ANTI-FXI/FXIA ANTIBODY AND USE THEREOF

Abstract

The present invention relates to the field of therapeutic monoclonal antibodies, and specifically provides an anti-FXI/FXIa antibody or an antigen-binding fragment thereof, nucleic acid molecules encoding same, and methods for preparing same. The anti-FXI/FXIa antibody or antigen-binding fragment thereof described in the present invention has specificity and high affinity to FXI/FXIa, and can effectively inhibit the activity of FXI/FXIa. Therefore, the present invention further provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, and a use thereof in the preparation of a drug which is used for the prevention and/or treatment of diseases or disorders related to coagulation or thromboembolism.

Claims

1. An antibody or antigen-binding fragment thereof capable of specifically binding to FXI and/or FXIa, wherein the antibody or antigen-binding fragment thereof comprises: (a) three heavy chain complementarity determining regions (CDRs) as follows: a CDR-H1, a CDR-H2 and a CDR-H3 contained in a heavy chain variable region (VH) set forth in any one of SEQ ID NOs: 15, 1, 16, 17, 29, 31; and/or three light chain complementarity determining regions (CDRs) as follows: a CDR-L1, a CDR-L2 and a CDR-L3 contained in a light chain variable region (VL) set forth in any one of SEQ ID NOs: 18, 2, 19, 20, 30, 32; or, (b) three heavy chain CDRs as follows: the CDR-H1 described in (a) or a variant comprising an amino acid mutation as compared thereto, the CDR-H2 described in (a) or a variant comprising an amino acid mutation as compared thereto, the CDR-H3 described in (a) or a variant comprising an amino acid mutation as compared thereto; and/or three light chain CDRs as follows: the CDR-L1 described in (a) or a variant comprising an amino acid mutation as compared thereto, the CDR-L2 described in (a) or a variant comprising an amino acid mutation as compared thereto, the CDR-L3 described in (a) or a variant comprising an amino acid mutation as compared thereto; wherein at least one CDR of the three heavy chain CDRs and/or three light chain CDRs described in (b) comprises an amino acid mutation as compared to the corresponding CDR in (a), and the amino acid mutation comprises a substitution, deletion or addition of one or several amino acids (e.g., a substitution, deletion or addition of 1, 2, or 3 amino acids); preferably, the substitution is a conservative substitution; preferably, the CDRs are defined according to Kabat, IMGT, Chothia or AbM numbering system; preferably, the antibody or antigen-binding fragment thereof further comprises framework regions (FRs) of immunoglobulin derived from a human or murine; preferably, the antibody or antigen-binding fragment thereof binds to a human FXI and/or human FXIa.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (1) the heavy chain variable region (VH) and/or the light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system: (1a) the heavy chain variable region (VH) comprising three CDRs as follows: the CDR-H1 comprising a sequence of SEQ ID NO: 3, the CDR-H2 comprising a sequence of SEQ ID NO: 4, and the CDR-H3 comprising a sequence of SEQ ID NO: 5; and/or, the light chain variable region (VL) comprising three CDRs as follows: the CDR-L1 comprising a sequence of SEQ ID NO: 6, the CDR-L2 comprising a sequence of SEQ ID NO: 7, and the CDR-L3 comprising a sequence of SEQ ID NO: 8; or (1b) the heavy chain variable region (VH) comprising three CDRs as follows: the CDR-H1 comprising a sequence of SEQ ID NO: 33, the CDR-H2 comprising a sequence of SEQ ID NO: 34, and the CDR-H3 comprising a sequence of SEQ ID NO: 35; and/or, the light chain variable region (VL) comprising three CDRs as follows: the CDR-L1 comprising a sequence of SEQ ID NO: 36, the CDR-L2 comprising a sequence of SEQ ID NO: 37, and the CDR-L3 comprising a sequence of SEQ ID NO: 38; or (2) the heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the AbM numbering system: (2a) a heavy chain variable region (VH) comprising three CDRs as follows: CDR-H1 comprising a sequence of SEQ ID NO: 9, CDR-H2 comprising a sequence of SEQ ID NO: 45, 46 or 10, and CDR-H3 comprising a sequence of SEQ ID NO: 11; and/or, the light chain variable region (VL) comprising three CDRs as follows: the CDR-L1 comprising a sequence of SEQ ID NO: 12, the CDR-L2 comprising a sequence of SEQ ID NO: 13, and the CDR-L3 comprising a sequence of SEQ ID NO: 14; or (2b) the heavy chain variable region (VH) comprising three CDRs as follows: the CDR-H1 comprising a sequence of SEQ ID NO: 39, the CDR-H2 comprising a sequence of SEQ ID NO: 40 or 47, and the CDR-H3 comprising a sequence of SEQ ID NO: 41; and/or, the light chain variable region (VL) comprising three CDRs as follows: the CDR-L1 comprising a sequence of SEQ ID NO: 42, the CDR-L2 comprising a sequence of SEQ ID NO: 43, and the CDR-L3 comprising a sequence of SEQ ID NO: 44; or (3) the heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein at least one CDR of the heavy chain variable region (VH) and/or light chain variable region (VL) comprises an amino acid mutation as compared with the heavy chain variable region and/or light chain variable region described in (1a), (1b), (2a) or (2b), and the amino acid mutation is a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof); preferably, the substitution is a conservative substitution; preferably, the VH and/or VL of the antibody or antigen-binding fragment thereof comprises framework regions (FRs) derived from a human or murine immunoglobulin; preferably, the antibody or antigen-binding fragment thereof binds to a human FXI and/or human FXIa.

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) the VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 15, 1, 16, 17, and/or the VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 18, 2, 19, 20; (b) the VH comprising an amino acid sequence set forth in any one of SEQ ID NOs: 29 and 31, and/or the VL comprising an amino acid sequence set forth in any one of SEQ ID NOs: 30 and 32; or (c) the VH having an identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the VH in (a) or (b); and/or the VL having an identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the VL in (a) or (b); or (d) the VH having a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) as compared to the VH in (a) or (b); and/or the VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) as compared to the VL in (a) or (b); preferably, the substitution is a conservative substitution.

4. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) the VH comprising the sequence set forth in SEQ ID NO: 15 and the VL comprising the sequence set forth in SEQ ID NO: 18; (b) the VH comprising the sequence set forth in SEQ ID NO: 1 and the VL comprising the sequence set forth in SEQ ID NO: 2; (c) the VH comprising the sequence set forth in SEQ ID NO: 15 and the VL comprising the sequence set forth in SEQ ID NO: 20; (d) the VH comprising the sequence set forth in SEQ ID NO: 16 and the VL comprising the sequence set forth in SEQ ID NO: 18; (e) the VH comprising the sequence set forth in SEQ ID NO: 17 and the VL comprising the sequence set forth in SEQ ID NO: 19; (f) the VH comprising the sequence set forth in SEQ ID NO: 17 and the VL comprising the sequence set forth in SEQ ID NO: 18; (g) the VH comprising the sequence set forth in SEQ ID NO: 29 and the VL comprising the sequence set forth in SEQ ID NO: 30; or (h) the VH comprising the sequence set forth in SEQ ID NO: 31, and the VL comprising the sequence shown in SEQ ID NO: 32; (i) a VH having an identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the VH in any one of (a) to (h); and/or a VL having an identity of at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the VL in any one of (a) to (h); or (j) a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) as compared to the VH in any one of (a) to (h); and/or a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids or any combination thereof) as compared to the VL in any one of (a) to (h); preferably, the substitution is a conservative substitution.

5. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof further comprises: (a) a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to a wild type sequence from which it is derived; and/or (b) a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to a wild type sequence from which it is derived; preferably, the heavy chain constant region is an IgG heavy chain constant region, such as an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the following: (1) a human IgG1 heavy chain constant region; or, (2) a human IgG4 heavy chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 21 or a variant thereof, and the variant has a conservative substitution of up to 20 amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to SEQ ID NO: 21; preferably, the light chain constant region is a κ light chain constant region; preferably, the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) as shown in SEQ ID NO: 22 or a variant thereof, and the variant has a conservative substitution of up to 20 amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to SEQ ID NO: 22; more preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in SEQ ID NO: 21 and a light chain constant region (CL) as shown in SEQ ID NO: 22.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof comprises: (a) a heavy chain, which comprises an amino acid sequence selected from: (i) a sequence comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21; (ii) a sequence having a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids, or any combination thereof) as compared to the sequence shown in (i); or (iii) a sequence having an identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the sequence shown in (i); and/or a light chain, which comprises an amino acid sequence selected from: (iv) a sequence comprising the VL sequence comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (v) a sequence having a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids, or any combination thereof) as compared to the sequence shown in (iv); or (vi) a sequence having an identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the sequence shown in (iv), or (b) a heavy chain, which comprises an amino acid sequence selected from: (i) a sequence comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 31 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21; (ii) a sequence having a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids, or any combination thereof) as compared to the sequence shown in (i); or (iii) a sequence having an identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the sequence shown in (i); and/or a light chain, which comprises an amino acid sequence selected from: (iv) a sequence comprising the VL sequence comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (v) a sequence having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g., a substitution, deletion or addition of 1, 2, 3, 4 or 5 amino acids, or any combination thereof) as compared to the sequence shown in (iv); or (vi) a sequence having an identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the sequence shown in (iv); preferably, the substitution described in (ii) or (v) is a conservative substitution.

8. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody comprises: (a) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (b) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 15 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 20 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (c) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 16 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) comprising the amino acid sequence set forth shown in SEQ ID NO: 22; (d) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 19 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (e) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 18 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22; (f) a heavy chain comprising the VH comprising the amino acid sequence set forth in SEQ ID NO: 31 and a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21, and, a light chain comprising the VL comprising the amino acid sequence set forth in SEQ ID NO: 32 and a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22.

9. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of scFv, Fab, Fab′, (Fab′).sub.2, Fv fragment, disulfide-ligated Fv(dsFv), and diabody.

10. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof is labeled; preferably, the antibody or antigen-binding fragment thereof bears a detectable label, such as an enzyme (e.g., horseradish peroxidase), a radionuclide, a fluorescent dye, a luminescent substance (e.g., a chemiluminescent substance) or a biotin.

11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof has at least one of the following characteristics: (a) binding to FXI and/or FXIa (e.g., human FXI and/or human FXIa) with a K.sub.D of less than about 100 nM, for example, less than about 10 nM, 1 nM, 0.1 nM or less; preferably, the K.sub.D is measured by biofilm interference technique (BLI); (b) binding to FXI and/or FXIa (e.g., human FXI and/or human FXIa) with an EC.sub.50 of less than about 500 nM, for example, less than about 100 nM, 10 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM or less; preferably, the EC.sub.50 is measured by flow cytometry or cell-based competitive ELISA technique; (c) the binding of the antibody or antigen-binding fragment thereof to FXI and/or FXIa inhibits or blocks the binding of FXI and/or FXIa to a substrate, thereby prolonging clotting time; (d) the binding of the antibody or antigen-binding fragment thereof to FXI and/or FXIa inhibits or blocks the catalytic effect of FXI and/or FXIa on a substrate; (e) the binding of the antibody or antigen-binding fragment thereof to FXI and/or FXIa does not affect exogenous coagulation; (f) the antibody or antigen-binding fragment thereof has reduced ADCC and/or CDC activity; (g) the antibody or antigen-binding fragment thereof does not have ADCC and/or CDC activity; (h) the binding of the antibody or antigen-binding fragment thereof to FXI and/or FXIa inhibits or blocks the formation of the dimers of FXI and/or the dimers of FXIa; (i) the binding of the antibody or antigen-binding fragment thereof to FXI and/or FXIa inhibits or blocks the formation of a complex between FXI and/or FXIa and high molecular weight kininogen (HK); (j) the antibody or antigen-binding fragment thereof binds to a catalytic domain of FXI and/or FXIa, and/or induces a conformational change thereof; (k) the antibody or antigen-binding fragment thereof inhibits or blocks the binding of FXI and/or FXIa to a platelet receptor; or (l) any combination of (a) to (k).

12. An isolated nucleic acid molecule, which encodes the antibody or antigen-binding fragment thereof according to claim 1, or a portion thereof.

13. The isolated nucleic acid molecule according to claim 12, comprising: (1) a nucleic acid molecule encoding an antibody heavy chain variable region, and/or a nucleic acid molecule encoding an antibody light chain variable region, wherein, the nucleic acid molecule encoding the antibody heavy chain variable region has a nucleotide sequence selected from the group consisting of: (1a) the nucleotide sequence set forth in SEQ ID NO: 23 or 27, (1b) a sequence that is substantially the same as the nucleotide sequence described in (1a) (e.g., a sequence having an identity of at least about 85%, 90%, 95%, 99% or more to the nucleotide sequence described in (1a), or a sequence having a substitution of one or more nucleotides as compared to the nucleotide sequence described in (1a)), or (1c) a sequence that does not differ from the nucleotide sequence described in (1a) by more than 3, 6, 15, 30 or 45 nucleotides; and/or the nucleic acid molecule encoding the antibody light chain variable region has a nucleotide sequence selected from the group consisting of: (1d) the nucleotide sequence set forth in SEQ ID NO: 24 or 28, (1e) a sequence that is substantially the same as the nucleotide sequence described in (1d) (e.g., a sequence having an identity of at least about 85%, 90%, 95%, 99% or more to the nucleotide sequence described in (1d), or a sequence having a substitution of one or more nucleotides as compared to the nucleotide sequence described in (1d)), or (1f) a sequence that does not differ from the nucleotide sequence described in (1d) by more than 3, 6, 15, 30 or 45 nucleotides; or (2) a nucleic acid molecule set forth in SEQ ID NO: 23, and/or a nucleic acid molecule set forth in SEQ ID NO: 24; or (3) a nucleic acid molecule set forth in SEQ ID NO: 27, and/or a nucleic acid molecule set forth in SEQ ID NO: 28; or (4) a nucleic acid molecule encoding an antibody heavy chain, and/or a nucleic acid molecule encoding an antibody light chain, wherein, the nucleic acid molecule encoding the antibody heavy chain has a nucleotide sequence selected from the following: (4a) the nucleotide sequence set forth in SEQ ID NO: 25, (4b) a sequence that is substantially the same as the nucleotide sequence described in (4a) (e.g., a sequence having an identity of at least about 85%, 90%, 95%, 99% or more to the nucleotide sequence described in (4a), or a sequence having a substitution of one or more nucleotides as compared with the nucleotide sequence described in (4a)), or (4c) a sequence that does not differ from the nucleotide sequence described in (4a) by more than 3, 6, 15, 30 or 45 nucleotides; and/or the nucleic acid molecule encoding the antibody light chain has a sequence selected from the group consisting of: (4d) the nucleotide sequence set forth in SEQ ID NO: 26, (4e) a sequence that is substantially the same as the nucleotide sequence described in (4d) (e.g., a sequence having an identity of at least about 85%, 90%, 95%, 99% or more to the nucleotide sequence described in (4d), or a sequence having a substitution of one or more nucleotides as compared with the nucleotide sequence described in (4d)), or (4f) a sequence that does not differ from the nucleotide sequence described in (4d) by more than 3, 6, 15, 30 or 45 nucleotides; or (5) a nucleic acid molecule set forth in SEQ ID NO: 25, and/or a nucleic acid molecule set forth in SEQ ID NO: 26.

14. (canceled)

15. A vector, which comprises an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of claim 1 or a portion thereof.

16. A host cell, which comprises an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof of claim 1 or a portion thereof, or a vector comprising the isolated nucleic acid molecule.

17. A method for preparing the antibody or antigen-binding fragment thereof according to claim 1, which comprises culturing a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof or a portion thereof under conditions that allow the expression of the antibody or antigen-binding fragment thereof or a portion thereof, and recovering the antibody or antigen-binding fragment thereof or a portion thereof from the culture of the cultured host cell.

18. A multispecific antibody comprising 1) the antibody or antigen-binding fragment thereof capable of specifically binding to FXI and/or FXIa according to claim 1, and 2) a second antibody or antigen-binding fragment thereof, or an antibody mimetic that specifically binds to a second antigen; preferably, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.

19. A conjugate comprising the antibody or antigen-binding fragment thereof capable of specifically binding to FXI and/or FXIa according to claim 1, and a conjugate moiety, wherein the conjugate moiety is a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme, or the conjugate moiety is a therapeutic agent; optionally, the therapeutic agent is an antiplatelet drug, an anticoagulant drug, or a thrombolytic drug; optionally, the therapeutic agent is bound to the antibody or antigen-binding fragment thereof capable of specifically binding to FXI and/or FXIa according to any one of claims 1 to 11 through a linker.

20. A pharmaceutical composition, which comprises a pharmaceutically acceptable carrier and/or an excipient and comprises one of the following: the antibody or antigen-binding fragment thereof according to claim 1, an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a multispecific antibody comprising the antibody or antigen-binding fragment thereof, and another antibody or antigen-binding fragment thereof, or an antibody mimetic, or a conjugate comprising the antibody or antigen-binding fragment thereof and a conjugate moiety; preferably, the pharmaceutical composition further comprises another pharmaceutically active agent, such as an antiplatelet drug, an anticoagulant drug or a thrombolytic drug; more preferably, the additional pharmaceutically active agent is aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, or any combination thereof.

21. The pharmaceutical composition according to claim 20, wherein the pharmaceutical composition comprises the antibody or antigen-binding fragment thereof in an effective dose that is sufficient to cause at least one of the following biological activities in a subject: (a) binding to a catalytic domain of FXI and/or FXIa, and/or inducing its conformational change; (b) inhibiting or blocking the binding of FXI and/or FXIa to a substrate; (c) inhibiting or blocking the binding of FXI and/or FXIa to a platelet receptor; (d) inhibiting or blocking the binding of FXI to coagulation factor XIIa (FXIIa), thereby inhibiting or blocking the conversion of FXI into active FXIa; (e) inhibiting or blocking the binding of FXIa to coagulation factor FIX, thereby inhibiting or blocking the conversion of FIX into active FIXa; (f) inhibiting or blocking the activation of FXI and/or FXIa-mediated endogenous coagulation pathway; (g) inhibiting or blocking the activity of FXI and/or FXIa in thrombosis; (h) prolonging FXI and/or FXIa-mediated clotting time; (i) inhibiting thrombosis; (j) preventing and/or treating a disease or disorder associated with coagulation or thromboembolism mediated by FXI and/or FXIa; or (k) any combination of (a) to (j).

22. The pharmaceutical composition according to claim 20 further comprising a second antibody or a nucleic acid encoding the second antibody, wherein the second antibody is another antibody capable of recognizing a different epitope of FXI or FXIa, or is an antibody capable of specifically binding to a receptor or ligand selected from the group consisting of: thrombin, antiplasmin, Factor XII, Factor VIII, Factor VII, Factor X, Factor IX, Factor II, tissue Factor, P-selectin and ligand thereof, L-selectin and ligand thereof, and any combination of the above antibodies.

23. A kit comprising an instruction for use and one of the following: the antibody or antigen-binding fragment thereof according to claim 1, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a multispecific antibody comprising the antibody or antigen-binding fragment thereof, and another antibody or antigen-binding fragment thereof, or an antibody mimetic, a conjugate comprising the antibody or antigen-binding fragment thereof and a conjugate moiety, or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, the vector, the host cell, the multispecific antibody, or the conjugate, and a pharmaceutically acceptable carrier and/or an excipient.

24.-26. (canceled)

27. A method for preventing and/or treating a disease or disorder associated with coagulation or thromboembolism, and/or delaying the occurrence of a disease or disorder associated with coagulation or thromboembolism, and/or reducing or inhibiting the recurrence of a disease or disorder associated with coagulation or thromboembolism, wherein the method comprises administering to a subject in need thereof an effective amount of one of the following: the antibody or antigen-binding fragment thereof according to claim 1, an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a vector comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a host cell comprising an isolated nucleic acid molecule encoding the antibody or antigen binding fragment thereof, a multispecific antibody comprising the antibody or antigen-binding fragment thereof, and another antibody or antigen-binding fragment thereof, or an antibody mimetic, a conjugate comprising the antibody or antigen-binding fragment thereof and a conjugate moiety, or a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, the vector, the host cell, the multispecific antibody, or the conjugate, and a pharmaceutically acceptable carrier and/or an excipient, the subject is a mammal; preferably, the subject is a human.

28. The method according to claim 27, wherein the method further comprises administering a second therapy to the subject, and the second therapy comprises administering one or more additional drugs, wherein the one or more additional drugs are selected from the group consisting of antiplatelet drugs, anticoagulant drugs, and thrombolytic drugs; or wherein the one or more additional drugs are selected from the group consisting of aspirin, clopidogrel, prasugrel, ticagrelor, abciximab, eptifibatide, vorapaxar, unfractionated heparin, heparin, low molecular weight heparin, warfarin, fondaparinux, edoxaban, betrixaban, rivaroxaban, apixaban, dabigatran etexilate, argatroban, bivalirudin, streptokinase, urokinase, alteplase, prourokinase, and any combination thereof.

29. The method according to claim 27, wherein the disease or disorder associated with coagulation or thromboembolism is selected from the group consisting of: thrombosis, thrombotic stroke, atrial fibrillation, stroke prevention associated with atrial fibrillation (SPAF), deep vein thrombosis, venous thromboembolism, acute coronary syndrome (ACS), ischemic stroke, acute limb ischemia, chronic thromboembolism pulmonary hypertension, systemic embolism, myocardial infarction (MI), acute myocardial infarction (AMI), stable angina pectoris, unstable angina pectoris, reocclusion and restenosis after coronary intervention, peripheral arterial occlusive disease (PAOD), renal vein thrombosis, transient ischemic attack (TIA), pulmonary thromboembolism, diffuse intravascular coagulation, thromboembolic disorder caused by medical device (e.g., catheter), severe systemic inflammatory response syndrome, metastatic cancer, infectious disease, organ failure (e.g., renal failure), toxicity caused by administering a therapeutic protein in body, multiple trauma, ischemia-reperfusion injury, local fibrin deposition, adult alveolar proteinosis, thromboembolic event (VTE) before and after joint replacement (TKA) surgery, coronary heart disease, thromboembolism after myocardial infarction, stroke in patient with non-valvular atrial fibrillation, thrombosis and thromboembolism in chronic kidney disease, thrombosis and thromboembolism in patient undergoing hemodialysis and patient undergoing extracorporeal membrane oxygenation, deep vein thrombosis (DVT), and pulmonary embolism (PE).

30. A method for detecting the presence or level of FXI and/or FXIa in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof according to claim 1 or a conjugate comprising the antibody or antigen-binding fragment thereof and a conjugate moiety, under a condition allowing the formation of a complex, and detecting the formation of the complex.

31. The antibody or antigen-binding fragment thereof of claim 1, comprising 1) a heavy chain constant region (CH) comprising the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof, wherein the variant has a conservative substitution of up to 20 amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to SEQ ID NO: 21; and/or, 2) a light chain constant region (CL) comprising the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof, wherein the variant has a conservative substitution of up to 20 amino acids (e.g., a substitution, deletion or addition of up to 20, up to 15, up to 10, up to 5, 4, 3, 2 or 1 amino acids, or any combination thereof) as compared to SEQ ID NO: 22.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0440] FIG. 1 shows the detection of APTT activity in hybridoma supernatant;

[0441] FIGS. 2A to 2B show the detection of clotting time of murine anti-human FXI/FIXa monoclonal antibody by APTT method;

[0442] FIG. 3 shows the detection of anticoagulant function of murine anti-human FXI/FXIa monoclonal antibody by BIOPHEN method;

[0443] FIG. 4 shows the biological activity of murine anti-human FXI/FXIa monoclonal antibody to specifically block FXI and FXIa;

[0444] FIG. 5 shows the APTT anticoagulant activity test of humanized anti-human FXI/FXIa antibody;

[0445] FIG. 6 shows the detection of anticoagulant function of humanized anti-human FXI/FXIa monoclonal antibody by BIOPHEN method;

[0446] FIG. 7A shows the detection of affinity of humanized anti-human FXI/FXIa antibody to FXIa by ELISA;

[0447] FIG. 7B shows the detection of affinity of humanized anti-human FXI/FXIa antibody to FXI by ELISA;

[0448] FIG. 8 shows the detection of humanized anti-human FXI/FXIa antibody competing with the control antibody BAY-1213790 for binding to FXIa;

[0449] FIG. 9A shows the detection of anticoagulant activity of humanized anti-human FXI/FXIa antibody in human blood by measuring activated partial thromboplastin time (APTT);

[0450] FIG. 9B shows the detection of anticoagulant activity of humanized anti-human FXI/FXIa antibody in monkey blood by measuring activated partial thromboplastin time (APTT);

[0451] FIG. 9C shows the detection of anticoagulant activity of humanized anti-human FXI/FXIa antibody in dog blood by measuring activated partial thromboplastin time (APTT);

[0452] FIG. 9D shows the detection of anticoagulant activity of humanized anti-human FXI/FXIa antibody in rabbit blood by measuring activated partial thromboplastin time (APTT);

[0453] FIG. 9E shows the detection of anticoagulant activity of humanized anti-human FXI/FXIa antibody in rat blood by measuring activated partial thromboplastin time (APTT);

[0454] FIG. 10 shows the accelerated stability test of humanized anti-human FXI/FXIa antibody;

[0455] FIG. 11A shows the in vivo pharmacodynamics (PD) detection of APTT activity of humanized anti-human FXI/FXIa antibody in cynomolgus monkeys;

[0456] FIG. 11B shows the in vivo pharmacodynamic (PD) detection of PT activity of humanized anti-human FXI/FXIa antibody in cynomolgus monkeys.

SEQUENCE INFORMATION

[0457] The information of the sequences involved in the present invention is described in the following table:

TABLE-US-00001 SEQ ID NO Description 1 Murine antibody 36G9.10 heavy chain variable region 2 Murine antibody 36G9.10 light chain variable region 3 IMGT 36G9.10 CDR-H1 4 IMGT 36G9.10 CDR-H2 5 IMGT 36G9.10 CDR-H3 6 IMGT 36G9.10 CDR-L1 7 IMGT 36G9.10 CDR-L2 8 IMGT 36G9.10 CDR-L3 9 AbM 36G9.10 CDR-H1 10 AbM 36G9.10 CDR-H2 11 AbM 36G9.10 CDR-H3 12 AbM 36G9.10 CDR-L1 13 AbM 36G9.10 CDR-L2 14 AbM 36G9.10 CDR-L3 15 Humanized antibody 36G9.10hz73/36G9.10hz74 heavy chain variable region 16 Humanized antibody 36G9.10hz43 heavy chain variable region 17 Humanized antibody 36G9.10hz92/36G9.10hz93 heavy chain variable region 18 Humanized antibody 36G9.10hz43/36G9.10hz73/36G9.10hz93 light chain variable region 19 Humanized antibody 36G9.10hz92 light chain variable region 20 Humanized antibody 36G9.10hz74 light chain variable region 21 Human IgG1 heavy chain constant region (N297A mutant) 22 Human κ light chain constant region 23 Humanized antibody 36G9.10hz73 heavy chain variable region nucleotide sequence 24 Humanized antibody 36G9.10hz73 light chain variable region nucleotide sequence 25 Humanized antibody 36G9.10hz73 heavy chain full length nucleotide sequence 26 Humanized antibody 36G9.10hz73 light chain full length nucleotide sequence 27 Murine antibody 36G9.10 heavy chain variable region nucleotide sequence 28 Murine antibody 36G9.10 light chain variable region nucleotide sequence 29 Murine antibody 7B2 heavy chain variable region 30 Murine antibody 7B2 light chain variable region 31 Humanized antibody 7B2hz11 heavy chain variable region sequence 32 Humanized antibody 7B2hz11 light chain variable region sequence 33 IMGT 7B2 CDR-H1 34 IMGT 7B2 CDR-H2 35 IMGT 7B2 CDR-H3 36 IMGT 7B2 CDR-L1 37 IMGT 7B2 CDR-L2 38 IMGT 7B2 CDR-L3 39 AbM 7B2 CDR-H1 40 AbM 7B2 CDR-H2 41 AbM 7B2 CDR-H3 42 AbM 7B2 CDR-L1 43 AbM 7B2 CDR-L2 44 AbM 7B2 CDR-L3 45 AbM 36G9.10hz73/36G9.10hz74 CDR-H2 46 AbM 36G9.10hz43 CDR-H2 47 AbM 7B2hz11 CDR-H2

EXAMPLES

[0458] The present invention will now be described with reference to the following examples which are intended to illustrate the present invention rather than limit the scope of the present invention.

[0459] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F. M. Ausubel et al., Short Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995. Those skilled in the art know that the examples describe the present invention by way of illustration, and are not intended to limit the scope sought to be protected by the present invention.

Example 1

Preparation of Murine Anti-Human FXI/FXIa Monoclonal Antibody

[0460] The murine anti-human FXI/FXIa monoclonal antibody was obtained by using protein to immunize wild-type Balb/c mice. Each Balb/c mouse was injected subcutaneously with 25 μg of human FXIa (manufacturer: Haematologic Technologies, catalog number HCXI-0150) and 25 μg of human FXI (manufacturer: Haematologic Technologies, catalog number HCXI-0160) emulsified with CFA (complete Freund's adjuvant, manufacturer: Sigma, catalog number F5506) for the first immunization, and booster immunization was carried out once every two weeks, in which emulsion prepared with IFA (incomplete Freund's adjuvant, manufacturer: Sigma, catalog number: F5881) was used in the booster immunization. After immunization for three times, the serum titer was measured by ELISA. Booster immunization was performed 3 to 5 days before the fusion, and mice with higher titer were selected for intraperitoneal immunization with 10 μg of FXIa and 10 μg of FXI. The standard fusion process was adopted to fuse mouse spleen cells and Sp2/0-Ag14 (ATCC, Cat #CRL-1581) mouse myeloma cells in the mode of PEG fusion, and then HAT (manufacturer: Sigma, catalog number: H0262-10VL) was used for stressed screening, and ELISA screening was performed 14 days later. The specific method for ELISA screening comprised: 100 μL/well of 0.5 μg/mL biotin-labeled FXI (manufacturer: Haematologic Technologies, catalog number: HCXI-0150-B) was coated on an ELISA plate (manufacturer: Thermo Fisher Sci., catalog number: 5129) at room temperature for 1 hour. 200 μL of washing buffer (1× TBS comprising 0.05% Tween-20) was used to perform washing for 3 times, 100 μL of hybridoma supernatant was added, and incubated at 37° C. for 1 hour. 200 μL of washing buffer was used to perform washing for 3 times, and 100 μL of Streptavidin-HRP (Pierce, Cat #21130) diluted in a ratio of 1:8000 was added and incubated at 37° C. for 1 hour. 200 μL of washing buffer was used to perform washing for 3 times, 100 μL of TMB (purchased from Thermo Fisher Sci., Cat #TMBW-1000-01) was added and allowed to develop for 10 minutes in the dark, and then 100 μL of stop solution (purchased from Thermo Fisher Sci., Cat #13361-100-10)was added, and reading was performed at 450 nm by a microplate reader.

[0461] The supernatants of 24,000 hybridoma clones were screened by ELISA, and 100 hybridoma clones capable of recognizing biotin-labeled FXI were obtained and transferred to 24-well plates. After 7 to 10 days, the hybridoma supernatants were tested by the APTT assay. The best 22 clones were subcloned by limiting dilution method to obtain monoclonal hybridomas. The monoclonal hybridomas were tested for their anticoagulant activity by the APTT assay by the activated partial thromboplastin time (APTT) kit (Activated Partial Thromboplastin Time Kit, manufacturer: Thermo Fisher Sci, catalog number 100402). The specific steps were as follows: 100 μL of normal human plasma (purchased from Innovative Research, Cat #IPLA-N) was added to a preheated test tube, then 100 μL of APTT reagent (purchased from Thermo Fisher Sci. Cat #100402TS) and 100 μL of the sample to be tested were added and mixed well, incubated at 37° C. for 5 minutes, then 100 μL of calcium chloride (purchased from Thermo Fisher Sci. Cat #100304, 20 mM) was added, the OD405 reading was measured, and the clotting time was calculated by curve fitting, in which Sp2/0-Ag14 cell supernatant was used as a negative control. 14E11 was used as a positive control antibody (prepared with reference to Aronora patent U.S. Pat. No. 8,388,959B2). The fold change shown in the APTT assay results was the ratio of the clotting time measured in the sample added with the experimental antibody to the clotting time measured in the control sample without the antibody. The fold change with a value of 1 or less indicated that the clotting time was not delayed or accelerated, while the fold change with a value of more than 1 indicated that the clotting time was prolonged.

[0462] As shown in FIG. 1, a total of 5 subclonal hybridoma supernatants were tested by APTT, in which 36G9.10 showed a prolonged APTT time, demonstrating a significant anticoagulant function, and could be used for further analysis, while the other clones 6B6.9, 28A8.3, 36C3 and 40F6.9 showed no significant difference in clotting time as compared to the negative control. Through the same screening method, hybridoma 7B2 with prolonged APTT time was obtained for further analysis.

[0463] The monoclonal hybridomas 36G9.10 and 7B2 were subjected to expanding culture without serum to 100-150 mL. The supernatants were purified by Protein G, and the purified murine antibodies were detected by HPLC-SEC, and their purity were all greater than 97%. The purified murine antibodies could be used for further function verification.

Example 2

Function Identification of Murine Anti-Human FXI/FXIa Monoclonal Antibody

[0464] 2.1 Detection of the Function of Murine Anti-Human FXI/FXIa Antibody to Prolong Coagulation

[0465] Using the APTT detection method in Example 1, under the conditions that antibodies were diluted to having a concentration of 2.00, 1.00, 0.50 and 0.25 μg/mL, 36G9.10 was compared to the positive control antibodies BAY-1213790 (prepared with reference to M007-H04 in Bayer patent WO2013167669) and 14E11, and PBS buffer was used as the negative control.

[0466] As shown in FIG. 2A, the candidate murine antibody 36G9.10 showed a significantly prolonged clotting time at each concentration as compared to the control antibodies BAY-1213790 and 14E11. Under the condition of antibody concentration of 5 μg/mL, 7B2 was compared with the positive control antibody 14E11, and PBS buffer was used as the negative control. The results were shown in FIG. 2B. The candidate murine antibody 7B2 showed a significantly prolonged clotting time as compared to PBS, and showed a prolonged clotting time equivalent to that of the positive control antibody 14E11.

[0467] 2.2 Detection of the Activity of Murine Anti-Human FXI/FXIa Antibody to Inhibit FXIa from Catalyzing the Production of FXa and the Anticoagulant Function Thereof

[0468] BIOPHEN Factor XIa kit (Hyphen BioMed, catalog number 220412) was used in the detection according to the kit instructions, and the release of para-nitroaniline (pNA) product was measured under OD405 nm to further confirm the activity of anti-FXI/FXIa antibody to inhibit FXIa from catalyzing the production of FXa. All antibodies had a detection concentration of 0.25 μg/mL. The stronger the function of anti-FXI/FXIa antibody blocking FXI/FXIa activity, the lower the OD405 nm signal value of the pNA product.

[0469] The results were shown in FIG. 3. The 36G9.10 murine antibody showed a significantly stronger anticoagulant function than the control antibodies BAY-1213790 and 14E11.

[0470] 2.3 Affinity Test of Murine Anti-Human FXI/FXIa Monoclonal Antibody

[0471] Octet ForteBio® was widely used in the detection of antibody-antigen dynamic affinity, and it was used to determine the dynamic affinity of the candidate murine antibody 36G9.10 and the control antibodies BAY1213790 and 14E11 to FXIa. The specific experimental steps were as follows: the streptavidin biosensors was first bound with the biotin-labeled antibody to be tested to reach a response signal value of 0.8 nm, then bound with FXIa protein (3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, and 0 μg/mL) for 5 minutes, then dissociation was performed for 7 minutes, and the bivalent analysis model was used for all fitting analysis.

[0472] As shown in Table 1, the 36G9.10 showed a faster binding rate than those of the control antibodies 14E11 and BAY-1213790 (as shown by the Kon values), a slower dissociation rate than that of BAY-1213790 (as shown by the Kdis values), and an affinity stronger by 1.7 and 2.5 times than those of the control antibodies 14E11 and BAY-1213790, respectively.

TABLE-US-00002 TABLE 1 Determination of dynamic affinity of murine anti-FXI/FXIa antibody to FXIa Antibody name KD (M) Kon (1/Ms) Kdis (1/s) R.sup.2 14E11 8.03E−10 7.65E+05 6.14E−04 0.9889 BAY-1213790 1.19E−09 9.18E+05 1.09E−03 0.9786 36G9.10 4.80E−10 1.50E+06 7.20E−04 0.9831

[0473] 2.4 Murine Anti-Human FXI/FXIa Antibody Specifically Blocks the Biological Activities of FXI and FXIa

[0474] In order to confirm that the murine antibody 36G9.10 specifically blocked the biological activities of FXI and FXIa, human plasma lacking FXI (purchased from Innovative Research, catalog number: 50-643-396) was used, and APTT clotting time was determined by adding FXI (0.2, 0.4, 0.8 μg/mL), FXIa (0.2, 0.4, 0.8 μg/mL), FXI (0.4 μg/mL) and 36G9.10 (0.4 μg/mL), FXIa (0.4 μg/mL) and 36G9.10 (0.4 μg/mL).

[0475] The results were shown in FIG. 4. The addition of FXI or FXIa could significantly shorten the clotting time of human plasma lacking FXI, and showed a dose-dependent relationship. When 36G9.10 murine antibody was added, FXI and FXIa showed a significantly prolonged clotting time, indicating that the murine antibody 36G9.10 specifically inhibited the biological activities of FXI and FXIa.

Example 3

Subtype Identification and Variable Region Amplification of Murine Anti-Human FXI/FXIa Antibody

[0476] In order to identify the antibody subtypes of the candidate hybridoma clones, Pierce Rapid Isotyping kit (purchased from Thermo Fisher Sci. Cat #26179) was used to identify the antibody subtypes of the candidate clones 36G9.10 and 7B2. The identification results showed that the candidate clones had a heavy chain of IgG1 subtype, and a light chain of Kappa subtype.

[0477] The hybridoma cells were cultured to have a number of about 8000, the cells were lysed and subjected to the synthesis of first strand cDNA by using a cDNA reverse transcription kit (purchased from Thermo Fisher Sci. Cat #18080-200). Special primers were used to amplify VH and VK genes from cDNA by PCR, and the PCR products were purified by DNA purification kit (purchased from Qiagen, Cat#28104) and ligated to TOPO vector (purchased from Thermo Fisher Sci. Cat #K457540). Approximately 12 clones were picked for each ligation reaction and sequenced. The sequences were analyzed by Vector NTI 11.5 (purchased from Thermo Fisher Sci.) and Sequencer 5.4.6 (purchased from Genecodes), and the variable region sequences and CDR sequences of the murine anti-FXI/FXIa antibodies were obtained as shown in the sequence listing, in which the 36G9.10 murine antibody had a heavy chain variable region as shown in SEQ ID NO: 1, and a light chain variable region as shown in SEQ ID NO: 2; the 7B2 murine antibody had a heavy chain variable region as shown in SEQ ID NO: 29, and a light chain variable region as shown in SEQ ID NO: 30. Further, the chimeric antibodies 36G9.10-hz00 and 7B2-hz00 were constructed respectively for 36G9.10 and 7B2, which were obtained by grafting the heavy chain variable region sequence of 36G9.10 or 7B2 to the mutant human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21), and grafting the light chain variable region sequence of 36G9.10 or 7B2 to the human Kappa light chain constant region (SEQ ID NO: 22).

Example 4

Humanization of Anti-Human FXI/FXIa Murine Antibody

[0478] The murine antibodies 36G9.10 and 7B2 were humanized by the humanization method of CDR grafting. In short, the humanization involves the following steps: the amino acid sequences of murine monoclonal antibody was compared with the amino acid sequence of human germline antibody to find a sequence with high homology and superior physical and chemical properties, which was used as a human germline framework sequence; analyzing and investigating HLA-DR affinity to select a human germline framework sequence with low affinity; then the six CDRs of the murine antibody were grafted into the selected heavy chain and light chain framework sequences.

[0479] Specifically, the heavy chain and light chain CDR regions of the murine antibodies 36G9.10 and 7B2 were respectively grafted into the corresponding humanized template FR. The heavy chain humanization templates for 36G9.10 were human germline gene sequence IGHV1-2*02 (see IMGT accession number X62106) and IGHV1-69-2*01 (see IMGT accession number KF698734), and the light chain humanization templates were human germline gene sequence IGKV1-33*01 (see IMGT accession number M64856) and IGKV1-16*01 (see IMGT accession number J00248). The heavy chain humanization template for 7B2 was human germline gene sequence IGHV1-69-2*01 (see IMGT accession number KF698734), and the light chain humanization template was human germline gene sequence IGKV1-39*01 (see IMGT accession number X59315).

[0480] Furthermore, by using computer simulation technology, the variable region and surrounding framework amino acid sequence thereof were analyzed by molecular docking so as to investigate its spatial binding form. By calculating the electrostatic force, van der Waals force, hydrophobicity and entropy value, the key amino acids in the amino acid sequence of the murine antibody that could interact with coagulation factor XIa and maintain the spatial structure were analyzed, and these murine amino acids were retained in the grafted antibody. That was, a series of back mutations were made to the FR region amino acid residues of the humanized template so that the humanized antibody retained the antigen-binding ability of the murine antibody as much as possible.

[0481] According to the above method, a total of 5 humanized antibodies were constructed on the basis of the CDRs of the murine antibody 36G9.10, where were named as 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, respectively; wherein the heavy chain constant region of each antibody was human IgG1 heavy chain constant region (N297A mutant) (SEQ ID NO: 21). Based on the CDRs of the murine antibody 7B2, the humanized antibody 7B2-hz11 was constructed, wherein the heavy chain constant region was mutant human IgG1 heavy chain constant regions (N297A mutant) (SEQ ID NO: 21). The light chain constant region sequence of the antibodies 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz43, 36G9.10-hz92, 36G9.10-hz93 and 7B2hz11 was SEQ ID NO: 22, none of which had ADCC and CDC effects.

[0482] The amino acid sequences of the variable and constant regions of the humanized antibodies were shown in Table 2.

TABLE-US-00003 TABLE 2 Amino acid sequences of variable and constant regions of humanized anti-human FXI/FXIa antibodies Heavy chain variable Light chain variable Heavy chain constant Light chain constant Name region (SEQ ID NO:) region (SEQ ID NO:) region (SEQ ID NO:) region (SEQ ID NO:) 36G9.10-hz43 16 18 21 22 36G9.10-hz73 15 18 36G9.10-hz74 15 20 36G9.10-hz92 17 19 36G9.10-hz93 17 18 7B2-hz11 31 32

Example 5

Affinity Determination of Anti-Human FXI/FXIa Antibody

[0483] Octet Fortebio® was used for the affinity determination of chimeric and humanized antibodies. The main steps for the determination were as follows: firstly the chimeric and humanized antibodies (having a concentration of 0.3 μg/mL) were immobilized to AHC (anti-human-Fc) sensor, then FXI was subjected to 1:2 gradient dilution with an initial concentration of 3.2 μg/mL, and the association and dissociation rates of the chimeric and humanized antibodies were determined. The data obtained were analyzed by Octet data analysis software.

[0484] The results were shown in Table 3, which indicated that the chimeric antibody 36G9.10-hz00 and the humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93 all had an affinity KD stronger than that of the positive control BAY-1213790.

TABLE-US-00004 TABLE 3 Determination of the dynamic affinity of humanized anti-human FXI/FXIa antibodies to human FXI Kinetic data Antibody KD (M) kon(1/Ms) kdis(1/s) BAY-1213790 3.74E−10 9.10E+05 3.40E−04 36G9.10-hz00 3.66E−12 6.52E+06 2.39E−05 36G9.10-hz43 4.41E−11 5.77E+06 2.55E−04 36G9.10-hz73 1.03E−11 6.31E+06 6.51E−05 36G9.10-hz74 7.98E−12 6.47E+06 5.16E−05 36G9.10-hz92 1.52E−11 5.07E+06 7.69E−05 36G9.10-hz93 8.81E−12 6.06E+06 5.34E−05

Example 6

Determination of APTT Anticoagulant Activity, Inhibition of the Activity of FXIa to Catalyze the Production of FXa and Anticoagulant Function of Chimeric and Humanized Anti-Human FXI/FXIa Antibodies

[0485] The anticoagulant activity of the humanized antibodies was detected using the APTT Coagulation Test Kit, and the specific detection method was shown in Example 1. The results were shown in FIG. 5. The mean value and standard deviation were calculated by performing the determination for 4 times. The chimeric antibody 36G9.10-hz00 and the humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93 all showed a prolonged clotting time greater than those of the positive control antibodies 14E11 and BAY-1213790.

[0486] Using Biophen Factor XIa kit (purchased from Aniara Cat #220412), the anticoagulant activity of the humanized antibodies was detected, and the specific method was shown in Example 2.

[0487] The detection results were shown in FIG. 6, in which the chimeric antibody 36G9.10-hz00, the humanized antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz74, 36G9.10-hz92, 36G9.10-hz93, the antibodies 14E11 and BAY-1213790 all could effectively reduce the production of FXa, and the antibodies 36G9.10-hz43, 36G9.10-hz73, 36G9.10-hz92, 36G9.10-hz93 could more effectively reduce the production of FXa as compared to the control antibodies 14E11 and BAY-1213790, indicating that they had superior anticoagulant activity.

Example 7

Determination of Affinity of Humanized Anti-Human FXI/FXIa Antibody to FXI/FXIa

[0488] ELISA method was used to detect the affinity of 36G9.10-hz73 to human FXIa. The specific steps were as follows: FXIa antigen (Haematologic Technologies, HCXIA-160) was diluted to 1 μg/mL with CBS coating solution (0.32 g of Na.sub.2CO.sub.3, 0.59 g of NaHCO.sub.3 were dissolved in deionized water, and diluted to 200 ml), and the coating of FXIa antigen at 100 μL/well was performed overnight at 4° C.; the liquid in the well was discarded on the next day, and washing was performed once with 300 μL of PBS; 100 μL of PBS (comprising 2% BSA, BOVOGEN, BSAS 1.0) was added, and subjected to blocking at 37° C. for 2 hours; PBS (comprising 2% BSA) was used to dilute antibodies 36G9.10-hz73 and BAY-1213790 (starting at 10 μg/mL, 4-fold dilution, 12 concentration points), 100 μL thereof was added to the corresponding well, and incubated at 37° C. for 2 hours; washing was performed with 300 μL of PBST for 3 times; HRP-labeled goat-anti-human secondary antibody (purchased from Jackson, 109-035-00) was subjected to 1:10000 dilution with PBS (comprising 2% BSA), and 100 μL thereof was added to the corresponding well, and incubated at 37° C. for 1 hour; washing was performed with 300 μL of PBST for 5 times; 100 μL of TMB color developing solution (purchased from BioPanda, TMB-S-004) was added to the corresponding well, and allowed to develop color at room temperature for 20 minutes; the color development was stopped with the addition of 50 μL of 2N H.sub.2SO.sub.4, and reading at OD450 nm was performed by using microplate reader (purchased from MD, SpectraMax M2) and the results were imported into Graphpad Prism for curve fitting.

[0489] The experimental results were shown in FIG. 7A, in which the affinity EC50 of 36G9.10-hz73 to FXIa was 2.488 ng/mL, and the affinity EC50 of BAY-1213790 to FXIa was 6.163 ng/mL, indicating that 36G9.10-hz73 was better than BAY-1213790.

[0490] ELISA method was used to detect the affinity of 36G9.10-hz73 to human FXI, in which FXI antigen (obtained by cloning PMD-F11 plasmid purchased from Sino Biological into PLVX-IRES-PURO vector and transfecting into 293F cell for expression) was diluted with CBS coating solution to 1 μg/mL, and the coating at 100 μL per well was performed overnight at 4° C. The remaining steps were as shown in the previous sections, except that the antibody had an initial concentration of 0.37 μg/mL, with 3-fold dilution and 9 concentration points.

[0491] The experimental results were shown in FIG. 7B, in which the affinity EC50 of 36G9.10-hz73 to FXI was 9.68 ng/mL, and BAY-1213790 did not bind to FXI, indicating that 36G9.10-hz73 could neutralize FXI and FXIa at the same time and exert better anticoagulant function.

Example 8

Detection of Anti-Human FXI/FXIa Antibody Competing with Control Antibody for Binding to FXIa

[0492] In order to determine whether 36G9.10-hz73, BAY-1213790 and 14E11 recognize the same epitope of FXIa, a competitive ELISA method was used for detection. The specific steps were as follows: FXIa antigen was diluted to 1 μg/mL with CBS coating solution, and the coating of FXIa antigen at 100 μL per well was performed at 4° C. overnight; the liquid in the well was discarded on the next day, and the washing was performed with 300 μL of PBS; 100 μL of PBS (comprising 2% BSA) was added and subjected to blocking at 37° C. for 2 hours; the biotin-labeled 36G9.10-hz73 antibody was diluted with PBS (comprising 2% BSA) to 10 ng/mL, and this was used as a mother solution to dilute the antibodies BAY-1213790 and 14E11 to 10 μg/mL, 100 μL thereof was added into the corresponding well, 10 μg/mL 36G9.10-hz73 antibody was used as a positive control, 10 ng/mL biotin-labeled 36G9.10-hz73 antibody was used as a negative control, a blank control was also set, and they were all incubated at 37° C. for 2 hours; the washing was performed with 300 μL of PBST for 3 times; HRP-Streptavidin secondary antibody (purchased from Proteintech, SA00001-0) was subjected to dilution at a ratio of 1: 3000 with PBS (comprising 2% BSA), 100 μL thereof was added to the corresponding well, and incubated at 37° C. for 1 hour; washing was performed with 300 μL of PBST for 5 times; 100 4, of TMB color developing solution (purchased from BioPanda, TMB-S-004) was added to the corresponding well and allowed to develop color at room temperature for 20 minutes; the color development was stopped with the addition of 50 μL of 2N H.sub.2SO.sub.4, and the reading at OD450 nm was performed by a microplate reader (purchased from MD, SpectraMax M2) and the results were imported into Graphpad Prism for plotting.

[0493] The experimental results were shown in FIG. 8, in which BAY-1213790 and 14E11 did not compete with 36G9.10-hz73 for binding to FXIa, indicating that the former two and 36G9.10-hz73 bound to different epitopes of FXIa.

Example 9

Inhibition of the Catalytic Effect of FXIa on Substrate by Anti-Human FXI/FXIa Antibody

[0494] In 1988, Shun-ichiro KAWABATA et al. screened out several FXIa-specific fluorescently labeled substrates for sensitive detection of human FIXa enzyme activity in vitro (see Eur. J. Biochem. 172, 17-25 (1988), for details). Therefore, by detecting the catalytic cleavage of the specific fluorescent substrate (1-1575, Bachem) by human FXIa, the activity of human FXIa and the effective neutralization of FXIa by the humanized anti-human FXI/FXIa antibody were determined. The specific steps were as follows: human FXIa was diluted to 1 nM with a buffer comprising 50 mM Tris/HCl, 100 mM NaCl, 5 mM CaCl.sub.2 and 0.1% BSA, and this was used as a mother solution to serially dilute the antibodies 36G9.10-hz73 and BAY-1213790, with an initiative concentration of 20 μg/mL, 4-fold dilution, 14 concentration points; 10 μL of the diluted antibody was added to each well of a 384-well plate (purchased from Coring, 4514), and incubated at 37° C. for 1 hour; after incubation, 10 μL of fluorescent substrate 1-1575 with a concentration of 2 μM was added into each well, mixed and immediately measured by a microplate reader to continuously read the fluorescence values at 360/465 nm, which were imported into Graphpad Prism for curve fitting.

[0495] The experimental results were shown in Table 4, in which the antibody 36G9.10-hz73 could effectively inhibit the catalytic effect of FXIa on the fluorescent substrate, and had IC50 of 26.215 ng/mL, and BAY-1213790 had IC50 of 69.96 ng/mL, indicating that 36G9.10-hz73 was better than BAY-1213790.

TABLE-US-00005 TABLE 4 Inhibition of the catalytic effect of FXIa on fluorescent substrate by humanized anti-human FXI/FXIa antibody Antibody name IC50 value (ng/mL) 36G9.10-hz73 26.215 ± 3.365 (n = 2) BAY-1213790 69.96 ± 7.44 (n = 2)

Example 10

Detection of Anticoagulant Activity of Anti-Human FXI/FXIa Antibody by Measuring Activated Partial Thromboplastin Time (APTT)

[0496] After APTT reagent was added to anticoagulated plasma, the endogenous coagulation pathway was activated to activate XI to XIa, and an antibody targeting FXIa could inhibit the activity of FXIa, thereby prolonging APTT. Therefore, the determination of APTT was used to detect the anticoagulant activity of humanized anti-human FXI/FXIa antibody. The specific steps were as follows: venous blood from human, monkey, dog, rabbit and rat were collected respectively, 2 to 3 samples were taken for each species, 4 tubes of blood were taken for each sample (1.8 ml per tube), subjected to anticoagulation using 3.2% sodium citrate and centrifugation at 2500×g, 4° C. for 10 minutes, then the resultant plasma was taken and stored at 4° C. for later use (all plasma of each sample were combined); the antibody to be tested was diluted with physiological saline, starting at 1500 μg/mL, 3 concentration points, and used as a 10× mother solution of the antibody to be tested; at high concentration, the sample detection system was 150 μl, in which 135 μl of plasma was taken and added with 15 μl of the mother solution of the antibody to be tested (i.e., the mother solution of the antibody to be tested was diluted by 10 times), and then a fully-automated blood coagulation analyzer (purchased from Sysmex CA1500) was used to detect APTT; at low concentration, the sample detection system was 75 μl, in which 67.5 μl of plasma was taken and added with 7.5 μl of the mother solution of the antibody to be tested (i.e., the mother solution of the antibody to be tested was diluted by 10 times), and then APTT was detected with the fully-automated blood coagulation analyzer.

[0497] The results of the experiment were shown in FIG. 9A, in which all of the 4 tested antibodies 36G9.10-hz73, BAY-1213790, BMS-962212 (prepared by referring to J Med Chem. 2017 Dec. 14; 60(23): 9703-9723. doi: 10.1021/acs.jmedchem.7b01171. Epub 2017 Nov. 17) and 14E11 showed a prolonged APTT in human plasma, the 36G9.10-hz73 showed the best effect with a prolonged APTT by 2.2 times at 6 μg/mL, which was better than those of the other three. As shown in FIGS. 9B to 9E, 36G9.10-hz73 only prolonged APTT clotting time in monkey plasma, while BAY-1213790 prolonged APTT clotting time in plasma of monkey and rabbit, and 14E11 prolonged APTT clotting time in plasma of monkey, rabbit and rat, indicating that 36G9.10-hz73 was different from BAY-1213790 and 14E11 in term of species cross, so that it was speculated that 36G9.10-hz73 bound to an epitope of FXI/FXIa different from those to which BAY-1213790 and 14E11 bound.

[0498] The prothrombin time (PT) test mainly reflected the condition of the exogenous coagulation system. The fold change shown in the PT test results was a ratio of the clotting time measured in the sample added with the experimental antibody to the clotting time measured in the control sample without the antibody. The fold change with a value of 1 or less indicated that the clotting time was not delayed or accelerated, while the fold change with a value of more than 1 indicated that the clotting time was prolonged.

[0499] Using the same treatment method as above, the effects of 36G9.10-hz73, BAY-1213790, BMS-962212 and 14E11 on PT were detected with the fully-automated blood coagulation analyzer. The detection results were shown in Table 5, in which all samples did not prolong the PT (in terms of fold change) in the plasma of 5 species as compared with PBS control even at the concentration of 150 μg/mL (1 μM), indicating that they had no effect on exogenous coagulation, and did not increase the risk of bleeding.

TABLE-US-00006 TABLE 5 Detection of PT activity of humanized anti-human FXI/FXIa antibody Antibody name Human Monkey Dog Rabbit Rat 36G9.10- 0.96 ± 0.04 1.00 ± 0.01 1.03 ± 0.01 0.99 ± 0.01 0.99 ± 0.01 hz73 BAY- 0.95 ± 0.05 1.00 ± 0.01 1.02 ± 0.01 0.99 ± 0.01 0.99 ± 0.01 1213790 14E11 0.95 ± 0.05 / / 1.00 ± 0.01 1.00 ± 0.01 BMS-962212 0.97 ± 0.06 0.99 ± 0.01 1.01 ± 0.01 1.00 ± 0.00 1.00 ± 0.01

Example 11

Detection of Accelerated Stability of Anti-Human FXI/FXIa Antibody

[0500] The antibody 36G9.10-hz73 was diluted with 20mM His-HCl (hydrochloric acid-histidine buffer) (comprising 0.03% Tween-20, pH5.5) to a concentration of 11.5 mg/mL, and allowed to stand at 40° C. and 25° C. for 14 days and 28 days, respectively, then the APTT detection method in Example 4 was used to detect the anticoagulant activity of the sample after acceleration, and the purity of the antibody after acceleration was detected by SEC.

[0501] The experimental results were shown in FIG. 10 and Table 6, in which the antibody 36G9.10-hz73 stood at 40° C. and 25° C. for 14 days and 28 days showed no significant change in anticoagulant activity and purity.

TABLE-US-00007 TABLE 6 Detection of accelerated stability of humanized anti-human FXI/FXIa antibody Conditions of Purity by SEC APTT detection placement detection (%) (EC50, nM) 0 days 99.4 18.36 25° C.-28 days 99.8 17.36 40° C.-14 days 97.9 20.33 40° C.-28 days 94.4 26.89

Example 12

Detection of Pharmacokinetics (PK) and Pharmacodynamics (PD) of Anti-Human FXI/FXIa Antibody

[0502] In order to detect the PK and PD of 36G9.10-hz73 in vivo, different doses of antibody 36G9.10-hz73 was administered to cynomolgus monkeys by a single intravenous injection (IV) or subcutaneous injection (SC), and blood samples were taken at different time points to detect PK and APTT activity. The specific administration groups and dosages were shown in Table 7, in which the volume of one single intravenous injection or subcutaneous administration was 2 mL/kg, the intravenous injection site was the veins of the limbs, and the subcutaneous injection site was the back of the neck.

TABLE-US-00008 TABLE 7 Dosage regimen of humanized anti-human FXI/FXIa antibody for detection of PK and PD Dose Number of Group (mg/kg) Administration mode animals Gender of animals 1 1 Intravenous injection 2 1 male + 1 female 2 3 Intravenous injection 3 1 male + 2 female 3 3 Subcutaneous injection 3 2 male + 1 female

[0503] After completing the blood collection at all time points, the antibody 36G9.10-hz73 was used as the standard, and the antibody concentration standard curve was drawn using the ELISA method in Example 7, and the blood concentration of the antibody 36G9.10-hz73 at each time point was determined by using the standard curve under the same conditions, and finally the measured blood concentrations were imported into Graphpad Prism for curve fitting, and the PK of 36G9.10-hz73 in cynomolgus monkey was calculated.

[0504] At the same time, immediately after each blood sampling, the method in Example 10 was used to detect APTT, and finally the data at all time points were imported into Graphpad Prism for curve fitting, and the PD of 36G9.10-hz73 in cynomolgus monkey was calculated.

[0505] The PK test results were shown in Table 8, in which the intravenous injection of 3 mg/kg 36G9.10-hz73 antibody showed a half-life of 290.15 hours in cynomolgus monkeys, and the subcutaneous injection of 3 mg/kg 36G9.10-hz73 antibody showed a half-life of 188.02 hours in cynomolgus monkeys. The subcutaneous injection of 3 mg/kg 36G9.10-hz73 antibody showed a bioavailability of 94%±2% (n=2).

TABLE-US-00009 TABLE 8 Determination of PK of humanized anti-human FXI/FXIa antibody Administration mode Half-life (hours) AUC (0-t, h*μg/mL) Intravenous injection-1 mg/kg 355.37 3997.79 Intravenous injection-3 mg/kg 290.15 5035.86 Subcutaneous injection-3 mg/kg 188.02 9581.64

[0506] The PD detection results showed that 36G9.10-hz73 can be used for intravenous injection and subcutaneous administration to achieve a good effect of prolonging APTT. As shown in FIG. 11A, the dose-effect relationship between the two intravenous injection dose groups was clearly shown, the 3 mg/kg subcutaneous injection group showed a slightly better effect on prolonging APTT than the intravenous injection group, having an effect of prolonging APTT by about 1.3 times at 1008 hours; as shown in FIG. 11B, the three dose groups did not prolong PT, indicating that they had no effect on exogenous coagulation. Therefore, the 36G9.10-hz73 antibody had the potential clinical advantage of not causing the risk of bleeding.

[0507] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been disclosed, and these changes are within the protection scope of the present invention. All of the present invention is given by the appended claims and any equivalents thereof.