FXII / FXIIa binding proteins and medical uses thereof

Abstract

The present disclosure relates to FXII / FXIIa binding proteins and medical uses thereof. In particular, the present disclosure relates to FXII / FXIIa binding proteins, polynucleotides encoding FXII / FXIIa binding proteins, vectors, pharmaceutical compositions, and medical uses thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to FXII / FXIIa binding protein, polynucleotides encoding FXII / FXIIa binding protein, vectors, pharmaceutical compositions and medical uses thereof. Background Art

[0002] Coagulation factor FXII is a serum glycoprotein with a molecular weight of about 80 kDa. It is self-activated by exposure to a negatively charged surface or hydrolyzed by kallikrein to form α-factor XIIa, which is then further converted to β-FXIIa by trypsin and activated. α-FXIIa consists of an N-terminal heavy chain (about 50 kDa) and a C-terminal light chain (about 28 kDa). The N-terminal heavy chain contains a contact binding domain, and the C-terminal light chain contains a catalytic center. The heavy chain and the light chain are connected by a disulfide bond. β-FXIIa is the active form of FXII, with a molecular weight of about 30 kDa, and is composed of a complete light chain and a 2000 Da heavy chain fragment linked by a disulfide bond (Weidmann H, et al. 2017, Bba-Mol Cell Res. 1864: 2118–2127; Davoine C, et al. 2020, Eur J Med Chem. 208: 112753.).

[0003] In the coagulation cascade, FXII drives the coagulation process of the intrinsic coagulation pathway (contact pathway) and is essential for this coagulation pathway. In the intrinsic coagulation pathway, FXII activates FXI to FXIa, and then FXIa activates FIX. The complex of FVIIIa and FXIa then activates FX and participates in the common coagulation pathway. In addition to participating in the intrinsic coagulation pathway, β-FXIIa promotes the cleavage of plasma prekallikrein and cuts the cofactor HK as part of the kallikrein-kinin system to release bradykinin to drive inflammation and regulate vascular permeability. β-FXIIa can also activate components of the complement system, so FXII is involved in thrombosis, vascular regulation and pro-inflammatory processes (Schmaier AH, Stavrou EX. 2019, Res Pract Thromb Haemost. 3 (4): 599–606). Under pathological conditions, the coagulation cascade can be inappropriately activated, subsequently leading to the formation of thromboembolism in blood vessels. Studies have shown that FXII knockout mice or pharmacological blockade of FXII function can significantly inhibit arterial thrombosis, and FXII deficiency or activity blockade has no effect on bleeding in vivo (Renné, Thomas et al. 2005. J Exp Med. 202(2): 271–81; Larsson, Magnus et al. 2014. Sci Transl Med. 6(222).). It is known that the deficiency of coagulation factor FXII is not associated with increased spontaneous injury-related bleeding complications, and people who lack FXII do not suffer abnormal bleeding even during major surgical procedures (Azaad et al. 2015. Open J Blood Dis. 05(04): 39–42; Girolami, A. et al. 2004. Clin Appl Thromb Hemost. 10(4): 351–55.). These findings suggest that FXII is central to the pathological thrombotic process and that interfering with or blocking FXII activation or inhibiting FXIIa activity may be appropriate to block pathological thrombosis and its clinical consequences without increasing the risk of bleeding.

[0004] Camelids (such as camels and alpacas) produce a unique heavy chain antibody (HcAb) lacking light chains. The variable region fragment (VHH) derived from this antibody is called a single domain antibody (sdAb). The molecular weight of a single domain antibody is only 12-15kDa, which is one-tenth of a traditional antibody (containing four chains). Its structure is 2.5nm in diameter and 4nm in length. It is the smallest antibody known to have complete antigen binding activity. Single domain antibodies also contain three CDRs, of which CDR3 plays a major role in affinity. Compared with human antibody VH, the CDR3 of single domain antibodies is longer and can form a bulge loop structure, which can penetrate deep into the antigen and thus better bind to the antigen. Therefore, VHH has the characteristics of high affinity and high specificity. In addition, the hydrophobic residues of FR2 in single domain antibodies are replaced by hydrophilic residues, which have better water solubility and are not easy to form aggregates. Compared with traditional antibodies, single domain antibodies have many advantages such as high binding force, high specificity, high solubility, high stability and high expression.

[0005] Currently, there is no single-domain antibody drug targeting FXII on the market worldwide. Therefore, the development of a single-domain antibody drug against FXII / FXIIa that can provide low immunogenicity and effectively inhibit thrombosis without increasing the risk of bleeding for the prevention of thrombosis and the treatment of related diseases is an important issue that needs to be solved in this field. Summary of the invention

[0006] The present invention discloses a FXII / FXIIa binding protein, a polynucleotide encoding the FXII / FXIIa binding protein, a vector, a pharmaceutical composition and medical uses thereof.

[0007] Serum albumin binding protein

[0008] The present disclosure provides serum albumin binding proteins comprising at least one immunoglobulin single variable domain that specifically binds serum albumin.

[0009] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 25, 32-41, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0010] In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0011] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein comprises CDR1 as shown in SEQ ID NO:26, and / or CDR2 as shown in SEQ ID NO:27, and / or CDR3 as shown in SEQ ID NO:28.

[0012] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 26-28.

[0013] In some embodiments, a serum albumin binding protein is provided, wherein the CDR1 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the CDR2 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the CDR3 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR3s.

[0014] In some specific embodiments, the above-mentioned amino acid mutations are conservative replacements, substitutions or modifications, and / or deletions and / or additions that do not affect the function.

[0015] In some embodiments, the immunoglobulin single variable domain in the present serum albumin binding protein includes any one or any combination (eg, a combination of any two or any three) of the aforementioned CDR1, CDR2, and CDR3.

[0016] In some embodiments, the aforementioned immunoglobulin single variable domain is of camelid origin.

[0017] In some embodiments, 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.

[0018] In some embodiments, the immunoglobulin single variable domain is humanized. In some specific embodiments, the humanization uses the heavy chain variable region germline genes IGHV3-23 and / or IGHV3-66.

[0019] In some embodiments, the humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: 20, 23, 27, 28, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84.

[0020] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one mutation as shown below:

[0021] 20V, 23V, 27L, 27Y or 27N, 28P, 28I, 28S or 28A, 29Y, 29F, 29P, 29G or 29S, 30L, 30A or 30T, 37F, 43E, 44E or 44Q, 45R, 47G, 49A, 71Q, 74A or 74G, 78V, 79S, 81E, 83Q, 84P, and 94A.

[0022] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0023] 1)27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0024] 2)20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0025] 3)23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0026] 4)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A;

[0027] 5)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V;

[0028] 6)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A;

[0029] 7)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P;

[0030] 8)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P;

[0031] 9)30L, 37F, 44E, 45R, 47G, 84P;

[0032] 10)30L, 37F, 44E, 45R, 47G.

[0033] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0034] 1)F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0035] 2)L20V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0036] 3)A23V, F27N, F29Y, S30L, V37F, G44E, L45R, W47G;

[0037] 4)F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S74A;

[0038] 5)F27N, F29Y, S30L, V37F, G44E, L45R, W47G, L78V;

[0039] 6)F27N, F29Y, S30L, V37F, G44E, L45R, W47G, S49A;

[0040] 7)F27N, F29Y, S30L, V37F, G44E, L45R, W47G, R83Q, A84P;

[0041] 8)F27N, F29Y, S30L, V37F, G44E, L45R, W47G, A84P;

[0042] 9)S30L, V37F, G44E, L45R, W47G, A84P;

[0043] 10)S30L, V37F, G44E, L45R, W47G.

[0044] The above mutation sites are defined according to the Kabat numbering system.

[0045] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 25, 32-41, or an amino acid sequence having at least 80% sequence identity thereto.

[0046] In the present 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 ranges between any two of the foregoing values, including integers and decimals.

[0047] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein provided by the present disclosure comprises three complementarity determining regions CDR1, CDR2 and CDR3 and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0048] In some embodiments, the serum albumin binding protein provided by the present disclosure is an antibody or an antigen-binding fragment thereof that binds to serum albumin, or a conjugate or fusion protein comprising the antibody or the antigen-binding fragment thereof.

[0049] In some embodiments, the aforementioned antibody is a camel antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0050] In some embodiments, the aforementioned antigen-binding fragment is an sdAb or a bispecific antibody or a multispecific antibody.

[0051] In some embodiments, the aforementioned immunoglobulin single variable domain is VHH.

[0052] In some embodiments, the aforementioned immunoglobulin single variable domain is a humanized VHH.

[0053] In some embodiments, the aforementioned immunoglobulin single variable domain is an affinity matured VHH.

[0054] In some embodiments, a serum albumin binding protein is provided, which comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains may be the same or different, and any two immunoglobulin single variable domains may be directly connected or connected through a linker.

[0055] In some embodiments, a serum albumin binding protein is provided that binds to or competes for binding to the same epitope as the aforementioned immunoglobulin single variable domains of the present disclosure.

[0056] In some embodiments, a serum albumin binding protein is provided that blocks the binding of the aforementioned immunoglobulin single variable domain of the present disclosure to serum albumin.

[0057] In some embodiments, a serum albumin binding protein is provided, the binding of which to serum albumin is blocked by the aforementioned immunoglobulin single variable domain of the present disclosure.

[0058] In some embodiments, the serum albumin binding protein comprises one or more (eg, 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, which may be the same or different.

[0059] In some embodiments, the serum albumin binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is an Fc region derived from human IgG1, IgG2 or IgG4. The Fc region may or may not contain a mutation.

[0060] In some embodiments, the immunoglobulin single variable domain in the serum albumin binding protein is connected to the Fc region directly or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, G4S (SEQ ID NO: 57), GS, GAP, (G4S) 2 (SEQ ID NO: 51), (G4S) 3 (SEQ ID NO: 52), (G4S) 4 (SEQ ID NO: 53), (G4S) 5 (SEQ ID NO: 54), ASGS (SEQ ID NO: 56), GGNGT (SEQ ID NO: 58), YGNGT (SEQ ID NO: 59), EPKSS (SEQ ID NO: 60), etc.

[0061] Exemplarily, the serum albumin binding protein linked to an Fc region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 30 and 42, or an amino acid sequence having at least 80% sequence identity thereto.

[0062] In some embodiments, the immunoglobulin single variable domain in the aforementioned serum albumin binding protein specifically binds to serum albumin, and the serum albumin can be rodent serum albumin (e.g., mouse serum albumin (mSA)), or primate serum albumin (e.g., macaque serum albumin (cySA), human serum albumin (HSA)). In some specific embodiments, the serum albumin is HSA.

[0063] In some alternatives, the serum albumin binding protein of the present invention may comprise any of the aforementioned complete immunoglobulin single variable domains; it may also comprise a functional portion of any of the aforementioned immunoglobulin single variable domains 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.

[0064] In some embodiments, the variant of the functional portion of the immunoglobulin single variable domain may be a polypeptide that retains the serum albumin 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%, at least 90% sequence homology therewith. For example, it may be a polypeptide that retains the serum albumin binding function of CDR3 and has a certain sequence homology therewith, for example, a polypeptide that has at least 80%, at least 90% sequence homology with any of the above-mentioned CDR3.

[0065] In some embodiments, in the serum albumin binding protein provided by the present disclosure, the immunoglobulin single variable domain can be covalently or non-covalently linked to any other macromolecular or small molecule compound as a carrier for prolonging the half-life.

[0066] In some embodiments, the serum albumin binding proteins of the present disclosure comprise one or more therapeutic or diagnostic agents, wherein the therapeutic or diagnostic agent is covalently or non-covalently linked to the immunoglobulin single variable domain.

[0067] In some embodiments, the therapeutic or diagnostic agent is selected from the group consisting of a therapeutic or diagnostic protein, a nucleic acid, and a small molecule compound.

[0068] In some embodiments, the serum albumin binding protein provided by the present disclosure or the immunoglobulin single variable domain thereof is ≤1×10 -7 M, for example, ≤2×10 -8 M, ≤1×10 -8 M, ≤9×10 -9 M, ≤8×10 -9 M,≤7×10 -9 M,≤6×10 -9 M, ≤5×10 -9 M, ≤4×10 -9 M, ≤3×10 -9 M, ≤2×10 -9 M, or ≤1×10 -9 M, or ≤9×10 -10 M, ≤8×10 -10 M,≤7×10 -10 M,≤6×10 -10 M, ≤5×10 -10 M, ≤4×10 -10 M, ≤3×10 -10 M, ≤2×10 -10M, ≤1×10 -10 M of K D The value binds to serum albumin, such as human serum albumin.

[0069] In some embodiments, the serum albumin binding protein provided by the present disclosure has at least one activity selected from the following:

[0070] (a)≤1×10 -7 M, for example, ≤1×10 -8 M of K D Value combined with HSA;

[0071] (b) does not detectably block the binding of serum albumin, such as HSA, to FcRn, such as human FcRn, in vivo and / or in vitro;

[0072] (c) when comprising a therapeutic agent or a diagnostic agent, prolonging the plasma half-life of the therapeutic agent or the diagnostic agent.

[0073] In some embodiments, the serum albumin binding proteins provided by the present disclosure retain the favorable properties of single domain antibodies (e.g., VHH) and have an extended life span in the individual circulation. Therefore, such molecules can circulate in the serum of the subject for several days, thereby reducing the frequency of treatment, the inconvenience brought to the subject, and reducing the cost of treatment.

[0074] In some embodiments, the half-life of the serum albumin binding proteins of the present disclosure can be controlled by the number of single variable domains that specifically bind serum albumin present in the molecule.

[0075] In some embodiments, the serum albumin binding proteins of the present disclosure promote the binding of serum albumin to FcRn in vivo and / or in vitro.

[0076] FXII / FXIIa binding protein

[0077] In one aspect, the present disclosure provides FXII / FXIIa binding proteins comprising at least one immunoglobulin single variable domain that specifically binds to FXII / FXIIa.

[0078] In the context of the present disclosure, "FXII / FXIIa" means FXII and / or FXIIa; that is, specifically binding to FXII / FXIIa means specifically binding to FXII, or specifically binding to FXIIa, or specifically binding to both FXII and FXIIa.

[0079] In some embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3-5, 17-23, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0080] In some specific embodiments, the CDRs are defined according to the Kabat numbering system.

[0081] In some embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 6, 45-46;

[0082] The amino acid sequence shown in SEQ ID NO:45 is CISWSGRX1X2X3HADSVKG, wherein X1 is selected from D or E, X2 is selected from T or I, and X3 is selected from H or I; the amino acid sequence shown in SEQ ID NO:46 is VGKGTSWYX4GX5YX6CPSX7GFDY, wherein X4 is selected from S or G, X5 is selected from A, S or N, X6 is selected from G or R, and X7 is selected from G or E.

[0083] In some embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises CDR1, CDR2, and CDR3 as shown in any one of the following:

[0084] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6-8;

[0085] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6, 9-10;

[0086] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6, 11-12.

[0087] In some embodiments, the CDR1 comprised by the immunoglobulin single variable domain that specifically binds to FXII / FXIIa has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR1s; and / or, the CDR2 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR2s; and / or, the CDR3 comprised by the immunoglobulin single variable domain has 0, 1, 2, 3, 4 or 5 amino acid mutations compared to any of the aforementioned CDR3s.

[0088] In some specific embodiments, the above-mentioned amino acid mutations are conservative replacements, substitutions or modifications, and / or deletions and / or additions that do not affect the function.

[0089] In some embodiments, the present disclosure provides an immunoglobulin single variable domain that specifically binds to FXII / FXIIa, comprising any one or any combination (eg, a combination of any two or any three) of the aforementioned CDR1, CDR2, and CDR3.

[0090] In some embodiments, the immunoglobulin single variable domain is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced.

[0091] In some embodiments, the immunoglobulin single variable domain is humanized. In some specific embodiments, the humanized heavy chain variable region germline genes IGHV3-23 and / or IGHV3-66 are used. In some specific embodiments, the framework region of the immunoglobulin single variable domain is derived from IGHV3-23; or, from IGHV3-66.

[0092] In some embodiments, the humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: 20, 23, 27, 28, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83, and 84.

[0093] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one mutation as shown below:

[0094] 20V, 23V, 27L, 27Y or 27N, 28P, 28I, 28S or 28A, 29Y, 29F, 29P, 29G or 29S, 30L, 30A or 30T, 37F, 43E, 44E or 44Q, 45R, 47G, 49A, 71Q, 74A or 74G, 78V, 79S, 81E, 83Q, 84P, and 94A.

[0095] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0096] 1)27L, 28P, 29F, 30A, 37F, 44E, 45R, 47G, 74A, 79S, 94A;

[0097] 2)27L, 28I, 29P, 30A, 37F, 44E, 45R, 47G, 74G, 94A;

[0098] 3)27L, 28A, 29G, 30A, 37F, 44E, 45R, 47G, 74A, 94A.

[0099] In some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0100] 1)F27L, T28P, V29F, S30A, V37F, G44E, L45R, W47G, S74A, Y79S, R94A;

[0101] 2)F27L, T28I, V29P, S30A, V37F, G44E, L45R, W47G, S74G, R94A;

[0102] 3)F27L, T28A, V29G, S30A, V37F, G44E, L45R, W47G, S74A, R94A.

[0103] In some specific embodiments, the above mutation sites are defined according to the Kabat numbering system.

[0104] In some embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 17-23, or an amino acid sequence having at least 80% sequence identity thereto.

[0105] In the present 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; "at least 90% (sequence) identity" encompasses 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.

[0106] In some embodiments, the immunoglobulin single variable domain in the FXII / FXIIa binding protein provided by the present disclosure comprises three complementarity determining regions CDR1, CDR2 and CDR3 and four FRs, arranged from amino terminus to carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0107] In some embodiments, the FXII / FXIIa binding protein provided by the present disclosure is an antibody or an antigen-binding fragment thereof that binds to FXII and / or FXIIa, or a conjugate, fusion protein, etc. comprising the antibody or an antigen-binding fragment thereof.

[0108] In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a camel antibody, a chimeric antibody, a humanized antibody, a fully human antibody or an antigen-binding fragment thereof. In some specific embodiments, the antibody or antigen-binding fragment thereof is, for example, a recombinant antibody or a fragment thereof.

[0109] In some specific embodiments, the antibody or its antigen-binding fragment is a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a peptibody, a domain antibody and a multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv) or its antigen-binding fragment.

[0110] In some embodiments, the FXII / FXIIa binding protein is an anti-FXII / FXIIa nanobody. In some specific embodiments, the immunoglobulin single variable domain in the FXII / FXIIa binding protein is a VHH. In some specific embodiments, the aforementioned immunoglobulin single variable domain is a humanized VHH. In some specific embodiments, the aforementioned immunoglobulin single variable domain is an affinity-matured VHH.

[0111] In some embodiments, a FXII / FXIIa binding protein is provided, which comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) the aforementioned immunoglobulin single variable domains, wherein the immunoglobulin single variable domains may be the same or different, and any two immunoglobulin single variable domains may be directly connected or connected through a linker.

[0112] In some specific embodiments, the FXII / FXIIa binding protein comprises at least two immunoglobulin single variable domains, wherein any two immunoglobulin single variable domains can be directly linked or linked via a linker.

[0113] Exemplarily, the linker can be a non-functional amino acid sequence of 1-20 or more amino acids in length and without secondary or higher structures. For example, the linker is G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, GGNGT, YGNGT, EPKSS, etc.

[0114] In some embodiments, the present disclosure provides anti-FXII / FXIIa antibodies or antigen-binding fragments thereof that bind to or compete for binding to the same FXII / FXIIa (eg, human FXII and / or human FXIIa) epitope as any of the aforementioned FXII / FXIIa binding proteins.

[0115] In some embodiments, anti-FXII / FXIIa antibodies or antigen-binding fragments thereof are provided that block the binding of any of the aforementioned FXII / FXIIa binding proteins to FXII / FXIIa (eg, human FXII and / or human FXIIa).

[0116] In some embodiments, an anti-FXII / FXIIa antibody or antigen-binding fragment thereof is provided, wherein the binding of the antibody to FXII / FXIIa (eg, human FXII and / or human FXIIa) is blocked by any of the aforementioned FXII / FXIIa binding proteins.

[0117] In some embodiments, the aforementioned FXII / FXIIa binding protein further comprises a human immunoglobulin Fc region; for example, the Fc region is an Fc region derived from human IgG1, IgG2 or IgG4. In some specific embodiments, the Fc region may be derived from natural or wild-type IgG1, IgG2 or IgG4. In some specific embodiments, the Fc region comprises a mutation that reduces effector function, for example, a reduction in effector function compared to natural or wild-type IgG1, IgG2 or IgG4. Exemplarily, effector functions include antibody-dependent cell-mediated cytotoxicity (ADCC), and / or antibody-dependent cell-mediated phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC).

[0118] For example, exemplary mutations that reduce ADCC effects are L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on IgG1, IgG2, IgG3 or IgG4, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331A / D3 on IgG1 8S on IgG4, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4. Hybrid IgG2 / 4 Fc domains can also be used, such as an Fc with residues 117-260 from IgG2 and residues 261-447 from IgG4. Exemplary mutations that increase affinity for FcγRIIb also include E233D / G237D / P238D / H268D / P271G / A330R on IgG1.

[0119] In the context of mutations contained in the Fc region in the present disclosure, " / " means "and / or", for example, "L234A / L235A" means "L234A and / or L235A", that is, the Fc contains at least one of the L234A and L235A mutations; the mutated amino acid positions are defined according to the EU numbering system.

[0120] In some embodiments, the immunoglobulin single variable domain in the aforementioned FXII / FXIIa binding protein is directly connected to the Fc region or through a linker. The linker can be a non-functional amino acid sequence with a length of 1-20 or more amino acids and no secondary or higher structure. For example, G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, GGNGT, YGNGT, EPKSS, etc.

[0121] Exemplarily, the FXII / FXIIa binding protein linked to an Fc region comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14-16, 47, or an amino acid sequence having at least 80% sequence identity thereto.

[0122] In some alternatives, the FXII / FXIIa binding protein of the present invention may comprise any of the aforementioned complete immunoglobulin single variable domains; it may also comprise a functional portion of any of the aforementioned immunoglobulin single variable domains 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.

[0123] In some embodiments, a variant of the functional portion of the FXII / FXIIa binding protein may be a polypeptide that retains the FXII and / or FXIIa protein 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%, at least 90% sequence homology thereto.

[0124] In some embodiments, in the FXII / FXIIa binding protein provided by the present disclosure, the immunoglobulin single variable domain can be covalently or non-covalently linked to any other macromolecular or small molecule compound as a carrier for prolonging the half-life.

[0125] In some embodiments, the FXII / FXIIa binding protein (anti-FXII / FXIIa antibody or antigen-binding fragment thereof) has at least one of the following properties:

[0126] 1)≤10 -7 K D Values ​​bind FXII and / or FXIIa;

[0127] 2) Inhibit FXIIa enzyme activity;

[0128] 3) Inhibit the activation of FXI by FXIIa;

[0129] 4) Prolongation of partial thromboplastin activity time (aPTT);

[0130] 5) Inhibit intrinsic coagulation;

[0131] 6) Inhibit the formation and / or growth of thrombus.

[0132] In some specific embodiments, the FXII / FXIIa binding protein is present in an amount of ≤10 -7 K D Values ​​for binding to FXII and / or FXIIa. For example, K D Value ≤ 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 or lower.

[0133] In some specific embodiments, the FXII / FXIIa binding protein inhibits the enzymatic activity of FXIIa. 50 (nM) ≤ 10nM, 9.5nM, 9nM, 8.5nM, 8nM or lower.

[0134] In some specific embodiments, the FXII / FXIIa binding protein inhibits the activation of FXI by FXIIa. 50 (nM) ≤ 10nM, 9.5nM, 9nM, 8.5nM, 8nM or lower.

[0135] Among them, IC in 2) or 3) 50 The determination method of (nM) can refer to the test method provided in Example 7.

[0136] In some specific embodiments, the FXII / FXIIa binding protein inhibits intrinsic coagulation and / or prolongs the partial thromboplastin time (aPTT); illustratively, aPTT 1.5 (μg / mL)≤3μg / mL, 2.8μg / mL, 2.5μg / mL, 2μg / mL, 1.5μg / mL, 1μg / mL or lower.

[0137] In another aspect, the present disclosure provides a FXII / FXIIa binding protein comprising a first domain and a second domain.

[0138] In some embodiments, the first domain comprises at least one immunoglobulin single variable domain that specifically binds FXII / FXIIa.

[0139] In some embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa is any one of the aforementioned immunoglobulin single variable domains that specifically bind to FXII / FXIIa.

[0140] Exemplary, in some specific embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises: CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3-5, 17-23, wherein the CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems.

[0141] Exemplary, in some specific embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6, 45-46.

[0142] Exemplary, in some specific embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises CDR1, CDR2, and CDR3 as shown in any of the following:

[0143] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6-8;

[0144] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6, 9-10;

[0145] CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 6, 11-12.

[0146] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced and / or antibody isomerization reduced.

[0147] Illustratively, in some specific embodiments, the immunoglobulin single variable domain is humanized. In some specific embodiments, the humanized heavy chain variable region germline genes IGHV3-23 and / or IGHV3-66 are used. In some specific embodiments, the humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: 20, 23, 27, 28, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84.

[0148] Exemplarily, in some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0149] 1)27L, 28P, 29F, 30A, 37F, 44E, 45R, 47G, 74A, 79S, 94A;

[0150] 2)27L, 28I, 29P, 30A, 37F, 44E, 45R, 47G, 74G, 94A;

[0151] 3)27L, 28A, 29G, 30A, 37F, 44E, 45R, 47G, 74A, 94A.

[0152] The above mutation sites are defined according to the Kabat numbering system.

[0153] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 17-23, or an amino acid sequence having at least 80% sequence identity thereto.

[0154] Exemplarily, in some specific embodiments, the single antigen binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) the aforementioned immunoglobulin single variable domains, which may be the same or different.

[0155] In some embodiments, the second domain comprises at least one of the following: HSA, an HSA binding domain, an immunoglobulin Fc region, an immunoglobulin single variable domain that specifically binds FXII / FXIIa.

[0156] In some embodiments, the HSA binding domain comprises at least one immunoglobulin single variable domain that specifically binds HSA.

[0157] In some embodiments, the immunoglobulin single variable domain that specifically binds to HSA is an immunoglobulin single variable domain in any of the serum albumin binding molecules provided by the disclosure.

[0158] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain in the HSA binding domain comprises CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 25, 32-41.

[0159] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain in the HSA binding domain comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 26-28.

[0160] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the HSA binding domain is humanized, affinity matured, T cell epitope removed, antibody deamidation reduced, and / or antibody isomerization reduced.

[0161] Illustratively, in some specific embodiments, the immunoglobulin single variable domain in the HSA binding domain is humanized. In some specific embodiments, the humanization uses the heavy chain variable region germline genes IGHV3-23 and / or IGHV3-66. In some specific embodiments, the humanized immunoglobulin single variable domain comprises an amino acid mutation at at least one of the following positions: 20, 23, 27, 28, 29, 30, 37, 44, 45, 47, 49, 74, 78, 83 and 84.

[0162] Exemplarily, in some specific embodiments, the humanized immunoglobulin single variable domain comprises at least one set of mutations as shown below:

[0163] 1)27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0164] 2)20V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0165] 3)23V, 27N, 29Y, 30L, 37F, 44E, 45R, 47G;

[0166] 4)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 74A;

[0167] 5)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 78V;

[0168] 6)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 49A;

[0169] 7)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 83Q, 84P;

[0170] 8)27N, 29Y, 30L, 37F, 44E, 45R, 47G, 84P;

[0171] 9)30L, 37F, 44E, 45R, 47G, 84P;

[0172] 10)30L, 37F, 44E, 45R, 47G.

[0173] The above mutation sites are defined according to the Kabat numbering system.

[0174] Exemplarily, in some specific embodiments, the immunoglobulin single variable domain in the HSA binding domain comprises an amino acid sequence as shown in any one of SEQ ID NOs: 25, 32-41, or an amino acid sequence having at least 80% sequence identity thereto.

[0175] Exemplarily, in some specific embodiments, the HSA binding domain comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8) of the aforementioned immunoglobulin single variable domains, and the immunoglobulin single variable domains may be the same or different.

[0176] In some embodiments, the FXII / FXIIa binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, etc.) immunoglobulin single variable domains that specifically bind to FXII / FXIIa. Any two immunoglobulin single variable domains that specifically bind to FXII / FXIIa may be the same or different.

[0177] In some embodiments, the FXII / FXIIa binding protein comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, etc.) immunoglobulin single variable domains that specifically bind to FXII / FXIIa; and, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, etc.) immunoglobulin single variable domains that specifically bind to HSA. Any two immunoglobulin single variable domains that specifically bind to FXII / FXIIa may be the same or different, and any two immunoglobulin single variable domains that specifically bind to HSA may be the same or different.

[0178] In some specific embodiments, any two immunoglobulin single variable domains can be directly linked or connected through a linker.

[0179] Exemplarily, linkers include, but are not limited to, (G m S n ) h or (GGNGT) h (SEQ ID NO: 61) or (YGNGT) h (SEQ ID NO: 62) or (EPKSS) h (SEQ ID NO: 63) wherein m, n are each independently selected from an integer of 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and h is independently selected from an integer of 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).

[0180] In some embodiments, the linker is (G x S) y A linker, wherein x is selected from an integer of 1-5 (e.g., 1, 2, 3, 4 or 5), and y is selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6); in some specific embodiments, the linker is selected from G4S, GS, GAP, (G4S)2, (G4S)3, (G4S)4, (G4S)5, ASGS, for example (G4S)2.

[0181] In some embodiments, in the FXII / FXIIa binding protein:

[0182] The first domain is located at the N-terminus of the second domain; and / or,

[0183] The first domain is located at the C-terminus of the second domain.

[0184] In some embodiments, in the FXII / FXIIa binding protein:

[0185] The immunoglobulin single variable domain that specifically binds to FXII / FXIIa is located at the N-terminus of the immunoglobulin single variable domain that specifically binds to HSA; and / or,

[0186] The immunoglobulin single variable domain that specifically binds to FXII / FXIIa is located at the C-terminus of the immunoglobulin single variable domain that specifically binds to HSA.

[0187] In some embodiments, the valency of the FXII / FXIIa binding protein is ≥ 2; that is, the FXII / FXIIa binding protein has at least 2 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 15, 20, etc.) immunoglobulin binding domains. In some specific embodiments, the FXII / FXIIa binding protein includes at least 2 immunoglobulin single variable domains that specifically bind to FXII / FXIIa. In some specific embodiments, the FXII / FXIIa binding protein includes at least 1 immunoglobulin single variable domain that specifically binds to FXII / FXIIa, and at least 1 immunoglobulin single variable domain that specifically binds to HSA.

[0188] In some embodiments, the FXII / FXIIa binding protein includes a first domain that specifically binds to FXII / FXIIa and a second domain that specifically binds to HSA. The valency of the first domain can be 1-valent, 2-valent, 3-valent, 4-valent, 5-valent, 6-valent, 7-valent, 8-valent, etc. The valency of the second domain can be 1-valent, 2-valent, 3-valent, 4-valent, 5-valent, 6-valent, 7-valent, 8-valent, etc.

[0189] In some specific embodiments, the valence ratio of the immunoglobulin single variable domain that specifically binds to FXII / FXIIa to the immunoglobulin single variable domain in the HSA binding domain is 5:1 to 1:5. Exemplarily, the valence ratio is 1:1, 1:2, 1:3, 3:1, 2:1, 3:2, 2:3.

[0190] In the present disclosure, "valency" is described as the number of antigen binding moieties present in an antigen binding molecule (e.g., antigen binding domain). Thus, a single binding molecule can bind to more than one binding site on a target molecule. Thus, the valency ratio of the immunoglobulin single variable domain of the first domain to the immunoglobulin single variable domain of the second domain is the ratio of the number of antigen binding sites in the first domain that bind FXII / FXIIa to the number of antigen binding sites in the second domain that bind HSA or bind FXII / FXIIa.

[0191] In some embodiments, FXII / FXII binding protein comprises any one or any combination of the following structures from N-terminus to C-terminus:

[0192] Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0193] Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0194] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0195] [Immunoglobulin single variable domain 1] c -[Connector] a -Immunoglobulin single variable domain 2-[Linker] b -[Immunoglobulin single variable domain 1] d

[0196] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] b - immunoglobulin single variable domain 2;

[0197] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a- immunoglobulin single variable domain 1;

[0198] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0199] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0200] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 2;

[0201] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0202] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0203] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 1;

[0204] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 1;

[0205] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker]b - immunoglobulin single variable domain 1;

[0206] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0207] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 2;

[0208] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 2;

[0209] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 2;

[0210] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0211] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 1;

[0212] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b - immunoglobulin single variable domain 1;

[0213] Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0214] Immunoglobulin single variable domain 2-[Linker] b -Immunoglobulin single variable domain 1-[Linker] a -Immunoglobulin single variable domain 1-[Linker] a - immunoglobulin single variable domain 2;

[0215] Wherein, - represents a peptide bond, any two linkers may be the same or different; a, b, c, d are independently selected from 0 or 1.

[0216] In some embodiments, the FXII / FXIIa binding protein comprises the following structure from the N-terminus to the C-terminus:

[0217] Immunoglobulin single variable domain 1-linker-immunoglobulin single variable domain 2-linker-immunoglobulin single variable domain 1.

[0218] In some embodiments, the immunoglobulin single variable domain 1 and the immunoglobulin single variable domain 2 are independently selected from: an immunoglobulin single variable domain that specifically binds to FXII / FXIIa, and an immunoglobulin single variable domain that specifically binds to HSA.

[0219] In some specific embodiments, immunoglobulin single variable domain 1 is any of the aforementioned immunoglobulin single variable domains that specifically binds to FXII / FXIIa, and immunoglobulin single variable domain 2 is any of the aforementioned immunoglobulin single variable domains that specifically binds to HSA.

[0220] In some specific embodiments, immunoglobulin single variable domain 1 is any of the above-mentioned immunoglobulin single variable domains that specifically bind to FXII / FXIIa, and immunoglobulin single variable domain 2 is any of the above-mentioned immunoglobulin single variable domains that specifically bind to FXII / FXIIa. Wherein, immunoglobulin single variable domain 1 and immunoglobulin single variable domain 2 may be the same or different.

[0221] In some specific embodiments, the linker is (G x S z ) yA linker, wherein x is each independently selected from an integer of 1-5 (e.g., 1, 2, 3, 4 or 5), y is each independently selected from an integer of 1-6 (e.g., 1, 2, 3, 4, 5 or 6), and z is each independently selected from 0-4 (e.g., 0, 1, 2, 3 or 4); exemplarily, the linker is selected from G4S, GS, GAP, (G4S)2 (SEQ ID NO: 51), (G4S)3 (SEQ ID NO: 52), (G4S)4 (SEQ ID NO: 53), (G4S)5 (SEQ ID NO: 54), G4SG3S (SEQ ID NO: 55), ASGS (SEQ ID NO: 56), for example, (G4S)2, G4SG3S.

[0222] In the context of this disclosure, "each independently selected from" encompasses x S z When there are multiple (i.e., y ≥ 2), each G x S z The choice of x in G can be x S z The choices of x in G are the same or different. x S z The choice of z in G can be x S z The choices of z are the same or different.

[0223] In some embodiments, the aforementioned FXII / FXIIa binding protein is an antibody or antigen-binding fragment thereof against FXII / FXIIa and HSA. In some embodiments, the aforementioned FXII / FXIIa binding protein is an antibody or antigen-binding fragment thereof against FXII / FXIIa A. The antibody or antigen-binding fragment includes but is not limited to: The antigen-binding fragment includes but is not limited to: Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single-chain antibody, scFv, sdAb, sdFv, nanobody, peptide antibody (peptibody), domain antibody and multispecific antibody (bispecific antibody, diabody, triabody and tetrabody, tandem two-scFv, tandem three-scFv).

[0224] In some specific embodiments, in the aforementioned anti-FXII / FXIIa antibody or antigen-binding fragment thereof: the immunoglobulin single variable domain that specifically binds to FXII / FXIIa is VHH.

[0225] In some specific embodiments, in the aforementioned anti-FXII / FXIIa and HSA antibodies or antigen-binding fragments thereof: the immunoglobulin single variable domain that specifically binds to FXII / FXIIa is VHH, and / or the immunoglobulin single variable domain that specifically binds to HSA is VHH.

[0226] In some specific embodiments, in the FXII / FXIIa binding protein:

[0227] The N-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to FXII / FXIIa, and / or, the C-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to FXII / FXIIa; and / or,

[0228] The N-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to HSA, and / or, the C-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to HSA; and / or,

[0229] The N-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to FXII / FXIIa, and / or, the C-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to FXII / FXIIa; and / or,

[0230] The N-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to HSA, and / or, the C-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to FXII / FXIIa; and / or,

[0231] The N-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to FXII / FXIIa, and / or, the C-terminus of the VHH that specifically binds to FXII / FXIIa is connected to one or more VHHs that specifically bind to HSA; and / or,

[0232] The N-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to FXII / FXIIa, and / or, the C-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to HSA; and / or,

[0233] The N-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to HSA, and / or the C-terminus of the VHH that specifically binds to HSA is linked to one or more VHHs that specifically bind to FXII / FXIIa.

[0234] In some embodiments, the FXII / FXIIa binding protein comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO: 24, 48-50, or an amino acid sequence having at least 80% sequence identity thereto.

[0235] In some embodiments, the FXII / FXIIa binding protein package (antibodies or antigen-binding fragments thereof against FXII / FXIIa and HSA) has at least one of the following properties:

[0236] 1)≤10 -7 K D Values ​​bind FXII and / or FXIIa;

[0237] 2) Inhibit FXIIa enzyme activity;

[0238] 3) Inhibit the activation of FXI by FXIIa;

[0239] 4) Prolongation of partial thromboplastin activity time (aPTT);

[0240] 5) Inhibit intrinsic coagulation;

[0241] 6) Inhibit the formation and / or growth of thrombus.

[0242] In some specific embodiments, the FXII / FXIIa binding protein is present in an amount of ≤10 -7 K D Values ​​for binding to FXII and / or FXIIa. For example, K D Value ≤ 10 -7 , 10 -8 , 10 -9 , 10 -10 , 10 -11 or lower.

[0243] In some specific embodiments, the FXII / FXIIa binding protein inhibits the enzymatic activity of FXIIa. 50 (nM) ≤ 10nM, 9.5nM, 9nM, 8.5nM, 8nM or lower.

[0244] In some specific embodiments, the FXII / FXIIa binding protein inhibits the activation of FXI by FXIIa. 50 (nM) ≤ 10nM, 9.5nM, 9nM, 8.5nM, 8nM or lower.

[0245] Among them, IC in 2) or 3) 50 The determination method of (nM) can refer to the test method provided in Example 7.

[0246] In some specific embodiments, the FXII / FXIIa binding protein inhibits intrinsic coagulation and / or prolongs the partial thromboplastin time (aPTT); illustratively, aPTT 1.5 (μg / mL)≤3μg / mL, 2.8μg / mL, 2.5μg / mL, 2μg / mL, 1.5μg / mL, 1μg / mL or lower.

[0247] In some embodiments, the present disclosure provides anti-FXII / FXIIa and HSA antibodies or antigen-binding fragments thereof that bind to or compete for binding to the same FXII / FXIIa (e.g., human FXIIa and / or human FXII) and / or HSA epitope as any of the aforementioned FXII / FXIIa binding proteins.

[0248] In some embodiments, anti-FXII / FXIIa and HSA antibodies or antigen-binding fragments thereof are provided, which block the binding of any of the aforementioned FXII / FXIIa binding proteins to FXII / FXIIa (eg, human FXIIa and / or human FXII) and / or HSA.

[0249] In some embodiments, an anti-FXII / FXIIa and HSA antibody or an antigen-binding fragment thereof is provided, wherein the binding of the antibody to FXII / FXIIa (eg, human FXIIa and / or human FXII) and / or HSA is blocked by any of the aforementioned FXII / FXIIa binding proteins.

[0250] In some embodiments, a conjugate is provided, comprising any of the aforementioned FXII / FXIIa binding proteins.

[0251] Nucleic acid molecules

[0252] The present disclosure provides polynucleotides encoding any one of the FXII / FXIIa binding proteins provided by the present disclosure.

[0253] In some embodiments, the nucleic acid of the present disclosure may be RNA, DNA or cDNA.According to some embodiments of the present disclosure, the nucleic acid of the present disclosure is a substantially isolated nucleic acid.

[0254] The nucleic acid of the present disclosure may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid, a YAC or a viral vector. The vector may be an expression vector in particular, i.e., a vector that provides expression of the FXII binding protein in vitro and / or in vivo (i.e., in a suitable host cell, host organism and / or expression system). The expression vector generally comprises at least one nucleic acid of the present disclosure, which is operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.). It is common sense for those skilled in the art to select the elements and their sequences for expression in a specific host. The regulatory elements and other elements useful or necessary for the expression of the FXII / FXIIa binding protein of the present disclosure are, for example, promoters, enhancers, terminators, integration factors, selection markers, leader sequences, reporter genes.

[0255] The nucleic acids of the present disclosure can be prepared or obtained by known means (eg, by automated DNA synthesis and / or recombinant DNA technology) based on the information of the amino acid sequence of the polypeptides of the present disclosure, and / or can be isolated from suitable natural sources.

[0256] Host cells

[0257] The present disclosure provides recombinant host cells that express or are capable of expressing any one of the FXII / FXIIa binding proteins, and / or contain a nucleic acid or vector of the present disclosure.

[0258] In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0259] Exemplarily, bacterial cells include cells of Gram-negative bacterial strains (e.g., Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (e.g., Bacillus strains, Streptomyces strains, Staphylococcus strains, and Lactococcus strains).

[0260] Exemplary fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or cells of species of Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces (e.g., Schizosaccharomyces pombe), Pichia (e.g., Pichia pastoris and Pichia methanolica), and Hansenula.

[0261] Exemplarily, mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

[0262] In addition, the present disclosure may also use amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins.

[0263] The cells of the present disclosure are incapable of developing into completed plants or animal individuals.

[0264] Production or preparation method

[0265] The present disclosure provides a method for preparing the FXII / FXIIa binding protein of the present disclosure, the method generally comprising the following steps:

[0266] - culturing a host cell of the present disclosure under conditions that allow expression of a binding protein of the present disclosure; and

[0267] - Recovering the target protein expressed by the host cell from the culture; and

[0268] -Optionally, further purification and / or modification of the protein of interest of the present disclosure is included.

[0269] The FXII / FXIIa-binding proteins of the present disclosure can be produced intracellularly in cells as described above (e.g., in the cytoplasm, in the periplasm, or in inclusion bodies), then isolated from the host cells and optionally further purified; or they can be produced extracellularly (e.g., in the culture medium in which the host cells are cultured), then isolated from the culture medium and optionally further purified.

[0270] Methods and reagents for recombinant production of polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selection markers, methods for inducing protein expression, culture conditions, etc. are known in the art. Similarly, protein separation and purification techniques suitable for use in the methods of making the binding proteins disclosed herein are well known to those skilled in the art. Methods for producing and purifying antibodies are well known and can be found in the prior art, such as the Cold Spring Harbor Guide to Antibody Experimental Techniques (Chapters 5-8 and 15). The engineered antibodies disclosed herein can also be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can be stably transfected into cells. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture fluid secreting the antibodies can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product needs to be immediately frozen, such as -70°C, or lyophilized.

[0271] However, the FXII / FXIIa binding proteins of the present disclosure may also be obtained by other methods of producing proteins known in the art, such as chemical synthesis, including solid phase or liquid phase synthesis.

[0272] Pharmaceutical composition

[0273] The present disclosure provides a pharmaceutical composition containing a preventively or therapeutically effective amount of the FXII / FXIIa binding protein of the present disclosure, its encoding polynucleotide or vector as described above, and one or more pharmaceutically acceptable diluents, buffers, excipients or adjuvants.

[0274] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of FXII / FXIIa binding protein in a unit dose. In other specific embodiments, the amount of FXII / FXIIa binding protein in a unit dose of the pharmaceutical composition is 0.1-2000 mg; in some specific embodiments, it is 1-1000 mg.

[0275] In some embodiments, an article or product is provided, comprising the aforementioned FXII / FXIIa binding protein, polynucleotide and / or vector. Optionally, the article comprises a container and a label. The container is, for example, a bottle, a syringe and a test tube. The container holds a composition effective for treating a condition. The label on or connected to the container indicates that the composition is used to treat a selected condition. The composition contains the aforementioned FXII / FXIIa binding protein, polynucleotide and / or vector.

[0276] Reagent test kit

[0277] The present disclosure provides a kit comprising the FXII / FXIIa binding protein, encoding polynucleotide, vector and / or composition of the present disclosure. In some embodiments, a diagnostic reagent comprising the above nucleic acid molecule is also provided, as well as related diagnostic uses.

[0278] Methods for treating diseases and pharmaceutical uses

[0279] The present disclosure provides uses and methods of the FXII / FXIIa binding protein, encoding polynucleotide, vector, and pharmaceutical composition of the present disclosure in preventing and / or treating diseases, wherein the diseases may be related to or unrelated to the FXII / FXIIa signaling pathway.

[0280] In some embodiments, the present disclosure provides for use of a FXII / FXIIa binding protein, encoding polynucleotide, vector, or pharmaceutical composition in at least one of the following:

[0281] 1) Used to inhibit FXIIa activity;

[0282] 2) preparing a drug for inhibiting FXIIa activity;

[0283] 3) Used to inhibit the activation of FXI by FXIIa;

[0284] 4) preparing a drug for inhibiting the activation of FXI by FXIIa;

[0285] 5) Used to inhibit intrinsic coagulation;

[0286] 6) Preparation of drugs for inhibiting intrinsic coagulation;

[0287] 7) Used to inhibit or prevent the formation and / or growth of thrombus;

[0288] 8) Preparation of drugs for inhibiting or preventing the formation and / or growth of thrombus;

[0289] 9) For preventing or treating thrombosis or diseases related to thrombosis;

[0290] 10) Preparation for preventing or treating thrombosis or diseases related to thrombosis.

[0291] In some embodiments, the present disclosure provides methods for preventing or treating diseases associated with FXII / FXIIa, comprising administering to a subject any one of the FXII / FXIIa binding proteins, encoding polynucleotides, vectors and / or pharmaceutical compositions provided by the present disclosure.

[0292] In some specific embodiments, the method for preventing or treating a disease associated with FXII / FXIIa comprises providing a subject in need thereof with a preventively effective amount of a FXII / FXIIa binding protein, encoding polynucleotide, vector and / or pharmaceutical composition.

[0293] In some specific embodiments, the method for preventing or treating a disease associated with FXII / FXIIa comprises providing a therapeutically effective amount of a FXII / FXIIa binding protein, encoding polynucleotide, vector and / or pharmaceutical composition to a subject in need thereof.

[0294] In some specific embodiments, the subject suffers from a disease such as thrombosis or a disease associated with thrombosis.

[0295] In some specific embodiments, thrombosis includes, but is not limited to, venous, arterial or capillary thrombosis, thrombosis in the heart, thromboembolism, thrombosis during and / or after contacting the blood of a human or animal subject with an artificial surface.

[0296] In some embodiments, the FXII / FXIIa binding proteins, encoding polynucleotides, vectors and / or pharmaceutical compositions of the present disclosure may be administered by any suitable method known in the art, and administration may be systemic or local. BRIEF DESCRIPTION OF THE DRAWINGS

[0297] Figure 1 Schematic diagram of the FXII protein structure.

[0298] Figure 2 The inhibitory effects of different FXII / FXIIa antibodies on FXIIa enzymatic activity are shown.

[0299] Figure 3A The inhibitory effects of 2207 and CSL-312 on FXIIa enzymatic activity are shown, n=3, IC of CSL-312 50 8.756nM, IC of 2207 50 It is 10.07nM,ns.

[0300] Figure 3B The inhibitory effects of 2207 and CSL-312 on FXIIa-mediated FXI activation are shown, n=3, IC of CSL-312 50 8.452nM, IC of 2207 50 It is 7.464nM,ns.

[0301] Figure 4A The aPTT effects of different antibodies in human plasma are shown.

[0302] Figure 4BThe aPTT effects of 2207 and CSL-312 in human plasma are shown.

[0303] Figure 4C The effects of different antibodies on PT in human plasma are shown.

[0304] Figure 4D The effect of 2207 on PT in human plasma is shown.

[0305] Figure 5 The effect of anti-FXIIa-HSA antibody 2207 on aPTT at different administration doses in rabbits is shown.

[0306] Figure 6 Schematic diagram of the physiological processes involved in FXII.

[0307] Fig. 7A and Figure 7B These are the experimental results of the effect of HSA antibody on the binding between FcRn and HSA.

[0308] Figure 8 This is the concentration-time curve of HSA antibody in plasma after intravenous administration in the pharmacokinetic study of mice.

[0309] Figures 9A-9B The results of the pharmacokinetic study of HSA antibody in mice are as follows: Fig.9A To calculate the T of HSA antibody in plasma using a non-compartmental model 1 / 2 Parameter comparison results, Fig. 9B This is the Cl_obs (mL / (min*kg)) parameter comparison result.

[0310] the term

[0311] In order to make the present disclosure more easily understood, certain technologies and sciences are specifically defined below. Unless otherwise clearly defined elsewhere in the present disclosure, all other technologies and sciences used in the present disclosure have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0312] The three letter codes and one letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0313] "FXII" is also called "coagulation factor FXII", and "factor XII" is also called Hageman Factor. A β-plasma globulin serine proteinase composed of an amino-terminal heavy chain and a carboxyl-terminal light chain. Its heavy chain contains two fiber-binding protein-like domains (type I and II), two epidermal growth factor-like domains, a kringle domain, and a proline-rich region, and its light chain contains a protein enzyme domain. Factor XII itself is activated to α-factor XIIa (also known as FXIIa) by plasma kallikrein, platelet or bacterial polyphosphates, extracellular DNA or RNA, heparin released from mast cells, amyloid protein peptides, misfolded protein aggregates, and negatively charged surfaces (such as glass). α-Factor XIIa is subsequently activated by further conversion to β-FXIIa by trypsin. Factor XII activates factor XI and prekallikrein in the coagulation cascade. An example of an amino acid sequence of a full-length FXII protein is the amino acid sequence provided in UniProtKB / Swiss-Prot under accession number P00748.3.

[0314] In the scope of the present disclosure, FXII and FXIIa should be understood in the broadest sense. The term covers the natural forms of FXI and FXIa in nature, naturally occurring variants, and also includes artificially expressed forms, recombinant proteins, functional variants, etc. When the context does not specifically indicate, in the case of antigen-antibody interaction, FXII (or FXIIa) covers the scope of the complete protein or its epitope.

[0315] "Functional variants" include but are not limited to homologs, fragments, truncations, mutants, modifications, etc. of wild-type proteins. Functional variants of proteins have improved, reduced or maintained protein activity compared to wild-type proteins.

[0316] "FXII / FXIIa" refers to one or both of Factor XII and activated Factor XII (FXIIa). Thus, "FXII / FXIIa binding protein" includes binding proteins of one or both of FXII and FXIIa.

[0317] "FXII / FXIIa binding protein" means any protein that can specifically bind to FXII and / or FXIIa or any molecule comprising the protein. FXII / FXIIa binding protein may include antibodies, antigen binding fragments thereof, or conjugates thereof as defined in the present disclosure for FXII. FXII binding protein also encompasses immunoglobulin superfamily antibodies (IgSF) or CDR-grafted molecules. "FXII / FXIIa binding protein" of the present disclosure may include at least one immunoglobulin single variable domain (such as VHH) that binds to FXII / FXIIa. In some embodiments, "FXII / FXIIa binding protein" may include 2, 3, 4 or more immunoglobulin single variable domains (such as VHH) that bind to FXII / FXIIa. In addition to binding to an immunoglobulin single variable domain comprising FXII / FXIIa, the FXII binding proteins of the present disclosure may also comprise a linker and / or a portion with effector function, such as a half-life extending portion (e.g., an immunoglobulin single variable domain that binds serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region.

[0318] In some embodiments, the "FXII binding protein" of the present disclosure also encompasses multispecific antibodies, which contain immunoglobulins that bind to different antigens (e.g., a first antibody that binds to a first antigen (e.g., FXII) and a second antibody that binds to a second antigen (e.g., HSA), optionally, including a third antibody that binds to a third antigen, and further optionally, including a fourth antibody that binds to a fourth antigen.

[0319] "Multispecific antibodies" refer to antibodies that bind to two or more different target antigens or different antigenic epitopes. The term "multispecific antibodies" includes bispecific, trispecific, tetraspecific, pentaspecific and hexaspecific. For example, "FXII binding protein" as a multispecific antibody against FXII / FXIIa and HSA can bind to FXII / FXIIa and HSA at the same time.

[0320] "Binding to FXII" means being able to interact with FXII or a fragment thereof or an epitope thereof, and the FXII or a fragment thereof or an epitope thereof may be of human origin.

[0321] “Antibody” or “immunoglobulin” in a broad sense covers traditional antibodies (a tetrapeptide chain structure antibody composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds), as well as Fab, Fv, sFv, F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, peptibodies, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies) and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv) with antigen-binding activity. Therefore, the “antibody” used in the present disclosure includes full-length antibodies, single chains thereof, and any part, domain or fragment thereof having antigen-binding activity, and multispecific antibodies comprising a single chain thereof and any part, domain or fragment thereof having antigen-binding activity (including but not limited to antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively). Traditional antibodies or immunoglobulins are usually tetrapeptide chains composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the heavy chain constant region are different, so their antigenicity is also different. Based on this, immunoglobulins can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain. The same class of Ig can be divided into different subclasses according to the difference in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains by different constant regions. Each of the five classes of Ig can have a κ (kappa) chain or a λ (lambda) chain. In some embodiments, the antibodies disclosed herein specifically or substantially specifically bind to FXII / FXIIa.

[0322] The "antibodies" of the present disclosure include, but are not limited to: (i) a Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) a Fd fragment consisting of VH and CH1 domains; (iii) a F(ab')2 fragment, a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv), in which the VH domain and the VL domain are linked by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883 242, incorporated herein by reference in its entirety); (iv) a "bifunctional antibody" or "trifunctional antibody", a multivalent or multispecific fragment constructed by gene fusion (Tomlinson et al., 2000, Methods of Enzymology 243:5879-5883). Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448, all of which are fully incorporated herein by reference); (v) "domain antibodies" or "dAbs" (sometimes referred to as "immunoglobulin single variable domains"), including immunoglobulin single variable domains from other species, such as rodents (e.g., as disclosed in WO00 / 29004), nurse sharks and camelid V-HH dAbs; (vi) SMIPs (small molecule immunopharmaceuticals), camelid antibodies, nanobodies and IgNARs; (vii) humanized antibodies of (i)-(vi) above.

[0323] "Antibody framework region (FR)" refers to the portion of a variable domain that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain.

[0324] For the determination or definition of "CDR", the definitive depiction of CDR and the identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. This can be accomplished by any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definitions, and conformational definitions. The Kabat numbering system is a standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the position of certain structural loop regions. (See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group that model antibody structure (see, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd.). The AbM numbering system uses a combination of knowledge databases and ab initio methods to model the tertiary structure of antibodies from primary sequence (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198). Contact definitions are based on analysis of available complex crystal structures (see, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45). In the conformational definition, the positions of the CDRs can be identified as residues that make enthalpic contributions to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).Still other CDR boundary definitions may not strictly follow one of the above methods, but still overlap with at least a portion of the Kabat CDRs, although they may be shortened or extended based on the prediction or experimental results that a particular residue or residue group does not significantly affect antigen binding. As used herein, CDRs may refer to CDRs defined by any method known in the art (including a combination of methods). The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment comprising more than one CDR, CDRs may be defined according to any of the Kabat, Chothia, extended, AbM, IMGT, contact and / or conformational definitions.

[0325] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which may be a heavy chain or light chain domain, including a VH, VHH or VL domain) 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 conventional four-chain monoclonal antibodies). Examples of "immunoglobulin single variable domains" include nanobodies (including VHH, humanized VHH and / or camelized VH, such as camelized human VH), IgNAR, domains, (single domain) antibodies that are VH domains or derived from VH domains (such as dAbs TM ) and (single domain) antibodies (such as dAbs) as VL domains or derived from VL domains TM ). 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 an immunoglobulin single variable domain is a "VHH domain" (or simply "VHH") as defined below.

[0326] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, 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 the variable domain from the heavy chain variable domain (which is referred to as a "VH domain" in the present disclosure) and the light chain variable domain (which is referred to as a "VL domain" in the present disclosure) present in conventional tetrapeptide chain structure antibodies. The VHH domain specifically binds to an epitope without the need for other antigen binding domains (this is in contrast to the VH or VL domains in conventional tetrapeptide chain antibodies, in which case the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", "VHH antibody" and "domain" ("Nanobody" is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. "VHH domain" includes, but is not limited to, natural antibodies produced by camelids, antibodies produced by camelids that are then humanized, or those obtained by screening using phage display technology.

[0327] As is known in the art for VH domains and VHH domains, the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed by the Kabat numbering). This means that, in general, the numbering according to Kabat may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or numbering systems include Chothia, IMGT, AbM.

[0328] The total number of amino acid residues in a VHH domain will generally range from 110 to 120, often between 112 and 115. It should be noted, however, that smaller and longer sequences may also be suitable for the purposes described in the present disclosure.

[0329] VHH domains (alone or as part of a larger polypeptide) offer a number of significant advantages over the use of conventional VH and VL domains, scFv or conventional antibody fragments (e.g. Fab- or F(ab')2-fragments):

[0330] - Only a single domain is required to bind the antigen with high affinity and selectivity, so that there is no need for the presence of two separate domains, nor is there a need to ensure that the two domains are present in the appropriate spatial conformation and configuration (e.g. scFv generally requires the use of a specially designed linker);

[0331] - VHH domains can be expressed from a single gene and do not require post-translational folding or modification;

[0332] - VHH domains can be easily engineered into multivalent and multispecific formats;

[0333] - VHH domains are highly soluble and have no tendency to aggregate;

[0334] - VHH domains are highly stable to heat, pH, proteases and other denaturing agents or conditions, and therefore can be prepared, stored or transported without the use of refrigeration equipment, thereby achieving cost, time and environmental savings;

[0335] - VHH domains are easy and relatively cheap to prepare, even on the scale required for production;

[0336] - The VHH domain is relatively small (approximately 15 kDa or 1 / 10 the size of a conventional IgG) compared to conventional tetrapeptide chain structure antibodies and antigen-binding fragments thereof, and therefore exhibits higher tissue penetration and can be administered at higher doses than conventional tetrapeptide chain structure antibodies and antigen-binding fragments thereof;

[0337] - VHH domains may display so-called cavity binding properties (particularly due to their extended CDR3 loop compared to conventional VH domains) and thus may reach targets and epitopes that are inaccessible to conventional tetrapeptide chain structured antibodies and antigen-binding fragments thereof.

[0338] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously 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.

[0339] Examples of "humanization" include "humanization" of a VHH domain derived from Camelidae by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence with one or more amino acid residues present at the corresponding positions in the VH domain of a human conventional tetrapeptide chain structure antibody (also referred to as "sequence optimization" in the present disclosure, in addition to humanization, "sequence optimization" may also cover other modifications to the sequence by one or more mutations that provide improved properties of VHH, such as removal of potential post-translational modification sites). The humanized VHH domain may contain one or more completely human framework region sequences, and in some specific embodiments, may contain human framework region sequences of IGHV3. Another example of "humanization" includes transplanting mouse CDR sequences to human antibody variable region frameworks, i.e., antibodies produced in different types of human germline antibody framework sequences. The strong antibody variable antibody response induced by chimeric antibodies due to carrying a large amount of mouse protein components can be overcome. Humanization methods such as protein surface amino acid humanization (resurfacing) and antibody humanization universal framework transplantation (CDR grafting to a universal framework), that is, CDR "grafting" on other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Scaffolds and techniques suitable for the CDR transplantation are known in the art. For example, the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database, as well as in Kabat, EA et al., 1991 Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed in the present invention also include humanized antibodies after affinity maturation of CDR by phage display. In addition, in order to avoid the decrease in immunogenicity and the decrease in activity caused by the decrease in activity, the human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain activity.

[0340] An "affinity matured" FXII / FXIIa binding protein or FXII / FXIIa antibody has one or more changes in one or more CDRs that result in an increase in affinity for the antigen compared to its parent antibody. Affinity matured antibodies can be prepared by methods known in the art, for example, as described in 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.

[0341] Typically, the FXII / FXIIa binding proteins of the present disclosure will be expressed as preferably 10 -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 mol / L, even more preferably 10 -9 Up to 10 -10 or lower dissociation constant (K D ), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (K A ) binds to the antigen to be bound (i.e. FXII / FXIIa). Any -4 KD values ​​of M are generally considered to be indicative of non-specific binding. Specific binding of an antigen binding protein to an antigen or epitope 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., radioimmunoassays (RIA), enzyme immunoassays (EIA), and sandwich competitive assays as described in the present disclosure.

[0342] When "competition" is used in the context of antigen binding proteins (e.g., neutralizing antigen binding proteins or neutralizing antibodies) that compete for the same epitope, it means competition between antigen binding proteins, which is determined by the following assay: the antigen binding protein (e.g., antibody or immunologically functional fragment thereof) to be tested prevents or inhibits (e.g., reduces) specific binding of a reference antigen binding protein (e.g., ligand or reference antibody) to a common antigen (e.g., FXII / FXIIa antigen or fragment thereof). Numerous types of competitive binding assays can be used to determine whether one antigen binding protein competes with another, such as: solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid phase direct label assays, solid phase direct label sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Manual). Press); solid phase direct labeling RIA with I-125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung et al., 1990, Virology 176: 546-552); and directly labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32: 77-82). Typically, the assay involves the use of purified antigen that binds to an unlabeled test antigen binding protein and a labeled reference antigen binding protein (the antigen is on a solid surface or on a cell surface). Competitive inhibition is measured by measuring the amount of label bound to the solid surface or cell in the presence of the test antigen binding protein. Typically, the test antigen binding protein is present in excess. Antigen binding proteins identified by competitive assays (competing antigen binding proteins) include: antigen binding proteins that bind to the same epitope as a reference antigen binding protein; and antigen binding proteins that bind to an epitope that is sufficiently close to the epitope bound by the reference antigen binding protein, and the two epitopes sterically hinder each other from binding. Additional details about methods for determining competitive binding are provided in the disclosed embodiments. Typically, when the competing antigen binding protein is present in excess, it will inhibit (e.g., reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or 75% or more of the specific binding of the reference antigen binding protein to the common antigen.In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

[0343] "Inhibit" or "block" are used interchangeably and encompass both partial and complete inhibition / blocking.

[0344] "Antigen" refers to a molecule used for immunization of an immunologically active vertebrate to produce antibodies that recognize the antigen, or to screen an expression library (e.g., especially a phage, yeast, or ribosome display library). In this article, antigen is more broadly defined and is generally intended to include a target molecule specifically recognized by an antibody, thus including a portion or mimetic of a molecule used in an immunization process for producing an antibody or in a library screening for selecting an antibody.

[0345] "Epitope" or "antigenic determinant" used interchangeably refers to any antigenic determinant on the antigen to which the paratope of an antibody binds. Antigenic determinants generally comprise chemically active surface groups of molecules, such as amino acids or sugar side chains, and generally have specific three-dimensional structural characteristics and specific charge characteristics. For example, an epitope generally comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which may be a "linear" epitope or a "conformational" epitope. In a linear epitope, all points of interaction between a protein and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the points of interaction exist across separate protein amino acid residues. Many epitope mapping techniques well known in the art can be used to identify the epitope of a given antigen (e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996), US4708871). Conventional techniques known to those skilled in the art can be used to competitively screen antibodies for binding to the same epitope. For example, competition and cross-competition studies can be performed to obtain antibodies that compete or cross-compete with each other for antigen binding (high throughput screening methods such as referring to WO03 / 48731). Therefore, conventional techniques known to those skilled in the art can be used to obtain antibodies and antigen-binding fragments thereof that compete with antibody molecules of the present disclosure for binding to the same epitope on FXII.

[0346] "Binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (e.g., an antibody or portion thereof and an antigen) and is used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules can be determined by determining the dissociation constant (K DK can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). D The rate constants corresponding to the association and dissociation of a monovalent complex are called the association rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through the equation K D = kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods (see Caceci et al., 1984, Byte 9: 340-362; Wong & Lohman, 1993, PNAS 90: 5428-5432). Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blot, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere herein. Similarly, the specificity of an interaction can be determined and compared to the K of the interaction of interest (e.g., a specific interaction between an antibody and an antigen). D The K values ​​are compared with those of non-target interactions (e.g., control antibodies known not to bind). D In some embodiments, an anti-FXII / FXIIa antibody of the present disclosure is capable of binding to its target with an affinity that is at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold greater than its affinity for binding to another non-FXII / FXIIa molecule, without limiting the definition herein.

[0347] "Conservative modification" or "conservative substitution" is applicable to amino acid and nucleotide sequences. For a specific nucleotide sequence, conservative modification or conservative substitution refers to the mutual replacement of those nucleic acids encoding the same or substantially identical amino acid sequence, or in the case where the nucleotide does not encode the amino acid sequence, it refers to substantially the same nucleotide sequence. For an amino acid sequence, conservative modification or conservative substitution refers to the replacement of an amino acid in a polypeptide with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently and have little or substantially no effect on the function, activity or other biological properties of the polypeptide. It is known to those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change biological activity (see, e.g., Watson et al. (1987) Molecullar Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)).

[0348] The above conservative amino acid substitutions are well known in the art. For example, conservative amino acid substitutions are preferably substituted by an amino acid residue in the following groups (i)-(v) with another amino acid residue in the same group:

[0349] (i) Small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro and Gly;

[0350] (ii) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln;

[0351] (iii) polar positively charged residues: His, Arg and Lys; (iv) larger aliphatic nonpolar residues: Met, Leu, Ile, Val and Cys; and

[0352] (v) Aromatic residues: Phe, Tyr and Trp.

[0353] Particularly preferred conservative amino acid substitutions are as follows: Ala is replaced by Gly or Ser; Arg is replaced by Lys; Asn is replaced by Gln or His; Asp is replaced by Glu; Cys is replaced by Ser; Gln is replaced by Asn; Glu is replaced by Asp; Gly is replaced by Ala or Pro; His is replaced by Asn or Gln; Ile is replaced by Leu or Val; Leu is replaced by Ile or Val; Lys is replaced by Arg, Gln or Glu; Met is replaced by Leu, Tyr or Ile; Phe is replaced by Met, Leu or Tyr; Ser is replaced by Thr; Thr is replaced by Ser; Trp is replaced by Tyr; Tyr is replaced by Trp or Phe; Val is replaced by Ile or Leu.

[0354] "Amino acid mutation" includes amino acid substitution, deletion, insertion, modification and any combination thereof to achieve a final construct so that the final construct has desired properties, such as enhanced stability and improved activity. Amino acid sequence deletions and insertions include amino and / or carboxyl terminal deletions and amino acid insertions. Preferred amino acid mutations are amino acid substitutions. In order to change, for example, the binding properties of anti-FXII / FXIIa antibodies, non-conservative amino acids can be substituted, i.e., one amino acid is replaced with another amino acid having different structural and / or chemical properties. Preferred amino acid substitutions include replacing hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art, including methods such as site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc. Amino acid mutations can occur in the CDR region, FR region or Fc region of the antibody.

[0355] Regarding amino acid mutations in the Fc region, mutations can be introduced into the wild-type Fc sequence of the antibody of the present disclosure to change Fc-mediated related activities, including but not limited to: a). mutations that change Fc-mediated CDC activity; b). mutations that change Fc-mediated ADCC activity; or c). mutations that change FcRn-mediated in vivo half-life (see Leonard G Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R. Hinton et al., J Immunol, 2006, 176: 346-356). Specifically, the invention includes: modifying the hinge region of CH1 so that the number of cysteine ​​residues in the hinge region is changed, for example, increased or decreased (see US5,677,425, which is incorporated herein in its entirety). Introducing mutations that enhance binding to FcγRIIIa (to induce enhanced ADCC), and mutations that weaken binding to FcγRIIb, such as 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 299T and 297N (see US11 / 124,620, US6,737,056, which are incorporated herein in their entirety). Fc modification is performed to increase its biological half-life, for example, one or more of the following mutations can be introduced: T252L, T254S, T256F (see US6,277,375); in order to increase the biological half-life, the antibody can be changed in the CH1 or CL region to contain a salvage receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG (see US5,869,046, US6,121,022); additional mutations for increasing serum half-life include 428L, 434A, 434S and 428L / 434S (see US8,883,973, US6,737,056, US7,371,826, which are incorporated herein in their entirety). The Fc region is altered by replacing at least one amino acid residue, thereby changing the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced to change the affinity of the antibody for the effector ligand, but retain the antigen binding ability of the parent antibody.The effector ligand with altered affinity can be, for example, an Fc receptor or C1 component of complement (see US5,624,821, US5,648,260, which are incorporated herein in their entirety). The ability of an antibody to fix complement is altered by changing one or more amino acid residues within amino acid positions 231 and 239 (see WO 94 / 29351, which is incorporated herein in its entirety). The Fc region is modified to increase the ability of an antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for Fcγ receptors by modifying one or more amino acids at the following positions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325 6, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 (see WO 00 / 42072, incorporated herein by reference in its entirety). In addition, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, RL et al. (2001) J. Biol. Chem. 276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 have been shown to improve binding to FcγRIII. In addition, the following combination mutants have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A and S298A / E333A / K334A. Furthermore, mutations such as M252Y / S254T / T256E or M428L / N434S improve binding to FcRn and increase antibody circulation half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10: 301-316).

[0356] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. Mammalian expression systems lead to glycosylation of antibodies, especially 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 expanded and cultured in serum-free culture medium in a bioreactor to produce antibodies. The culture fluid that secretes antibodies can be purified and collected by conventional techniques. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be immediately frozen, such as -70°C, or freeze-dried.

[0357] "Sequence" (for example, 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 sequence or nucleotide sequence encoding the sequence, unless the present disclosure requires further limited explanation.

[0358] "Polynucleotide" or "nucleic acid" refers to a nucleotide chain of any length, including DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and analogs thereof. If present, modifications to the nucleotide structure may be imparted before or after chain assembly. Polynucleotides may also contain similar forms of ribose or deoxyribose sugars generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lyxose, pyranose, furanose, sedoheptulose), acyclic analogs, and abasic nucleoside analogs such as methyl riboside.

[0359] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if every position of the 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 positions compared × 100%. For example, when the sequences are optimally aligned, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. In general, comparison is made when the two sequences are aligned to obtain the maximum percentage of homology.

[0360] "Vector" means a construct capable of delivering and, in some embodiments, expressing one or more target genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0361] "Host cell" includes individual cells or cell cultures that may be or have been recipients of vectors for incorporation of polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of the present 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 the same in terms of DNA content. Included are mutant progeny with the same function or biological activity as screened in the initially transformed cell. Host cells may include microorganisms (e.g., bacteria), plants, or animal cells. Bacteria that are susceptible to transformation include members of the family Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese Hamster Ovary cell line), NS0 cells, 293 cells.

[0362] "Pharmaceutical composition" means a mixture containing one or more antibodies or antigen-binding fragments described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, as well as other components such as physiologically / pharmaceutically acceptable adjuvants and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0363] "Pharmaceutically acceptable excipients" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate buffered saline solution, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and R Remington, The Science and Practice of Pharmacy 20th edition Mack Publishing, 2000).

[0364] "Administering," "applying," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid, such as for treatment, pharmacokinetic, diagnostic, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "applying," and "treating" also means in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a combination composition, or by another cell. When applied to humans, veterinary medicine, or research subjects, it refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0365] "Treatment" means administering an internal or external therapeutic agent, such as any of the antibodies disclosed herein or a pharmaceutical composition thereof, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or symptoms thereof, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing regression of such symptoms or inhibiting the development of such symptoms to any clinically measurable extent. The amount of the therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") may vary according to a variety of factors, such as the disease state, age, and weight of the subject, and the ability of the drug to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been alleviated can be evaluated by any clinical detection method commonly used by doctors or other professional health care personnel to evaluate the severity or progression of the symptoms. Although an embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating symptoms of a target disease in a subject, it should alleviate symptoms of the target disease in a statistically significant number of subjects as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0366] An "effective amount" includes an amount sufficient to improve or prevent the symptoms or symptoms of a medical condition. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects. The subject of the present disclosure may be an animal or a human subject.

[0367] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and where it does not occur.

[0368] As used in this disclosure, "subject", "patient" means a mammal, particularly a primate, especially a human. DETAILED DESCRIPTION

[0369] The following examples are used to further describe the present disclosure, but these examples are not intended to limit the scope of the present disclosure. Experimental methods in the examples of the present disclosure that do not specify specific conditions are usually carried out according to conventional conditions, such as the Cold Spring Harbor Antibody Technology Laboratory Manual and the Molecular Cloning Manual; or according to the conditions recommended by the raw material or product manufacturer. Reagents that do not specify specific sources are conventional reagents purchased from the market.

[0370] Example 1. Preparation of FXII antigen and detection protein

[0371] Human coagulation factor XII / XIIa / βXIIa (Uniprot Accession No P00748) as the antigen and detection protein involved in the present disclosure were purchased from Enzyme research laboratories (FXII: Product No. HFXII 1212; FXIIa: Product No. HFXIIa 1212a; βFXIIa: Product No. HFXIIab). The following FXII / FXIIa / βFXIIa antigens refer to human FXII / FXIIa / βFXIIa unless otherwise specified. The amino acid sequence is as follows:

[0372] >Amino acid sequence of FXII / FXIIa:

[0373] IPPWEAPKEHKYKAEEHTVVLTVTGEPCHFPFQYHRQLYHKCTHKGRPGPQPW

[0374] CATTPNFDQDQRWGYCLEPKKVKDHCSKHSPCQKGGTCVNMPSGPHCLCPQH

[0375] LTGNHCQKEKCFEPQLLRFFHKNEIWYRTEQAAVARCQCKGPDAHCQRLASQA

[0376] CRTNPCLHGGRCLEVEGHRLCHCPVGYTGAFCDVDTKASCYDGRGLSYRGLA

[0377] RTTLSGAPCQPWASEATYRNVTAEQARNWGLGGHAFCRNPDNDIRPWCFVLNR

[0378] DRLSWEYCDLAQCQTPTQAAPPTPVSPRLHVPLMPAQPAPPKPQPTTRTPPQSQT

[0379] PGALPAKREQPPSLTRNGPLSCGQRLRKSLSSMTRVVGGLVALRGAHPYIAALY

[0380] WGHSFCAGSLIAPCWVLTAAHCLQDRPAPEDLTVVLGQERRNHSCEPCQTLAVR

[0381] SYRLHEAFSPVSYQHDLALLRLQEDADGSCALLSPYVQPVCLPSGAARPSETTL

[0382] CQVAGWGHQFEGAEEYASFLQEAQVPFLSLERCSAPDVHGSSILPGMLCAGFLE

[0383] GGTDACQGDSGGPLVCEDQAAERRLTLQGIISWGSGCGDRNKPGVYTDVAYYL

[0384] AWIREHTVS

[0385] SEQ ID NO: 1> Amino acid sequence of βFXIIa:

[0386] NGPLSCGQRVVGGLVALRGAHPYIAALYWGHSFCAGSLIAPCWVLTAAHCLQD

[0387] RPAPEDLTVVLGQERRNHSCEPCQTLAVRSYRLHEAFSPVSYQHDLALLRLQED

[0388] ADGSCALLSPYVQPVCLPSGAARPSETTLCQVAGWGHQFEGAEEYASFLQEAQ

[0389] VPFLSLERCSAPDVHGSSILPGMLCAGFLEGGTDACQGDSGGPLVCEDQAAERR

[0390] LTLQGIISWGSGCGDRNKPGVYTDVAYYLAWIREHTVS

[0391] SEQ ID NO: 2

[0392] At the same time, a commercial kit (Cat. No.: 21955, Thermo) was used to prepare biotin-coupled βFXIIa protein for phage library screening, and the specific operation steps were carried out according to the instruction manual of the kit.

[0393] Example 2. Screening of single domain antibodies that specifically bind to FXII / FXIIa

[0394] Healthy Bactrian camels were immunized with human coagulation factor XIIa protein (Cat. No.: HFXIIa 1212a, Enzyme research). 5 mL of camel serum before immunization was taken and the serum was separated. After Freund's complete or incomplete adjuvant was mixed with the antigen in a volume ratio of 1:1, the camels were immunized subcutaneously at multiple points (immunization dose was 100 μg protein / mouse / each time). Booster immunization was performed every two weeks, and the titer was measured after four immunizations. Plates were coated with 1 μg / mL human coagulation factor XIIa (Costar, Cat. No.: 9018), 100 μL / well, and placed at 4°C overnight. The next day, PBST (0.05% Tween 20) was used to wash three times, 300 μL / well, and PBS solution containing 4% skimmed milk powder was added for blocking after washing, and incubated at 37°C for 2 hours. After washing three times with PBST, gradient dilutions of immune camel serum were added and incubated at 37°C for 1 hour. Negative control pre-immunization serum and blank PBS solution. After incubation, wash three times with PBST, add horseradish peroxidase (HRP)-labeled goat anti-alpaca Fc polyclonal antibody (Thermo, catalog number: A16060) at a dilution of 1:5000, and incubate at 37°C for 1 hour. After washing again, add TMB colorimetric solution for color development, terminate with 1M sulfuric acid, and use SpectraMax M5 microplate reader to read the absorbance value at OD450nm wavelength. After the fourth immunization, the titer was detected after a dilution of 1:656100 times. After the fifth immunization, the titer was detected after a dilution of 1:1968300 times. The titer was qualified, and camel peripheral blood was collected. Peripheral blood lymphocytes (PBMC) were isolated, RNA was extracted, and cDNA was obtained by reverse transcription for the construction of phage library.

[0395] The single domain antibody with high affinity to the βFXIIa antigen protein was obtained by screening the phage library. 10 μg of βFXIIa-biotin protein was combined with 1 mg Dynabeads MyOne Streptavidin T1 (Invitrogen, Cat. No.: 11206D). After standing at 37°C for one hour, it was blocked with 2% skim milk at room temperature for 2 hours. The camel single domain antibody phage display library was added and allowed to act at room temperature for 1 hour. Wash 10 times with PBST (0.05% Tween-20) solution to remove unbound phages. Phages that specifically bind to βFXIIa-biotin were eluted with 1 mg / mL Trypsin and infected with Escherichia coli TG1 in logarithmic phase growth to produce and purify phages for the next round of screening. After the same screening process was repeated 2-3 times, positive clones were enriched.

[0396] 96 monoclonal colonies were selected from the positive clones screened and enriched, and packaged into phage single-chain antibodies for phage ELISA test. 1 μg / mL FXIIa protein was coated on the ELISA plate, and phage supernatant diluted with 4% BSA blocking solution was added, and anti-M13 HRP antibody was used for detection. The ELISA binding test was carried out to the middle OD 450 The clones with values ​​greater than 1.5 were sequenced and 39 specific sequences were obtained.

[0397] Example 3. Construction of complete antibodies

[0398] The 39 specific sequences obtained by phage library screening in Example 2 were used to construct complete antibodies, and then ELISA binding experiments, SPR protein interaction experiments and enzyme activity inhibition experiments were performed to determine that 5 of the antibodies had strong binding and could inhibit the enzymatic activity of FXIIa. The complete VHH sequences of antibodies 0001, 0002 and 0015 are as follows, and the CDR sequences are underlined:

[0399] >0001

[0400] HVQLVESGGGLVQAGGSLRLSCTASGLPFA NTMG WFRQAPGKEREGVA CISWSGRDTHHADSVKG RFTISRDNAKNTLSLQMNSLKPEDTAMYYCAA VGKGTSWYSGAYGCPSEGFDY WGQGTQVTVSS

[0401] SEQ ID NO: 3

[0402] >0002

[0403] DVQLVESGGALVQAGGSLLRLSCAASGLIPA NTMG WFRQAPGKEREGVA CISWSGRDTYHADSVKG RFTISRDNGKNTLYLQMNSLKPEDTAMYYCAA VGKGTSWYGGSYGCPSEGFDY WGQGTQVTVSS

[0404] SEQ ID NO: 4

[0405] >0015

[0406] HVQLVESGGGLVQAGGSLRLSCTASGLAGA NTMG WFRQAPGKEREGVA CISWSGREIYHADSVKG RFTISRDNAKNTLYLQMNSLKPEDTAMYYCAA VGKGTSWYSGNYRCPSGGFDY WGQGTQVTVSS

[0407] SEQ ID NO: 5

[0408] In the above sequences SEQ ID NO:3-7, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underlined sequences are CDR1, CDR2, and CDR3, respectively. The antibody numbering system disclosed in the present invention is Kabat. The CDR sequences are summarized in Table 1.

[0409] Table 1. CDR sequences of FXII single domain antibodies

[0410]

[0411] The above 0001, 0002, and 0015 antibodies have the following CDR sequence formula:

[0412] CDR1: NTMG (SEQ ID NO: 6);

[0413] CDR2: CISWSGRX1X2X3HADSVKG (SEQ ID NO: 45), wherein X1 is selected from D or E, X2 is selected from T or I, and X3 is selected from H or I;

[0414] CDR3: VGKGTSWYX4GX5YX6CPSX7GFDY (SEQ ID NO: 46), wherein X4 is selected from S or G, X5 is selected from A, S or N, X6 is selected from G or R, and X7 is selected from G or E.

[0415] The VHH sequence was connected to the human IgG4 Fc (CH2-CH3, containing S228P) sequence and constructed into the pTT5 expression vector, and the cells were transiently transfected to express the antibody and purify it. After detection, the target antibody was obtained.

[0416] The sequence of the linked IgG4 may be as follows:

[0417] >hIgG4 Fc(S228P)

[0418] ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0419] SEQ ID NO: 13

[0420] The following is the full protein sequence of the VHH sequence fused to human Fc (CH2-CH3), the italic single underline is the hIgG4Fc (S228P) sequence (shown in SEQ ID NO: 13). The protein sequence is as follows:

[0421]

[0422]

[0423]

[0424]

[0425] Example 4. Humanization of single domain antibodies

[0426] By performing three-dimensional structural homology modeling on the selected specific FXII single-domain antibody molecule, combined with the results of comparison with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the heavy chain variable region germline gene IGHV3-66 with high homology to the screened antibody was selected as a template, and the CDR of the camel-derived single-domain antibody was transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The transplanted single-domain antibody was simulated and analyzed again in three dimensions, and the specific sites in the FR region that affect the structural morphology of the CDR region were reversed. The reverse mutations are shown in Table 2, and the numbering is the Kabat numbering system:

[0427] Table 2. Germline gene templates and back mutation sites of antibodies

[0428]

[0429] The specific humanized sequences are as follows (Note: the underlined sequences are CDR sequences):

[0430] >0001_Hu-1

[0431] EVQLVESGGGLVQPGGSLRLSCAASGLPFA NTMG WFRQAPGKEREGVS CISWSGRDTHHADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYSGAYGCPSEGFDY WGQGTLVTVSS

[0432] SEQ ID NO: 17

[0433] >0001_Hu-2

[0434] EVQLVESGGGLVQPGGSLRLSCAASGLPFA NTMG WFRQAPGKEREGVS CISWSGRDTHHADSVKG RFTISRDNSKNTLSLQMNSLRAEDTAVYYCAA VGKGTSWYSGAYGCPSEGFDY WGQGTLVTVSS

[0435] SEQ ID NO:18

[0436] >0001_Hu-3

[0437] EVQLVESGGGLVQPGGSLRLSCAASGLPFA NTMG WFRQAPGKEREGVS CISWSGRDTHHADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYSGAYGCPSEGFDY WGQGTLVTVSS

[0438] SEQ ID NO:19

[0439] >0002_Hu-1

[0440] EVQLVESGGGLVQPGGSLRLSCAASGLIPA NTMG WFRQAPGKEREGVS CISWSGRDTYHADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYGGSYGCPSEGFDY WGQGTLVTVSS

[0441] SEQ ID NO:20

[0442] >0002_Hu-2

[0443] EVQLVESGGGLVQPGGSLRLSCAASGLIPA NTMG WFRQAPGKEREGVS CISWSGRDTYHADSVKG RFTISRDNGKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYGGSYGCPSEGFDY WGQGTLVTVSS

[0444] SEQ ID NO:21

[0445] >0015_Hu-1

[0446] EVQLVESGGGLVQPGGSLRLSCAASGLAGA NTMG WFRQAPGKEREGVS CISWSGREIYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYSGNYRCPSGGFDY WGQGTLVTVSS

[0447] SEQ ID NO: 22

[0448] >0015_Hu-2

[0449] EVQLVESGGGLVQPGGSLRLSCAASGLAGA NTMG WFRQAPGKEREGVS CISWSGREIYHADSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA VGKGTSWYSGNYRCPSGGFDY WGQGTLVTVSS

[0450] SEQ ID NO: 23

[0451] The VHH sequence was connected to the hIgG4 Fc (S228P) sequence, a vector was constructed, cells were transiently transfected, antibodies were expressed, and purified. After testing, the target antibody was obtained. The 0002_Hu-2 fusion hIgG4 Fc (S228P) sequence is provided below as an example, and the italic single underline is the hIgG4 Fc (S228P) sequence (shown in SEQ ID NO: 13). The protein sequence is as follows:

[0452]

[0453]

[0454] Example 5. Screening and preparation of HSA antibodies

[0455] 5.1 Screening of antibodies that specifically bind to human serum albumin (HSA)

[0456] 1. Camel Immunization

[0457] A natural Bactrian camel from Inner Mongolia was immunized using serum albumin extracted from commercial human blood (Sigma, Cat No.126658). 5 mL of camel serum before immunization was taken and the serum was separated. After Freund's complete or incomplete adjuvant was mixed with the antigen in a volume ratio of 1:1, the camel was immunized subcutaneously at multiple points (immunization dose was 100 μg protein / mouse / each time). Reinforced immunization was performed every two weeks, and the titer was measured after four immunizations. The plate was coated with 5 μg / mL human serum albumin (HSA) (Costar, Cat.No, 9018), 100 μL / well, and placed at 4°C overnight. The next day, PBST (0.05% Tween20) was used to wash three times, 300 μl / well, and then 4% skim milk powder was added for blocking, 37°C, 2h. After washing, the serum of the immune camel with gradient dilution was added and incubated at 37°C for 1h. The negative control was the same gradient dilution of pre-immunization serum and blank PBS solution. After incubation, wash three times with PBST, add horseradish peroxidase-labeled goat anti-camel Fc polyclonal antibody (Thermo, Cat No. A16060, 1:5000 dilution), and incubate at 37°C for 1 hour. After washing again, add TMB colorimetric solution for color development, terminate with 1M sulfuric acid, and read the absorbance value at OD450nm wavelength using SpectraMax M5 microplate reader. The titer was detected after 1:51200 dilution. If the titer is qualified, collect camel peripheral blood for library construction.

[0458] 2. Phage library establishment

[0459] 100-200 mL of camel peripheral blood was collected and peripheral blood lymphocytes (PBMC) were separated using the Ficoll method. The cell count was 1.2 × 10 8 Add Trizol reagent and resuspend (1×10 7 cells / mL Trizol) to lyse the cells, and place on ice for 5 minutes; centrifuge at 13000rpm for 3 minutes, take the supernatant, and discard the precipitate; add 1 / 5 volume of chloroform, shake vigorously for 30-60s, and let stand in an ice bath for 2 minutes; centrifuge at 13000rpm for 10 minutes, and draw the upper aqueous phase into a new 1.5mL tube; add an equal volume of isopropanol, mix well, and let stand at -20℃ for 30 minutes; centrifuge at 13000rpm for 10 minutes, remove the supernatant, and retain the precipitate; add pre-cooled 75% ethanol to wash the precipitate, and let stand at room temperature for 5-10 minutes; add 600μL of deionized water from which RNase has been removed, re-dissolve, obtain RNA, reverse transcribe to obtain cDNA, and construct a phage library.

[0460] 3. Screening of phage libraries

[0461] HSA protein lacking some amino acids of the third domain at the C-terminus (also known as truncated HSA) was expressed and purified in HEK293 cells, and this protein was used to screen the phage library. Antibodies with affinity to HSA were obtained through screening.

[0462] Use 10 μg of the above-mentioned truncated HSA-avi-biotin protein to bind 1 mg of Dynabeads M-280 streptavidin (Invitrogen, Cat No. 11206D), place at room temperature for one hour, wash 3 times with 1×PBS, block with 2% skim milk at room temperature for 2 hours, add camel heavy chain single domain antibody phage display library, and act at room temperature for 1 hour. Wash 9 times with PBST (0.05% Tween-20) solution to remove unbound phages. Use 1 mg / mL of trypsin to elute the phages that specifically bind to the truncated HSA, and infect Escherichia coli TG1 in the logarithmic phase to produce and purify phages for the next round of screening. After repeating the same screening process for 1-2 rounds, the positive clone sequences are enriched.

[0463] 4. Phage ELISA Identification

[0464] From the clones obtained in the last round of screening, monoclonal colonies were selected and packaged into phage single-chain antibodies for phage ELISA testing. 2 μg / mL of human, monkey, and mouse serum albumin (HSA, CySA, mSA) were coated on the ELISA plate overnight, and 4% skim milk was blocked at 37°C for 1 hour. Then, the phage supernatant blocked with 2% skim milk was added, and the plate was allowed to react for 1 hour at room temperature, washed 3 times with PBST (0.05% Tween 20), and anti-M13 HRP was added for detection. The clones with OD450 values ​​greater than 0.5 with the three albumins in the ELISA binding test were sequenced to obtain 50 specific sequences. Only some antibodies that showed high OD450 values ​​with human and monkey serum albumin at the same time were sequenced to obtain another 7 new specific sequences. A total of 57 new specific sequences were obtained by phage screening.

[0465] 5.2 Construction of antibody mammalian cell expression vector

[0466] The 57 specific sequences obtained by phage library screening in 5.1 were classified and sorted, 19 of which were cloned, and the phage supernatant ELISA binding of these 19 HSA antibodies was tested. The OD of clone #7 was 450 The values ​​are shown in Table 3.

[0467] Table 3. HSA antibody phage supernatant ELISA test results

[0468] serial number <![CDATA[HSA OD 450 ]]> <![CDATA[mSA OD 450 ]]> <![CDATA[cySAOD 450 ]]> #7aHSA 1.6929 1.1185 1.7477

[0469] Some of the complete VHH sequences are as follows:

[0470] >#7aHSA

[0471]

[0472] The above sequence is SEQ ID NO:6, the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in the sequence are FR sequences, and the underlines are CDR1, CDR2, and CDR3 sequences respectively. The CDR sequences of the HSA antibodies provided in the present disclosure according to Kabat encoding are summarized in Table 4.

[0473] Table 4. CDR sequences of HSA antibodies (Kabat numbering system)

[0474]

[0475] The VHH sequence was fused to human IgG1-Fc (CH2-CH3) and constructed into the PTT5 expression vector. The sequence of the connected human IgG1-Fc is as follows:

[0476] >Human IgG1-Fc

[0477] EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:29)

[0478] The following is the full protein sequence of the VHH sequence fused to the human IgG1-Fc (CH2-CH3) segment. The single underline is the human IgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 29), and the double underline is the linker sequence. The full protein sequence is as follows:

[0479] >#7aHSA-IgG1Fc:

[0480]

[0481] 5.3 Expression, screening and detection of HSA antibodies

[0482] The #7aHSA antibody fused with human IgG1-Fc was transiently expressed in HEK293 cells and purified by Protein A affinity, and then tested by ELISA binding assay and Biacore protein interaction assay.

[0483] The in vitro binding ability of #7aHSA antibody to human, monkey and mouse serum albumin was tested by ELISA binding experiment, and the test antigens were human serum albumin (Sigma, Cat No.126658), monkey serum albumin (Abcam, Cat No.ab184894) and mouse serum albumin (Abcam, Cat No.ab183228). Among them: the negative control was PBS, and the positive control used ALB8 fused with human IgG1-Fc (the variable region sequence was from the sequence of ALB8 in Ablynx patent publication WO 2008 / 028977 A2), the sequence is as follows:

[0484] >ALB8-IgG1Fc

[0485]

[0486] (Note: The single underline is the Fc region of IgG1, and the double underline is the linker region)

[0487] Human, monkey, and mouse albumin were diluted to 1 μg / mL with PBS buffer at pH 7.4, added to a 96-well ELISA plate (Corning, Cat No. 9018) at a volume of 100 μL / well, and placed at 4°C overnight for 16-20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, add 2% skim milk powder blocking solution diluted with PBS buffer (300 μL / well), and incubate at 37°C for 1 hour for blocking. After the blocking is completed, discard the blocking solution, wash the plate 3 times with PBST buffer, add HSA antibody with an initial concentration of 2 μg / mL, perform 11 gradients of three-fold dilutions with PBS buffer, and incubate at 25°C for 1 hour. After the incubation, the reaction solution in the plate was discarded, the plate was washed 3 times with PBST, 100 μL of HRP-labeled goat anti-human IgGFc secondary antibody (Jackson immu, 109-035-190) (1:5000 dilution) was added to each well, and incubated at 25°C for 1 hour. After washing the plate 3 times with PBST, 100 μL of TMB colorimetric substrate was added for color development, 1M sulfuric acid was used to terminate the reaction, and the absorption value was read at OD450nm using a SpectraMax M5 microplate reader. GraphPad prism was used to plot the absorption value and antibody concentration using a 4-parameter fitting to calculate the EC50 value of antibody binding to the antigen. The results are shown in Table 5.

[0488] Table 5. ELISA binding EC50 (μg / mL) of HSA antibodies to human, monkey and mouse albumin

[0489] Antibody ID <![CDATA[EC binding to HSA 50 > <![CDATA[EC combined with monkey SA 50 > <![CDATA[EC combined with murine SA 50 > #7aHSA-IgG1Fc 0.04552 0.04256 0.04502 Positive control (ALB8-IgG1Fc) 0.009926 0.010134 0.01061 Negative control (PBS) / / /

[0490] (Note: “ / ” means not detected)

[0491] In addition, the affinity constants of HSA antibody fused with human IgG1Fc and human, monkey, and mouse albumin (HSA, CySA, and mSA) were determined by the Biacore T200 (GE Healthcare) instrument. First, protein A was covalently coupled to a CM5 S series chip (GE, Cat. 29-1049-88) using an amino coupling kit (GE, Cat. BR-1000-50), and the HSA antibody was captured by affinity onto the chip coupled with protein A. Then, different concentrations of human, monkey, and mouse albumin were passed over the chip surface, and the reaction signal was detected in real time using the Biacore instrument to obtain the binding and dissociation curves. The affinity constants were obtained by fitting the Langmuir 1:1 binding model using the BIAevaluationversion 4.1 and GE software. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat.#BR-1006-69). After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (GE, Cat.#BR-1003-54). The affinity results are shown in Table 6.

[0492] Table 6. Affinity results of HSA antibodies to human, monkey and mouse albumin

[0493]

[0494] 5.4 Humanization of HSA Antibody

[0495] By performing three-dimensional structural homology modeling on the selected specific HSA antibody molecule #7aHSA, combined with the results of comparison with the V-base human germline sequence database and the IMGT human antibody heavy chain variable region germline gene database, the heavy chain variable region germline gene IGHV3-23 with high homology to the selected antibody was selected as a template, and the CDR of the camel-derived single-domain antibody was transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The transplanted single-domain antibody was simulated and analyzed again in three dimensions. The specific sites in the FR region that affect the structural morphology of the CDR region were reversed. The modified antibody has higher stability. The amino acid residues are determined and annotated by the Kabat numbering system. Among them, the FRs of #7_Hu-9 and #7_Hu-10 retain more human germLine sequences, making them have lower immunogenic mutations.

[0496] The design of the reverse mutation site and mutation method is shown in Table 7.

[0497] Table 7. Reverse mutation sites and mutation patterns

[0498]

[0499]

[0500] The specific humanized sequences obtained are as follows, the CDR regions are underlined, and the numbering system is the Kabat code:

[0501] >#7_Hu_1

[0502] EVQLLESGGGLVQPGGSLRLSCAASGNTYL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS (SEQ ID NO: 32)

[0503] >#7_Hu_2

[0504] EVQLLESGGGLVQPGGSLRVSCAASGNTYL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS (SEQ ID NO: 33)

[0505] >#7_Hu_3

[0506] EVQLLESGGGLVQPGGSLRLSCVASGNTYL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS(SEQ ID NO:34)

[0507] >#7_Hu_4

[0508] EVQLLESGGGLVQPGGSLRLSCAASGNTYL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS(SEQ ID NO:35)

[0509] >#7_Hu_5

[0510] EVQLLESGGGLVQPGGSLRLSCAASGNTYL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTVYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS(SEQ ID NO:36)

[0511] >#7_Hu_6

[0512] EVQLLESGGGLVQPGGSLRLSCAASGNTYL TSCMG WFRQAPGKEREGVA SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS(SEQ ID NO:37)

[0513] >#7_Hu_7

[0514] EVQLLESGGGLVQPGGSLRLSCAASGNTY LTSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLQPEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS(SEQ ID NO:38)

[0515] >#7_Hu_8

[0516] EVQLLESGGGLVQPGGSLRLSCAASGNTY LTSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRPEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS (SEQ ID NO: 39)

[0517] >#7_Hu_9

[0518] EVQLLESGGGLVQPGGSLRLSCAASGFTF LTSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKGR FTISRDNSKNTLYLQMNSLRPEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS (SEQ ID NO: 40)

[0519] >#7_Hu_10

[0520] EVQLLESGGGLVQPGGSLRLSCAASGFTFL TSCMG WFRQAPGKEREGVS SIYTVGGSTFYTNSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAA GSPARCRLRDPTTFDY WGQGTLVTVSS (SEQ ID NO: 41)

[0521] The method in 5.4 is used to construct a fusion protein of humanized HSA antibody and the Fc (CH2-CH3) segment of hIgG1 or a protein fused with a 6His tag. The single underline is the hIgG1-Fc (CH2-CH3) segment sequence (shown in SEQ ID NO: 29), the italic is the 6His tag sequence, and the double underline is the linker sequence. The protein sequence is as follows (taking #7_hu_1-hIgG1Fc and #7_hu_10-6His as examples, and the same is true for other humanized HSA antibodies). For the antibody numbering system, #7_hu_10-6His is the C-terminal fusion of #7_Hu_10 (SEQ ID NO: 41). (SEQ ID NO: 64), and the same is true for other antibodies.

[0522] >#7_hu_1-hIgG1Fc:

[0523]

[0524] >#7_Hu_10-6his

[0525]

[0526] >ALB8-6his

[0527]

[0528] The plasmid was transiently transfected into HEK293 cells, and the expression supernatant was collected after 5 days of cell culture, centrifuged at 4000 rpm for 15 minutes to remove the cells, the supernatant was harvested and filtered using a 0.45 μM filter, and then purified using a Protein A column or a nickel column or other conventional methods in the art to obtain the HSA antibody disclosed in the present invention.

[0529] 5.5 Affinity determination of humanized HSA antibody with human, monkey and mouse serum albumin

[0530] The affinity constants of humanized HSA antibody fused with 6His tag and human, monkey and mouse albumin were determined by Biacore T200 (GE Healthcare). First, amino coupling kit (GE, Cat.BR-1000-50) and His capture kit (GE, Cat.28-9950-56) were used to amino couple CM5 S series chip (GE, Cat.29-1049-88) according to the kit instructions, and the surface response value reached about 10000RU, and then blocked with ethanolamine for standby use; humanized HSA antibody fused with 6His tag was captured onto the anti-his chip by affinity, and then different concentrations of human, monkey and mouse albumin were passed on the chip surface, and the reaction signal was detected in real time by Biacore instrument to obtain the binding and dissociation curves, and the affinity constants were obtained by fitting with Langmuir 1:1 binding model by BIAevaluation version 4.1 and GE software. The buffer used in the experiment was HBS-EP solution (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4, GE, Cat. # BR-1006-69). After each cycle, the chip was regenerated using pH 1.5 glycine-hydrochloric acid (GE, Cat. # BR-1003-54). The affinity results of some humanized antibodies are shown in Table 8. The results show that the affinity of the antibodies obtained by the present disclosure screening to HSA is comparable to or slightly better than that of the positive control.

[0531] Table 8. Affinity of humanized HSA antibodies to human, monkey and mouse albumin

[0532]

[0533] 5.6 Effect of HSA antibody on the binding of FcRn and biotin-HSA in cells stably expressing hFcRn The blocking effect of HSA antibody on the binding of HSA to FcRn was detected by the change in the fluorescence of random biotinylated HSA bound to the surface of HEK293 cells (293-hFcRn-mut cells) stably expressing hFcRn mutants. This experiment used HEK293 cells stably expressing hFcRn mutants (L320A, L321A), which can be well localized and maintained on the cell membrane surface. Cells expressing hFcRn mutants were incubated with different concentrations of HSA antibody and Biotin-HSA, and then SA-FITC secondary antibody was added for incubation. The degree of reduction of the secondary antibody fluorescence signal (MFI) was used as a measure of the strength of the HSA antibody's blocking effect on the binding of biotin-HSA to FcRn. The specific experimental method is as follows:

[0534] After 293-hFcRn-mut cells were washed twice with pH 5.5 PBS, 5 × 10 5 cells. After centrifugation and removal of the supernatant, 90 μL of HSA antibody diluted with PBS (pH 5.5) and 10 μL of biotin-HSA (final concentration of 250 μg / mL) were added to each well and incubated on ice for 40 minutes. After washing twice with 200 μL PBS (pH 5.5), 100 μL of streptavidin-FITC secondary antibody (eBioscience, Cat No.11-4317-87) diluted with PBS (pH 5.5) was added to each well at a dilution ratio of 1:200, and incubated on ice for 40 minutes. After washing twice with 200 μL PBS (pH 5.5), 200 μL PBS (pH 5.5) was added to each well, and the fluorescence value was measured using a flow cytometer (BD Biosciences, BD Accuri C6) and the FL1 channel, and the data was analyzed using Flowjo software, and the analysis results were plotted using Prism6 software. The inhibitory test results of some antibodies are shown in Fig. 7A and 7B .

[0535] Result analysis: The fluorescence signal value (MFI) was used to determine whether the binding of HSA antibody to HSA blocked the binding of HSA to FcRn. If the fluorescence signal decreased, it indicated that the binding was blocked.

[0536] like Fig. 7AAs shown, we used the biotin-HSA+FcRn stably transfected cells+SA-FITC group (non-blocking group) as the baseline; the biotin-HSA+HSA+FcRn stably transfected cells+SA-FITC group (HSA self-competing group) as the positive control; Nc is the isotype control antibody. The results show that the HSA antibodies #7_Hu_6-6his and #7_Hu_7-6his disclosed in the present invention do not block the binding of HSA to FcRn.

[0537] like Figure 7B As shown, #7_Hu_10-6his and #7_Hu_10 did not block the binding of HSA to FcRn (detected at a concentration of 1 mg / mL).

[0538] This indicates that the HSA antibody disclosed herein will not affect the pH-dependent binding of FcRn to HSA and can well maintain the FcRn-mediated serum albumin recycling mechanism.

[0539] 5.7 Pharmacokinetics of HSA Antibody in Mice

[0540] The pharmacokinetic characteristics of the disclosed HSA antibodies (#7_Hu_6-6his; #7_Hu_7-6his; #7_Hu_10-6his), positive control antibodies (ALB8-6his) and isotype control antibodies (Isotype, non-HSA binding VHH molecules) were studied in CD-1 male mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). Three mice in each group were administered by tail vein injection at a dose of 4 mg / kg on day 0, and blood samples were collected before administration, 5 minutes, 1 hour, 7 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, and 144 hours after administration. Plasma was prepared by anticoagulation with EDTA and centrifugation for pharmacokinetic determination.

[0541] PK assay: Coated with 5 μg / mL anti-his antibody (Thermo, MAI-21315-1mg), added to a 96-well ELISA plate (Corning, Cat No. 9018) at a volume of 100 μL / well, and placed at 4°C overnight for 16-20 hours. After discarding the liquid, wash the plate three times with PBST (pH 7.4, 0.05% Tween-20) buffer, add Casin Blocker (Thermo, 37528) blocking solution (300 μL / well), and incubate in a 37°C incubator for 2 hours for blocking. After the blocking is completed, discard the blocking solution, wash the plate 3 times with PBST buffer, add mouse plasma at different time points appropriately diluted with diluent (Casin Blocker containing 1% CD1 mouse plasma), and incubate at 37°C for 1.5 hours. After the incubation, the reaction solution in the plate was discarded, the plate was washed 3 times with PBST, 100 μL of HRP-labeled rabbit anti-camel Cocktail VHH secondary antibody (Genescript, A02016, without BSA) (1:5000 dilution) was added to each well, and incubated at 37°C for 1 hour. After washing the plate 3 times with PBST, 100 μL of TMB colorimetric substrate was added for color development, 1M sulfuric acid was used to terminate the reaction, and the absorbance value was read at OD450nm using a SpectraMax M5 microplate reader. The VHH antibody concentration in the plasma sample was calculated according to the standard curve, and the results were as follows: Figure 8 The calculated concentration was used to calculate the half-life using PK solver, and the result is as follows Fig.9A , 9B and as shown in Table 9.

[0542] Table 9. Mouse plasma half-life of HSA antibodies

[0543]

[0544] (Note: *, p<0.05; **, p<0.01; ***, p<0.0001; ****, p<0.00001; all compared with the positive control ALB8-6his)

[0545] The results showed that the HSA antibodies #7_Hu_6-6his, #7_Hu_7-6his, and #7_Hu_10-6his disclosed in the present invention had similar pK curves, half-lives were within the half-life range of mouse serum albumin, and were superior to the positive control ALB8-6his antibody, and the clearance rates were also lower than that of the ALB8-6his antibody.

[0546] Example 6. Construction and preparation of multivalent tandem single domain antibodies

[0547] The FXII / FXIIa humanized single domain antibody molecules 0002_Hu-2, C004_hu-1 and C018_hu-3 were combined with the human serum albumin HSA binding single domain antibody #7_Hu_10 (double underline sequence) through a linker (single wavy line sequence) to obtain a bivalent or trivalent tandem single domain antibody molecule, the sequence of which is as follows:

[0548]

[0549]

[0550]

[0551]

[0552] The anti-FXII / FXIIa antibody disclosed in the present invention was expressed using a mammalian cell system, and the ExpiCHO Expression System (Cat. No.: A29133) was used for transfection at a ratio of 1 μg DNA / mL transfected cells. The operation was carried out according to the reagent instructions, using standard methods, placed in a 37°C shaker for shaking culture (8% CO2), and the cell culture fluid was collected on the 8th day, centrifuged at 4000 rpm, and the supernatant was taken and filtered using a 0.45 μM filter membrane. Protein A filler (GE, MabSelect TM SuRe TM , Catalog No.: 17543801) or Amsphere A3 filler (Catalog No.: BP-AMS-A3-0100, JSR) was used for affinity purification to obtain the target antibody.

[0553] Example 7. Detection of antibody binding ability to FXIIa

[0554] Protein A biosensor chip (Cat. No. 29127556, GE) or anti-VHHs antibody (MonoRab TMRabbit Anti-Camelid VHH Cocktail, Genscript, A02014) coupled to a CM5 biosensor chip (Cat. No. BR100530, GE) to capture a certain amount of the antibody to be tested, and then a series of FXIIa concentration gradients flowed through the chip surface, and the reaction signal was detected in real time using a Biacore instrument (Biacore T200, GE) to obtain the binding and dissociation curves. After each cycle of dissociation was completed, the biochip was washed and regenerated with the regeneration solution configured in the human anti-capture kit or the pH 1.5 glycine-HCl regeneration solution (Cat. No. BR-1003-54, GE). The buffer used in the experiment was HBS-EP + 10× buffer solution (Cat. # BR-1006-69, GE) diluted to 1× (pH 7.4) with DIWater.

[0555] The experimental data were fitted with the (1:1) Langmuir model using BIAevaluation version 4.1, GE software to obtain affinity values. Some antibody test results are shown in Table 10.

[0556] Table 10. Affinity determination of antibodies to FXIIa antigen

[0557] Antibody antigen Ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> CSL-312 FXIIa 6.29E+04 3.66E-05 5.83E-10 0001-Fc FXIIa 2.37E+05 1.14E-04 4.81E-10 0002-Fc FXIIa 2.02E+05 9.97E-05 4.95E-10 0015-Fc FXIIa 2.02E+05 1.11E-04 5.51E-10 0001_Hu-1-Fc FXIIa 5.37E+05 3.71E-04 6.92E-10 0001_Hu-2-Fc FXIIa 5.26E+05 3.15E-04 5.99E-10 0001_Hu-3-Fc FXIIa 5.45E+05 3.05E-04 5.59E-10 0002_Hu-1-Fc FXIIa 4.38E+05 3.00E-04 6.86E-10 0002_Hu-2-Fc FXIIa 4.62E+05 3.04E-04 6.58E-10 0015_Hu-1-Fc FXIIa 4.66E+05 3.14E-04 6.74E-10 0015_Hu-2-Fc FXIIa 4.97E+05 2.32E-04 4.66E-10 2207 FXIIa 4.23E+05 7.63E-05 1.80E-10

[0558] Example 8. In vitro FXIIa enzyme activity inhibition test of anti-FXII / FXIIa antibodies

[0559] As a functional experiment for in vitro screening of candidate anti-FXII / FXIIa antibodies, the ability of anti-FXII / FXIIa antibodies to bind to FXIIa and inhibit FXIIa from cleaving specific substrates was tested.

[0560] The SpectraMax M5 microplate reader was pre-set to 37°C, and a 384-well plate (thermo, Cat.#94410153) was pre-cooled on ice. 5 μL of the test antibody (final concentration 3.333 μg / mL) and 5 μL of FXIIa (Enzyme Research, HFXII 1212a, final concentration 3.333 μg / mL) diluted in phosphate buffer were added, incubated at 37°C for 5 min, then placed on ice, and 5 μL of S-2302 (Chromogenix, final concentration 1.333 mM) was added. The absorbance at 405 nm was immediately read at 37°C, once every minute, and a kinetic curve was drawn with absorbance (vertical axis) versus time (horizontal axis). The slope of the linear interval of the curve was statistically analyzed using GraphPad Prism 9; one-way ANOVA was used for statistical analysis, and Tukey's post-hoc test was used to analyze the differences between the groups. Some antibody test results are available at Figure 2 , where human IgG isotype was used as a negative control.

[0561] For some antibody tests, the concentration gradient method was used. The operation process is the same as above, in which the antibody uses a gradient concentration (final concentration 100ng / mL to 10μg / mL), the final concentration of FXIIa is 6.667μg / mL, and the rest remains unchanged. When analyzing the data, the slope of the kinetic curve at each antibody concentration is used for analysis, and the slope (vertical axis) is plotted against the final concentration of the antibody (horizontal axis). The curve is fitted with a four-parameter S-shaped dose-effect relationship curve (sigmoidal, 4PL), and the half-maximal inhibitory concentration (IC 50 ), as shown in Table 11. The fitting curves of positive antibodies and antibody 2207 are shown in Figure 3A .

[0562] The antibody's inhibitory effect on FXIIa activity is also reflected in its inhibition of downstream activation of FXI. Under normal circumstances, FXIIa can activate FXI to generate FXIa, and FXIa can bind to its substrate S-2366 and change color (see the attached for the specific activation pathway). Figure 6). The basic process of this enzymatic test remains the same as above. First, add 5 μL of FXIIa (final concentration 2 μg / mL) and 5 μL of the test antibody to each well of a 384-well plate, wherein the test antibody is a gradient concentration (final concentration 1.481 nM120 nM), incubate at 37°C for 5 min, put the well plate back on ice, add 5 μL of FXI (final concentration 2 μg / mL) to each well, incubate at 37°C for 5 min, put the well plate back on ice, add an excess of the positive control antibody CSL-312 (monoclonal antibody shown in WO2021108862, the amino acid sequences of the heavy chain and light chain of CSL-312 are sequence 9 and sequence 10 shown in WO2021108862, respectively) to each well, with a final concentration of 20 nM, incubate at 37°C for the third time for 5 min, put the well plate back on ice, and add S-2366 (final concentration 400 μM) to each well. Immediately read the absorbance of each well at 405 nm, once every minute for 30 minutes. Continue to plot the slope of the kinetic curve against the final concentration of the antibody, and fit the four-parameter S-shaped good-effect relationship curve to calculate the IC 50 , as shown in Table 12. The fitting curves of positive antibodies and antibody 2207 are shown in Figure 3B .

[0563] Table 11. Inhibitory effect of antibodies on FXIIa enzyme activity

[0564] Antibody <![CDATA[IC 50 (nM)]]> CSL-312 8.756 2207 10.07

[0565] Table 12. Inhibitory effect of antibodies on activation of FXI by FXIIa

[0566] Antibody <![CDATA[IC 50 (nM)]]> CSL-312 8.452 2207 7.464

[0567] The above results indicate that the anti-FXII / FXIIa antibodies disclosed herein can directly inhibit the activity of FXIIa, and can also inhibit the activation of FXI, a downstream enzyme in the signaling pathway, by FXIIa.

[0568] Example 9. Detection of aPTT / PT anticoagulant activity in human blood

[0569] The in vitro anticoagulant activity was tested on human plasma, which was commercially available lyophilized human plasma powder (Dade Behring, ORKL17) and was freshly prepared according to the instructions.

[0570] The activated partial thromboplastin time (aPTT) in the presence of different concentrations of candidate antibodies (diluted with phosphate buffer) was measured using a Sysmex kit. The candidate antibody to be tested was mixed with plasma 1:1 (volume ratio) and incubated at 37°C for 3 minutes; then 50 μL was taken and 50 μL of aPTT reagent was added, and incubated at 37°C for 3 minutes; finally, 50 μL of CaCl2 was added to initiate coagulation, and the timing was started to measure the time when coagulation occurred. The concentration of the candidate antibody that prolonged aPTT by 50% was determined (aPTT 1.5 ), where the antibody concentration is expressed as the final concentration of the antibody in the system. Some antibody test results are shown in Figure 4A , 4B and Table 13.

[0571] The Sysmex kit was used to measure the prothrombin time (PT) in the presence of different concentrations of candidate antibodies (diluted with phosphate buffer). The candidate antibodies to be tested were mixed with plasma 1:1 (volume ratio) and incubated at 37°C for 3 minutes; then 100 μL was taken and 50 μL of PT reagent was added to start coagulation, and the timer was started to measure the time when coagulation occurred. Some antibody test results are shown in Figure 4C and Figure 4D .

[0572] Table 13. aPTT assay of anti-FXII / FXIIa antibodies in human plasma

[0573] Antibody <![CDATA[aPTT 1.5 (μg / mL)]]> <![CDATA[EC 50 (μg / mL)]]> <![CDATA[E max (%)]]> Hill Slope CSL-312 1.637 5.809 442.6 1.463 0001_Hu-3-Fc 1.462 1.807 703.7 11.65 0002_Hu-2-Fc 0.977 1.584 739.1 5.131 0015_Hu-1-Fc 1.018 1.125 752.6 25.09 2207 2.525 4.133 818.5 5.190

[0574] The above results show that the anti-FXII / FXIIa antibodies provided by the present disclosure can significantly prolong aPTT. The prolongation of aPTT reflects that the antibodies can reduce the amount of thrombus formation by inhibiting the intrinsic coagulation pathway. At the same time, all antibodies have no significant effect on PT, indicating that they do not affect the extrinsic coagulation pathway and do not cause bleeding risks.

[0575] like Figure 4A and 4B, with the increase of dosage, each antibody can quickly prolong aPTT and quickly reach an effect close to Emax; while the increase of CSL-312 is relatively slow, and with the increase of dosage, the effect of prolonging aPTT is gradually reflected. This result shows that the antibody provided by the present disclosure has a more sensitive effect on the prolongation of aPTT compared with CSL-312, which is specifically reflected in the value of Hill Slope in Table 13. The larger the Hill Slope, the faster the antibody can prolong aPTT. The Hill Slope values ​​of the antibodies in the present disclosure are all greater than those of CSL-312. In addition, the Emax values ​​of the antibodies provided by the present disclosure are all greater than those of CSL-312, indicating that the antibodies in the present disclosure have a stronger effect of prolonging aPTT, which can be reflected in better thrombus inhibition and elimination effects.

[0576] Example 10. In vivo pharmacodynamics and pharmacokinetic studies in New Zealand rabbits

[0577] The experiment examined the pharmacodynamics and pharmacokinetics of the test antibody in New Zealand rabbits.

[0578] First, the anticoagulant efficacy of the antibody to be tested in rabbits was detected. One healthy adult New Zealand rabbit was included in each group. Blood was collected according to the method of Example 9 before administration and the aPTT before administration was measured. Each rabbit was given a dose of 0.15-5 mg / kg, and blood was collected 15 minutes after administration and aPTT was measured. The percentage compared with before administration was calculated. The relative percentage of aPTT (vertical axis) was plotted against the dose (horizontal axis), and aPTT was calculated using a three-parameter nonlinear fitting (agonist vs response, three parameters). 1.5 The results are shown in Figure 5 Table 14, aPTT of 2207 in New Zealand rabbits to prolong aPTT 1.5 The efficacy of 2207 in New Zealand rabbits is dose-dependent, and the aPTT prolongation effect becomes more obvious with increasing doses.

[0579] Table 14. Dose-effect relationship of 2207 for prolonging aPTT in New Zealand rabbits

[0580] Dosage (mg / kg) aPTT (15 minutes after administration relative to the percentage before administration) 0.15 132.9 0.5 184.8 1.5 267.9 5.0 739.6 .

Claims

1. A FXII / FXIIa binding protein comprising an immunoglobulin single variable domain that specifically binds to FXII / FXIIa, wherein the immunoglobulin single variable domain comprises a CDR1, a CDR2 and a CDR3 selected from any one of 1) to 4): 1) CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6, 45 and 46, respectively; 2) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 17-19; 3) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4, 20-21; 4) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5, 22-23; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 are respectively as follows: As shown in SEQ ID NO: 6, 7, 8; SEQ ID NO: 6, 9, 10; or As shown in SEQ ID NO: 6, 11, 12.

2. The FXII / FXIIa binding protein according to claim 1, wherein The immunoglobulin single variable domain is humanized, affinity matured, T cell epitopes removed, antibody deamidation reduced and / or antibody isomerization reduced; Preferably, the framework region of the immunoglobulin single variable domain is derived from human heavy chain variable region germline genes IGHV3-23 and / or IGHV3-66; Preferably, the immunoglobulin single variable domain comprises at least one amino acid mutation as shown below: 27L, 27Y or 27N, 28P, 28I, 28S or 28A, 29Y, 29F, 29P, 29G or 29S, 30L, 30A or 30T, 44E or 44Q, 74A or 74G, 20V, 23V, 37F, 43E, 45R, 47G, 49A, 71Q, 78V, 79S, 81E, 83Q, 84P, and 94A; The above mutation sites are defined according to the Kabat numbering system; More preferably, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises amino acid mutations in any of the following groups: 1)27L, 28P, 29F, 30A, 37F, 44E, 45R, 47G, 74A, 79S, 94A; 2)27L, 28I, 29P, 30A, 37F, 44E, 45R, 47G, 74G, 94A; 3)27L, 28A, 29G, 30A, 37F, 44E, 45R, 47G, 74A, 94A.

3. The FXII / FXIIa binding protein according to claim 1 or 2, wherein The immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 17-23, or having at least 80% or at least 90% identity thereto; Preferably, the FXII / FXIIa binding protein is an anti-FXII / FXIIa antibody or an antigen-binding fragment thereof; preferably, the anti-FXII / FXIIa antibody or an antigen-binding fragment thereof is selected from a linear antibody, a single-chain antibody, a nanobody, a peptide antibody, a domain antibody, a multispecific antibody or an antigen-binding fragment of any of the foregoing.

4. A FXII / FXIIa binding protein comprising a first domain and a second domain; in, The first domain comprises at least one immunoglobulin single variable domain that specifically binds to FXII / FXIIa; The immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises CDR1, CDR2 and CDR3 as shown in any of the following: 1) CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 6, 45 and 46, respectively; 2) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 3, 17-19; 3) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 4, 20-21; 4) CDR1, CDR2 and CDR3 in the amino acid sequence shown in any one of SEQ ID NOs: 5, 22-23; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems; Preferably, the CDR1, CDR2 and CDR3 are respectively as follows: As shown in SEQ ID NO: 6, 7, 8; SEQ ID NO: 6, 9, 10; or As shown in SEQ ID NO: 6, 11, 12; More preferably, the immunoglobulin single variable domain that specifically binds to FXII / FXIIa comprises an amino acid sequence as shown in any one of SEQ ID NOs: 3-5, 17-23, or having at least 80% or at least 90% identity thereto.

5. The FXII / FXIIa binding protein according to claim 4, wherein The FXII / FXIIa binding protein comprises a human immunoglobulin Fc region; preferably, the human immunoglobulin Fc region is an Fc region derived from human IgG1, IgG2 or IgG4.

6. The FXII / FXIIa binding protein according to any one of claims 4 to 5, wherein The second domain comprises at least one of the following: HSA, an HSA binding domain, an immunoglobulin Fc region, an immunoglobulin single variable domain that specifically binds to FXII / FXIIa; Preferably, the HSA binding domain comprises at least one immunoglobulin single variable domain that specifically binds to HSA; the immunoglobulin single variable domain that specifically binds to HSA comprises CDR1, CDR2 and CDR3 as shown in any one of the following: 1) CDR1, CDR2 and CDR3 in the amino acid sequence of any one of SEQ ID NOs: 25, 32-41; 2) CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 26-28, respectively; The CDRs are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering systems, preferably according to the Kabat numbering system.

7. The FXII / FXIIa binding protein according to claim 6, wherein The immunoglobulin single variable domain that specifically binds to HSA comprises an amino acid sequence as shown in any one of SEQ ID NOs: 25, 32-41, or a sequence having at least 80% or at least 90% identity thereto.

8. The FXII / FXIIa binding protein according to any one of claims 4 to 7, wherein: The first domain is located at the N-terminus of the second domain; and / or, The first domain is located at the C-terminus of the second domain; Preferably, the FXII / FXIIa binding protein comprises at least two immunoglobulin single variable domains, wherein any two immunoglobulin single variable domains are directly connected or connected via a linker; more preferably, the linker is (G x S z ) y A linker, wherein each x is independently selected from an integer of 1-5, each y is independently selected from an integer of 1-6, and each z is independently selected from 0-4; more preferably, x is 3 or 4, z is selected from 0 or 1, and y is 1, 2 or 3; Preferably, the valency of the FXII / FXIIa binding protein is ≥2; more preferably, the valency ratio of the first domain to the second domain is 5:1 to 1:5, preferably 1:1, 1:2, 1:3, 3:1, 2:1, 3:2, 2:

3.

9. The FXII / FXIIa binding protein of any one of claims 4 to 8, comprising an amino acid sequence as shown in any one of SEQ ID NOs: 14 to 16, 24, 47 to 50, or having at least 80%, at least 90% identity thereto.

10. A polynucleotide encoding the FXII / FXIIa binding protein according to any one of claims 1 to 9. A vector comprising the polynucleotide according to claim 10.

12. A host cell comprising or expressing the polynucleotide according to claim 10, or the vector according to claim 11.

13. A pharmaceutical composition comprising the FXII / FXIIa binding protein of any one of claims 1 to 9, the polynucleotide of claim 10, or the vector of claim 11; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, diluent or auxiliary material.

14. A method for preparing a FXII / FXIIa binding protein, comprising: Expressing the FXII / FXIIa binding protein of any one of claims 1 to 9 in a host cell, and isolating the expressed FXII / FXIIa binding protein from the host cell; Optionally, the method further comprises a step of purifying the FXII / FXIIa binding protein.

15. Use of the FXII / FXIIa binding protein of any one of claims 1 to 9, the polynucleotide of claim 10, the vector of claim 11, or the pharmaceutical composition of claim 13 in at least one of the following (1) to (5): (1) For inhibiting FXIIa activity, or for preparing a drug for inhibiting FXIIa activity; (2) Used to inhibit the activation of FXI by FXIIa, or to prepare a drug used to inhibit the activation of FXI by FXIIa; (3) Used for inhibiting intrinsic coagulation, or for preparing a drug for inhibiting intrinsic coagulation; (4) Used to inhibit or prevent the formation and / or growth of thrombus, or to prepare a drug for inhibiting or preventing the formation and / or growth of thrombus; (5) For the prevention or treatment of thrombosis or diseases related to thrombosis.

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