglobulin single variable domain, which includes CDR3 in the amino acid sequence shown in SEQ ID NO: 42; Preferably, the immunoglobulin has a single variable domain that binds to TLT-1 37mer.

3. The fusion protein of claim 2, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any of the amino acid sequences shown in SEQ ID NO: 15, 40-43, and 48-64, or CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NO: 81, 82, and 37, respectively. in, The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, preferably according to the Kabat numbering system.

4. The fusion protein of claim 2 or 3, wherein the immunoglobulin single variable domain comprises: (1) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 36 and 37 respectively; (2) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 65, 36 and 37 respectively; (3) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 36 and 37 respectively; (4) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 67, 36 and 37 respectively; (5) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 68, 36 and 37 respectively; (6) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 69 and 37 respectively; (7) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 70 and 37 respectively; (8) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 71 and 37 respectively; (9) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 72 and 37 respectively; (10) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 73 and 37 respectively; (11) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 70 and 37 respectively; (12) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 73 and 37 respectively; or, (13) CDR1, CDR2 and CDR3, respectively, with amino acid sequences as shown in SEQ ID NO: 67, 70 and 37.

5. The fusion protein as described in any of the preceding claims, wherein the immunoglobulin single variable domain is humanized, reversed mutation, affinity matured, T cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced; Preferably, the heavy-chain framework region of the genus template used in the humanization process is derived from IGHV3-23 and / or IGJH4.

6. The fusion protein according to any one of the preceding claims, wherein the amino acid sequence of the single variable domain of the immunoglobulin is as shown in any one of SEQ ID NO: 15, 40-43, 48-64 or has at least 80% or at least 90% sequence identity with it; Preferably, the single variable domain of the immunoglobulin is an anti-TLT-1 nanobody or VHH.

7. The fusion protein according to any one of the preceding claims, wherein the anticoagulant is a plasminogen activator; Preferably, the plasminogen activator is selected from urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (t-PA), glucosamine, and streptokinase (SK); More preferably, the uPA is selected from LMW-scuPA, HMW-UK, LMW-UK, and Pro-UK; More preferably, the amino acid sequence of the uPA is as shown in SEQ ID NO: 74-75 or has at least 90% identity with it.

8. The fusion protein according to any of the preceding claims, wherein the TLT-1 binding domain and the anticoagulant are directly or via a linker; Preferably, the amino acid sequence of the linker is as follows (G m S n ) h Or (GGNGT) h Or (YGNGT) h As shown, where, m and n are each independently selected from integers from 1 to 8, and h is selected from integers from 1 to 20; More preferably, the connector is the connector shown in (G4S)2 or (G4S)3.

9. The fusion protein according to any of the preceding claims, wherein the TLT-1 binding domain is located at the N-terminus or C-terminus of the anticoagulant.

10. The fusion protein according to any one of the preceding claims, having a function or property selected from one or more of the following: (a) Platelet targeting; (b) Combining TLT-1 in a steady-state mode of fast binding and fast dissociation; (c) Thrombus targeting; (d) Anticoagulant activity, preferably thrombolytic activity.

11. A conjugate comprising the TLT-1 binding domain as defined in any one of claims 1-10 and the anticoagulant.

12. A TLT-1 binding protein comprising an immunoglobulin single variable domain, wherein the immunoglobulin single variable domain comprises CDR3 in the amino acid sequence shown in SEQ ID NO: 42; Preferably, the immunoglobulin has a single variable domain that binds to TLT-1 37mer.

13. The TLT-1 binding protein of claim 12, wherein the immunoglobulin single variable domain comprises CDR1, CDR2, and CDR3 of any of the amino acid sequences shown in SEQ ID NO: 15, 40-43, and 48-64, or comprises CDR1, CDR2, and CDR3 of the amino acid sequences shown in SEQ ID NO: 81, 82, and 37, respectively; wherein, The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, with the Kabat numbering system being preferred.

14. The TLT-1 binding protein of claim 12 or 13, wherein the immunoglobulin single variable domain comprises: (1) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 36 and 37 respectively; (2) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 65, 36 and 37 respectively; (3) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 36 and 37 respectively; (4) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 67, 36 and 37 respectively; (5) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 68, 36 and 37 respectively; (6) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 69 and 37 respectively; (7) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 70 and 37 respectively; (8) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 71 and 37 respectively; (9) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 72 and 37 respectively; (10) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 35, 73 and 37 respectively; (11) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 70 and 37 respectively; (12) CDR1, CDR2 and CDR3 of the amino acid sequences shown in SEQ ID NO: 66, 73 and 37 respectively; or, (13) CDR1, CDR2 and CDR3, respectively, with amino acid sequences as shown in SEQ ID NO: 67, 70 and 37.

15. The TLT-1 binding protein according to any one of claims 12 to 14, wherein the immunoglobulin single variable domain is humanized, reversed mutation, affinity maturation, T cell epitope removal, antibody deamidation reduction, and / or antibody isomerization reduction modification; Preferably, the heavy-chain framework region of the genus template used in the humanization process is derived from IGHV3-23 and / or IGJH4.

16. The TLT-1 binding protein according to any one of claims 12 to 15, wherein the amino acid sequence of the single variable domain of the immunoglobulin is as shown in any one of SEQ ID NO: 15, 40-43, 48-64 or has at least 80% or at least 90% sequence identity with it; Preferably, the single variable domain of the immunoglobulin is an anti-TLT-1 nanobody or VHH.

17. The TLT-1 binding protein according to any one of claims 12 to 16, having a function or property selected from one or more of the following: (f) Combined with TLT-1ECD, preferably combined with TLT-1 37mer; (g) Combining TLT-1 in a steady-state mode with fast binding and fast dissociation; (h) Targets platelets; (i) Targeting thrombi and exerting thrombolytic effects.

18. A polynucleotide encoding a fusion protein according to any one of claims 1 to 10, a TLT-1 binding domain in the conjugate of claim 11, or a TLT-1 binding protein according to any one of claims 12 to 17.

19. A vector comprising or expressing the polynucleotide of claim 18.

20. A host cell comprising or expressing the polynucleotide of claim 18 or the vector of claim 19.

21. A method for producing or preparing the fusion protein according to any one of claims 1 to 10, the TLT-1 binding domain in the conjugate of claim 11, or the TLT-1 binding protein according to any one of claims 12 to 17, comprising the following steps: Culturing the host cells of claim 20; The fusion protein, TLT-1 binding domain, or TLT-1 binding protein were recovered, and Optionally, the fusion protein, TLT-1 binding domain, or TLT-1 binding protein may be purified.

22. A pharmaceutical composition comprising one or more of the following: the fusion protein of any one of claims 1 to 10, the conjugate of claim 11, the TLT-1 binding domain in the conjugate of claim 11, the TLT-1 binding protein of any one of claims 12 to 17, the polynucleotide of claim 18, and the carrier of claim 19; Preferably, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

23. Use of the fusion protein according to any one of claims 1 to 10, the TLT-1 binding domain in the conjugate according to claim 11, the conjugate according to claim 11, the TLT-1 binding protein according to any one of claims 12 to 17, the polynucleotide according to claim 18, and the carrier according to claim 19 in the preparation of a medicament for treating diseases; Preferably, the drug has thrombolytic activity; Preferably, the disease is a cardiovascular disease; More preferably, the diseases include stroke, myocardial infarction, pulmonary embolism, and deep vein thrombosis.

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