Nanobody for enhancing activity of fix and fixa

Abstract

A binding molecule for enhancing the activity of FIX and FIXa. Specifically provided is a FIX and / or FIXa nanobody or a mutant library thereof. The binding molecule comprises an anti-FIX and / or FIXa single-domain antibody (VHH), and complementarity determining regions CDRs of the single-domain antibody comprise CDR1, CDR2 and CDR3. The nanobody can promote the enzymatic activity of FIX and / or FIXa.

Description

Nanobodies that enhance FIX and FIXa activity

[0001] This application claims priority to the Chinese Invention Patent Application No. 202411352574.5, filed on September 26, 2024, entitled "Nanobodies that enhance FIX and FIXa activity", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of biotechnology, in particular to a nanobody targeting FIX and its application. BACKGROUND

[0003] Coagulation factors are various protein components involved in the blood clotting process, including coagulation factors IX-XIII, etc. During the hemostasis process, plasma coagulation factors are activated, and through positive feedback or autocatalysis, the reaction of substrate and enzyme is amplified and enhanced, ultimately promoting the formation of fibrin clots. If the corresponding components are lacking, it will lead to coagulation dysfunction, and bleeding or thrombotic events will occur. Hemophilia is a bleeding disorder caused by a deficiency of coagulation factors, among which FVIII deficiency is hemophilia A and FIX deficiency is hemophilia B.

[0004] Activated coagulation factor FIX, i.e. FIXa, whose activity is a key rate-limiting step in the hemostasis process. In the intrinsic coagulation pathway, FⅨa and FⅧa assemble on the phospholipid surface in the presence of calcium ions to form an intrinsic FX activation complex (Tenase), which then binds and efficiently activates FX to produce a large amount of FXa, ultimately promoting the explosive production of thrombin and the formation of stable blood clots, achieving physiological hemostasis. FIXa itself has very low enzymatic activity on FX, while FⅧa as a cofactor of FⅨa can increase the reaction rate of FⅨa catalyzed FXa formation by about 100,000 times. Therefore, FIXa as a key rate-limiting step affects the body's coagulation function.

[0005] In the current treatment of hemophilia, direct infusion of blood-derived and recombinant FVIII or FIX is still the standard means of preventing and / or treating hemophilic bleeding in the World Federation of Hemophilia (WFH) guidelines for the management of hemophilia. However, due to the short half-life of FVIII and FIX products through intravenous injection, in order to approach the normal life level, the guidelines recommend that FVIII and FIX intravenous injection is needed every 2-3 days, and lifelong medication. High-frequency injection regimen can cause serious, lifelong intravenous infection risk and heavy diagnosis and treatment burden; at the same time, about 30% of patients with infusion of coagulation factors will develop antibodies against coagulation factors themselves, leading to drug failure. These reasons have prompted the continuous development of different coagulation factor replacement therapy methods in the field. For example, Emicizumab, a FVIII replacement bispecific antibody marketed by Roche and China Foreign Pharmaceutical in 2017, targets the combination of FIX and FX and improves the enzyme catalytic activity of FIX activation, and supports subcutaneous 1-week, 2-week, and 4-week administration, greatly reducing the administration frequency of patients, but the treatment cost of up to 1.2 million yuan per year in China and 480,000 US dollars in the United States still brings heavy economic burden to patients; in terms of gene therapy, hemophilia B gene therapy enhances the enzyme activity of mutant FIX sequence, and the effective period of the drug is up to 10 years or more, but the treatment cost of up to 3.2 million US dollars per injection makes it difficult for most patients to accept. It can be seen that in the body of patients with hemophilia A caused by FVIII deficiency or hemophilia B caused by FIX deficiency, improving the enzyme activity of FIX is the key to hemophilia treatment. Therefore, there is an urgent need in the art to explore new FIXa enzyme activity promoting molecules for improving FIX and downstream enzyme-linked reactions, thereby laying a foundation for the development of more efficient and more accessible hemophilia treatment drugs. SUMMARY

[0006] In view of the defects of the prior art, the present application provides a nanobody comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3, and having the function of recognizing and binding FIX and / or FIXa and promoting the enzyme activity thereof.

[0007] The first aspect of the present application provides a mutation library of a coding sequence of a FIX and / or FIXa single-domain antibody, the mutation library comprising a sequence encoding CDR1 as shown in SEQ ID NO: 1 (GRTFSSYD), CDR2 as shown in SEQ ID NO: 2 (ISSGGST), and CDR3 as shown in SEQ ID NO: 3 (NVRGTAY), and optionally a coding sequence of a CDR region of a single-domain antibody with FIX and / or FIXa binding activity modified by one or more of the following ways:

[0008] (a) CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR3 as shown in SEQ ID NO: 3; and / or

[0009] (b) CDR1 as shown in SEQ ID NO: 1, a CDR2 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; and / or

[0010] (c) a CDR1 variant obtained after 1 -4 amino acid mutations and / or deletions of CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3; preferably 1, 2, 3, 4 mutations; and / or

[0011] (d) a CDR1 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR1 as shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained after 1 or 2 amino acid mutations and / or deletions of CDR3 as shown in SEQ ID NO: 3; and / or

[0012] (e) CDR1 as shown in SEQ ID NO: 1, a CDR2 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained after 1 or 2 amino acid mutations and / or deletions of CDR3 as shown in SEQ ID NO: 3;

[0013] (f) a CDR1 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR1 as shown in SEQ ID NO: 1, a CDR2 variant obtained after 1 -4, preferably 1 or 2, amino acid mutations and / or deletions of CDR2 as shown in SEQ ID NO: 2, and CDR3 as shown in SEQ ID NO: 3.

[0014] In one or more embodiments, the mutations are preferably single amino acid random mutations.

[0015] In one or more embodiments, the mutations are selected from the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y.

[0016] In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 1, 2, or 3.

[0017] In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 1.

[0018] In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 2.

[0019] In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 3.

[0020] In one or more embodiments, CDR1 has the sequence set forth in SEQ ID NO: 1 or a variant thereof having 1, 2, 3, 4, or 5 amino acid mutations,

[0021] CDR2 has the sequence set forth in SEQ ID NO: 2 or a variant thereof having 1, 2, 3, or 4 amino acid mutations,

[0022] CDR3 has the sequence set forth in SEQ ID NO: 3 or a variant thereof having 1 or 2 amino acid mutations.

[0023] In one or more embodiments, the mutation is a substitution mutation or a deletion mutation, preferably a substitution mutation.

[0024] In one or more embodiments, the variant of CDR1 has 1, 2, 3, or 4 amino acid mutations or deletions in amino acids 1-8 of SEQ ID NO: 1.

[0025] In one or more embodiments, the variant mutation site of CDR1 is selected from 1 or 2, preferably 1, of the following:

[0026] (1a) G1 of SEQ ID NO: 1, the mutation is selected from amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y; preferably S or P;

[0027] (1b) R2 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of I, S, H, T, P, F, G, Q, Y, and L;

[0028] (1c) T3 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of V, I, A, L, W, F, D, and Y;

[0029] (1d) F4 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of S, G, Y, and V;

[0030] (1e) S5 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of R, D, K, F, G, P, N, L, and P;

[0031] (1f) S6 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of Y, D, A, V, G, I, P, K, F, L, and M;

[0032] (1g) Y7 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of K, Q, H, L, V, C, L, and A;

[0033] (1h) D8 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of V, A, E, and S.

[0034] In one or more embodiments, the variant of CDR2 has 1, 2, 3, or 4 mutations or deletions in 1, 2, 3, or 4 of amino acids 1-8 of SEQ ID NO: 2.

[0035] In one or more embodiments, the variant of CDR2 has 1, 2, 3, or 4 mutations or deletions in 1, 2, 3, or 4 of amino acids 1-8 of SEQ ID NO: 2.

[0036] (2a) I1 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably M, N, or T;

[0037] (2b) S2 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of N, D, R, A;

[0038] (2c) S3 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of R, Y, V, H, K, M and L is deleted;

[0039] (2d) G4 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of D, A, E, I, R, T, L, K and S is deleted;

[0040] (2e) G5 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of L, R, Q, S, A, K and T is deleted;

[0041] (2f) S6 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of Y, M, G, H, N, L, R, V, M, Q, I, W and T;

[0042] (2g) T7 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of S, K, H, Y and V.

[0043] In one or more embodiments, the variant of CDR3 is mutated or deleted in 1 or 2 of amino acids 1-7 of SEQ ID NO: 3. Preferably, the variant of CDR3 is mutated or deleted in 1 or 2 of amino acids 1-3 and 6-7 of SEQ ID NO: 3.

[0044] In one or more embodiments, the variant of CDR3 is mutated at 1 or 2, preferably 1, of the following positions:

[0045] (3a) N1 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably T;

[0046] (3b) V2 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion, or any 1 of A, L, I, Y, R, and T;

[0047] (3c) R3 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably C or E;

[0048] (3d) G4 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion;

[0049] (3e) T5 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably N;

[0050] (3f) A6 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of D, L, and S;

[0051] (3g) Y7 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of E, H, S, L, K, M, F, R, V, I, or N.

[0052] In one or more embodiments, the variant mutation site of the CDR3 is preferably Y7 of SEQ ID NO: 3, preferably any 1 of E, H, S, L, K, M, F, R, V, I, or N.

[0053] In one or more embodiments, in the mutant library, the complementarity determining region CDR of the single-domain antibody comprises a variant of CDR1, a variant of CDR2, and a variant of CDR3. In one or more embodiments, the variant of CDR1 has a sequence as set forth in any one of SEQ ID NOs: 4-56. In one or more embodiments, the variant of CDR2 has a sequence as set forth in any one of SEQ ID NOs: 57-109. In one or more embodiments, the variant of CDR3 has a sequence as set forth in any one of SEQ ID NOs: 110-133, 273.

[0054] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated; preferably, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in any one of SEQ ID NOs: 110-116 and 129-131, 273.

[0055] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated (preferably at the 7th amino acid); preferably, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in any one of SEQ ID NOs: 57-60, CDR3 is as set forth in any one of SEQ ID NOs: 117-119.

[0056] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3 and 1 amino acid thereof is mutated (preferably at the 7th amino acid); CDR1 is as set forth in SEQ ID NO: 4, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 117.

[0057] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1 and 1 or 2 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 4, 5, 13, 39-56, CDR2 is as set forth in any one of SEQ ID NO: 57, 58, 61, 67, 68, 76, 96-108, CDR3 is as set forth in SEQ ID NO: 3.

[0058] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 1, CDR2 is as set forth in any one of SEQ ID NO: 75-98, CDR3 is as set forth in SEQ ID NO: 3.

[0059] In one or more embodiments, in the mutant library, CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 4, 5, 13, 39-56, CDR2 is as set forth in any one of SEQ ID NO: 57, 58, 61, 67, 68, 76, 96-108, CDR3 is as set forth in SEQ ID NO: 3.

[0060] In another preferred embodiment, the single domain antibody in the mutant library can promote the activity of FIX and / or FIXa.

[0061] In another aspect of the present application, there is provided a single domain antibody produced or screened from the mutant library according to any one of the embodiments of the present application, the single domain antibody has the ability to bind to FIX and / or FIXa, and the single domain antibody promotes the enzymatic activity of FIX and / or FIXa.

[0062] In one or more embodiments, the enzymatic activity is the activation of FX to form FXa. In one or more embodiments, the enzymatic activity is the serine protease activity.

[0063] In one or more embodiments, the CDRs of the single domain antibody are as set forth in any one of the embodiments of the first aspect of the present application.

[0064] In one or more embodiments, the single domain antibody is a monoclonal antibody.

[0065] The present disclosure also provides a FIX and / or FIXa binding molecule comprising an anti-FIX and / or FIXa single domain antibody (VHH), wherein the CDRs of the single domain antibody comprise CDR1, CDR2 and CDR3. The binding molecule is capable of promoting the enzymatic activity of FIX and / or FIXa.

[0066] In one or more embodiments, the enzymatic activity is the serine protease activity.

[0067] In one or more embodiments, the FR of the antibody in the antibody library is selected from the group consisting of: human-derived FR, murine-derived FR, camelid or llama-derived FR.

[0068] In one or more embodiments, the CDR1, CDR2, CDR3 of the single domain antibody is as shown in any of the rows of Table 1. In one or more embodiments, the FR of the single domain antibody is replaced by the FR of one or more germlines of one or more species, the genes of which include but are not limited to human IGHV3-15*01, human IGHV3-23*01, human IGHV3-23*04, human IGHJ3*01, llama IGHV3-3*01, llama IGHV3S4*01, llama IGHV3S3*01, llama IGHJ6*01, camel IGHV3S42*01, camel IGHV3S27*01.

[0069] In one or more embodiments, the FR1, FR2, FR3 and FR4 of the single domain antibody is as shown in the FR1, FR2, FR3 and FR4 of the VHH as shown in any of SEQ ID NOs: 134-272 and 433-585.

[0070] In one or more embodiments, the single domain antibody has an amino acid sequence as shown in any of SEQ ID NOs: 134-272, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence as shown in any of SEQ ID NOs: 134-272.

[0071] In one or more embodiments, the FIX and / or FIXa binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody comprising one, two or more of the single domain antibodies.

[0072] In one or more embodiments, the multivalent single domain antibody or multispecific single domain antibody links multiple single domain antibodies via a linker. The linker consists of 1-15 amino acids selected from the group consisting of G and S.

[0073] In one or more embodiments, the FIX and / or FIXa binding molecule or single domain antibody is a chimeric antibody.

[0074] The present application also provides a library of biomolecules comprising:

[0075] (1) CDR1, CDR2 and CDR3 of a binding molecule as described in any embodiment herein,

[0076] (2) a single domain antibody or binding molecule as described in any embodiment herein, or

[0077] (3) a nucleic acid sequence encoding (1) or (2).

[0078] In one or more embodiments, the library comprises CDR1, CDR2 and CDR3 as shown in any one or more or all of the rows of Table 1.

[0079] In one or more embodiments, the library comprises VHH as shown in any one or more or all of the rows of Table 1.

[0080] In one or more embodiments, the library is a nucleic acid library comprising nucleic acid sequences encoding CDR1, CDR2 and CDR3 as shown in any one or more or all of the rows of Table 1, or comprising nucleic acid sequences encoding VHH as shown in any one or more or all of the rows of Table 1.

[0081] In one or more embodiments, the nucleic acid library has a capacity of at least 1*10 7 , 1*10 8 , 1*10 9 , 1*10 10 , 1*10 11 , 1*10 12 or 1*10 13 .

[0082] In one or more embodiments, the nucleic acid library is a phage display library or a yeast display library.

[0083] The present application also provides a nucleic acid molecule encoding:

[0084] (1) a FIX and / or FIXa binding molecule as described in any embodiment herein, and / or

[0085] (2) a complement of (1).

[0086] The present application also provides a nucleic acid construct comprising a nucleic acid molecule as described herein.

[0087] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

[0088] The present application also provides a host cell selected from the group consisting of:

[0089] (1) expressing and / or secreting a FIX and / or FIXa binding molecule according to any of the embodiments herein;

[0090] (2) comprising a nucleic acid molecule according to herein; and / or

[0091] (3) comprising a nucleic acid construct according to herein.

[0092] The present application also provides a method of making a FIX and / or FIXa binding molecule according to any of the embodiments herein, comprising: culturing a host cell according to herein under conditions suitable for production of the FIX and / or FIXa binding molecule, and optionally purifying the FIX and / or FIXa binding molecule from the culture.

[0093] The present application also provides a pharmaceutical composition comprising a FIX and / or FIXa binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell according to any of the embodiments herein, and a pharmaceutically acceptable excipient.

[0094] In one or more embodiments, the pharmaceutical composition is for use in promoting coagulation.

[0095] The present application also provides the use of a FIX and / or FIXa binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell according to any of the embodiments herein for the manufacture of a medicament or a kit for the prevention or treatment of a bleeding disorder.

[0096] In one or more embodiments, the animal is a mammal, preferably the animal is a mouse or a human.

[0097] In one or more embodiments, the bleeding disorder is a FVIII and / or FIX deficiency bleeding disorder.

[0098] In one or more embodiments, the bleeding disorder is selected from the group consisting of hemophilia A, hemophilia B, e.g. hemophilia A with mild, moderate, or severe FVIII deficiency, hemophilia B with mild, moderate, or severe FIX deficiency.

[0099] The present application also provides a method of increasing FIX and downstream enzymatic reactions, preventing or treating a bleeding disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of a FIX and / or FIXa binding molecule, a nucleic acid molecule, a nucleic acid construct, a host cell or a pharmaceutical composition according to any of the embodiments herein.

[0100] In one or more embodiments, the bleeding disorder is a FVIII and / or FIX deficiency bleeding disorder.

[0101] In one or more embodiments, the bleeding disorder is selected from hemophilia A, hemophilia B, e.g. hemophilia A with mild, moderate, or severe FVIII deficiency, hemophilia B with mild, moderate, or severe FIX deficiency.

[0102] The present application also provides a kit for detecting FIX, comprising the FIX and / or FIXa binding molecule, the nucleic acid molecule, the nucleic acid construct or the host cell according to any one of the embodiments of the present application.

[0103] In one or more embodiments, the kit further comprises a reagent for detecting the binding of FIX to the FIX and / or FIXa binding molecule. For example, the reagent for detecting the binding is an enzyme-linked immunoassay.

[0104] In one or more embodiments, the reagent for detecting the binding is a detectable label that can be attached to the FIX and / or FIXa binding molecule, e.g. biotin. The detectable label can be attached to the FIX and / or FIXa binding molecule or present separately in the kit.

[0105] The present application also provides a non-diagnostic method for detecting the presence of FIX in a sample, the method comprising: incubating the sample with the FIX and / or FIXa binding molecule according to any one of the embodiments herein, and detecting the binding of FIX to the FIX and / or FIXa binding molecule, thereby determining the presence of FIX in the sample. The detection is by enzyme-linked immunoassay.

[0106] The present application also provides the use of the FIX and / or FIXa binding molecule according to any one of the embodiments herein in the manufacture of a kit for detecting FIX in a sample.

[0107] Advantages of the present application: The present application relates to a class of nanobodies against FIXa, which have strong binding affinity to FIX / IXa, can promote the activation of FIX, and enhance the enzymatic activity of FIXa on FX. The activated and enzymatically active FIXa will be suitable for intervention in different bleeding disorders, such as hemophilia A, hemophilia B. DETAILED DESCRIPTION

[0108] The present inventors have found a class of nanobodies against FIX / FIXa, which have strong binding affinity to FIX / IXa, can promote the activation of FIX, and enhance the enzymatic activity of FIXa on FX, producing more FX, thereby promoting the generation of the coagulation cascade, and shortening the APTT time of plasma. Wherein FIX is coagulation factor IX, and FIXa is activated FIX.

[0109] As used herein, a "FIX and / or FIXa binding molecule" is a protein having the function of recognizing and binding to FIX, including but not limited to, antibodies, antigen-binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target-binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, and chemokines.

[0110] The FIX and / or FIXa binding molecules described herein can be monovalent or multivalent nanobodies or single domain antibodies, or multispecific nanobodies or single domain antibodies, comprising one, two or more nanobodies or single domain antibodies described herein. The multispecificity can be against FIX and or FIXa and another antigen, or against two different epitopes of FIX.

[0111] As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, and single-chain molecules, as well as antibody fragments, particularly antigen-binding fragments, such as Fab, F(ab')2, and Fv. In some embodiments herein, the terms "immunoglobulin" (Ig) and "antibody" are used interchangeably. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains.

[0112] As used herein, a "heavy chain antibody" is an antibody derived from a Camelid or a cartilaginous fish. In contrast to the above 4-chain antibodies, heavy chain antibodies lack a light chain and a heavy chain constant region 1 (CH1), and comprise only two heavy chains, each consisting of a variable region (VHH) and other constant regions, the variable region being connected to the constant region by a hinge-like region structure. Each heavy chain of a Camelid heavy chain antibody comprises one variable region (VHH) and two constant regions (CH2 and CH3), and each heavy chain of a cartilaginous fish heavy chain antibody contains one variable region and five constant regions (CH1-CH5). An antigen-binding fragment of a heavy chain antibody includes VHH.

[0113] A heavy chain antibody further comprises a heavy chain constant region. In one or more embodiments, the heavy chain constant region is the constant region of a Camelid heavy chain antibody, comprising CH2 and CH3. By fusion with the constant region of a human IgG Fc, a heavy chain antibody can have CH2 and CH3 of a human IgG Fc, such as CH2 and CH3 of IgG4.

[0114] As used herein, the terms "single domain antibody", "VHH", "nanobody" are used interchangeably and all refer to a nanobody that specifically recognizes and binds to FIX and or FIXa. A nanobody is the variable region of a heavy chain antibody. Typically, a nanobody contains three CDRs and four FRs. A nanobody is the smallest functional antigen binding fragment. Typically, a nanobody is constructed by cloning the variable region of the heavy chain of an antibody that was previously obtained naturally lacking the light chain and the heavy chain constant region 1 (CH1) of the antibody.

[0115] Binding molecules comprising two or more nanobodies are multivalent nanobodies; binding molecules comprising two or more nanobodies of different specificity are multispecific nanobodies. Multivalent nanobodies or multispecific nanobodies comprise multiple nanobodies linked by a linker. The linker typically consists of 1-15 amino acids selected from G and S, for example (G 4S)3. The single domain antibody of the present application can form a heavy chain antibody with a constant region, which can be a human constant region.

[0116] Herein, heavy chain antibody and antibody are intended to distinguish between different combinations of antibodies. Due to the similarity in structure of both, the following description of the structure of an antibody applies to a heavy chain antibody as well, except where indicated otherwise.

[0117] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of an antibody. The variable domains of the heavy chain and light chain can be referred to as "VH" and "VL", respectively. The variable domain of a heavy chain antibody is referred to as VHH. These domains are generally the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen binding sites.

[0118] The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies, and are used to confer antigen binding specificity and particular antigenic specificity of a particular antibody. However, the variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in three segments called hypervariable regions (HVRs) both in the light chain and the heavy chain variable domains, namely HCDR1, HCDR2, HCDR3 in the variable domain of the heavy chain and LCDR1, LCDR2, and LCDR3 in the variable domain of the light chain. The hypervariable regions of a heavy chain antibody are CDR1, CDR2, CDR3. The more highly conserved parts of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains and heavy chain antibodies each comprise four FR regions, FR1, FR2, FR3, and FR4, mostly adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. In general, the structure of a light chain variable domain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, the structure of a heavy chain variable domain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4, and the structure of a heavy chain antibody variable domain is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0119] The CDRs are the only structural elements of the antigen binding site that determine the specificity and affinity of an antibody. The FRs, as framework regions, mainly play a supporting role in the structure of the CDRs, therefore, the CDRs can be grafted between different sequences of FRs of different species, and still retain their antigen binding ability. This is also widely recognized in the field, and the humanization of antibodies is based on this principle. In the present application, the FR1 of the single domain antibody has the FR1 of the VHH shown in any one of SEQ ID NOs: 134-272. The FR2 of the single domain antibody has the FR2 of the VHH shown in any one of SEQ ID NOs: 134-272. The FR3 of the single domain antibody has the FR3 of the VHH shown in any one of SEQ ID NOs: 134-272. The FR4 of the single domain antibody has the FR4 of the VHH shown in any one of SEQ ID NOs: 134-272.

[0120] An "Fc region" (fragment, crystallizable region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors including binding to Fc receptors (FcRs) located on various cells of the immune system (e.g., effector cells) or to the first component (Clq) of the classical complement system. In IgG antibody isotypes, the Fc region is composed of two identical copies of a stretch of protein from the CH2 and CH3 domains of both heavy chains of an antibody. While the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc region is usually defined as spanning from an amino acid residue located about 6 or 10 residues C-terminal to the heavy chain variable domain C-terminus (C226 or P230, respectively) to the carboxyl terminus. As used herein, the Fc region can be a native sequence Fc or a variant Fc. The Fc domain does not participate directly in binding of an antibody to an antigen but exhibits various effector functions, such as participation of the antibody in antibody-dependent cell-mediated cytotoxicity, or binding to the cell membrane surface FcRn to participate in the circulation of the antibody and prolong the half-life of the antibody.

[0121] An "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute most of the antigen binding specificity and diversity of an antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. "Single-chain Fv" also abbreviated as "sFv" or "scFv" are antibody fragments that comprise the VHand VLdomains of antibody, linked by a synthetic linker from one to the other as a contiguous polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VHand VLdomains, which enables the sFv to form the desired structure for antigen binding. The scFv of a heavy chain antibody is VHH.

[0122] An antibody herein also includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with, or homologous to, corresponding sequences in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with, or homologous to, corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

[0123] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by human-derived polynucleotides or is identical to that of the antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems.

[0124] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

[0125] In this document, "library," "biomolecular library," or "mutant library" refers to a group of recombinant peptides (e.g., antibodies or their antigen-binding fragments, such as VHHs) or their encoding nucleic acids obtained through innate immunity, artificial synthesis, or mutation (e.g., phage display) technology. Typically, the library selects known framework sequences from known species and obtains highly diverse CDR regions through randomization design of CDR regions. VHHs usually have the same or similar CDRs or CDR groups. Preferably, the VHHs obtainable in the library are recombinant peptides obtained from at least the same base sequence source and after specific randomization design targeting CDR1-CDR3; in a preferred embodiment, the VHHs obtainable from the library have one identical CDR, with the remaining CDRs being similar. "Similar" CDRs or CDR groups refer to sequences where the corresponding CDRs (e.g., CDR1, CDR2, or CDR3) or CDR groups of two VHHs differ by no more than 5 amino acids, for example, 1, 2, 3, or 4 amino acids. In a preferred embodiment, the VHHs obtained from the library have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity. In one or more embodiments, the library described in this application is a nucleic acid library containing nucleic acid sequences encoding any or more rows or all of CDR1, CDR2, and CDR3 shown in Table 1, or containing nucleic acid sequences encoding any or more rows or all of VHHs shown in Table 1. In one or more embodiments, the nucleic acid library has a capacity of at least 1*10^6. 7 1*10 8 1*10 9 1*10 10 1*1011 1*10 12 1*10 13 Exemplarily, the nucleic acid library is a phage display library or a yeast display library.

[0126] Enzymatic activity

[0127] FIX is a serine protease consisting of 416 amino acid residues, activated by FXI of the endogenous coagulation pathway, releasing a peptide fragment upon activation, forming two peptide chains linked by a disulfide bond, activated FIX, i.e. FIXa. Activated FIXa activates FX to FXa in the presence of Ca 2+ In the presence of platelet phospholipids, it forms a complex with FVIII, which activates FX to FXa. Under normal physiological conditions, FIXa plays an enzymatic catalytic role in this reaction, while FVIII only plays a regulatory role, since FIX itself can bind to FX, thereby increasing the local substrate concentration and activating FX, but the reaction rate can increase by several thousand times in the presence of factor FVIII.

[0128] The FIX / FIXa enzymatic activity according to the present application refers to the activation of FX to FXa by FIX / FIXa in the presence of a nanobody obtained from a binding molecule or a library according to the present application. Chromogenic substrate is an artificially synthesized compound containing a cleavage point for the active enzyme FIXa, which corresponds to the FX substrate fragment in the FX-FIXa enzyme reaction. In the detection process, this compound is catalytically cleaved by FIXa to dissociate the color-producing substance (p-nitroaniline, PNA), resulting in a color change, from which the FIXa-FX enzymatic activity in the sample can be inferred.

[0129] The present application provides a FIX and / or FIXa binding molecule, said binding molecule comprising an anti-FIX and / or FIXa single-domain antibody (VHH), said single-domain antibody comprising complementarity determining regions CDRs comprising CDR1, CDR2 and CDR3. Said binding molecule is capable of promoting the enzymatic activity of FIX and / or FIXa. Said enzymatic activity is the activation of FX to FXa. In one or more embodiments, said enzymatic activity is a serine protease activity.

[0130] The FIX and / or FIXa binding molecules described herein comprise a CDR1 as set forth in SEQ ID NO: 1 (GRTFSSYD), a CDR2 as set forth in SEQ ID NO: 2 (ISSGGST), and a CDR3 as set forth in SEQ ID NO: 3 (NVRGTAY), and optionally a single-domain antibody CDR region with FIX and / or FIXa binding activity engineered in one or more of the following ways: (a) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR3 as set forth in SEQ ID NO: 3; and / or (b) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; and / or (c) a CDR1 variant obtained by 1-4 amino acid mutations and / or deletions of the CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; preferably 1, 2, 3, 4 mutations; and / or (d) a CDR1 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained by 1 or 2 amino acid mutations and / or deletions of the CDR3 as set forth in SEQ ID NO: 3; and / or (e) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained by 1 or 2 amino acid mutations and / or deletions of the CDR3 as set forth in SEQ ID NO: 3; (f) a CDR1 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3. The mutations are preferably single amino acid random mutations. In one or more embodiments, the mutations are selected from the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y.

[0131] In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 1, 2, or 3. In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 1. In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 2. In one or more embodiments, the mutant is a single point random mutation or deletion of any one amino acid in the sequence set forth in SEQ ID NO: 3.

[0132] In one or more embodiments, CDR1 has the sequence set forth in SEQ ID NO: 1 or a variant thereof having 1, 2, 3, 4, or 5 amino acid mutations, CDR2 has the sequence set forth in SEQ ID NO: 2 or a variant thereof having 1, 2, 3, or 4 amino acid mutations, and CDR3 has the sequence set forth in SEQ ID NO: 3 or a variant thereof having 1 or 2 amino acid mutations.

[0133] In one or more embodiments, the mutation is a substitution mutation or a deletion mutation, preferably a substitution mutation.

[0134] In one or more embodiments, the variant of CDR1 has 1, 2, 3, or 4 amino acid mutations or deletions in amino acids 1-8 of SEQ ID NO: 1.

[0135] In one or more embodiments, the variant mutation site of CDR1 is selected from 1 or 2, preferably 1, of the following:

[0136] (1a) G1 of SEQ ID NO: 1, the mutation is selected from amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y; preferably S or P;

[0137] (1b) R2 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of I, S, H, T, P, F, G, Q, Y, and L;

[0138] (1c) T3 of SEQ ID NO: 1, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of V, I, A, L, W, F, D, and Y;

[0139] (1d) F4 of SEQ ID NO: 1, with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of S, G, Y, and V;

[0140] (1e) S5 of SEQ ID NO: 1, with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of R, D, K, F, G, P, N, L, and P;

[0141] (1f) S6 of SEQ ID NO: 1, with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of Y, D, A, V, G, I, P, K, F, L, and M;

[0142] (1g) Y7 of SEQ ID NO: 1, with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of K, Q, H, L, V, C, L, and A;

[0143] (1h) D8 of SEQ ID NO: 1, with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of V, A, E, and S.

[0144] In one or more embodiments, the variant of CDR2 has 1, 2, 3, or 4 amino acid mutations or deletions in 1, 2, 3, or 4 of the amino acids in positions 1-8 of SEQ ID NO: 2.

[0145] In one or more embodiments, the variant of CDR2 has 1, 2, 3, or 4 amino acid mutations or deletions in 1, 2, 3, or 4 of the amino acids in positions 1-8 of SEQ ID NO: 2.

[0146] (2a) I1 of SEQ ID NO: 2; with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably M, N, or T;

[0147] (2b) S2 of SEQ ID NO: 2; with a mutation selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of N, D, R, A;

[0148] (2c) S3 of SEQ ID NO: 2; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or selected from any 1 of R, Y, V, H, K, M and L;

[0149] (2d) G4 of SEQ ID NO: 2; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or selected from any 1 of D, A, E, I, R, T, L, K and S;

[0150] (2e) G5 of SEQ ID NO: 2; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or selected from any 1 of L, R, Q, S, A, K and T;

[0151] (2f) S6 of SEQ ID NO: 2; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of Y, M, G, H, N, L, R, V, M, Q, I, W and T;

[0152] (2g) T7 of SEQ ID NO: 2; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of S, K, H, Y and V.

[0153] In one or more embodiments, the variant of CDR3 is mutated or deleted in 1 or 2 of the amino acids in positions 1-7 of SEQ ID NO: 3.

[0154] In one or more embodiments, the variant of CDR3 is mutated at a site selected from 1 or 2, preferably 1, of:

[0155] (3a) N1 of SEQ ID NO: 3; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably T;

[0156] (3b) V2 of SEQ ID NO: 3; the mutation is selected from any 1 of the amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or selected from one of A, L, I, Y, R and T;

[0157] (3c) R3 of SEQ ID NO: 3, with a mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably C or E;

[0158] (3d) G4 of SEQ ID NO: 3, with a mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion;

[0159] (3e) T5 of SEQ ID NO: 3, with a mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably N;

[0160] (3f) A6 of SEQ ID NO: 3, with a mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of D, L, and S;

[0161] (3g) Y7 of SEQ ID NO: 3, with a mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of E, H, S, L, K, M, F, R, V, I, or N.

[0162] In one or more embodiments, the variant mutation site of the CDR3 is Y7 of SEQ ID NO: 3, preferably any 1 of E, H, S, L, K, M, F, R, V, I, or N.

[0163] In one or more embodiments, the variant of CDR1 has a sequence as set forth in any one of SEQ ID NOs: 4-56. The variant of CDR2 has a sequence as set forth in any one of SEQ ID NOs: 57-109. The variant of CDR3 has a sequence as set forth in any one of SEQ ID NOs: 110-133, 273.

[0164] In one or more embodiments, in the binding molecule, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3 with 1 or 2 amino acid mutations; preferably, CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in any one of SEQ ID NOs: 110-116 and 129-131, 273.

[0165] In one or more embodiments, the binding molecule has CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 2 and 1 or 2 amino acid mutations thereof, CDR3 as set forth in SEQ ID NO: 3 and 1 or 2 amino acid mutations thereof (preferably at the 7th amino acid); preferably, CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in any one of SEQ ID NOs: 57-60, CDR3 as set forth in any one of SEQ ID NOs: 117-119.

[0166] In one or more embodiments, the binding molecule has CDR1 as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acid mutations thereof, CDR2 as set forth in SEQ ID NO: 2, CDR3 as set forth in SEQ ID NO: 3 and 1 amino acid mutation thereof (preferably at the 7th amino acid); CDR1 as set forth in SEQ ID NO: 4, CDR2 as set forth in SEQ ID NO: 2, CDR3 as set forth in SEQ ID NO: 117.

[0167] In one or more embodiments, the binding molecule has CDR1 as set forth in SEQ ID NO: 1 and 1 or 2 amino acid mutations thereof, CDR2 as set forth in SEQ ID NO: 2, CDR3 as set forth in SEQ ID NO: 3; CDR1 as set forth in any one of SEQ ID NOs: 18-38, CDR2 as set forth in SEQ ID NO: 2, CDR3 as set forth in SEQ ID NO: 3.

[0168] In one or more embodiments, the binding molecule has CDR1 as set forth in SEQ ID NO: 1, CDR2 as set forth in SEQ ID NO: 2 and 1 or 2 amino acid mutations thereof, CDR3 as set forth in SEQ ID NO: 3; CDR1 as set forth in any one of SEQ ID NO: 1, CDR2 as set forth in any one of SEQ ID NOs: 75-98, CDR3 as set forth in SEQ ID NO: 3.

[0169] In one or more embodiments, in the binding molecule, CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3, or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, and CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NOs: 4, 5, 13, 39-56, CDR2 is as set forth in any one of SEQ ID NOs: 57, 58, 61, 67, 68, 76, 96-108, and CDR3 is as set forth in SEQ ID NO: 3.

[0170] The present application also includes derivatives and analogs of the various antibodies (e.g., nanobodies, heavy chain antibodies or antigen binding fragments thereof, multivalent nanobodies, multispecific nanobodies, antibodies or antigen binding fragments thereof) described herein. By "derivative" and "analog" is meant a polypeptide that substantially retains the same biological function or activity of the various antibodies of the present application. Derivatives or analogs of the present application can be (i) a polypeptide having a substitution group at one or more amino acid residues, or (ii) a polypeptide formed by fusing the mature polypeptide to another compound, such as a compound that increases the half-life of the polypeptide, e.g., a polyethylene glycol, or (iii) a polypeptide formed by adding an additional amino acid sequence to the polypeptide sequence, such as a leader or secretion sequence, or a sequence or protein that is used to purify the polypeptide, or a proprotein sequence, or a fusion protein with a 6His tag. These derivatives and analogs are within the scope of one skilled in the art in light of the teachings herein.

[0171] Without materially affecting the activity of the antibody, one skilled in the art can make one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acid changes to the sequences of the present application to obtain variants of the sequences of the antibody or functional fragments thereof. These variants include, but are not limited to, deletion, insertion, and / or substitution of one or more (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acids, and addition of one or a few (typically within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and / or N-terminus. In the art, conservative substitutions with similar or identical properties are often made without altering the function of the protein. For example, substitutions are made with amino acids having similar properties in the FR and / or CDR regions of the variable region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues can or can not be encoded by the genetic code. For another example, addition of one or a few amino acids at the C-terminus and / or N-terminus also typically does not alter the function of the protein. All of these are considered to be within the scope of the present application.

[0172] Variants of the antibodies described herein include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that hybridizes to the DNA encoding the antibodies of the application under conditions of high or low stringency, and polypeptides or proteins obtained using antisera against the antibodies of the application.

[0173] In some embodiments, the sequences of the variants described herein can have at least 95%, 96%, 97%, 98%, or 99% identity to the sequences from which they are derived. Sequence identity as described herein can be measured using sequence analysis software, for example, the computer program BLAST, particularly BLASTP or TBLASTN, using default parameters. The application also includes molecules having an antibody heavy chain variable region with CDRs, provided that the CDRs have greater than 90% (more preferably greater than 95%, most preferably greater than 98%) homology to the CDRs identified herein.

[0174] The antibodies of the application can be prepared using methods conventional in the art, such as hybridoma technology, which is well known in the art. The nanobodies of the application can be prepared using methods conventional in the art, such as phage display technology, which is well known in the art. Alternatively, the antibodies of the application can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the application. Transformation can be performed using any known method, for example, including packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used will depend on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide in liposomes, and direct microinjection of the DNA into nuclei, among others. Mammalian cell lines that can be used as hosts for expression are well known in the art, including, but not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with the essential ActRIIB binding properties.

[0175] The application also provides polynucleotides encoding the antibodies or fragments thereof described above. The polynucleotides of the application can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be the coding strand or the non-coding strand. The application also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences that encode the same amino acid sequences but have different nucleotide sequences.

[0176] As will be appreciated by those of ordinary skill in the art, a very large number of nucleic acids can be made that all encode the antibodies or antigen-binding fragments thereof of the present application due to the degeneracy of the genetic code. Thus, given a particular amino acid sequence, one of ordinary skill in the art can make any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the encoded protein. Accordingly, the present application also relates to polynucleotides that hybridize to the above-described polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The present application specifically relates to polynucleotides that hybridize to the polynucleotides described herein under stringent conditions. In the present application, "stringent conditions" means: (1) hybridization and washing under lower ionic strength and higher temperature, such as 0.2 x SSC, 0.1% SDS, 60°C; or (2) hybridization with the addition of a denaturant, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably 95% or more. Moreover, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0177] The nucleotide full-length sequences of the various antibodies of the present application or fragments thereof can generally be obtained by PCR amplification, recombination or artificial synthesis. One feasible method is to synthesize the relevant sequences by artificial synthesis, especially when the length of the fragments is relatively short. Generally, longer fragments can be obtained by first synthesizing a plurality of small fragments and then ligating them together. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0178] Once the relevant sequences are obtained, recombination can be used to obtain the relevant sequences in large quantities. This is generally achieved by cloning them into vectors, transferring them into cells, and then isolating the relevant sequences from the proliferated host cells by conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in the present application include biomolecules that exist in isolated form. At present, it is possible to obtain the DNA sequences encoding the proteins (or fragments thereof, or derivatives thereof) of the present application entirely by chemical synthesis. The DNA sequences can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. In addition, mutations can be introduced into the protein sequences of the present application by chemical synthesis.

[0179] Accordingly, the present application also relates to nucleic acid constructs comprising the appropriate DNA sequences as described above and an appropriate promoter or control sequence, such as expression and recombination vectors. These vectors can be used to transform appropriate host cells to enable them to express proteins. The vectors typically contain sequences for plasmid propagation and for cloning and expression of foreign nucleotide sequences. The sequences, which in certain embodiments are collectively referred to as "flanking sequences," typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of nucleic acids encoding antibodies to be expressed, and optional marker elements.

[0180] The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples of which are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells like yeast; insect cells like Drosophila S2 and Sf9; animal cells like CHO, COS7, 293 cells, etc.

[0181] In certain embodiments, the host cell can be various functional cells known in the art, such as various killer cells, including but not limited to cytokine-induced killer cells (CIK), dendritic cell-stimulated cytokine-induced killer cells (DC-CIK), cytotoxic T lymphocytes (CTL), gamma delta T cells, natural killer cells (NK), tumor infiltrating lymphocytes (TIL), lymphokine-activated killer cells (LAK), CD3AK cells (killer cells of anti-CD3 mAb), and CAR-T / TCR-T cells. In certain embodiments, the killer cell is a T cell or a NK cell. Exemplary NK cells include but are not limited to primary NK cells, NK cell lines (such as NK92), and NKT cells. In certain embodiments, the NK cell is a primary NK cell. Exemplary T cells include but are not limited to peripheral blood T lymphocytes, cytotoxic killer T cells (CTL), helper T cells, suppressor / regulatory T cells, gamma delta T cells, and T cells of mixed cell populations such as cytokine-induced killer cells (CIK), tumor infiltrating lymphocytes (TIL), etc. In certain embodiments, the T cell is a peripheral blood T lymphocyte and a T cell derived from TIL.

[0182] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known in the art. When the host is a prokaryote, such as E. coli, the transformation of the host cell can be effected by the use of techniques such as calcium chloride precipitation. If necessary, the transformation can be performed by electroporation. When the host is a eukaryote, transformation techniques such as calcium phosphate co-precipitation, conventional mechanical procedures such as microinjection, electroporation, and liposome packaging can be used.

[0183] The resulting transformant can be cultured in conventional media using standard techniques, the expression of the polypeptide encoded by the genes of the application being induced as appropriate. The culture conditions, such as temperature, pH and the like, can be selected by the skilled artisan to facilitate the growth of the host cells. When the host cells have reached an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period.

[0184] The polypeptides in the above methods can be expressed intracellularly, on the cell membrane, or secreted from the cell. If desired, the recombinant proteins can be isolated and purified using various separation and purification methods well known in the art based on their physical, chemical and other properties. Examples of such methods include, but are not limited to, conventional renaturation procedures, treatment with protein precipitants (salting out procedures), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

[0185] All aspects of the various antibodies described herein can be used to make a medicament to promote FIX activation, enhance the enzymatic activity of FIXa on FX, treat a bleeding disorder.

[0186] The pharmaceutical compositions herein contain the binding molecules described herein, as well as a pharmaceutically acceptable excipient, including but not limited to diluents, carriers, solubilizers, emulsifiers, preservatives, and / or adjuvants. The excipient is preferably nontoxic to recipients at the dosages and concentrations employed. Such excipients include, but are not limited to, saline, buffers, dextrose, water, glycerol, ethanol, and combinations thereof. In certain embodiments, the pharmaceutical composition can contain substances that improve, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorbability, or permeability of the composition. Such substances are known in the art. The optimal pharmaceutical composition will be determined by one of skill in the art, taking into account the intended route of administration, mode of delivery, and desired dosage.

[0187] Pharmaceutical compositions for in vivo administration are typically provided in the form of a sterile preparation. Sterilization is accomplished by filtration through a sterile filtration membrane. This method can be used in sterilizing the composition when it is lyophilized, either prior to or subsequent to lyophilization and rehydration. The pharmaceutical compositions of the present application can be selected for parenteral delivery. Compositions for parenteral administration can be stored in a lyophilized form or in solution. They are prepared for administration by conventional means, such as with physiological saline or aqueous solutions containing glucose and other adjuncts. Parenteral compositions are typically placed in a container having a sterile access port, such as a vial stopper pierceable by a hypodermic injection needle. Alternatively, compositions can be selected for delivery by inhalation or through the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising the antibody in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome vehicles, bioerodible micro-particles or porous beads, and depot injections, are also known to those skilled in the art.

[0188] Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulations can be stored either in ready-to-use form or in a form (e.g., lyophilized) that is reconstituted prior to administration. The present application also provides kits for producing single dose administration units. The kits of the present application can each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the present application, kits containing single and multiple chamber pre-filled syringes (e.g., liquid syringes and lyophilized syringes) are provided.

[0189] The present application also provides methods for promoting FIX activation by administering a binding molecule according to any of the embodiments of the present application or a pharmaceutical composition thereof. Herein, the terms "subject," "individual," "subject," are used interchangeably herein and include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human.

[0190] The therapeutically effective amount of a pharmaceutical composition containing a binding molecule of the present application to be employed will depend, for example, on the therapeutic effect desired and the target. Those skilled in the art will appreciate that appropriate dosage levels for treatment will vary depending on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and / or condition (age and general health) of the patient. In certain embodiments, the clinician can titer the dosage and modify the route of administration as required by a particular patient to obtain the optimum therapeutic response. For example, about 10 micrograms per kilogram body weight to about 50 milligrams per kilogram body weight per day.

[0191] The frequency of dosage will depend on the pharmacokinetic parameters of the binding molecule in the formulation being used. Clinicians will typically administer the composition until a dosage is reached that achieves the desired effect. The composition can therefore be administered as a single dose, or over time as two or more doses (which can or can not contain the same amounts of the desired molecule), or by implantation devices or catheters in a continuous infusion.

[0192] The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, by intravenous, intraperitoneal, intracerebral (intra-parenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional routes of injection; by sustained release systems or by implantation devices.

[0193] The present application also provides a test kit comprising a nanobody recognizing a FIX protein, a FIX and / or FIXa binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell, a lysis medium for lysing a sample, general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc.

[0194] Particular embodiments

[0195] Item 1. A mutagenized library of coding sequences of a FIX and / or FIXa single-domain antibody, said mutagenized library comprising a coding sequence of a CDR1 as shown in SEQ ID NO: 1, a coding sequence of a CDR2 as shown in SEQ ID NO: 2, and a coding sequence of a CDR3 as shown in SEQ ID NO: 3, and optionally a coding sequence of a CDR region of a single-domain antibody having FIX and / or FIXa binding activity designed after one or more of the following:

[0196] (a) a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained after 1-4, preferably 1 or 2, amino acid mutations and / or deletions of a CDR3 as shown in SEQ ID NO: 3; and / or

[0197] (b) a CDR1 as shown in SEQ ID NO: 1, a CDR2 variant obtained after 1-4, preferably 1 or 2, amino acid mutations and / or deletions of a CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 3; and / or

[0198] (c) a CDR1 variant obtained after 1-4, preferably 1, 2, 3 or 4, amino acid mutations and / or deletions of a CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 3; and / or

[0199] (d) a CDR1 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR1 as shown in SEQ ID NO: 1, a CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained by 1 or 2 amino acid mutations and / or deletions of the CDR3 as shown in SEQ ID NO: 3; and / or

[0200] (e) a CDR1 as shown in SEQ ID NO: 1, a CDR2 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR2 as shown in SEQ ID NO: 2, and a CDR3 variant obtained by 1 or 2 amino acid mutations and / or deletions of the CDR3 as shown in SEQ ID NO: 3; and / or

[0201] (f) a CDR1 variant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR1 as shown in SEQ ID NO: 1, a CDR2 mutant obtained by 1-4, preferably 1 or 2, amino acid mutations and / or deletions of the CDR2 as shown in SEQ ID NO: 2, and a CDR3 as shown in SEQ ID NO: 3,

[0202] Preferably, the mutations are single amino acid random mutations, preferably the mutations are selected from the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y,

[0203] Preferably, the mutations are single point random mutations or deletions of any one of the amino acids in the sequences shown in SEQ ID NO: 1, 2 or 3 only,

[0204] Preferably, the mutations are substitution mutations or deletion mutations, preferably substitution mutations.

[0205] Item 2. The mutation library according to item 1, characterized in that,

[0206] the variant mutation sites of the CDR1 are selected from 1 or 2, preferably 1, of the following:

[0207] (1 a) G1 of SEQ ID NO: 1, the mutations are selected from the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y; preferably S or P;

[0208] (1 b) R2 of SEQ ID NO: 1, the mutations are selected from any 1 of the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of I, S, H, T, P, F, G, Q, Y and L;

[0209] (1c) T3 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of V, I, A, L, W, F, D, and Y;

[0210] (1d) F4 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of S, G, Y, and V;

[0211] (1e) S5 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of R, D, K, F, G, P, N, L, and P;

[0212] (1f) S6 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of Y, D, A, V, G, I, P, K, F, L, and M;

[0213] (1g) Y7 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of K, Q, H, L, V, C, L, and A;

[0214] (1h) D8 of SEQ ID NO: 1, the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably 1 of V, A, E, and S, and / or

[0215] the variant mutation site of the CDR2 is selected from 1 or 2, preferably 1, of:

[0216] (2a) I1 of SEQ ID NO: 2; the mutation selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably M, N, or T;

[0217] (2b) S2 of SEQ ID NO: 2; the mutation selected from 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of N, D, R, A;

[0218] (2c) S3 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of R, Y, V, H, K, M and L;

[0219] (2d) G4 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of D, A, E, I, R, T, L, K and S;

[0220] (2e) G5 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of L, R, Q, S, A, K and T;

[0221] (2f) S6 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of Y, M, G, H, N, L, R, V, M, Q, I, W and T;

[0222] (2g) T7 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of S, K, H, Y and V, and / or

[0223] the variant mutation site of the CDR3 is selected from 1 or 2, preferably 1 of:

[0224] (3a) N1 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably T;

[0225] (3b) V2 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of A, L, I, Y, R and T;

[0226] (3c) R3 of SEQ ID NO: 3, the mutation being selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably C or E;

[0227] (3d) G4 of SEQ ID NO: 3, the mutation being selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion;

[0228] (3e) T5 of SEQ ID NO: 3, the mutation being selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably N;

[0229] (3f) A6 of SEQ ID NO: 3, the mutation being selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of D, L and S;

[0230] (3g) Y7 of SEQ ID NO: 3, the mutation being selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of E, H, S, L, K, M, F, R, V, I or N.

[0231] Item 3. The mutation library according to item 1, wherein in the mutation library CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated; preferably CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in any one of SEQ ID NOs: 110-116 and 129-131, 273, or

[0232] CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated; preferably CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in any one of SEQ ID NOs: 57-60, CDR3 is as set forth in any one of SEQ ID NOs: 117-119, or

[0233] CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3 and 1 amino acid thereof is mutated; preferably, CDR1 is as set forth in SEQ ID NO: 4, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 117, or

[0234] CDR1 is as set forth in SEQ ID NO: 1 and 1 or 2 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 18-38, CDR2 is as set forth in SEQ ID NO: 2, CDR3 is as set forth in SEQ ID NO: 3, or

[0235] CDR1 is as set forth in SEQ ID NO: 1, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 1, CDR2 is as set forth in any one of SEQ ID NO: 75-98, CDR3 is as set forth in SEQ ID NO: 3, or

[0236] CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 4, 5, 13, 39-56, CDR2 is as set forth in any one of SEQ ID NO: 57, 58, 61, 67, 68, 76, 96-108, CDR3 is as set forth in SEQ ID NO: 3.

[0237] Item 4. The mutant library of item 1, wherein the complementarity determining regions CDR of the single-domain antibody of the mutant library comprise a variant of CDR1, a variant of CDR2 and a variant of CDR3, wherein the variant of CDR1 has a sequence as set forth in any one of SEQ ID NO: 4-56, the variant of CDR2 has a sequence as set forth in any one of SEQ ID NO: 57-109, and the variant of CDR3 has a sequence as set forth in any one of SEQ ID NO: 110-133, 273.

[0238] Item 5. A single-domain antibody produced or screened from the mutant library of any one of items 1-4, said single-domain antibody having FIX and / or FIXa binding ability, and said single-domain antibody facilitating FIX and / or FIXa enzymatic activity,

[0239] Preferably, the enzymatic activity is an activation of FX to form FXa, more preferably, the enzymatic activity is a serine protease activity,

[0240] Preferably, the single-domain antibody is a monoclonal antibody.

[0241] Item 6. A FIX and / or FIXa binding molecule, said binding molecule comprising an anti-FIX and / or FIXa single-domain antibody VHH, the complementarity determining regions CDRs of said single-domain antibody comprising CDR1, CDR2 and CDR3, the CDR1, CDR2, CDR3 of said single-domain antibody being as shown in any one of the rows of Table 1,

[0242] Preferably, the FR of said single-domain antibody is selected from the group consisting of: a human-derived FR, a murine-derived FR, a camelid or a llama-derived FR,

[0243] Preferably, the FR1, FR2, FR3 and FR4 of said single-domain antibody are as shown in the FR1, FR2, FR3 and FR4 of a VHH as shown in any one of SEQ ID NOs: 134-272,

[0244] Preferably, said single-domain antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 134-272, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence as shown in any one of SEQ ID NOs: 134-272,

[0245] Preferably, said FIX and / or FIXa binding molecule is a monovalent or multivalent single-domain antibody, a multi-specific single-domain antibody comprising one, two or more of said single-domain antibodies.

[0246] Item 7. A nucleic acid molecule encoding

[0247] (1) the FIX and / or FIXa binding molecule of item 6, and / or

[0248] (2) the reverse complement of (1).

[0249] Item 8. A nucleic acid construct comprising the nucleic acid molecule of item 7,

[0250] Preferably, said nucleic acid construct is a cloning vector, an expression vector or an integration vector.

[0251] Item 9. A host cell selected from the group consisting of:

[0252] (1) expressing and / or secreting the FIX and / or FIXa binding molecule of item 6;

[0253] (2) comprising the nucleic acid molecule of item 7; and / or

[0254] (3) comprising the nucleic acid construct of item 8.

[0255] Item 10. A method of producing the FIX and / or FIXa binding molecule of any of the embodiments herein, comprising: culturing the host cell of item 9 under conditions suitable for production of the FIX and / or FIXa binding molecule, and optionally purifying the FIX and / or FIXa binding molecule from the culture.

[0256] Item 11. A pharmaceutical composition comprising the FIX and / or FIXa binding molecule of item 6, the nucleic acid molecule of item 7, the nucleic acid construct of item 8, or the host cell of item 9, and a pharmaceutically acceptable excipient.

[0257] Item 12. Use of the FIX and / or FIXa binding molecule of item 6, the nucleic acid molecule of item 7, the nucleic acid construct of item 8, or the host cell of item 9, for the manufacture of a medicament or a kit for the prevention or treatment of a bleeding disorder,

[0258] Preferably, the animal is a mammal, more preferably, the animal is a mouse or a human,

[0259] Preferably, the bleeding disorder is selected from the group consisting of hemophilia A, hemophilia B.

[0260] Item 13. A method of increasing FIX and downstream enzyme-linked reactions, the method comprising administering to a patient in need thereof a therapeutically effective amount of the FIX and / or FIXa binding molecule of item 6, the nucleic acid molecule of item 7, the nucleic acid construct of item 8, the host cell of item 9, or the pharmaceutical composition of item 10.

[0261] Item 14. A kit for detecting FIX, comprising the FIX and / or FIXa binding molecule of item 6, the nucleic acid molecule of item 7, the nucleic acid construct of item 8, or the host cell of item 9,

[0262] Preferably, the kit further comprises a reagent for detecting the binding of FIX to the FIX and / or FIXa binding molecule, preferably a reagent for detecting the binding by an enzyme-linked immunoassay, preferably the detection reagent is a detectable label that is attached to the FIX and / or FIXa binding molecule, preferably biotin, the detectable label being attached to the FIX and / or FIXa binding molecule or being present separately in the kit.

[0263] Item 15. A non-diagnostic method for detecting the presence of FIX in a sample, said method comprising: incubating a sample with the FIX and / or FIXa binding molecule of item 6, and detecting the binding of FIX to the FIX and / or FIXa binding molecule, thereby determining the presence of FIX in the sample, preferably said detecting is by an enzyme-linked immunoassay.

[0264] Item 16. Use of the FIX and / or FIXa binding molecule of item 6 in the manufacture of a kit for detecting FIX in a sample.

[0265] The application is further illustrated by the following examples without, however, being restricted thereto within the scope of the examples. The experimental methods in the following examples, for which no specific conditions are indicated, are carried out according to routine methods and conditions, or according to the instructions of the commercial suppliers.

[0266] Example

[0267] Example 1 : Discovery and screening of anti-FIX Nanobodies

[0268] 1.1 Immunization of animals and construction of anti-FIX Nanobody yeast display library

[0269] The llama was immunized with human FIXa protein molecule (Human Factor IXa Beta, enzyme research laboratories, HFIXa 1080) to stimulate the immune B cells to produce specific antibodies against the antigen. After 4-5 times of immunization, the peripheral blood of the animal was collected, and the peripheral lymphocytes were isolated and the cell RNA was extracted. After reverse transcription and two rounds of nested PCR (polymerase chain reaction) amplification, the diversified VHH gene fragments were obtained. Then they were ligated to the vector, and a yeast library was successfully constructed by electroporation, and the library capacity was 2.14 x 1010 8 .

[0270] 1.2 Screening of anti-FIX Nanobody yeast display library

[0271] Yeast library uses two core subunits Agal and Aga2 in the alpha-agglutinin system of yeast to express antibody library proteins on the surface of yeast cells, and uses magnetic beads and flow cytometry sorting technology for screening to obtain high-affinity or high-stability antibody sequences. The display library cells are sequentially incubated with magnetic beads and target protein FIX, and the products of a round of magnetic bead enrichment are identified by flow cytometry, and then multiple rounds of flow sorting and identification are performed. In order to specifically enrich antibodies against FIX, the use of excess FIX during incubation is identified. Finally, the library products enriched in each round are plated, monoclonal identified, and NGS (next generation sequencing) is used to obtain monoclonal sequences which are then translated into protein sequences. Unique sequences are selected for alignment and phylogenetic tree construction. The obtained monoclonal sequences are classified according to CDR3 (complementary determining region 3), and unique clones are selected for human FIX, human FIXa, and cynomolgus monkey FIX flow cytometry identification, obtaining dozens of unique sequences that are positive for human FIX protein staining, including FIXa preferentially binding clones and cynomolgus monkey FIX cross-binding clones. After FIX enzyme activity identification, single clones with FIX enzyme catalytic activity are screened as follows:

[0272] Example 2: Construction and sequence screening of phage mutation library

[0273] In order to further optimize the FIX enzyme catalytic activity of the above-mentioned monoclonal sequences, a phage display amino acid mutation library is used to construct a diversity library of CDR regions of the above-mentioned clones. The amino acid mutation library is a trimer primer library containing any specified type and proportion of codons synthesized in the specified mutation region. These primer libraries are spliced to obtain a gene mutant library with rich diversity. This library construction method reduces bias by controlling the proportion of amino acids, avoids unnecessary synonymous codons and amino acids (such as stop codons and cysteine), and can achieve precise control of amino acids at multiple diversity mutation sites, reduce library capacity in a targeted manner without affecting library diversity, and improve the success rate of screening. This scheme constructs amino acid mutation libraries for CDR1, CDR2, and CDR3 respectively to investigate the effects of different amino acids at different positions on the enzyme activity of nanobodies.

[0274] 2.1 CDR3 has the greatest impact on antigen-antibody binding, so single and more than two combinations of CDR3 mutations are performed, and at the same time, the effects of changes in CDR3 on the affinity and enzyme activity of FIX are identified by combining different sequences of CDR1 and CDR2.

[0275] Constructing CDR3 amino acid single-site and double-site mutation libraries while fixing CDR1 and CDR2

[0276] The anti-FIX / FIXa antibody CDR3 was randomly mutated at amino acid single sites and double sites using trimer phosphoramidites. A CDR3 mutation library was constructed using 36 trimer primers designed and synthesized by NCBI.

[0277] Mutation library construction:

[0278] Linearized vector preparation: 20 ng of template plasmid, 10 pmol of F3 primer, 10 pmol of R3 primer, 25 μL of Primestar DNA polymerase (Takara), and water to 50 μL; PCR program: 95°C for 2 min, 95°C for 30 s, 55°C for 5 s, 72°C for 20 s, 25 cycles, and 72°C for 3 min; 1% agarose gel detection and recovery of linearized vector.

[0279] Amino acid mutation CDR3 preparation: 100 ng of CDR3 with 36 different site amino acid mutations mixed in equal proportions was used as a template, 10 pmol of F3 primer, 10 pmol of R3 primer, 25 μL of Primestar DNA polymerase (Takara), and water to 50 μL; PCR program: 95°C for 2 min, 95°C for 30 s, 55°C for 5 s, 72°C for 5 s, 25 cycles, and 72°C for 1 min; 2% agarose gel detection and recovery of CDR3 fragments.

[0280] Recombinant plasmid preparation: 0.05 pmol of linearized vector, 0.1 pmol of amino acid mutation CDR3, 10 μL of NEBuilder HiFi DNA Assembly Master Mix, and water to 20 μL; 50°C for 15 min, then stored at -20°C.

[0281] Library construction: 2 μL of recombinant plasmid was added to 25 μL of Lucigen TG1 and mixed, then transferred to a 1 mm electrotransformation cup, and electrotransformed at 1.8 kv, then 975 μL of SOC recovery medium was added, and incubated at 37°C at 220 rpm for 1 h, to obtain an amino acid mutation library with a capacity of 2.4 x 10 5 cfu / mL.

[0282] Mutation library screening:

[0283] Phage packaging: The prepared mutant library bacterial solution was added to 2xYT medium containing 4% glucose, and incubated at 37°C until OD600 was about 0.5, then 2.5 x 10 11pfu KM13 helper phage was incubated at 37°C for 1 h. The infected phage was centrifuged at 5000 g for 5 min, resuspended with 2xYT medium containing 100 μg / mL, 50 μg / mL kanamycin, and cultured at 30°C at 220 rpm overnight.

[0284] Phage purification: 500 μL of the bacterial solution was taken, and 50 mL of 2xYT medium without antibiotics was added to culture to an OD600 of 0.5. The supernatant was removed by centrifugation at 3200 g for 20 min. 5xPEG6000 concentrate was added, and the solution was allowed to stand for 1 h before centrifugation at 3200 g for 30 min. The supernatant was removed, and the solution was resuspended with 5 mL of PBS. 1 mL of 5xPEG6000 concentrate was added, and the solution was allowed to stand for 10 min on ice before centrifugation at 3200 g for 30 min. The supernatant was removed, and the solution was resuspended with 2 mL of PBS. The solution was centrifuged at 3200 g for 5 min, and the supernatant was filtered with a 0.45 μm filter membrane before being stored at 4°C.

[0285] Antibody-induced expression:

[0286] 1 μL of the phage supernatant was taken, and 10 mL of the TG1 strain in the logarithmic growth phase was incubated at 37°C at 70 rpm for 1 h. After the infection was completed, the solution was centrifuged at 3200 g for 5 min, resuspended with 4% glucose in 2xYT resistant medium, and 100 μL of the gradient dilution was applied to an Amp-resistant YTE plate. 600 clones were randomly selected from the colonies growing on the plate and cultured in 600 μL of 2xYT resistant medium at 37°C overnight. 20 μL of the bacterial solution was transferred and cultured until the OD600 was 0.6. Then, 1 mM of IPTG was added, and the solution was induced at 25°C at 220 rpm overnight.

[0287] Mutant antibody activity screening:

[0288] The bacterial solution induced for expression was centrifuged at 2500 rpm for 30 min, and the supernatant was collected for Elisa affinity detection. Seven clones that met the affinity requirements were selected. The bacterial solution before the transfer of the seven clones was sequenced to obtain the screened CDR3 mutation and CDR1, CDR2 combination sequence. Anti-FIX nanobodies were prepared by expression in mammalian cells.

[0289] Similarly, using the above method, the monoclonal antibodies were sequentially banked according to the following table rules, and active CDR combination mutation sequences were obtained.

[0290] Example 3 Recombinant expression of anti-FIX nanobodies

[0291] The sequences obtained after library construction of Example 2 were selected for codon optimization, and the anti-FIXa nanobody gene sequences were synthesized, subcloned into an expression vector, and plasmids were extracted and sequenced. The expression vector has a signal peptide to promote nanobody secretion, and the vector encodes a HA, His tag at the C-terminus of the antibody sequence. CHO (Chinese hamster ovary) suspension cells were revived in advance in a shaker flask to be in the logarithmic growth phase at the time of transfection, and then the plasmid / PEI (polyethylenimine) mixture was directly added to the prepared cell suspension for transient transfection. After 5-6 days of culture, the cell culture supernatant was collected by centrifugation and filtration.

[0292] The cell culture supernatant was loaded onto a Ni-IDA-Sepharose Cl-6B affinity chromatography column pre-equilibrated with Ni-IDA (nickel-iminodiacetic acid) binding buffer at a flow rate of 0.5 mL / min using a low-pressure chromatography system. The flow-through OD280 value reached the baseline after washing with Ni-IDA binding buffer. The flow-through OD280 value reached the baseline after washing with Ni-IDA wash buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH 8.0). The target protein was eluted with Ni-IDA elution buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0), and the flow-through was collected. The collected protein solution was added to a dialysis bag and dialyzed overnight using 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0. The molecular weight and purity of the recombinantly expressed anti-FIX nanobody were detected by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), Coomassie blue staining, and SEC (size exclusion chromatography), and the final purity of the different antibodies was greater than 95%. The sequences of the antibodies are shown in Table 1.

[0293] Table 1: Antibody sequences

[0294] Example 4: Affinity of anti-FIX nanobodies to FIX and its activated form, FIXa

[0295] To determine the specific binding of the purified antibody to the target protein FIXa, an enzyme linked immunosorbent assay (ELISA) was performed on the anti-FIXa antibody. ELISA is a highly sensitive detection technique based on immunological reactions, combining the specific reaction of antigen and antibody with the high catalytic effect of enzyme on substrate. Antigen and antibody undergo a binding reaction in the wells of a polystyrene microtiter plate. After incubation with each reagent, the excess free reactants are removed by washing, ensuring the specificity and stability of the test results.

[0296] First, the antigen FIXa was diluted to 1 ug / ml with an alkaline buffer (0.05 M NaHCO3 solution, pH 9.6) and coated onto an enzyme-labeled plate at 4°C overnight. After washing with PBST for 3 times, 5% BSA was added for blocking, and after incubation at 37°C for 2 h, washing was performed, followed by the addition of different concentrations of anti-FIXa nanobodies (primary antibody), with antibody diluent PBS as a negative control, and incubation at 37°C for 1.5 h. After washing again, Anti-HA HRP conjugated secondary antibody diluted 1:10000 in PBS was added, and incubation was performed at 37°C for 0.5 h. After washing, the chromogenic substrate TMB (3,3',5,5'-tetramethylbenzidine) was added, and the reaction was carried out at room temperature in the dark. The color change was observed, and the positive wells were blue, then the color development was terminated with 2M H2SO4. The absorbance value shown at 450 nm can reflect the degree of antigen-antibody binding, thereby judging the specific affinity of the antibody to FIXa. The results are shown in Table 2.

[0297] Example 5: Effect of anti-FIX nanobodies on the catalytic activity of FIXa

[0298] Chromogenic substrate was used to determine the activity of FIXa.

[0299] ​The microplate reader was set to 37°C preheating, selecting the kinetic cycle mode, measuring the dynamic change value of absorbance, the total time was set to 10 min, the interval was 5 s, the cycle number was 121 times, and the reading interval was oscillated. In the enzyme-labeled plate, 92.5 uL of Tris Buffer (50 mM Tris, 100 mM NaCl, 5 mM CaCl2, 20% EG, pH 7.4), 10 ul 2uM FIXa, 2.5ul different concentration gradient of the antibody to be tested were added, then 12.5ul 10uM chromogenic substrate was added, and the reaction was triggered. Immediately, the enzyme-labeled plate was placed into the enzyme-labeled instrument, and the change rate of absorbance at 405 nm was measured. The results are shown in Table 2. Compared with the blank (Elisa OD 0.08, enzyme activity rate 0.01464 OD / min), the antibody to be tested increased the enzyme activation rate (ΔOD / min, ΔOD = OD-OD 空白 ) of FIXa by multiple times.

[0300] Table 2

[0301] Example 6 Activated Partial Thromboplastin Time APTT Detection

[0302] Activated partial thromboplastin time (APTT) is the most commonly used sensitive screening test in clinic to reflect the coagulation activity of the endogenous coagulation system, and has a wide range of uses in the detection of endogenous coagulation factor defects and related inhibitors, screening of activated protein C resistance, monitoring of heparin therapy, early diagnosis of disseminated intravascular coagulation (DIC), preoperative examination, etc. It evaluates the degree of activation of the endogenous coagulation pathway by adding an activator that initiates the endogenous coagulation pathway.

[0303] Therefore, the coagulation promoting condition of the antibody of the application was studied by APTT detection (APTT assay kit (coagulation method), Shanghai Sunshine Bio, Instrumentation Laboratory Co.). The APTT kit (tannic acid) and factor VIII (FVIII) deficient plasma were purchased from Sunshine Bio. Using a STAGO Emo Step 4 semi-automatic coagulation analyzer, 100 uL of APTT reagent and 100 uL of FVIII-deficient plasma containing the antibody to be tested (final concentration of 2 ug / ml) were added to the reaction cup, and FVIII-deficient plasma without sample was set as negative control, 37°C incubation for 5 min, 100 uL of 37°C pre-warmed reagent 0.025M CaCl2 was added, and the coagulation time was recorded, i.e. activated partial thromboplastin time APTT. The APTT of FVIII-deficient plasma without sample was 110 s, and the APTT time of the antibody shown in Example 5 is shown in Table 3:

[0304] Table 3

[0305] The results show that the original sequence antibody can shorten the APTT of the FVIII-deficient plasma from 110 seconds by about 1 / 3, and the antibody produced by the library after random mutation can further shorten the APTT, even close to the normal range. The antibody constructed by the library not only can directly promote the enzyme activity of FIX / FIXa, but also can promote the endogenous coagulation pathway in the APTT study, that is, even in the case of FVIII-deficient plasma (FVIII content is less than 1%), it can directly promote the FIXa activity to shorten the APTT. Therefore, the antibody of the present application can have a strong pro-coagulation effect in patients with hemophilia A with mild, moderate and severe FVIII deficiency (corresponding to FVIII content of 6-40%, 1-5%, <1% of normal people); similarly, in patients with severe FIX deficiency, the general FIX content is <1%, and reaching 5% of the FIX content can improve the bleeding symptoms and even not bleed, therefore, for such patients, the enhancement of FIXa enzyme activity can not only have a therapeutic effect, but also reduce the risk of thrombosis. Therefore, the antibody of the present application has the potential to treat anti-hemorrhagic diseases and is beneficial to balance the bleeding-thrombosis risk.

Claims

1. A library of mutations of a coding sequence of a single domain antibody to FIX and / or FIXa, comprising a coding sequence of a CDR1 as set forth in SEQ ID NO: 1, a coding sequence of a CDR2 as set forth in SEQ ID NO: 2, and a coding sequence of a CDR3 as set forth in SEQ ID NO: 3, and optionally a coding sequence of a CDR region of a single domain antibody to FIX and / or FIXa designed after one or more of the following: (a) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR3 as set forth in SEQ ID NO: 3; and / or (b) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; and / or (c) a CDR1 variant obtained after 1-4, preferably 1, 2, 3 or 4 amino acid mutations and / or deletions of a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3; and / or (d) a CDR1 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR1 as set forth in SEQ ID NO: 1, a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained after 1 or 2 amino acid mutations and / or deletions of a CDR3 as set forth in SEQ ID NO: 3; and / or (e) a CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 variant obtained after 1 or 2 amino acid mutations and / or deletions of a CDR3 as set forth in SEQ ID NO: 3; and / or (f) a CDR1 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR1 as set forth in SEQ ID NO: 1, a CDR2 variant obtained after 1-4, preferably 1 or 2 amino acid mutations and / or deletions of a CDR2 as set forth in SEQ ID NO: 2, and a CDR3 as set forth in SEQ ID NO: 3, preferably the mutations are single amino acid random mutations, preferably the mutations are selected from the group consisting of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y, preferably the mutations are single point random mutations or deletions of any one of the amino acids in the sequence set forth in SEQ ID NO: 1, 2 or 3 only, preferably the mutations are substitution mutations or deletion mutations, preferably substitution mutations.

2. The mutant library of claim 1, wherein, the variant mutation site of CDR1 is selected from one or two, preferably one, of: (1a) G1 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, or Y; preferably S or P; (1b) R2 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of I, S, H, T, P, F, G, Q, Y and L; (1c) T3 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of V, I, A, L, W, F, D and Y; (1d) F4 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of S, G, Y and V; (1e) S5 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of R, D, K, F, G, P, N, L and P; (1f) S6 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of Y, D, A, V, G, I, P, K, F, L and M; (1g) Y7 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of K, Q, H, L, V, C, L and A; (1h) D8 of SEQ ID NO: 1, the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably one of V, A, E and S, and / or the variant mutation site of CDR2 is selected from one or two, preferably one, of: (2a) I1 of SEQ ID NO: 2; the mutation is selected from any one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably M, N or T; (2b) S2 of SEQ ID NO: 2; the mutation is selected from one of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any one of N, D, R, A; (2c) S3 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of R, Y, V, H, K, M and L; (2d) G4 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of D, A, E, I, R, T, L, K and S; (2e) G5 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of L, R, Q, S, A, K and T; (2f) S6 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of Y, M, G, H, N, L, R, V, M, Q, I, W and T; (2g) T7 of SEQ ID NO: 2; the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of S, K, H, Y and V, and / or the variant mutation site of the CDR3 is selected from 1 or 2, preferably 1, of: (3a) N1 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably T; (3b) V2 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion or any 1 of A, L, I, Y, R and T; (3c) R3 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably C or E; (3d) G4 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably a deletion; (3e) T5 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably N; (3f) A6 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of D, L and S; (3g) Y7 of SEQ ID NO: 3, the mutation is selected from any 1 of amino acids A, I, L, M, P, V, G, N, Q, S, T, D, E, H, K, R, F, W, and Y; preferably any 1 of E, H, S, L, K, M, F, R, V, I or N.

3. The library of claim 1, wherein In the mutation library, CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated; preferably CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in any one of SEQ ID NOs: 110-116 and 129-131, 273, or CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as shown in SEQ ID NO: 3 and 1 or 2 amino acids thereof are mutated; preferably CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in any one of SEQ ID NOs: 57-60, CDR3 is as shown in any one of SEQ ID NOs: 117-119, or CDR1 is as shown in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 3 and 1 amino acid thereof is mutated; preferably CDR1 is as shown in SEQ ID NO: 4, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 117, or CDR1 is as shown in SEQ ID NO: 1 and 1 or 2 amino acids thereof are mutated, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 3; preferably CDR1 is as shown in any one of SEQ ID NOs: 18-38, CDR2 is as shown in SEQ ID NO: 2, CDR3 is as shown in SEQ ID NO: 3, or CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as shown in SEQ ID NO: 3; preferably CDR1 is as shown in any one of SEQ ID NO: 1, CDR2 is as shown in any one of SEQ ID NOs: 75-98, CDR3 is as shown in SEQ ID NO: 3, or CDR1 is as shown in SEQ ID NO: 1, CDR2 is as shown in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as shown in SEQ ID NO: 3; preferably CDR1 is as shown in any one of SEQ ID NO: 1, CDR2 is as shown in any one of SEQ ID NOs: 75-98, CDR3 is as shown in SEQ ID NO: 3, or CDR1 is as set forth in SEQ ID NO: 1 and 1, 2, 3 or 4 amino acids thereof are mutated, CDR2 is as set forth in SEQ ID NO: 2 and 1 or 2 amino acids thereof are mutated, CDR3 is as set forth in SEQ ID NO: 3; preferably, CDR1 is as set forth in any one of SEQ ID NO: 4, 5, 13, 39-56, CDR2 is as set forth in any one of SEQ ID NO: 57, 58, 61, 67, 68, 76, 96-108, CDR3 is as set forth in SEQ ID NO:

3.

4. The library of claim 1, wherein The complementarity determining regions CDR of the single domain antibody of the mutant library comprise a variant of CDR1, a variant of CDR2 and a variant of CDR3, wherein the variant of CDR1 has a sequence as set forth in any one of SEQ ID NO: 4-56, the variant of CDR2 has a sequence as set forth in any one of SEQ ID NO: 57-109, the variant of CDR3 has a sequence as set forth in any one of SEQ ID NO: 110-133, 273.

5. The single domain antibody produced or selected from the mutant library of any one of claims 1-4, which has FIX and / or FIXa binding ability, and which promotes FIX and / or FIXa enzymatic activity, preferably, the enzymatic activity is serine protease activity, preferably, the single domain antibody is a monoclonal antibody.

6. A FIX and / or FIXa binding molecule, which comprises an anti-FIX and / or FIXa single domain antibody VHH, the complementarity determining regions CDR of the single domain antibody comprising CDR1, CDR2 and CDR3, the CDR1, CDR2, CDR3 of the single domain antibody being as set forth in any one of the rows of Table 1, preferably, the FR of the single domain antibody is selected from the group consisting of: a human-derived FR, a murine-derived FR, a camelid or a llama-derived FR, preferably, the FR1, FR2, FR3 and FR4 of the single domain antibody are as set forth in the FR1, FR2, FR3 and FR4 of a VHH as set forth in any one of SEQ ID NO: 134-272, preferably, the single domain antibody has an amino acid sequence as set forth in any one of SEQ ID NO: 134-272, or has at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence as set forth in any one of SEQ ID NO: 134-272, preferably, the FIX and / or FIXa binding molecule is a monovalent or multivalent single domain antibody, a multispecific single domain antibody comprising one, two or more of the single domain antibodies.

7. A nucleic acid molecule encoding (1) the FIX and / or FIXa binding molecule of claim 6, and / or (2) the reverse complement of (1).

8. A nucleic acid construct comprising the nucleic acid molecule of claim 7, preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

9. A host cell selected from the group consisting of: (1) the FIX and / or FIXa binding molecule of claim 6, and / or (2) the reverse complement of (1).

8. A nucleic acid construct comprising the nucleic acid molecule of claim 7, preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.

9. A host cell selected from the group consisting of: (1) expressing and / or secreting the FIX and / or FIXa binding molecule of claim 6; (2) comprising the nucleic acid molecule of claim 7; and / or (3) comprising the nucleic acid construct of claim 8.

10. A method of making the FIX and / or FIXa binding molecule of claim 6, comprising: culturing the host cell of claim 9 under conditions suitable for production of the FIX and / or FIXa binding molecule, and optionally purifying the FIX and / or FIXa binding molecule from the culture.

11. A pharmaceutical composition comprising the FIX and / or FIXa binding molecule of claim 6, the nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, or the host cell of claim 9, and a pharmaceutically acceptable excipient.

12. Use of the FIX and / or FIXa binding molecule of claim 6, the nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, or the host cell of claim 9 in the manufacture of a medicament or a kit for preventing or treating a bleeding disorder, preferably, the animal is a mammal, more preferably, the animal is a mouse or a human, preferably, the bleeding disorder is selected from the group consisting of hemophilia A, hemophilia B.

13. A method of increasing FIX and downstream enzymatic reactions, the method comprising administering to a patient in need thereof a therapeutically effective amount of the FIX and / or FIXa binding molecule of claim 6, the nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, the host cell of claim 9, or the pharmaceutical composition of claim 10.

14. A kit for detecting FIX, comprising the FIX and / or FIXa binding molecule of claim 6, the nucleic acid molecule of claim 7, the nucleic acid construct of claim 8, or the host cell of claim 9, preferably, the kit further comprises reagents for detecting the binding of FIX to the FIX and / or FIXa binding molecule, preferably reagents for detecting the binding by an enzyme-linked immunoassay, preferably the detection reagents are detectable labels attached to the FIX and / or FIXa binding molecule, preferably biotin, which are attached to the FIX and / or FIXa binding molecule or are present separately in the kit.

15. A non-diagnostic method of detecting the presence of FIX in a sample, the method comprising: incubating the FIX and / or FIXa binding molecule of claim 6 with a sample, and detecting the binding of FIX to the FIX and / or FIXa binding molecule, thereby determining the presence of FIX in the sample, preferably the detection is by an enzyme-linked immunoassay.

16. Use of the FIX and / or FIXa binding molecule of claim 6 in the manufacture of a kit for detecting FIX in a sample.

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