WO2022002249A1 - 抗FXI/FXIa抗体、其抗原结合片段及医药用途 - Google Patents

抗FXI/FXIa抗体、其抗原结合片段及医药用途 Download PDF

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WO2022002249A1
WO2022002249A1 PCT/CN2021/104253 CN2021104253W WO2022002249A1 WO 2022002249 A1 WO2022002249 A1 WO 2022002249A1 CN 2021104253 W CN2021104253 W CN 2021104253W WO 2022002249 A1 WO2022002249 A1 WO 2022002249A1
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seq
fxi
antibody
set forth
antigen
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PCT/CN2021/104253
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English (en)
French (fr)
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王雷
贺旭刚
张瑾
刘潇
胡冬梅
杜延平
吴然
申晨曦
杨阳
杨昌永
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北京拓界生物医药科技有限公司
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Priority to CN202311474389.9A priority Critical patent/CN117487017A/zh
Priority to KR1020237003848A priority patent/KR20230034361A/ko
Priority to AU2021302202A priority patent/AU2021302202A1/en
Priority to CA3184718A priority patent/CA3184718A1/en
Priority to US18/013,524 priority patent/US20230220111A1/en
Priority to BR112022026533A priority patent/BR112022026533A2/pt
Priority to CN202180004913.9A priority patent/CN114269790B/zh
Priority to CN202311469265.1A priority patent/CN117487016A/zh
Priority to JP2022579856A priority patent/JP2023532251A/ja
Priority to MX2022015959A priority patent/MX2022015959A/es
Priority to EP21832002.6A priority patent/EP4177274A4/en
Publication of WO2022002249A1 publication Critical patent/WO2022002249A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present application relates to anti-FXI/FXIa antibodies, antigen-binding fragments thereof, pharmaceutical compositions comprising the anti-FXI/FXIa antibodies and antigen-binding fragments thereof, and their medicinal uses for treating or preventing thrombosis-related diseases.
  • FXI is a key coagulation factor in the endogenous pathway.
  • FXIa is the activated state of FXI. In the coagulation cascade, it can not only be activated by the endogenous coagulation factor FXIIa to form FXIa, but also be activated by thrombin II, thereby amplifying the coagulation cascade and forming more thrombin and fibrin . During this process, excess thrombin and fibrin may be formed, leading to thrombotic diseases.
  • the mild bleeding phenotype in human FXI deficiency (hemophilia C) patients showed that the risk of bleeding was lower when FXI was inhibited. Further research found that in FXI-deficient patients, the incidence of ischemic stroke and deep vein thrombosis was significantly reduced, indicating that inhibition of FXI is beneficial to reduce the risk of ischemic stroke and deep vein thrombosis.
  • the FXI gene is located on human chromosome 4 and encodes a secreted protein consisting of 607 amino acids.
  • the FXI protein molecule contains four apple domains and one catalytic domain.
  • FXI exists as a homodimer in human blood, and the concentration of circulating FXI in human plasma that forms a non-covalent complex with HK is about 30 nM (15-45 nM).
  • Human FXI molecules share 88%, 67% and 58% (two species, three homology data) homology with monkey and mouse FXI molecules, respectively.
  • the present disclosure provides an anti-FXI/FXIa antibody or an antigen-binding fragment thereof, a nucleic acid encoding it, a vector, a host cell, a pharmaceutical composition, and a method for treating or delaying thrombosis or thromboembolism-related diseases or disorders or complications thereof , and its detection purposes.
  • an anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises:
  • a heavy chain HCDR1 comprising a sequence shown in X 1 X 2 X 3 MH (SEQ ID NO: 63), wherein X 1 is selected from E, S, G or D, and X 2 is selected from L, I, V or D, X 3 is selected from S, F, L, or the Y; and / or
  • Heavy chain HCDR2 which comprises the sequence shown in X 4 X 5 DPX 6 X 7 GX 8 TX 9 YAX 10 KFQG (SEQ ID NO: 64), wherein X 4 is selected from G or W, X 5 is selected from F or I, X 6 is selected from E or Q, X 7 is selected from D or N, X 8 is selected from E or D, X 9 is selected from I, R, V or E; X 10 is selected from Q or S; and/or
  • a heavy chain HCDR3 comprising the sequence shown in DPHRTWWRYFDWLYPRGMDV (SEQ ID NO: 9) or GNFYYFDY (SEQ ID NO: 39); and/or
  • a light chain LCDR1 comprising the sequence shown in RASQTVGKNYLA (SEQ ID NO: 10) or SASSSINYMH (SEQ ID NO: 40); and/or
  • Light chain LCDR2 which comprises the sequence shown in X 11 X 12 SX 13 X 14 AX 15 (SEQ ID NO: 65), wherein X 11 is selected from G, E or D, X 12 is selected from A or T, X 13 is selected from From N, V or K, X 14 is selected from R or L, X 15 is selected from T, L or S; and/or
  • Light chain LCDR3 it comprises the sequence shown in X 17 QX 18 X 19 X 20 X 21 PX 22 T (SEQ ID NO: 66), X 17 is selected from Q or H, X 18 is selected from F or R, X 19 is selected from R or S, X 20 is selected from S or F, X 21 is selected from Y or S, and X 22 is selected from Y or L.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises:
  • a heavy chain HCDR1 comprising the sequence shown in one of SEQ ID NOs: 7, 22, 24, 26, 28, 37;
  • a heavy chain HCDR2 comprising the sequence shown in one of SEQ ID NOs: 8, 23, 25, 27, 38;
  • a heavy chain HCDR3 comprising the sequence shown in one of SEQ ID NOs: 9 and 39;
  • a light chain LCDR1 comprising a sequence shown in one of SEQ ID NOs: 10 and 40;
  • a light chain LCDR2 comprising the sequence shown in one of SEQ ID NOs: 11, 29, 41;
  • a light chain LCDR3 comprising the sequence shown in one of SEQ ID NOs: 12 and 42.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises:
  • heavy chain HCDR1, HCDR2, HCDR3 respectively comprise the sequence shown in SEQ ID NO: 7, 8, 9, light chain LCDR1, LCDR2, LCDR3 respectively comprise the sequence shown in SEQ ID NO: 10, 11, 12;
  • the heavy chain HCDR1, HCDR2 and HCDR3 respectively comprise the sequences shown in SEQ ID NOs: 22, 23 and 9, and the light chains LCDR1, LCDR2 and LCDR3 respectively comprise the sequences shown in SEQ ID NOs: 10, 29 and 12;
  • the heavy chain HCDR1, HCDR2 and HCDR3 respectively comprise the sequences shown in SEQ ID NOs: 24, 25 and 9, and the light chains LCDR1, LCDR2 and LCDR3 respectively comprise the sequences shown in SEQ ID NOs: 10, 29 and 12;
  • the heavy chain HCDR1, HCDR2 and HCDR3 respectively comprise the sequences shown in SEQ ID NOs: 26, 27 and 9, and the light chains LCDR1, LCDR2 and LCDR3 respectively comprise the sequences shown in SEQ ID NOs: 10, 29 and 12;
  • heavy chain HCDR1, HCDR2, HCDR3 respectively comprise the sequence shown in SEQ ID NO: 28, 25, 9, light chain LCDR1, LCDR2, LCDR3 respectively comprise the sequence shown in SEQ ID NO: 10, 29, 12;
  • heavy chain HCDR1, HCDR2, HCDR3 respectively comprise the sequence shown in SEQ ID NO: 37, 38, 39, and light chain LCDR1, LCDR2, LCDR3 comprise the sequence shown in SEQ ID NO: 40, 41, 42 respectively;
  • an anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises a CDR sequence of (a), (b), (c), (d), (e) or a related (g) scheme, it is selected Binds sexually to FXIa, but not to FXI.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises the CDR sequences of (f) or related (g) schemes, it binds FXI and also binds FXIa; Binds to FXIa, but does not affect FXIa activity.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody, a humanized antibody, a human antibody or a fragment thereof; antibodies or fragments thereof. It can be a full-length antibody or a fragment thereof.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof is a humanized antibody or fragment thereof
  • the humanized light chain template may be IGKV3-11*01
  • the heavy chain template may be IGHV1-69-2* 01.
  • the humanization process further includes backmutation of VH, VL.
  • the VH has a back mutation of any one or any combination of Y27F, T28N, F29I, T30K, A93L, R94Y, E73T, R66K, V67A, T75A, T76N.
  • the VL has a back mutation of any one or any combination of R45K, L46R, L47W, I58V, F71Y.
  • the heavy chains HCDR1, HCDR2 and HCDR3 of the above-mentioned humanized antibodies or fragments thereof respectively comprise the sequences shown in SEQ ID NOs: 37, 38 and 39
  • the light chains LCDR1, LCDR2 and LCDR3 respectively comprise SEQ ID NOs: Sequences shown at 40, 41, 42.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises:
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises:
  • VH as set forth in SEQ ID NO: 20 or having at least 80% identity
  • VL as set forth in SEQ ID NO: 21 or having at least 80% identity
  • the VH of the anti-FXI/FXIa antibody or antigen-binding fragment thereof is linked to human or mouse CH1 and the VL is linked to human or mouse CL or CK.
  • the human CH is shown in SEQ ID NO: 13 or 59, and the C ⁇ is shown in SEQ ID NO: 14, for example.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof is a murine antibody or fragment thereof.
  • the light chain variable region thereof comprises the light chain FR region and/or the light chain constant region of the murine kappa, lambda chain or variants thereof.
  • the murine anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises a heavy chain FR region and/or a heavy chain constant region of a murine IgGl, IgG2, IgG3, IgG4 or variant thereof.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof is a chimeric antibody or fragment thereof. It comprises light chain FR regions and/or light chain constant regions of human kappa, lambda chains or variants thereof, and/or heavy chain FR regions and/or heavy chains of human IgG1, IgG2, IgG3 or IgG4 or variants thereof. chain constant region.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises a constant region Fc, eg, the Fc of IgGl, IgG2, IgG3, IgG4, IgG4P (ie, the S241P mutant of IgG4).
  • the Fc sequence of the IgG1 is shown in, for example, SEQ ID NO: 67
  • the sequence of the IgG4P Fc ie, the IgG4 Fc comprising S241P
  • SEQ ID NO: 60 is shown in, for example, SEQ ID NO: 60.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof has a heavy chain as set forth in or at least 80% identity to SEQ ID NO: 15 and a light chain as set forth in or with SEQ ID NO: 16 at least 80% identical; or
  • the heavy chain is set forth in or at least 80% identical to SEQ ID NO: 61 and the light chain is set forth in or at least 80% identical to SEQ ID NO: 62.
  • the antigen-binding fragment of an anti-FXI/FXIa antibody is a Fab, Fv, sFv, Fab', F(ab') 2 , linear antibody, single-chain antibody, scFv, sdAb, sdFv, Nanobody, peptidobody ( peptibody), domain antibodies and multispecific antibodies (bispecifics, diabodies, triabodies and tetrabodies, tandem di-scFv, tandem tri-scFv), e.g. Specifically scFv, Fv, Fab or Fab' fragments.
  • an anti-FXI/FXIa antibody or antigen-binding fragment thereof that binds the same epitope as the aforementioned anti-FXI/FXIa antibody or antigen-binding fragment thereof.
  • anti-FXI/FXIa antibodies or antigen-binding fragments thereof are provided that cross-block the binding of the aforementioned anti-FXI/FXIa antibodies or antigen-binding fragments thereof to human FXI/FXIa.
  • anti-FXI/FXIa antibodies or antigen-binding fragments thereof are provided whose binding to human FXI/FXIa is cross-blocked by the aforementioned anti-FXI/FXIa antibodies or antigen-binding fragments thereof.
  • anti-FXI/FXIa antibodies or antigen-binding fragments thereof are provided that are at least 80% identical to the heavy and/or light chains of the aforementioned anti-FXI/FXIa antibodies or antigen-binding fragments thereof.
  • At least 80% encompasses 80% and above, such as at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%.
  • an anti-FXI/FXIa antibody or antigen-binding fragment variant thereof comprising 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid changes.
  • the amino acid changes may be conservative substitutions of amino acid residues in the variable regions.
  • the above-described anti-FXI/FXIa antibodies or antigen-binding fragments thereof may have any or any of the following properties:
  • FXI and/or FXIa Block the binding of FXI and/or FXIa to a member of the coagulation pathway (preferably, the member is selected from one or more of coagulation factor IX, coagulation factor XIIa, thrombin);
  • the measurement method of the above-mentioned affinity is, for example, BIACORE TM .
  • the present disclosure provides a polynucleotide encoding any of the aforementioned anti-FXI/FXIa antibodies or antigen-binding fragments thereof or anti-FXI/FXIa antibodies or antigen-binding fragments thereof, the polynucleotide may be DNA or RNA .
  • the present disclosure provides an expression vector containing the above-mentioned polynucleotide, and the expression vector is selected from the group consisting of: eukaryotic expression vector, prokaryotic expression vector, and viral vector.
  • the present disclosure provides a host cell transformed with an expression vector as described above, which may be a eukaryotic cell, or a prokaryotic cell.
  • the host cell is selected from the group consisting of: bacteria, yeast, mammalian cells. In some specific embodiments, the host cell is selected from: Escherichia coli, Pichia pastoris, Chinese hamster ovary (CHO) cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure provides a method for preparing an anti-FXI/FXIa antibody or antigen-binding fragment thereof, comprising: expressing the antibody or antigen-binding fragment thereof in a host cell as previously described, and extracting the antibody or antigen-binding fragment thereof from the host cell The antibody or antigen-binding fragment thereof is isolated.
  • the present disclosure provides a composition, such as a pharmaceutical composition, comprising: a pharmaceutically acceptable excipient, dilution or carrier; and a therapeutically or prophylactically effective amount of an anti-FXI/FXIa antibody as described above or Antigen-binding fragments.
  • the unit dose of the pharmaceutical composition may contain 0.01 to 99% by weight of an anti-FXI/FXIa antibody or an antigen-binding fragment thereof, or a unit dose of the pharmaceutical composition may contain an anti-FXI/FXIa antibody or The amount of antigen-binding fragment thereof is 0.1-2000 mg, in some embodiments 1-1000 mg.
  • the above-mentioned pharmaceutical composition is in the form of subcutaneous administration or intravenous administration.
  • a pharmaceutical composition for subcutaneous administration comprising a therapeutically or prophylactically effective amount of the aforementioned anti-FXI/FXIa antibody or antigen-binding fragment thereof of the present disclosure, e.g., the heavy chain variable region of the antibody comprises SEQ ID The sequence shown in NO: 58, the light chain variable region comprises the sequence shown in SEQ ID NO: 51; for example, the full length of the heavy chain of the antibody comprises the sequence shown in SEQ ID NO: 61, and the full length of the light chain comprises the sequence shown in SEQ ID NO: 61 : the sequence shown in 62.
  • the present disclosure provides the use of any one or a combination selected from the group consisting of: an anti-FXI/FXIa antibody or antigen-binding fragment thereof according to the present disclosure, a pharmaceutical composition according to the present disclosure.
  • the medicament is for the treatment or prevention of thrombosis or a thromboembolic-related disease or disorder, such as ischemic stroke and/or deep vein thrombosis associated with atrial fibrillation.
  • the present disclosure provides a method of treating or preventing or delaying progression of a thrombosis or thromboembolism-related disease or disorder (or its complications), the method comprising administering to a subject or an anti-FXI/FXIa antibody or antigen-binding fragment thereof according to the present disclosure, or a pharmaceutical composition according to the present disclosure, in an amount effective to treat or delay the disease.
  • anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure can be applied to clot formation, thrombosis, or thromboembolism-related diseases or disorders.
  • thrombosis or thromboembolism-related diseases or conditions include, but are not limited to, cardiac coronary disease (such as acute coronary syndrome (ACS)), myocardial infarction with ST-segment elevation (STEMI), and myocardium without ST-segment elevation Infarction (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention (such as angioplasty, stenting, or aortocoronary bypass), peripheral arterial occlusive disease , pulmonary embolism, venous thromboembolism, venous thrombosis (especially in the deep and renal veins of the lower extremities), transient ischemic attack, and thrombotic and thromboembolic stroke, caused by chronic thromboembolism (CTEPH) pulmonary disease or pulmonary hypertension.
  • CTEPH chronic thromboembolism
  • Stimulation of the coagulation system can occur through a variety of triggers or related conditions.
  • the coagulation system can be highly activated and there may be thrombotic complications, especially venous thrombosis. Therefore, the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present disclosure can be used to prevent thrombus in the case of surgical intervention, especially for patients suffering from cancer or undergoing orthopaedic surgery such as hip or knee replacement ) patients.
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present disclosure are also useful for preventing thrombus in patients with activated coagulation systems, eg, in the irritating conditions described.
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure are for use in the treatment and/or prevention of acute, intermittent, or persistent arrhythmias (eg, atrial fibrillation), or undergoing cardioversion, or suffering from cardiac arrhythmias Valve disease, or cardiogenic thromboembolism (eg, cerebral arterial ischemia, stroke, and systemic and local thromboembolism) in patients with prosthetic heart valves.
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure are used for the treatment and/or prevention of disseminated intravascular coagulation (DIC).
  • DIC disseminated intravascular coagulation
  • the occurrence of the disease may be particularly associated with sepsis, but may also be attributed to surgical intervention, neoplastic disease, burns or other injuries, and may lead to severe organ damage through microthrombosis.
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure are used for the treatment and/or prevention of thromboembolic complications.
  • Such complications may occur, for example, in microangiopathic hemolytic anemia and, in the case of extracorporeal circulation, due to blood contact with foreign surfaces (eg, hemodialysis, ECMO ("Extracorporeal Membrane Oxygenation") , LVAD (“left ventricular assist device”) and similar methods).
  • Anti-FXI/FXIa antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present disclosure are useful for the treatment and/or prevention of diseases involving microclot formation or fibrin deposition in cerebral blood vessels, which may lead to dementia, such as blood vessels Dementia or Alzheimer's disease.
  • Anti-FXI/FXIa antibodies or antigen-binding fragments thereof, pharmaceutical compositions of the present disclosure are useful for the prevention and/or treatment of thrombotic and/or thromboembolic complications in cancer patients, eg, venous thromboembolism, especially those experienced Those patients undergoing major surgical intervention or chemotherapy or radiotherapy.
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure are used for the treatment and/or prevention of disseminated intravascular coagulation in the context of the following diseases, including but not limited to: infectious diseases and/or systemic inflammatory syndrome ( SIRS), septic organ dysfunction, septic and multiple organ failure, acute respiratory distress syndrome (ARDS), acute lung injury (ALI), septic stroke and/or septic organ failure.
  • SIRS systemic inflammatory syndrome
  • ARDS acute respiratory distress syndrome
  • ALI acute lung injury
  • septic stroke and/or septic organ failure In the case of infection, there may be general activation of the coagulation system (disseminated intravascular coagulation active consumptive coagulation, also known as "DIC"), with microthrombosis in multiple organs and secondary hemorrhagic complications.
  • DIC disseminated intravascular coagulation active consumptive coagulation
  • Endothelial damage may be present, with increased vascular permeability and diffusion of fluids and proteins such as extravasation spaces.
  • organ failure eg, renal failure, liver failure, respiratory failure, central nervous system deficit, and cardiovascular failure
  • multiple organ failure eg, multiple organ failure.
  • DIC CAD
  • coagulation factor X eg factor X, prothrombin and fibrinogen
  • platelets eg factor X, prothrombin and fibrinogen
  • the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure are used for the treatment and/or prevention of thrombotic or thromboembolic disease and/or inflammatory disease and/or increased vascular permeability in the following patients Disease:
  • the patient has a genetic mutation that results in increased enzyme activity or increased zymogen levels, as determined by relevant experiments/measurements of enzyme activity or zymogen concentration.
  • the present disclosure provides the use of the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure for the preparation of medicines for the treatment and/or prevention of diseases, especially the above-mentioned diseases, for example, for the preparation of drugs for the treatment and/or prevention of thrombosis Drugs for sexual or thromboembolic disease.
  • the present disclosure provides methods for treating and/or preventing the above-mentioned diseases by using the anti-FXI/FXIa antibodies or antigen-binding fragments thereof, and pharmaceutical compositions of the present disclosure.
  • the present disclosure provides a composition for detecting FXI/FXIa, the composition comprising an anti-FXI/FXIa antibody or antigen-binding fragment thereof.
  • the present disclosure also provides methods, systems, or devices for detecting FXI/FXIa in vivo or in vitro, comprising treating anti-FXI/FXIa antibodies.
  • an in vitro detection method, system or device may include, for example:
  • a change eg, a statistically significant change in complex formation in a test sample or subject as compared to a control sample or subject indicates the presence of FXI/FXIa in the test sample.
  • the in vivo detection method, system or device may include:
  • Detection can include determining where or when complexes are formed.
  • Antibodies that bind to FXI/FXIa can be directly or indirectly labeled with a detectable substance to facilitate detection of bound or unbound antibody. Suitable detectable substances include various enzymes, prosthetic groups, fluorescent substances, luminescent substances and radioactive substances.
  • Complex formation between an antibody or antigen-binding fragment thereof that binds FXI/FXIa and FXI/FXIa can be detected by measuring or visualizing the antibody that binds or does not bind to FXI/FXIa.
  • Conventional detection assays can be used, eg, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or histoimmunohistochemistry.
  • samples are analyzed for the presence of FXI/FXIa by a competitive immunoassay using a standard labeled with a detectable substance and an unlabeled antibody that binds FXI/FXIa.
  • the antibodies or fragments thereof of the present disclosure may be labeled with fluorophores and chromophores for detection purposes.
  • kits comprising an anti-FXI/FXIa antibody or antigen-binding fragment thereof; optionally, diagnostic instructions.
  • the kit may also contain at least one additional reagent, such as a label or additional diagnostic agent.
  • the antibodies can be formulated as pharmaceutical compositions.
  • Figures 1A-1B SPR detection of FXI/FXIa antibody binding to human FXI/FXIa protein.
  • Figure 1A is the result of SPR analysis of the binding of 3882 molecules to FXI;
  • Figure 1B is the result of SPR analysis of the binding of 1209 molecules to FXIa.
  • Figures 2A-2B In vitro FXIa enzymatic activity inhibition assay of FXI/FXIa antibodies.
  • Figure 2A shows the results of the in vitro FXIa enzyme activity inhibition test of anti-FXI/FXIa antibodies;
  • Figure 2B shows the in vitro FXIIa-mediated inhibition of FXI activating enzyme activity of anti-FXI/FXIa antibodies.
  • FIG. 3A to Figure 3B Detection of anticoagulant activity of aPTT and PT in human blood.
  • FIG. 3A and FIG. 3B are the results of aPTT and PT tests of anti-FXI/FXIa antibodies on human blood, respectively.
  • Figure 4A to Figure 4B Detection of anticoagulant activity of aPTT and PT in monkey blood.
  • Figure 4A and Figure 4B are the results of aPTT and PT tests of anti-FXI/FXIa antibodies on monkey blood, respectively.
  • Figures 5A to 5D Animal assays of FXI/FXIa antibodies in cynomolgus monkeys.
  • Figure 5A is the change curve of aPTT, plasma drug concentration, FXI:C% and free FXI of antibody 3882 in cynomolgus monkeys;
  • Figure 5B is the thrombosis inhibition effect of 3882 in cynomolgus monkeys;
  • Figure 5C and Figure 5D are 3882 In vivo safety testing in cynomolgus monkeys, bleeding time measurement and PT test results, respectively.
  • Figures 6A-6B Results of in vivo pharmacodynamic (PD) tests of FXI/FXIa antibodies in cynomolgus monkeys.
  • Figure 6A is the test result of APTT (s);
  • Figure 6B is the test result of FXI:C (%), 3882 (1mg/kg) is administered intravenously and subcutaneously, and the control BAY1213790 (1mg/kg) is administered intravenously way of medicine.
  • Fractor XI also referred to herein as “coagulation factor XI,” “FXI,” or “fXI,” is a double-chain glycoprotein with a molecular weight of about 160 kilodaltons (kD).
  • the two chains can be the same polypeptide with a molecular weight of about 80,000 Daltons; the two chains are connected by a disulfide bond.
  • FXI contains 4 "apple domains" (A1-A4 from the N-terminus, heavy chain) and a C-terminal catalytic domain (light chain).
  • the four apple domains contain binding sites for other proteins, such as Al for thrombin; A2 for HK; A3 for factor IX (FIX), GPIb and heparin; A4 for FXIIa.
  • FXI can be converted to its active form factor XIa (FXIa) by factor XIIa (FXIIa).
  • the serine protease FXIa converts factor IX to IXa, which in turn activates factor X (Xa).
  • Xa can subsequently mediate factor II/thrombin activation.
  • FXI and FXIa are to be understood in their broadest sense.
  • the term encompasses native forms of FXI and FXIa in nature, naturally occurring variants, as well as artificially expressed forms.
  • FXI or FXIa encompasses the entire protein and its epitopes in the context of antigen-antibody interactions.
  • Antibody is used in the broadest sense to encompass a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), full-length antibodies, and antibody fragments ( or antigen-binding fragments, or antigen-binding portions), as long as they exhibit the desired antigen-binding activity.
  • Antibody can refer to immunoglobulin, which is a tetrapeptide chain structure composed of two heavy chains and two light chains connected by interchain disulfide bonds. The amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, whose corresponding heavy chains are ⁇ , ⁇ , and ⁇ chains, respectively. , alpha chains and epsilon chains.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (CDRs) and four relatively conserved framework regions (FRs). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • Each light chain variable region (VL) and heavy chain variable region (VH) consists of 3 CDR regions and 4 FR regions.
  • the order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the definitive delineation of the CDRs and identification of residues comprising the binding site of the antibody can be accomplished by resolving the structure of the antibody and/or resolving the structure of the antibody-ligand complex. This can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography.
  • a variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, Chothia numbering system, AbM numbering system, IMGT numbering system, contact definitions, conformational definitions.
  • the Kabat numbering system is the standard for numbering residues in antibodies, and is often used to identify CDR regions (see, eg, Johnson & Wu, 2000, Nucleic Acids Res., 28:214-8).
  • the Chothia numbering system is similar to the Kabat numbering system, but the Chothia numbering system takes into account the positions of certain structural loop regions (see, eg, Chothia et al., 1986, J. Mol. Biol., 196:901-17; Chothia et al., 1989, Nature , 342:877-83).
  • the AbM numbering system uses an integrated suite of computer programs produced by the Oxford Molecular Group for modeling antibody structures (see, eg, Martin et al., 1989, ProcNatl Acad Sci (USA), 86:9268-9272; "AbMTM, A Computer Program for ModelingVariable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd).
  • the AbM numbering system uses a combination of knowledge databases and De-novo methods to model the tertiary structure of antibodies from basic sequences (see Samudrala et al., 1999, in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198 " Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach").
  • CDR boundary definitions may not strictly follow one of the above approaches, but still overlap at least a portion of the Kabat CDRs. Although they may be shortened or lengthened depending on the predicted or experimental results of specific residues or groups of residues that do not significantly affect antigen binding.
  • a CDR can refer to a CDR defined by any method known in the art, including combinations of methods.
  • the CDR amino acid residues of the VL and VH regions of the antibodies or antigen-binding fragments of the present disclosure conform to the known Kabat numbering system in number and position.
  • Kabat numbering system the skilled artisan can determine the corresponding equivalent positions in different numbering conventions compared to the amino acid positions in the CDRs of the present disclosure.
  • skilled artisans can also determine such equivalent sites through alignment analysis of amino acid sequences.
  • CH CH
  • CH region and “CH domain” are used interchangeably herein to refer to the heavy chain constant region and include “CH1", “CH2” and “CH3" regions or domains.
  • Human antibody or “recombinant human antibody” includes human antibodies produced, expressed, created or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as:
  • an antibody isolated from a host cell transformed to express the antibody such as a transfectoma
  • Antibodies prepared, expressed, created or isolated by methods such as splicing human immunoglobulin gene sequences into other DNA sequences.
  • Such recombinant human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • murine antibody in the present disclosure is a monoclonal antibody directed against human FXI/FXIa or an epitope thereof prepared according to knowledge and skill in the art. In preparation, test subjects are injected with the FXI/FXIa antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-human FXI/FXIa antibody or its antigen-binding fragment may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine constant region.
  • human antibody includes antibodies having variable and constant regions of human germline immunoglobulin sequences.
  • Human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody” does not include humanized antibodies.
  • humanized antibody also known as CDR-grafted antibody, refers to an antibody produced by grafting CDR sequences of a non-human species into the framework of a human antibody variable region.
  • the strong immune response induced by chimeric antibodies can be overcome because they carry a large number of heterologous protein components.
  • the variable regions of the human antibody may be subjected to minimal reverse mutations to maintain activity.
  • chimeric antibody is an antibody obtained by fusing the variable region of the antibody of the first species with the constant region of the second species, which can alleviate the immune response induced by the antibody of the first species.
  • a hybridoma that secretes a mouse-specific monoclonal antibody is selected, and then the variable region gene is cloned from the mouse hybridoma cell, and then the constant region gene of the human antibody is cloned according to the needs.
  • the variable region gene is linked with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, preferably comprising the heavy chain constant region of human IgG2 or IgG4, or without ADCC (antibody-dependent) after amino acid mutation. cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxicity of IgG1.
  • Antigen-binding fragments include single chain antibodies (ie, heavy or light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, mono Domain antibodies (eg VH or VL or VHH), scFvs, bivalent or trivalent or tetravalent antibodies, Bis-scFv, diabodies, tribodies, tetrabodies and the above An epitope-binding fragment of any one (see, e.g., Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews-Online 2(3), 209-217).
  • Fab-Fv form was first disclosed in WO2009/040562 and its disulfide stabilized form, Fab-dsFv, was first disclosed in WO2010/035012.
  • Antigen-binding fragments of the present disclosure also include Fab and Fab' fragments described in WO2005/003169, WO2005/003170 and WO2005/003171.
  • Multivalent antibodies may comprise multispecific, eg bispecific or monospecific (see eg WO92/22583 and WO05/113605).
  • Variant refers to a polypeptide that contains at least one amino acid modification (such as a substitution, deletion or insertion) compared to the "parent" amino acid sequence, provided that the variant is still capable of binding FXI/FXIa, particularly as in SEQ ID NO: 1 Human FXI/FXIa shown.
  • the variants show similar or even improved properties compared to antibodies 0012, 1209, 1267, 3807, 3871, 3882 in the embodiments of the present disclosure.
  • Variants of binding molecules (eg, antibodies or antigen-binding fragments thereof) of the present disclosure are typically prepared by introducing appropriate nucleotide changes into nucleic acid encoding the antibody or antibody fragment or by peptide synthesis.
  • amino acid modifications can be introduced into the variable or constant regions, for example, compared to the anti-FXI/FXIa antibodies in the embodiments of the present disclosure, amino acid modifications can be introduced to modulate antibody properties that affect drug development, such as thermodynamic stability, solubility or viscosity ("sequence optimization").
  • Amino acid modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of a binding molecule, preferably an antibody or antigen-binding fragment. Any combination of deletions, insertions and substitutions can be introduced into the "parental" amino acid sequence to obtain the final variant. Amino acid modifications also include post-translational processes that can alter the binding molecule, such as altering the number or location of glycosylation sites.
  • 1, 2, 3, 4, 5, or 6 amino acids can be inserted into or deleted from each CDR, 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids were inserted into or deleted from each FR.
  • Insertional variants of binding molecules (particularly antibodies or antibody fragments) of the present disclosure include antibodies or antibody fragments with an enzyme or another functional polypeptide (eg, which increases the serum half-life of the binding molecule (eg, antibody or antibody fragment)) fusion products.
  • amino acid substitutions can be introduced into the CDR, VH or FR regions of the heavy and/or light chains. Conservative substitutions are preferred, eg, which can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic properties of the residues involved.
  • Constant substitution refers to the replacement of another amino acid residue with similar properties to the original amino acid residue.
  • lysine, arginine and histidine have similar properties in that they have basic side chains
  • aspartic acid and glutamic acid have similar properties in that they have acidic side chains.
  • glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains
  • alanine , valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have non-polar side chains.
  • modifications can also be introduced into the Fc portion of a binding molecule, preferably an antibody or antigen-binding fragment thereof. Such modifications can be used to modulate the functional properties of the antibody, for example, interactions with complement proteins on other immune cells (such as C1q and/or Fc receptors), or to modulate serum half-life or antigen-dependent cellular cytotoxicity (ADCC) ). Mutations that alter effector function can be introduced into the Fc domain using routine methods known in the art.
  • Exemplary modifications include: Asn297 ⁇ Ala297 and Asn297 ⁇ Gln297 leading to IgGl glycosylation, or Lys322 ⁇ Ala322 and optionally Leu234 ⁇ Ala234 and Leu235 ⁇ Ala234, which have been reported to reduce or eliminate antibody-derived cell-mediated Cytotoxicity (ADCC) and/or complement-derived cytotoxicity (CDC).
  • ADCC antibody-derived cell-mediated Cytotoxicity
  • CDC complement-derived cytotoxicity
  • Examples of means of extending serum half-life include additional peptide or protein domains that bind to other proteins in the human body, such as serum albumin, the Fc region of immunoglobulins, or the neonatal Fc receptor (FcRn).
  • Other conceivable ways of extending serum half-life include amino-terminal extension with polypeptide chains of different lengths (eg, XTEN technology or ), conjugation to non-protein polymers or carbohydrates including, but not limited to, various polyols such as polyethylene glycol (PEGylated), polypropylene glycol, polyoxyalkylene or polyethylene glycol Copolymers of alcohol and polypropylene glycol; or carbohydrates such as hydroxyethyl starch (eg, ) or polysialic acid (eg, ).
  • amino acid substitutions can be made at various positions in the binding molecule to facilitate addition of the polymer, as known in the art.
  • binding to FXI/FXIa refers to being able to interact with FXI/FXIa or an epitope thereof, which may be of human origin.
  • antigen-binding site refers to a discrete three-dimensional spatial site on an antigen that is recognized by an antibody or antigen-binding fragment of the present disclosure.
  • epitopes refers to the site on an antigen to which an immunoglobulin or antibody binds.
  • Epitopes may be formed by adjacent amino acids, or non-adjacent amino acids (which are brought into spatial proximity to each other by tertiary folding of the protein). Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically exist in unique spatial conformations that include at least 3-15 amino acids.
  • Methods for determining epitopes are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • Specific binding and “selective binding” refer to the binding of an antibody to a predetermined epitope on an antigen.
  • the antibody is measured in an instrument by surface plasmon resonance (SPR) techniques, and the antibody is measured at about less than 10-7 M or even less Equilibrium dissociation constant (K D ) binds to a predetermined antigen or its epitope, and its affinity for binding to the predetermined antigen or its epitope is nonspecific other than its binding to the predetermined antigen or its epitope (or a closely related antigen)
  • Antigens such as BSA, etc.
  • Binding affinity refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity” refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its ligand Y can generally be represented by the equilibrium dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein.
  • kassoc or "ka” refers to the on-rate of a particular antibody-antigen interaction
  • kdis or “kd” as used herein is intended to refer to the rate of dissociation of a particular antibody-antigen interaction
  • KD refers to the equilibrium dissociation constant, which is obtained from the ratio of kd to ka (ie, kd/ka) and expressed as molar concentration (M).
  • the KD value of an antibody can be determined using methods known in the art, e.g., methods for determining the KD of an antibody include measuring surface plasmon resonance using a biosensing system such as a system, or measuring KD in solution by solution equilibrium titration (SET). Affinity.
  • Cross-reactivity refers to the ability of an antibody (or fragment thereof) of the present disclosure to bind FXI/FXIa from different species.
  • an antibody of the present disclosure that binds human FXI/FXIa can also bind FXI/FXIa of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR or ELISA), or binding or functional interaction with cells that physiologically express FXI/FXIa.
  • binding assays eg SPR or ELISA
  • Methods for determining cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance analysis, or flow cytometry.
  • Inhibition or blocking are used interchangeably and encompass both partial and complete inhibition or blocking. Inhibition or blocking of FXI/FXIa preferably reduces or alters the normal level or type of activity that occurs when FXI/FXIa binding occurs without inhibition or blocking. Inhibition and blocking are also intended to include any measurable reduction in FXI/FXIa binding affinity when contacted with an anti-FXI/FXIa antibody compared to FXI/FXIa not contacted with an anti-FXI/FXIA antibody.
  • “Inhibition of growth” (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.
  • mice can be immunized with human FXI/FXIA or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions. Human FR germline sequences can be obtained from ImMunoGeneTics (IMGT), or from J. Immunoglobulin, 2001 ISBN012441351.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant expression vector can stably transfect CHO cells.
  • Mammalian expression systems result in glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified and collected by conventional techniques.
  • Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, and ion exchange.
  • the obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • Antibodies of the present disclosure refer to monoclonal antibodies (mAbs), which refer to antibodies obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic or phage clonal cell lines.
  • Monoclonal antibodies or antigen-binding fragments can be obtained recombinantly using, eg, hybridoma technology, recombinant technology, phage display technology, synthetic technology (eg, CDR-grafting), or other existing techniques.
  • Antibodies can be screened competitively for binding to the same epitope using routine techniques known to those of skill in the art. For example, competition and cross-competition studies can be performed to obtain antibodies that compete with each other or cross-compete for binding to the antigen. A high-throughput method for obtaining antibodies that bind the same epitope based on their cross-competition is described in International Patent Publication WO03/48731. Thus, antibodies and antigen-binding fragments thereof that compete for binding to the same epitope on FXI/FXIa with the antibody molecules of the present disclosure can be obtained using conventional techniques known to those skilled in the art.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , human, subject, cell, tissue, organ or biological fluid contact.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as a composition comprising any of the antibodies or antigen-binding fragments thereof of the present disclosure, to a subject who has, is suspected of having, or is prone to having There are one or more diseases or symptoms thereof for which the therapeutic agent is known to have a therapeutic effect.
  • a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population to be treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • embodiments of the present disclosure may be ineffective in alleviating symptoms of a target disease in a subject, according to any statistical test known in the art, such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • any statistical test known in the art such as Student's t-test, chi-square test, based on Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matched or homologous. In general, comparisons are made when the two sequences are aligned for the greatest percent homology.
  • Cell Cell
  • cell line cell line
  • cell culture all such designations include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly the same in DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included.
  • “Pharmaceutical composition” means a mixture comprising one or more of the antibodies or antigen-binding fragments described herein, or a physiological/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable salts or prodrugs.
  • Pharmaceutical carriers and excipients The purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • Human FXI/FXIa protein (Uniprot Acc No. P03951), as the antigen and detection protein involved in the present disclosure, was purchased from Enzyme research laboratories (FXI:Cat.HFXI 1111; FXIa:HFXIa 1111a). The following FXI/FXIa antigens without special description refer to human FXI/FXIa.
  • ProteinG packing For the purification of mouse hybridoma supernatant, ProteinG packing (KANEKA:Cat.KanCapTMG) is the first choice for affinity chromatography.
  • the cultured hybridomas were centrifuged to take the supernatant, and a gravity column packed with Protein G was used for purification.
  • 3-5 column volumes of 6M guanidine hydrochloride Simga:Cat.G3272-1KG
  • wash with 3-5 column volumes of pure water then use 3-5 column volumes of 1 ⁇ PBS (pH7.4) (Sangon Biotech (Shanghai) co., Ltd.: Cat.
  • E607016-0500 buffer system was used as an equilibration buffer to equilibrate the chromatography column; the supernatant was loaded at a low flow rate for binding, and the flow rate was controlled to make The retention time is about 1min or longer; use 3-5 column volumes of 1 ⁇ PBS (pH7.4) to wash the column until the UV absorption falls back to the baseline; use 0.1M Glycine (pH3.0) (Sigma:Cat. 410225-250G) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) (Vetec, Cat.V900483) to quickly adjust the pH of the eluted product to 7-8 and temporarily store it.
  • 1M Tris-HCl pH8.0
  • solution replacement can be carried out using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube, and solution replacement to the desired buffer system, or molecular exclusion (such as G-25 desalting) Replace with the desired buffer system.
  • the cell culture supernatant expressing the Fc fusion protein or antibody is subjected to high-speed centrifugation to collect the supernatant.
  • ProteinA GE, Cat: 17-5474-99
  • affinity column was regenerated with 5 column volumes of 0.1M NaOH (Sigma: Cat: 71687-500g), followed by 5 column volumes of 1 ⁇ PBS (pH 7.4) (Sangon Biotech (Shanghai) co., Ltd.: Cat. E607016-0500) wash equilibration.
  • the supernatant was loaded at a low flow rate for binding, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the sample was diluted with equilibration buffer 20 mM Tris pH 8.0 (Vetec, Cat. V900483) so that the conductivity was less than 3 ms/cm, and a Q HP (GE, Cat: 17-1014-01) anion chromatography column was used for 5 column volumes was regenerated with 0.5M NaOH (Sigma Cat: 71687-500g) and then equilibrated with 15 column volumes of 20mM Tris pH 8.0 (Vetec, Cat. V900483). The supernatant was loaded at a low flow rate for binding, and the flow rate was controlled so that the retention time was about 2 min or longer.
  • the eluted product can be replaced by solution using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or using molecular size exclusion (such as G-25 desalting) to replace required buffer system.
  • Antibodies with high affinity to FXIa were obtained by screening a fully human single-chain antibody phage library.
  • 100 ⁇ g Dynabeads MyOne Streptavidin T1 was bound with 2 ⁇ g randomly biotinylated FXIa protein for 1 hour at room temperature.
  • PBST 0.05% Tween-20
  • the skim milk was dissolved in 1 ⁇ PBS, the final concentration was 2% as a blocking agent, added to the system, and blocked at room temperature for 1 hour.
  • a fully human single-chain antibody phage display library blocked with 2% milk for 1 hour at room temperature was added and allowed to act at room temperature for 1 hour.
  • PBST 0.05% Tween-20
  • pH 7.4 solution was washed 11 times, and each time was turned upside down 20 times to remove unbound phage.
  • the remaining phages that specifically bind to FXIa were eluted with 0.5 mL of trypsin at 1 mg/mL, respectively infected E. coli TG1 in log phase growth, and phages were produced and purified for the next round of screening.
  • the same screening process was repeated for 2-3 rounds, and the usage of FXIa was gradually decreased to 0.5 ⁇ g (for the second round of screening) and 0.1 ⁇ g (for the third round of screening). After the third round of screening, positive clones were enriched.
  • ELISA plates were coated with 1 ⁇ g/mL FXIa protein, placed at 4°C overnight, washed 3 times with PBST (0.05% Tween-20), blocked with 2% skim milk at room temperature for 1 hour, PBST (0.05% Tween-20) After washing 3 times, add the phage supernatant diluted with blocking solution, react at room temperature for 1 hour, wash 6 times with PBST (0.05% Tween-20), add anti-M13HRP (Sino Biological Inc., 11973-MM05T-H), and react at room temperature After 1 hour, wash 3 times with PBST (0.05% Tween-20), add 100 ⁇ L of TMB chromogenic substrate, stop the reaction with 100 ⁇ L of 1M sulfuric acid, and use a SpectraMax M5 microplate reader to read the absorbance at 450nm for detection.
  • PBST 0.05% Tween-20
  • Example 2 The high-affinity specific sequences screened in Example 2 were used to construct a complete recombinant antibody. Then, through ELISA binding experiment, ForteBio protein interaction experiment and FXIa enzyme activity inhibition experiment, it was determined that two of the antibodies have strong binding ability and can effectively inhibit FXIa enzyme activity.
  • the complete variable region sequence is as follows:
  • the sequence is: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underlined sequences are CDR1, CDR2, and CDR3, respectively.
  • the heavy and light chain CDR sequences are summarized in Table 1.
  • the three-dimensional structure of the antibody molecule 0012 was simulated to simulate the combination with the known antigen structure (PDB ID: 6AOD Chain: C). With reference to the mutation hotspots, three-dimensional structure and binding simulation results of human germline genes, some amino acid residues were selected to establish several random mutation phage libraries. Using phage library display technology, functional antibodies with improved affinity were screened. The new amino acid residues obtained from different libraries are combined and verified to obtain functional antibodies with improved affinity and function.
  • the obtained antibody molecule variable region heavy chain and light chain sequences are respectively as follows:
  • sequence numbering rule is Kabat
  • sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
  • underlines in the sequence are CDR1, CDR2, and CDR3 sequences, respectively.
  • variable regions of heavy and light chains of 0012-F3 are F-VH and 3-VL, respectively; the variable regions of heavy and light chains of 0012-H3 are H-VH and 3-VL, respectively; the heavy and light chains of 0012-J3
  • the variable regions are J-VH and 3-VL, respectively; the heavy and light chain variable regions of 0012-K3 (ie, 1209) are K-VH and 3-VL, respectively.
  • the heavy chain and light chain variable region germline genes with high homology to the selected antibodies are used as templates to transform the FR region and CDR region of the original monoclonal antibody, so that the sequence is closer to the human germline gene while retaining the function.
  • the three-dimensional structure of the transplanted antibody is simulated and analyzed again, and the specific sites in the FR region that affect the structure and shape of the CDR region are backmutated. Amino acid residues are identified and annotated by the Kabat numbering system.
  • the engineered antibodies have higher stability and lower immunogenicity.
  • the human germline gene VH1-24 in the Vbase database was used as the heavy chain template, and the human germline gene VKIIIA27 in the Vbase database was used as the light chain template.
  • antibodies Fg3g i.e. 1268
  • Hg3g Hg3g
  • Jg3g Jg3g
  • Kg3g i.e. 1267
  • the CDR numbering system for the above antibodies is Kabat.
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the underlines in the sequence are CDR1, CDR2, and CDR3 sequences, respectively.
  • Each of the above heavy chain variable regions is fused with CH1 (SEQ ID NO: 13) of the corresponding human antibody, and then fused with IgG1 Fc (SEQ ID NO: 67), and the light chain variable region is fused with human kappa (SEQ ID NO: 67). 14) Fusion to form recombinant antibodies for subsequent detection.
  • mice with high antibody titers in serum and titers approaching a plateau were selected for splenocyte fusion.
  • Booster immunization was performed 3 days before splenocyte fusion, intraperitoneal (IP) injection of 50 ⁇ g/head of the antigen solution prepared in normal saline.
  • Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma cells Sp2/0-Ag14 cells using an optimized PEG-mediated fusion procedure.
  • the fused hybridoma cells were seeded in 96-well plates at 1 ⁇ 10 ⁇ 4 to 1 ⁇ 10 ⁇ 5 cells per well, incubated at 37°C, 5% CO 2 and supplemented with 100 ⁇ l/well of HAT complete medium, 10-14 ELISA test was performed after a few days.
  • the hybridoma culture supernatant was detected by combined ELISA method.
  • the cell supernatant of the positive wells detected by ELISA was purified, cell binding experiment and cell blocking experiment were carried out.
  • the well cells with positive binding and blocking experimental results were expanded, cryopreserved and sequenced in time.
  • the hybridoma culture supernatant was detected by combined ELISA method.
  • the supernatant of cells from positive wells detected by ELISA was used for enzyme activity inhibition experiment. Cells from wells that were positive for binding and function were subcloned one or two times until single-cell clones were obtained.
  • FXI/FXIa combined ELISA and FXIa enzyme activity inhibition experiments are also required for each subcloned cells.
  • Hybridoma clones are obtained through the above experiments and expanded to further prepare antibodies.
  • the antibodies are purified according to the purification example for use in the detection example.
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and sequenced.
  • the positive clone 3807 was obtained, and the corresponding amino acid sequence of the antibody variable region is as follows:
  • the selected antibodies have high homology.
  • the germline gene of the heavy chain variable region was used as a template, and the CDRs of the mouse-derived monoclonal antibody were grafted into the corresponding human-derived modules.
  • the three-dimensional structure of the transplanted antibody is simulated and analyzed again, and the specific sites in the FR region that affect the structure and shape of the CDR region are restored and mutated.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • the humanized light chain template of murine antibody 3807 is IGKV3-11*01, the heavy chain template is IGHV1-69-2*01, and the sequence of the humanized variable region is as follows (CDRs are underlined):
  • the VH of antibody 3871 is 3807-VH1 and the VL is 3807-VL3; the VH of antibody 3875 is 3807-VH5 and the VL is 3807-VL3.
  • 3871 and 3875 were genetically modified according to the method of Example 5, so that the antibodies have higher stability and lower immunogenicity. Only the FR region of the heavy chain was modified, and the variable region of the light chain was unchanged.
  • the sequence of the heavy chain variable region of the humanized antibody 3875 genetic modification is as follows (the light chain remains unchanged):
  • the heavy chain variable region sequence of the humanized antibody 3871 genetically modified is as follows (the light chain remains unchanged):
  • Antibody 3882 has a VH of 3807-VH1 (GTFS) and a VL of 3807-VL3.
  • the above heavy chain variable regions are fused with the corresponding human antibody heavy chain CH1 (SEQ ID NO: 59) and the Fc (containing S241P mutation) (SEQ ID NO: 60) of human antibody IgG4, and the light chain variable region is fused with The constant region CL1 (SEQ ID NO: 14) of human kappa was fused to form a recombinant chimeric antibody for subsequent detection.
  • the full-length antibody sequence is exemplified with 3882.
  • the heavy chain variable region of the anti-FXI/FXIa murine or humanized antibody of the present disclosure is cloned into a mammalian cell expression vector containing human antibody heavy chain IgG1, and the light chain variable region is cloned into a human antibody kappa Mammalian cell expression vector for light chain constant region C ⁇ 1; ExpiCHO cells were transfected with the expression vector. According to the ratio of 1 ⁇ g DNA/mL transfected cells, the ExpiCHO Expression System (Cat. no. A29133) was used for transfection according to the reagent instructions.
  • the cells were incubated at 37°C with shaking (8% CO 2 ), and the cell culture medium was collected on the 8th day, centrifuged at 4000 rpm, and the supernatant was collected and filtered through a 0.45 uM filter. After detection, the target antibody was obtained.
  • a certain amount of the antibody to be tested was affinity captured with a Protein A biosensor chip (Cat. #29127556, GE), and then a series of concentration gradients of human FXI/FXIa were passed on the surface of the chip. Reaction signals were detected in real time using a Biacore instrument (Biacore T200, GE) to obtain binding and dissociation curves. After each cycle of dissociation, the biochip was washed and regenerated with the regeneration solution prepared in the human anti-capture kit or the pH 1.5 glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE). The buffer used in the experiment was HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE) diluted to 1 ⁇ (pH 7.4) with double distilled water.
  • anti-FXI/FXIa antibodies To test the function of anti-FXI/FXIa antibodies, the ability of anti-FXI/FXIa antibodies to bind to trypsin FXIa and inhibit the cleavage of specific substrates by FXIa, as well as the ability of anti-FXI/FXIa antibodies to bind to trypsinogen FXI and block FXIIa on FXI digestion ability.
  • the SpectraMax M5 microplate reader Pre-set the SpectraMax M5 microplate reader to 37°C, pre-cool a 384-well plate (thermo, Cat. 94410153) on ice, and add 20 ⁇ L of the antibody to be tested (10 ⁇ g/mL) and 10 ⁇ L of FXIa trypsin (5 ⁇ g) diluted with 1 ⁇ PBS respectively. /mL), incubate at 37°C for 5min, then incubate on ice for 5min, add 10 ⁇ L of S-2366 (2mM) (Chromogenix, S821090), immediately use a SpectraMax M5 microplate reader to read the kinetic curve at 405nm at 37°C (Read once a minute, 60min).
  • FXIIa To detect the ability of anti-FXI/FXIa antibody to block the cleavage of FXI by FXIIa, after pre-cooling the 384-well plate on ice, add 10 ⁇ l of FXI diluted with buffer (20 mM HEPES, pH 7.4, 150 mM NaCl and 0.1% BSA).
  • Example 11 Detection of aPTT and PT anticoagulant activity in human blood/monkey blood
  • Activated partial thromboplastin time in the presence of various concentrations of the candidate antibody (diluted in PBS) was determined with the Sysmex kit, the candidate antibody to be tested was incubated with plasma for 3 min at 37°C, and then treated by adding 25 mM chlorine Calcium reagents initiate coagulation, and the time to which coagulation occurs is measured.
  • concentration of the candidate antibody that prolongs aPTT by 50% relative to PBS ie, aPTT1.5
  • Prothrombin time (PT) in the presence of various concentrations of candidate antibodies was determined with the Sysmex kit, the candidate antibodies to be tested were incubated with plasma at 37°C for 3 minutes, and then by adding thromboplastin Coagulation was initiated and the time to which coagulation occurred was measured. Part of the antibody test results are shown in Figure 3B and Figure 4B.
  • the bleeding time was observed at 15 minutes (min) and 3 hours (h) after administration, respectively; plasma was collected and aPTT and PT were observed at 15 minutes, 3 hours, 6 hours and 1 day (d) after administration, respectively.
  • the bleeding time of the administration group was observed at 15min, 3h, 2d, 4d, 1 week (w), 2w and 3w after administration; at 15min, 3h, 6h, 1d, 2d, 4d, 1w, 2w, 3w after administration , 4w, 5w and 6w, respectively, collected plasma according to the above method and observed aPTT, PT, plasma drug concentration, plasma FXI:C% and plasma free FXI concentration.
  • Both groups of animals underwent AV-shunt thrombectomy 1 day after administration, and the thrombectomy time was 10 min.
  • aPTT, PT and plasma FXI:C% were determined by coagulation analyzer and corresponding kit, and plasma free FXI concentration and plasma drug concentration were determined by ELISA method.
  • the thrombus weight was compared between the administration group and the control group, and the t test was used for statistical analysis.
  • the antibody test results are shown in Figures 5A-5D, wherein the negative control (NC) is no drug administration.
  • NC negative control
  • the results showed that 5mpk inhibited the formation of thrombus well, and at the same time, prolonged the intrinsic coagulation time, but had no significant effect on the bleeding time and extrinsic coagulation time of animals.
  • the PK results showed that the half-life was about 20 days.
  • "**" in Figure 5B represents a P value ⁇ 0.01, which is a significant difference.
  • Animals in the intravenous administration group were administered before administration and 5min, 1h, 1d, 2d, 3d, 5d, 1w, 2w, 3w, and 4w after administration; , 3d, 5d, 1w, 2w, 3w, 4w, blood samples were collected with sodium citrate blood collection tubes, and aPTT and plasma FXI:C% were determined with coagulation analyzer and corresponding kits.

Abstract

提供了抗FXI/FXIa抗体、其抗原结合片段及医药用途，以及包含抗FXI/FXIa抗体或其抗原结合片段的药物组合物及治疗、预防疾病的方法，特别是治疗血栓形成或血栓栓塞相关疾病或病症的方法。

Description

抗FXI/FXIa抗体、其抗原结合片段及医药用途
本申请要求2020年07月02日提交的中国专利申请202010633951.8的优先权。
技术领域
本申请涉及抗FXI/FXIa抗体、其抗原结合片段，包含所述抗FXI/FXIa抗体及其抗原结合片段的药物组合物，及其用于治疗或预防血栓形成相关疾病的医药用途。
背景技术
近年来，随着全球人口老龄化以及生活方式的改变，血栓栓塞类疾病成为全球致死率和致残率最高的疾病之一。现有的抗血栓药物/抗凝药物所造成的不希望的出血事件，仍然是一个很大的问题(Katherine et al，2015，West J Emerg Med；16(1):11–17)。
现有动物实验和临床数据证明，抑制内源性途径，特别是凝血因子FXI，能够在降低血栓形成的同时不会造成明显的出血事件(Felicitas Müller et al，Curr Opin Hematol.2011 Sep；18(5):349–355)。
FXI是内源途径中的关键凝血因子。FXIa是FXI的活化状态，在凝血级联反应中，不仅能够被内源凝血因子FXIIa激活形成FXIa，而且能够被凝血酶II激活，从而放大凝血级联反应，形成更多的凝血酶和纤维蛋白。该过程中，可能形成过多的凝血酶和纤维蛋白，从而导致血栓类疾病。从人类FXI缺乏症(C型血友病)病人出血表型温和的特点发现，FXI被抑制时出血风险较小。进一步的研究发现，在FXI缺陷病人中，缺血性脑卒中及深静脉血栓的发病率明显降低，表明抑制FXI有利于减少缺血性脑卒中及深静脉血栓发病风险。
FXI基因位于人类第4号染色体，编码由607个氨基酸组成的分泌蛋白。FXI蛋白分子包含4个苹果结构域(apple domains)和1个催化结构域(catalytic domain)。FXI在人血液内是以同二聚体的形式存在，循环在人血浆中与HK形成非共价复合物的FXI的浓度为约30nM(15-45nM)。人FXI分子与猴和小鼠的FXI分子分别具有88％、67％和58％(两个物种，三个同源性数据)的同源性。
目前已经有多篇文献和临床试验表明，抑制FXI及其转化成的FXIa的活性能够有效的降低血栓的形成，并且不会造成可见的出血风险。从目前披露的临床结果来看，相对于现有的口服抗凝剂(NOAC)，抗FXI/FXIa抗体有着相似或更好的抗血栓效果的同时，出血风险更低，患者能够得到更安全的治疗。
提供低免疫原性、有效抑制血栓形成而不增加出血风险的抗FXI/FXIa抗体药物，从而使得抗血栓治疗更安全，仍然是医药领域亟需的。
发明内容
本公开提供一种抗FXI/FXIa抗体或其抗原结合片段，其编码核酸、载体、宿主细胞、药物组合物、其用于治疗或延缓血栓形成或血栓栓塞相关疾病或病症或其并发症的方法，及其检测用途。
一方面，抗FXI/FXIa抗体或其抗原结合片段，包含：
重链HCDR1，其包含X 1X 2X 3MH(SEQ ID NO：63)所示序列，其中，X 1选自E、S、G或D，X 2选自L、I、V或D，X 3选自S、F、L或Y；和/或
重链HCDR2，其包含X 4X 5DPX 6X 7GX 8TX 9YAX 10KFQG(SEQ ID NO：64)所示序列，其中，X 4选自G或W，X 5选自F或I，X 6选自E或Q，X 7选自D或N，X 8选自E或D，X 9选自I、R、V或E；X 10选自Q或S；和/或
重链HCDR3，其包含DPHRTWWRYFDWLYPRGMDV(SEQ ID NO：9)或GNFYYFDY(SEQ ID NO：39)所示序列；和/或
轻链LCDR1，其包含RASQTVGKNYLA(SEQ ID NO：10)或SASSSINYMH(SEQ ID NO：40)所示序列；和/或
轻链LCDR2，其包含X 11X 12SX 13X 14AX 15(SEQ ID NO：65)所示序列，其中，X 11选自G、E或D，X 12选自A或T，X 13选自N、V或K，X 14选自R或L，X 15选自T、L或S；和/或
轻链LCDR3，其包含X 17QX 18X 19X 20X 21PX 22T(SEQ ID NO：66)所示序列，X 17选自Q或H，X 18选自F或R，X 19选自R或S，X 20选自S或F，X 21选自Y或S，X 22选自Y或L。
在一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段包含：
重链HCDR1，其包含SEQ ID NO：7、22、24、26、28、37之一所示序列；
重链HCDR2，其包含SEQ ID NO：8、23、25、27、38之一所示序列；
重链HCDR3，其包含SEQ ID NO：9、39之一所示序列；
轻链LCDR1，其包含SEQ ID NO：10、40之一所示序列；
轻链LCDR2，其包含SEQ ID NO：11、29、41之一所示序列；和
轻链LCDR3，其包含SEQ ID NO：12、42之一所示序列。
在一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段中，包含：
(a)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：7、8、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、11、12所示序列；
(b)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：22、23、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
(c)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：24、25、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
(d)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：26、27、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
(e)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：28、25、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
(f)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：37、38、39所示 序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：40、41、42所示序列；
(g)重链HCDR，其与(a)-(f)中任一重链的HCDR相比，具有0-10个氨基酸突变，轻链CDR，其与(a)-(f)中任一轻链的LCDR相比具有0-10个氨基酸突变。
一些实施方案中，当抗FXI/FXIa抗体或其抗原结合片段包含(a)、(b)、(c)、(d)、(e)或相关的(g)方案的CDR序列时，其选择性的结合FXIa，而不结合FXI。
另一些实施方案中，当抗FXI/FXIa抗体或其抗原结合片段包含(f)或相关的(g)方案的CDR序列时，其结合FXI，也结合FXIa；一些具体的实施方案中，其虽然结合FXIa，但不影响FXIa活性。
在一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段；一些具体实施方案中，为人源化抗体、人抗体或其片段。其可以为全长抗体或其其片段。
在一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段为人源化抗体或其片段，人源化的轻链模板可以为IGKV3-11*01，重链模板可以为IGHV1-69-2*01。
在一些实施方案中，人源化过程还包括对VH、VL的回复突变。一些具体实施方案中，例如，VH具有Y27F、T28N、F29I、T30K、A93L、R94Y、E73T、R66K、V67A、T75A、T76N中任一或任意组合的回复突变。一些具体实施方案中，VL具有R45K、L46R、L47W、I58V、F71Y中任一或任意组合的回复突变。
一些具体实施方案中，上述人源化抗体或其片段的重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：37、38、39所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：40、41、42所示序列。
在一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段包含：
如SEQ ID NO：5、17-20、30-33、35、43、45-49、53-58之一所示或与之具有至少之具有至少80％同一性的VH，和/或
如SEQ ID NO：6、21、34、36、44、50-52之一所示或与之具有至少80％同一性的VL。
一些具体实施方案中，抗FXI/FXIa抗体或其抗原结合片段包含：
(h)如SEQ ID NO：5所示或与之具有至少80％同一性的VH，和如SEQ ID NO：6所示或具有至少80％同一性的VL；
(i)如SEQ ID NO：17所示或具有至少80％同一性的VH，和如SEQ ID NO：21所示或具有至少80％同一性的VL；
(j)如SEQ ID NO：18所示或具有至少80％同一性的VH，和如SEQ ID NO：21所示或具有至少80％同一性的VL；
(k)如SEQ ID NO：19所示或具有至少80％同一性的VH，和如SEQ ID NO：21所示或具有至少80％同一性的VL；
(l)如SEQ ID NO：20所示或具有至少80％同一性的VH，和如SEQ ID NO：21所示或具有至少80％同一性的VL；
(m)如SEQ ID NO：30所示或具有至少80％同一性的VH，和如SEQ ID NO：34所示或具有至少80％同一性的VL；
(n)如SEQ ID NO：31所示或具有至少80％同一性的VH，和如SEQ ID NO：34所示或具有至少80％同一性的VL；
(o)如SEQ ID NO：32所示或具有至少80％同一性的VH，和如SEQ ID NO：34所示或具有至少80％同一性的VL；
(p)如SEQ ID NO：35所示或具有至少80％同一性的VH，和如SEQ ID NO：36所示或具有至少80％同一性的VL；
(q)如SEQ ID NO：43所示或具有至少80％同一性的VH，和如SEQ ID NO：44所示或具有至少80％同一性的VL；
(r)如SEQ ID NO：45所示或具有至少80％同一性的VH，和如SEQ ID NO：51所示或具有至少80％同一性的VL；
(s)如SEQ ID NO：49所示或具有至少80％同一性的VH，和如SEQ ID NO：51所示或具有至少80％同一性的VL；
(t)如SEQ ID NO：58所示或具有至少80％同一性的VH，和如SEQ ID NO：51所示或具有至少80％同一性的VL。
一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段的VH与人源或小鼠CH1连接，VL与人源或小鼠CL或Cκ连接。所述人源CH如SEQ ID NO：13或59所示，所述Cκ例如SEQ ID NO：14所示。
一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段为鼠源抗体或其片段。其轻链可变区包含鼠源κ、λ链或其变体的轻链FR区和/或轻链恒定区。一些具体实施方案中，所述鼠源抗FXI/FXIa抗体或其抗原结合片段包含鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段为嵌合抗体或其片段。其包含人源κ、λ链或其变体的轻链FR区和/或轻链恒定区，和/或人源IgG1、IgG2、IgG3或IgG4或其变体的重链FR区和/或重链恒定区。
一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段包含恒定区Fc，例如IgG1、IgG2、IgG3、IgG4、IgG4P(即IgG4的S241P突变体)的Fc。所述IgG1的Fc序列例如SEQ ID NO：67所示，所述IgG4P Fc(即包含S241P的IgG4Fc)的序列例如SEQ ID NO：60所示。
一些实施方案中，抗FXI/FXIa抗体或其抗原结合片段的重链如SEQ ID NO：15所示或与之具有至少80％同一性，轻链如SEQ ID NO：16所示或与之具有至少80％同一性；或
重链如SEQ ID NO：61所示或与之具有至少80％同一性，轻链如SEQ ID NO：62所示或与之具有至少80％同一性。
一些实施方案中，抗FXI/FXIa抗体的抗原结合片段为Fab、Fv、sFv、Fab’、F(ab’) 2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体(peptibody)、结构域抗体和多特异性抗体(双特异性抗体、双链抗体(diabody)、三链抗体(triabody)和四链抗体(tetrabody)、串联二-scFv、串联三-scFv)，例如具体为 scFv、Fv、Fab或Fab’片段。
一些实施方案中，提供一种抗FXI/FXIa抗体或其抗原结合片段，其结合与前述抗FXI/FXIa抗体或其抗原结合片段相同的表位。
另一些实施方案中，提供抗FXI/FXIa抗体或其抗原结合片段，其交叉阻断前述抗FXI/FXIa抗体或其抗原结合片段与人FXI/FXIa的结合。
另一些实施方案中，提供抗FXI/FXIa抗体或其抗原结合片段，其与人FXI/FXIa的结合被前述抗FXI/FXIa抗体或其抗原结合片段交叉所阻断。
一些实施方案中，提供抗FXI/FXIa抗体或其抗原结合片段，与前述抗FXI/FXIa抗体或其抗原结合片段的重链和/或轻链具有至少80％同一性。
本公开上下文中，“至少80％”涵盖80％及以上，例如至少80％、至少81％、至少82％、至少83％、至少84％、至少85％、至少86％、至少87％、至少88％、至少89％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％。
一些实施方案中，提供抗FXI/FXIa抗体或其抗原结合片段变体，在前述抗FXI/FXIa抗体或其抗原结合片段的重链可变区和/或轻链可变区包含0、1、2、3、4、5、6、7、8、9或10个氨基酸变化。所述氨基酸变化可以是可变区中的氨基酸残基保守性取代。
一些实施方案中，上述抗FXI/FXIa抗体或其抗原结合片段可以具有以下任一或任几项的特性：
防止内在凝血途径或共同凝血途径的活化；
阻断FXI和/或FXIa结合至凝血途径中的成员(优选地，所述成员选自：凝血因子IX、凝血因子XIIa、凝血酶中的一个或多个)；
阻断FIX、FXI及FXIa中的一个或多个结合至血小板受体；
具有以≤10 -9KD结合至人FXI和/或FXIa蛋白；
结合至FXI/FXIa时，阻止FXI/FXIa催化结构域呈现活性构象；
可用于皮下给药或静脉给药。
上述亲和力的测定方式例如是BIACORE TM。
另一方面，本公开提供一种多核苷酸，其编码前述任一抗FXI/FXIa抗体或其抗原结合片段或抗FXI/FXIa抗体或其抗原结合片段，所述多核苷酸可以是DNA或RNA。
再一方面，本公开提供一种含有如上所述的多核苷酸的表达载体，表达载体选自：真核表达载体、原核表达载体、病毒载体。
再一方面，本公开提供一种用如上所述的表达载体转化的宿主细胞，其可以是真核细胞、或原核细胞。
一些实施方案中，所述宿主细胞选自：细菌、酵母菌、哺乳动物细胞。一些具体实施方案中，所述宿主细胞选自：大肠杆菌、毕赤酵母、中国仓鼠卵巢(CHO)细胞或人胚肾(HEK)293细胞。
再一方面，本公开提供一种用于制备抗FXI/FXIa抗体或其抗原结合片段的方法，包括：在如前所述的宿主细胞中表达该抗体或其抗原结合片段，并自该宿主 细胞中分离该抗体或其抗原结合片段。
再一方面，本公开提供一种组合物，例如药物组合物，其含有：可药用的赋形剂、稀释或载体；和治疗或预防有效量的如上所述的抗FXI/FXIa抗体或其抗原结合片段。在一些具体实施方式中，所述药物组合物的单位计量中可含有0.01至99重量％的抗FXI/FXIa抗体或其抗原结合片段，或药物组合物的单位剂量中含抗FXI/FXIa抗体或其抗原结合片段的量为0.1-2000mg，在一些具体实施方式中为1-1000mg。
一些具体实施方式中，上述药物组合物为皮下给药剂型，或静脉给药剂型。
一些实施方案中，提供皮下给药的药物组合物，含有治疗或预防有效量的前述本公开的抗FXI/FXIa抗体或其抗原结合片段，例如，所述抗体的重链可变区包含SEQ ID NO：58所示序列，轻链可变区包含SEQ ID NO：51所示序列；例如，所述抗体的重链全长包含SEQ ID NO：61所示序列，轻链全长包含SEQ ID NO：62所示序列。
再一方面，本公开提供选自以下的任一项或组合在制备药物中的用途：根据本公开的抗FXI/FXIa抗体或其抗原结合片段、根据本公开的药物组合物。
一些实施方案中，所述药物用于治疗或预防血栓形成或血栓栓塞相关疾病或病症，例如与心房纤维性颤动有关的缺血性中风和/或深层静脉血栓形成。
本公开提供一种治疗或预防血栓形成或血栓栓塞相关疾病或病症(或其并发症)或延缓血栓形成或血栓栓塞相关疾病或病症(或其并发症)进展的方法，该方法包括给予受试者治疗或延缓疾病有效量的根据本公开的抗FXI/FXIa抗体或其抗原结合片段、或根据本公开的药物组合物。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物可以应用于凝块形成、血栓形成或血栓栓塞相关疾病或病症。
上述血栓形成或血栓栓塞相关疾病或病症包括但不限于：心脏冠状动脉疾病(诸如急性冠状动脉综合征(ACS))、有ST段升高的心肌梗死(STEMI)和无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入(诸如血管成形术、支架植入术或主动脉冠状动脉分流术)后的再闭塞和再狭窄，、外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成(特别是在下肢深静脉和肾静脉中)、短暂性脑缺血发作以及血栓形成性脑卒中和血栓栓塞性脑卒中、由慢性血栓栓塞(CTEPH)引起的肺病或肺动脉高压。
凝血***的刺激可通过多种诱因或相关病症发生。在外科手术干预、无法移动、卧床、感染、炎症或者癌症或癌症治疗等的情况下，可高度激活凝血***，并且可能有血栓形成性并发症，特别是静脉血栓。因此，本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物可用于预防外科手术干预情况下的血栓，特别是对于患癌症的患者或者接受整形外科手术(如髋关节或膝关节置换)的患者。因此，本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物还用于预防具有激活的凝血***的患者的血栓，例如在所述的刺激情况下。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防急性、间歇性或持续性心律失常(例如，心房颤动)，或者经历心脏复律，或者患 有心脏瓣膜疾病，或者带有人工心脏瓣膜的患者的心源性血栓栓塞(例如，脑动脉缺血、脑卒中以及全身性血栓栓塞和局部血栓栓塞)。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防弥漫性血管内凝血(DIC)。该疾病的发生可能与败血症特别相关，但也可能归因于外科手术干预、肿瘤疾病、烧伤或其它损伤，并且可能通过微血栓导致严重的器官损伤。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防血栓栓塞性并发症。例如，所述并发症可能发生在微血管病性溶血性贫血中以及，因为在体外循环的情况下，血液与外源表面接触而导致并发症(如血液透析、ECMO(“体外膜氧合”)、LVAD(“左心室辅助装置”)和类似方法)。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防涉及脑血管中微凝块形成或纤维蛋白沉积的疾病，所述疾病可能会导致痴呆症，诸如血管性痴呆或阿尔茨海默病。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于预防和/或治疗癌症患者的血栓形成性和/或血栓栓塞性并发症，例如，静脉血栓栓塞，特别是那些经历了重大外科手术干预或者化疗或放疗的那些患者。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防以下疾病背景下的弥漫性血管内凝血，包括但不限于：传染病和/或全身炎症综合征(SIRS)、脓毒性器官功能障碍、脓毒性器官衰竭和多器官衰竭、急性呼吸窘迫综合征(ARDS)、急性肺损伤(ALI)、脓毒性脑卒中和/或脓毒性器官衰竭。在感染的情况中，可能有凝血***的普遍激活(弥漫性血管内凝血活消耗性凝血，又称“DIC”)，伴有多器官中微血栓形成和继发性出血性并发症。可能存在内皮损伤，伴有血管通透性升高以及体液和蛋白质扩散如外渗空间。随着感染发展，可能有器官衰竭(例如，肾衰竭、肝衰竭、呼吸衰竭、中枢神经功能缺损和心血管衰竭)或多器官衰竭。在DIC的情况中，在受损内皮细胞表面、异物表面或交联的血管外组织表面上存在凝血***的大量激活。因此，在患有缺血和随后的器官功能障碍的多个器官的小血管中存在凝血。继发效应是凝血因子(例如因子X、凝血酶原和纤维蛋白原)和血小板的消耗，这降低了血液的凝固性并且可能导致严重出血。
本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防如下患者的血栓形成性或血栓栓塞性疾病和/或炎性疾病和/或血管通透性升高的疾病：所述患者的基因突变导致酶活性增强或者酶原水平提高，这些通过酶活性或酶原浓度的相关实验/测量来确定。
本公开提供本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于制备治疗和/或预防疾病，尤其是上述疾病的药物的用途，例如用于制备治疗和/或预防血栓形成性或血栓栓塞性疾病的药物。
本公开提供本公开的抗FXI/FXIa抗体或其抗原结合片段、药物组合物用于治疗和/或预防上述疾病的方法。
再一方面，本公开提供检测FXI/FXIa的组合物，所述组合物包含抗FXI/FXIa 抗体或其抗原结合片段。本公开还提供用于体内或体外检测FXI/FXIa的方法、***或装置，其包括处理抗FXI/FXIa抗体。
一些实施方案中，体外检测方法、***或装置可能例如包括：
(1)使待测样品与结合FXI/FXIa的抗体或其抗原结合片段接触；
(2)检测在结合FXI/FXIa的抗体或其抗原结合片段和待测样品之间形成的复合物；和/或
(3)使参比样品(例如，对照样品)与抗体接触；和
(4)通过与参比样品比较，确定抗体和待测样品之间复合物形成的程度。
如与对照样品或受试者中相比，待测样品或受试者中复合物形成的变化(例如，统计学上的显著变化)表示待测样品中存在FXI/FXIa。
另一些实施方案中，体内检测方法、***或装置可以包括：
(1)向受试者施用结合FXI/FXIa的抗体或其抗原结合片段；和
(2)检测在结合FXI/FXIa的抗体或其抗原结合片段和待测物之间复合物的形成。
检测可以包括确定形成复合物的位置或时间。结合FXI/FXIa的抗体可以直接或间接地用可检测物质标记，以促进所结合的或未结合的抗体的检测。合适的可检测物质包括多种酶、辅基、荧光物质、发光物质和放射性物质。可以通过测量与FXI/FXIa结合或不结合的抗体或使其可视化，检测在结合FXI/FXIa的抗体或其抗原结合片段和FXI/FXIa之间的复合物形成。可以使用常规检测测定法，例如，酶联免疫吸附测定(ELISA)、放射免疫测定(RIA)或组织免疫组织化学。
一些实施方案中，通过竞争免疫测定法分析样品中FXI/FXIa的存在，所述竞争免疫测定法使用以可检测物质标记的标准物和未标记的结合FXI/FXIa的抗体。
一些实施方案中，出于检测目的，本公开的抗体或其片段可以用荧光团和发色团标记。
一些实施方案中，还提供试剂盒，所述试剂盒包含抗FXI/FXIa抗体或其抗原结合片段；任选地还可以包含诊断使用说明。试剂盒还可以含有至少一种额外的试剂，如标记物或额外的诊断剂。对于体内使用，抗体可以配制为药物组合物。
附图说明
图1A至图1B：FXI/FXIa抗体结合人FXI/FXIa蛋白的SPR检测。图1A为SPR分析3882分子对FXI的结合结果；图1B为SPR分析1209分子对FXIa的结合结果。
图2A至图2B：FXI/FXIa抗体的体外FXIa酶活抑制试验。图2A为抗FXI/FXIa抗体的体外FXIa酶活抑制试验结果；图2B为抗FXI/FXIa抗体的体外FXIIa介导的FXI活化酶活抑制试验。
图3A至图3B：人血的aPTT、PT抗凝血活性检测。图3A、图3B分别为抗FXI/FXIa抗体在人血上的aPTT和PT试验结果。
图4A至图4B：猴血的aPTT、PT抗凝血活性检测。图4A、图4B分别为抗FXI/FXIa抗体在猴血上的aPTT和PT试验结果。
图5A至图5D：FXI/FXIa抗体在食蟹猴上的动物实验检测。图5A为抗体3882在食蟹猴体内的aPTT、血浆药物浓度、FXI:C％和游离FXI的变化曲线；图5B为3882在食蟹猴体内的血栓生成抑制作用；图5C和图5D为3882在食蟹猴体内的安全性测试，分别出血时间测定和PT检测结果。
图6A至图6B：FXI/FXIa抗体在食蟹猴上体内药效学(PD)试验结果。图6A为APTT(s)检测结果；图6B为FXI：C(％)检测结果，3882(1mg/kg)为静脉给药和皮下给药两种方式，对照BAY1213790(1mg/kg)为静脉给药方式。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“因子XI”在本文中也称为“凝血因子XI”、“FXI”或“fXI”，其是分子量为约160千道尔顿(kD)的双链糖蛋白。两条链可为相同的分子量为约80,000道尔顿的多肽；通过二硫键连接两条链。FXI含有4个“苹果结构域(apple domains)”(来自N-末端的A1-A4，重链)和C-末端催化结构域(轻链)。不希望受限于特定理论，认为四个苹果结构域包含对其它蛋白的结合位点，诸如A1对凝血酶；A2对HK；A3对因子IX(FIX)、GPIb和肝素；A4对FXIIa。FXI可通过因子XIIa(FXIIa)转化为其活性形式凝血因子XIa(FXIa)。丝氨酸蛋白酶FXIa将凝血因子IX转化为IXa，IXa随后激活凝血因子X(Xa)。Xa随后可介导凝血因子II/凝血酶激活。
在本公开的范畴内，FXI和FXIa应做最广泛的理解。该术语涵盖FXI和FXIa在自然界中的天然形式、天然存在的变体，也包括人工表达的形式。当上下文没有专门说明时，涉及抗原-抗体相互作用的情形下，FXI(或FXIa)涵盖完整蛋白及其表位的范畴。
“抗体”以最广义使用，涵盖各种抗体结构，包括但不限于单克隆抗体，多克隆抗体；单特异性抗体，多特异性抗体(例如双特异性抗体)，全长抗体和抗体片段(或抗原结合片段，或抗原结合部分)，只要它们展现出期望的抗原结合活性。抗体可以指免疫球蛋白，是由两条重链和两条轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中第每类Ig都可以有κ链或λ链。抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(CDR)和4个序列相对保守的骨 架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(VL)和重链可变区(VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1，LCDR2，和LCDR3；重链的3个CDR区指HCDR1，HCDR2和HCDR3。
对于CDR的确定或定义，能够通过分辨抗体的结构和/或分辨抗体-配体复合物的结构来完成CDR的确定性描绘和包含抗体的结合位点的残基的鉴定。这可通过本领域技术人员已知的各种技术中的任一种，例如X射线晶体学来实现。多种分析方法可用于鉴定CDR，包括但不限于Kabat编号***、Chothia编号***、AbM编号***、IMGT编号***、接触定义、构象定义。
Kabat编号***是用于编号抗体中残基的标准，并且通常用于鉴定CDR区域(参见例如Johnson&Wu，2000，Nucleic Acids Res.，28：214-8)。
Chothia编号***与Kabat编号***类似，但Chothia编号***考虑了某些结构环区域的位置(参见例如Chothia等，1986，J.Mol.Biol.，196：901-17；Chothia等人，1989，Nature，342：877-83)。
AbM编号***使用建模抗体结构的由Oxford Molecular Group生产的计算机程序集成套件(参见例如Martin等，1989，ProcNatl Acad Sci(USA)，86：9268-9272；“AbMTM，A Computer Program for ModelingVariable Regions of Antibodies，”Oxford，UK；Oxford Molecular，Ltd)。AbM编号***使用知识数据库和De-novo方法的组合，从基本序列建模抗体的三级结构(参见Samudrala等，1999，在PROTEINS，Structure，Function and Genetics Suppl.，3：194-198中的“Ab Initio Protein Structure Prediction Using a Combined HierarchicalApproach”描述的那些)。
对于接触定义，其是基于可用复杂晶体结构的分析(参见例如MacCallum等，1996，J.Mol.Biol.，5：732-45)。构象定义中，CDR的位置可鉴定为对抗原结合做出焓贡献的残基(参见例如Makabe等，2008，Journal ofBiological Chemistry，283：1156-1166)。
另外其它的CDR边界定义可能不严格遵循上述方法之一，但仍然与Kabat CDR的至少一部分重叠。尽管根据特定残基或残基组不显著影响抗原结合的预测或实验结果，它们可缩短或延长。如本公开使用的，CDR可指通过本领域已知的任何方法(包括方法的组合)定义的CDR。
本公开的抗体或抗原结合片段的VL区和VH区的CDR氨基酸残基在数量和位置符合已知的Kabat编号***。在未采用Kabat编号***时，相较于本公开CDR中的氨基酸位点，技术人员可以确定不同编号规则中对应的等同位点。再比如，技术人员通过氨基酸序列的比对分析，也可以确定这样的等同位点。
“CL”、“CL区”和“CL结构域”在本文中可互换使用以指代轻链恒定区。
“CH”、“CH区”和“CH结构域”在本文中可互换使用以指代重链恒定区，并且包括“CH1”、“CH2”和“CH3”区或结构域。
“人抗体”或“重组人抗体”包括通过重组方法制备、表达、创建或分离的人抗 体，所涉及的技术和方法在本领域中是熟知的，诸如：
(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；
(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；
(3)从重组组合人抗体文库中分离的抗体；以及
(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。
此类重组人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的针对人FXI/FXIa或其表位的单克隆抗体。制备时用FXI/FXIa抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在本公开一个具体的实施方案中，所述的鼠源抗人FXI/FXIa抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区。
术语“人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本公开的人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“人抗体”不包括人源化抗体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人物种CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
术语“嵌合抗体(chimeric antibody)”，是将第一物种抗体的可变区与第二物种的恒定区融合而成的抗体，可以减轻第一物种抗体诱发的免疫应答反应。作为一个示例，建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“抗原结合片段”包括单链抗体(即重链或轻链)；Fab、修饰的Fab、Fab’、修饰的Fab’、F(ab’)2、Fv、Fab-Fv、Fab-dsFv、单结构域抗体(例如VH或VL或VHH)、scFv、二价或三价或四价抗体、Bis-scFv、双链抗体(diabody)、三链抗体(tribody)、四链抗体(tetrabody)和上述任意一种的表位结合片段(参见例如Holliger and Hudson，2005，Nature Biotech.23(9):1126-1136；Adair and Lawson， 2005，Drug Design Reviews-Online 2(3)，209-217)。产生和制备这些抗体片段的方法在本领域是公知的(参见例如Verma等人，1998，Journal ofImmunological Methods，216，165-181)。Fab-Fv形式首先公开于WO2009/040562，其二硫键稳定化形式Fab-dsFv首先公开于WO2010/035012。本公开的抗原结合片段还包括描述于WO2005/003169、WO2005/003170和WO2005/003171中的Fab和Fab’片段。多价抗体可包含多特异性，例如双特异性或单特异性(参见例如WO92/22583和WO05/113605)。
“变体”是指与“亲本”氨基酸序列相比，含有至少一个氨基酸修饰(如取代、缺失或***)的多肽，只要该变体依然能够结合FXI/FXIa，特别是如SEQ ID NO：1所示的人FXI/FXIa。优选，所述变体与本公开实施例中抗体0012、1209、1267、3807、3871、3882相比，显示相似甚至改善的特性的。本公开的结合分子(如抗体或其抗原结合片段)的变体通常通过向编码抗体或抗体片段的核酸中引入合适的核苷酸变化或通过肽合成来制备。通常，上述氨基酸修饰可被引入可变区或恒定区，例如，与本公开实施例中抗FXI/FXIa抗体相比，可引入氨基酸修饰以调节影响药品开发的抗体特性，如热力学稳定性、溶解度或粘度(“序列优化”)。
氨基酸修饰包括，例如，结合分子(优选抗体或抗原结合片段)的氨基酸序列中残基的缺失和/或***和/或取代。可向“亲本”氨基酸序列中引入缺失、***和取代的任何组合以得到最终变体。氨基酸修饰还包括可改变结合分子的翻译后过程，诸如改变糖基化位点的数量或位置。例如，取决于CDR和FR本身的长度，可将1、2、3、4、5或6个氨基酸***到每个CDR中或从其中缺失，可将1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20或25个氨基酸***每个FR或从其中缺失。本公开的结合分子(特别是抗体或抗体片段)的***变体包括抗体或抗体片段与酶或另一种功能性多肽(例如，其提高结合分子(例如，抗体或抗体片段)的血清半衰期)的融合产品。以及，可将氨基酸取代引入重链和/或轻链的CDR、VH或FR区。优选保守性取代，例如，其可基于所涉及的残基的极性、电荷、溶解度、疏水性、亲水性和/或两亲性特质的相似性而做出。
“保守性取代”指替换为具有与原始氨基酸残基相似的特性的另一个氨基酸残基。例如，赖氨酸、精氨酸和组氨酸具有相似的特性，在于它们具有碱性侧链，并且天冬氨酸和谷氨酸具有相似的特性，在于它们具有酸性侧链。此外，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸和色氨酸具有相似的特性，在于它们具有不带电荷极性侧链，并且丙氨酸、缬氨酸、亮氨酸、苏氨酸、异亮氨酸、脯氨酸、苯丙氨酸和甲硫氨酸具有相似的特性，在于它们具有非极性侧链。另外，酪氨酸、苯丙氨酸、色氨酸和组氨酸具有相似的特性，在于它们具有芳族侧链。因此，本领域技术人员将显而易见，甚至当取代如上文所述的显示相似特性的组中的氨基酸残基时，它将不显示特性的特定变化。
除了CDR和FR之外，也可向结合分子(优选抗体或其抗原结合片段)的Fc部分中引入修饰。这种修饰可用于调节抗体的功能性特性，例如，与其它免疫细胞上的补体蛋白(诸如C1q和/或Fc受体)的相互作用，或者用于调节血清半衰 期或抗原依赖性细胞毒性(ADCC)。可使用本领域已知的常规方法将改变效应子功能的突变引入到Fc结构域中。示例性的修饰包括：导致IgG1糖基化的Asn297→Ala297和Asn297→Gln297，或者Lys322→Ala322以及任选地Leu234→Ala234和Leu235→Ala234，其被报道减少或消除源自抗体的细胞介导的细胞毒性(ADCC)和/或源自补体的细胞毒性(CDC)。
延长血清半衰期的方式的实例包括附加结合人体内其它蛋白质(诸如血清白蛋白、免疫球蛋白Fc区或新生儿Fc受体(FcRn))的肽或蛋白结构域。其它可想到的延长血清半衰期的方式包括：用不同长度的多肽链延长氨基端(例如，XTEN技术或
Figure PCTCN2021104253-appb-000001
)、与非蛋白聚合物或者糖类的缀合，所述非蛋白聚合物包括但不限于：各种多元醇，诸如聚乙二醇(PEG化)、聚丙二醇、聚氧化烯烃或聚乙二醇与聚丙二醇的共聚物；或者糖类，诸如羟乙基淀粉(例如，
Figure PCTCN2021104253-appb-000002
)或聚唾液酸(例如，
Figure PCTCN2021104253-appb-000003
)。此外，如本领域已知的，可在结合分子中的多个位置进行氨基酸取代以促进所述聚合物的加成。
本公开的术语“与FXI/FXIa结合”，指能与FXI/FXIa或其表位相互作用，所述FXI/FXIa或其表位可以是人源的。
本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“表位”是指抗原上与免疫球蛋白或抗体结合的位点。表位可以由相邻的氨基酸、或不相邻的氨基酸(通过蛋白的三级折叠而使不相邻的氨基酸在空间上互相接近)形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象存在，其包括至少3-15个氨基酸。确定表位的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”是指抗体与预定的抗原上的表位结合。通常，用人FXI/FXIa或其表位作为分析物，并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于10 -7M或甚至更小的平衡解离常数(K D)与预定的抗原或其表位结合，并且其与预定抗原或其表位结合的亲和力是其与预定抗原或其表位(或紧密相关的抗原之外的非特异性抗原，如BSA等)结合的亲和力的至少两倍。
“亲和力”是指分子(例如，抗体)的单个结合部位与其结合配体(例如，抗原)之间非共价相互作用的总体的强度。除非另外指明，如本文所用，“结合亲和力”是指内部结合亲和力，其反映出结合对(例如，抗体与抗原)的成员之间1:1相互作用。分子X对其配体Y的亲和力通常可以由平衡解离常数(KD)表示。亲和力可以通过本领域已知的常规方法(包括本文所述的那些)测量。术语“kassoc”或“ka”指特定抗体-抗原相互作用的缔合速率，而如本文所使用的术语“kdis”或“kd”意在是指特定抗体-抗原相互作用的解离速率。如本文所使用的，术语“KD”指平衡解离常数，其获得自kd与ka的比率(即kd/ka)并且表示为摩尔浓度(M)。可以使用本领域已知的方法测定抗体的KD值，例如：用于测定抗体KD的方法包 括使用生物传感***例如***测量表面等离子体共振，或通过溶液平衡滴定法(SET)测量溶液中的亲和力。“交叉反应”是指本公开的抗体(或其片段)与来自不同物种的FXI/FXIa结合的能力。例如，结合人FXI/FXIa的本公开的抗体也可以结合另一物种的FXI/FXIa。交叉反应性是通过在结合测定(例如SPR或ELISA)中检测与纯化抗原的特异性反应性，或与生理表达FXI/FXIa的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振分析，或流式细胞术。
“抑制”或“阻断”可互换使用，并涵盖部分和完全抑制或阻断。对FXI/FXIa的抑制或阻断优选地降低或改变无抑制或阻断的情况下发生FXI/FXIa结合时出现活性的正常水平或类型。抑制和阻断也旨在包括与抗FXI/FXIa抗体接触时，与未与抗FXI/FXIA抗体接触的FXI/FXIa相比，任何可测量的FXI/FXIa结合亲和力降低。
“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南(5-8章和15章)。如，可以用人FXI/FXIA或其片段免疫小鼠，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至表达载体。重组表达载体可以稳定地转染CHO细胞。哺乳动物表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本公开的抗体指单克隆抗体(mAb)，指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的、原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-嫁接)，或其它现有技术进行重组得到。
可使用本领域技术人员已知的常规技术，就与相同表位的结合竞争性筛选抗体。例如，可进行竞争和交叉竞争研究，以获得彼此竞争或交叉竞争与抗原结合的抗体。基于它们的交叉竞争来获得结合相同表位的抗体的高通量方法描述于国际专利公开WO03/48731中。因此，可使用本领域技术人员已知的常规技术，获得与本公开的抗体分子竞争结合FXI/FXIa上的相同表位的抗体及其抗原结合片段。
“给予”、“施用”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、 细胞、组织、器官或生物流体的接触。“给予”、“施用”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”、“施用”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如包含本公开的任一种抗体或其抗原结合片段的组合物，所述受试者已经患有、疑似患有、倾向于患有一种或多种疾病或其症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽本公开的实施方案(例如治疗方法或制品)在缓解某个受试者中目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学病症的症状的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。
“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括其后代。还应当理解的是，由于有意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“药物组合物”表示含有一种或多种本文所述抗体或抗原结合片段或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，以及其他组分例如生理学/可 药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
实施例
以下结合实施例用于进一步描述，但这些实施例并非限制的范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.人FXI/FXIa抗原及检测蛋白的制备
1.人FXI/FXIa蛋白
人FXI/FXIa蛋白(Uniprot Acc No.P03951)作为本公开涉及的抗原及检测用蛋白，购自Enzyme research laboratories(FXI:Cat.HFXI 1111；FXIa:HFXIa 1111a)。以下FXI/FXIa抗原未特殊说明的均指人FXI/FXIa。
>人FXI/FXIa的氨基酸序列：
Figure PCTCN2021104253-appb-000004
人工合成以下KLH偶联的FXIa特异性多肽用于小鼠免疫。
KLH-CFYGVESPKILRVYSGIL(SEQ ID NO：2)
KLH-CGYRKLRDKIQNTLQKAKIPL(SEQ ID NO：3)
KLH-CGVQEIIIHDQYKMAESGYDI(SEQ ID NO：4)。
2.FXI/FXIa相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
(1)鼠杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清的纯化，首选ProteinG填料(KANEKA:Cat.KanCapTMG)进行亲和层析。将培养所得杂交瘤离心取上清，装有ProteinG填料的重力柱用来纯化。首先，用3-5倍柱体积的6M盐酸胍(Simga:Cat.G3272-1KG)进行再生，然后利用3-5倍柱体积的纯水清洗；再用3-5倍柱体积的1×PBS(pH7.4)(Sangon Biotech(Shanghai)co.，Ltd.:Cat.E607016-0500)缓冲体系作为平衡缓冲液对层析柱平衡；将上清低流速进行上样，以便结合，控制流速使保留时间约1min或更长 时间；利用3-5倍柱体积的1×PBS(pH7.4)洗涤层析柱至紫外吸收回落至基线；利用0.1M Glycine(pH3.0)(Sigma:Cat.410225-250G)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)(Vetec，Cat.V900483)快速调节pH至7-8，暂存。对于洗脱产物，可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩，及溶液置换至所需的缓冲体系，或者利用分子排阻(如G-25脱盐)替换成所需的缓冲体系。
(2)通过Protein A亲和层析，提取带人Fc标签的融合蛋白或者抗体：
首先，将表达Fc融合蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA(GE，Cat:17-5474-99)亲和柱利用5倍柱体积的0.1M NaOH(Sigma:Cat:71687-500g)再生，然后用5倍柱体积的1×PBS(pH7.4)(Sangon Biotech(Shanghai)co.，Ltd.:Cat.E607016-0500)清洗平衡。将上清低流速进行上样，以便结合，控制流速使保留时间约1min或更长时间，结合完毕后利用5倍柱体积的1×PBS(pH7.4)冲洗层析柱至紫外吸收回落至基线。利用0.1M Glycine(pH3.0)(Sigma:Cat.410225-250G)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)(Vetec，Cat.V900483)快速调节pH至7-8，暂存。对于洗脱产物，可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩，及溶液置换至所需的缓冲体系，或者利用分子排阻(如G-25脱盐)替换成所需的缓冲体系。
(3)阴离子层析精纯抗体：
首先，用平衡缓冲液20mM Tris pH 8.0(Vetec，Cat.V900483)稀释样品，使得电导率小于3ms/cm，Q HP(GE，Cat:17-1014-01)阴离子层析柱用5倍柱体积的0.5M NaOH(Sigma Cat:71687-500g)再生，然后用15倍柱体积的20mM Tris pH 8.0(Vetec，Cat.V900483)清洗平衡。将上清低流速进行上样，以便结合，控制流速使保留时间约2min或更长时间，结合完毕后利用10倍柱体积的20mM Tris pH 8.0(Vetec，Cat.V900483)冲洗层析柱至紫外吸收回落至基线。用洗脱缓冲液20mM Tris pH 8.0(Vetec，Cat.V900483)0.5M NaCl(Vetec，Cat.V900058)进行0-100％的梯度洗脱；根据SEC-UPLC(Column：Waters
Figure PCTCN2021104253-appb-000005
Protein BEH SEC column
Figure PCTCN2021104253-appb-000006
1.7μm，Cat.186005225)纯度，检测收集洗脱峰。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻(如G-25脱盐)替换成所需的缓冲体系。
实施例2.筛选特异性结合FXI/FXIa的抗体
通过全人源单链抗体噬菌体库的筛选来获得与FXIa具有高亲和力的抗体。用2μg随机生物素化的FXIa蛋白结合100μg Dynabeads MyOne Streptavidin T1，室温1小时。PBST(0.05％Tween-20)洗3遍，将脱脂牛奶溶于1×PBS，终浓度2％作为封闭剂，加入体系，室温封闭1小时。加入用2％的牛奶室温封闭1小时的全人源单链抗体噬菌体展示文库，在室温下作用1小时。PBST(0.05％Tween-20)，pH 7.4溶液洗11遍，每遍上下翻转20次，去除不结合的噬菌体。用1mg/mL的 0.5mL的胰蛋白酶将与FXIa特异性结合的剩余噬菌体洗脱，分别感染处于对数期生长的大肠杆菌TG1，产生并纯化噬菌体用于下一轮筛选。相同筛选过程重复2-3轮，FXIa使用量逐渐下降为0.5μg(针对第二轮筛选)和0.1μg(针对第三轮筛选)。第三轮筛选后，阳性的克隆被富集。
从筛选富集的克隆中，挑取单克隆菌落包装成单链抗体噬菌体，用于噬菌体ELISA测试。ELISA板上包被1μg/mL的FXIa的蛋白，4℃放置过夜，用PBST(0.05％Tween-20)洗3遍，用2％脱脂牛奶室温封闭1个小时，PBST(0.05％Tween-20)洗3遍后，加入封闭液稀释的噬菌体上清，室温反应1小时，PBST(0.05％Tween-20)洗6遍，加入抗-M13HRP(Sino Biological Inc.，11973-MM05T-H)，室温反应1小时，PBST(0.05％Tween-20)洗3遍，加入100μL TMB显色底物，并用100μL 1M硫酸终止反应，用SpectraMax M5酶标仪在450nm处读取吸收值检测。将ELISA结合测试中OD450值大于背景值3倍的克隆进行测序，得到高亲和力的单链抗体。
实施例3.构建完整的抗FXI/FXIa单抗
将实施例2筛选得到的高亲和力的特异性序列，构建完整重组抗体。然后，通过ELISA结合实验、ForteBio蛋白相互作用实验和对FXIa酶活抑制实验，确定其中2个抗体的结合力强并能有效抑制FXIa酶活。其完整的可变区序列如下：
>0012-VH：
Figure PCTCN2021104253-appb-000007
>0012-VL：
Figure PCTCN2021104253-appb-000008
(注：抗体编号规则为kabat)。
以上抗体重链和轻链可变区序列中，顺序为：FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，下划线分别为CDR1、CDR2、CDR3序列。各重链及轻链CDR序列总结如表1。
表1.重链及轻链CDR区序列
Figure PCTCN2021104253-appb-000009
实施例4.抗FXI/FXIa单抗的亲和力成熟
将抗体分子0012进行三维结构模拟，模拟与已知抗原结构(PDB ID:6AOD  Chain:C)的结合。参考人种系基因的突变热点、三维结构与结合模拟结果，选定部分氨基酸残基，建立若干随机突变噬菌体文库。利用噬菌体文库展示技术，筛选出亲和力有所提高的功能性抗体。对不同文库获得的新氨基酸残基进行组合与验证，获得亲和力与功能均有提高的功能性抗体。得到的抗体分子可变区重链和轻链序列分别如下:
>F-VH:
Figure PCTCN2021104253-appb-000010
>H-VH:
Figure PCTCN2021104253-appb-000011
>J-VH:
Figure PCTCN2021104253-appb-000012
>K-VH:
Figure PCTCN2021104253-appb-000013
>3-VL:
Figure PCTCN2021104253-appb-000014
以上序列编号规则为Kabat，顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中下划线分别为CDR1、CDR2、CDR3序列。
0012-F3的重链轻链可变区分别为F-VH和3-VL；0012-H3的重链轻链可变区分别为H-VH和3-VL；0012-J3的重链轻链可变区分别为J-VH和3-VL；0012-K3(即1209)的重链轻链可变区分别为K-VH和3-VL。
表2.抗体0012亲和力成熟后的重链和轻链CDR序列
Figure PCTCN2021104253-appb-000015
Figure PCTCN2021104253-appb-000016
实施例5.降低抗FXI/FXIa全人源单抗免疫原性的改造
通过对选定的全人源特异性抗体分子进行三维结构同源建模，结合与V-base人种系序列数据库，IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与筛选出来的抗体同源性高的重链和轻链可变区种系基因作为模板，将原单克隆抗体FR区与CDR区进行改造，在保留功能的同时使序列更接近人种系基因。对移植后的抗体再次进行三维结构模拟并分析，将FR区中影响CDR区结构形态的特定位点进行回复突变。氨基酸残基由Kabat编号***确定并注释。改造后的抗体具有更高的稳定性，更低的免疫原性。
亲和力成熟抗体种系基因架构选择
经分析，对于抗体0012，用Vbase数据库中人源种系基因VH1-24作为重链模板，用Vbase数据库中人源种系基因VKIIIA27作为轻链模板。
基因改造后，获得抗体Fg3g(即1268)、Hg3g、Jg3g和Kg3g(即1267)，其重轻链可变区序列如下：
>Fg3g的VH：
Figure PCTCN2021104253-appb-000017
>Hg3g的VH：
Figure PCTCN2021104253-appb-000018
>Jg3g的VH：
Figure PCTCN2021104253-appb-000019
>Kg3g的VH：
Figure PCTCN2021104253-appb-000020
>Fg3g或Hg3g或Jg3g或Kg3g的VL：
Figure PCTCN2021104253-appb-000021
Figure PCTCN2021104253-appb-000022
上述抗体的CDR编号***为Kabat。顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中下划线分别为CDR1、CDR2、CDR3序列。
以上各重链可变区与对应的人源抗体的CH1(SEQ ID NO：13)融合，再与IgG1Fc(SEQ ID NO：67)融合，轻链可变区与人源kappa(SEQ ID NO：14)融合，组成重组抗体进行后续检测。
>人抗体的CH1：
Figure PCTCN2021104253-appb-000023
>人抗体的IgG1Fc：
Figure PCTCN2021104253-appb-000024
>人抗体轻链Cκ：
Figure PCTCN2021104253-appb-000025
以1209示例抗体的全长重轻链序列：
>1209-HC：
Figure PCTCN2021104253-appb-000026
>1209-LC：
Figure PCTCN2021104253-appb-000027
实施例6.抗FXI/FXIa的杂交瘤单克隆抗体的筛选和制备
1.免疫小鼠
委托上海睿智化学进行免疫及脾细胞融合实验。取5只SJL白小鼠，5只Balb/c白小鼠，分别用25μg的FXIa抗原和3个KLH偶联的多肽与佐剂混合免疫。时间为第0、14、35天。第0天腹膜内(IP)注射25μg/只的乳化后抗原。第14、35天注射12.5μg/只。于第21、42天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射50μg/只的生理盐水配制的抗原溶液。
2.脾细胞融合
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0-Ag14细胞进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以每孔细胞数1×10^4至1×10^5种于96孔板中，37℃，5％CO 2孵育并补充HAT完全培养基100μl/孔，10-14天后进行ELISA检测。
3.杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并将结合ELISA检测的阳性孔细胞上清进行纯化、细胞结合实验和细胞阻断实验。结合和阻断的实验结果均为阳性的孔细胞及时进行扩增冻存和测序。
4.杂交瘤细胞进一步筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测。并将结合ELISA检测的阳性孔细胞上清进行酶活抑制实验。结合和功能的实验结果均为阳性的孔细胞进行了一到二次亚克隆稀释直至获得单细胞克隆。
每次亚克隆细胞也均需进行FXI/FXIa结合ELISA和FXIa酶活抑制实验，通过以上实验筛选得到杂交瘤克隆，进行扩大培养进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
5.杂交瘤阳性克隆序列测定
收集对数生长期杂交瘤细胞，用RNeasy Micro试剂盒(Qiagen，Cat No.74004)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara，Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen，TB326Rev.B 0503)进行PCR扩增后测序。获得阳性克隆3807，对应的抗体可变区氨基酸序列如下：
>3807-VH：
Figure PCTCN2021104253-appb-000028
>3807-VL：
Figure PCTCN2021104253-appb-000029
表3.抗体3807的重链及轻链CDR区序列
Figure PCTCN2021104253-appb-000030
实施例7.抗FXI/FXIa杂交瘤抗体的人源化
通过对抗体分子3807进行三维同源建模，结合V-base人种系序列数据库，IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与筛选出来的抗体同源性高的重链可变区种系基因作为模板，将小鼠来源单克隆抗体的CDR移植到相应的人源模块中。对移植后的抗体再次进行三维结构模拟并分析，将FR区中影响CDR区结构形态的特定位点进行恢复突变。其中氨基酸残基由Kabat编号***确定并注释。
1.杂交瘤克隆3807人源化架构选择
鼠源抗体3807的人源化轻链模板为IGKV3-11*01，重链模板为IGHV1-69-2*01，人源化可变区序列如下(下划线为CDR)：
>3807VH-CDR移植
Figure PCTCN2021104253-appb-000031
>3807VL-CDR移植
Figure PCTCN2021104253-appb-000032
2.杂交瘤克隆3807的回复突变
位点和突变方式设计如表4。
表4.各重链可变区及轻链可变区序列
Figure PCTCN2021104253-appb-000033
具体序列如下。
>3807-VH1：
Figure PCTCN2021104253-appb-000034
>3807-VH2：
Figure PCTCN2021104253-appb-000035
>3807-VH3：
Figure PCTCN2021104253-appb-000036
>3807-VH4：
Figure PCTCN2021104253-appb-000037
>3807-VH5：
Figure PCTCN2021104253-appb-000038
>3807-VL1：
Figure PCTCN2021104253-appb-000039
>3807-VL2：
Figure PCTCN2021104253-appb-000040
>3807-VL3：
Figure PCTCN2021104253-appb-000041
>3807-VL4：
Figure PCTCN2021104253-appb-000042
抗体3871的VH为3807-VH1，VL为3807-VL3；抗体3875的VH为3807-VH5，VL为3807-VL3。
实施例8.降低抗FXI/FXIa人源化单抗免疫原性的改造
按照实施例5的方法对3871、3875进行基因改造，使抗体具有更高的稳定性，更低的免疫原性。仅对重链FR区进行了改造，轻链可变区未变动。
1.人源化抗体3875基因改造的重链可变区序列如下(轻链不变)：
>3807-VH5(ESAN)
Figure PCTCN2021104253-appb-000043
>3807-VH5(TSTN)
Figure PCTCN2021104253-appb-000044
>3807-VH5(ESTN)
Figure PCTCN2021104253-appb-000045
2.人源化抗体3871基因改造的重链可变区序列如下(轻链不变)：
>3807-VH1(AR)
Figure PCTCN2021104253-appb-000046
>3807-VH1(YTFT)
Figure PCTCN2021104253-appb-000047
>3807-VH1(GTFS)
Figure PCTCN2021104253-appb-000048
Figure PCTCN2021104253-appb-000049
抗体3882的VH为3807-VH1(GTFS)，VL为3807-VL3。
将以上各重链可变区与对应的人源抗体重链CH1(SEQ ID NO：59)和人抗体IgG4的Fc(含S241P突变)(SEQ ID NO：60)融合，轻链可变区与人源kappa的恒定区CL1(SEQ ID NO：14)融合，组成重组嵌合抗体进行后续检测。
>人抗体重链CH1：
Figure PCTCN2021104253-appb-000050
>人抗体IgG4PFc(即含S241P突变)：
Figure PCTCN2021104253-appb-000051
以3882示例抗体全长序列。
>3882-HC：
Figure PCTCN2021104253-appb-000052
>3882-LC：
Figure PCTCN2021104253-appb-000053
表5.本公开抗体的CDR通式结构
Figure PCTCN2021104253-appb-000054
Figure PCTCN2021104253-appb-000055
实施例9.抗FXI/FXIa抗体制备和对人FXI/FXIa的结合能力检测
将本公开的抗FXI/FXIa鼠源或者人源化抗体的重链可变区克隆进含有人源抗体重链IgG1的哺乳动物细胞表达载体，将轻链可变区克隆进含有人源抗体kappa轻链的恒定区Cκ1的哺乳动物细胞表达载体；用表达载体转染ExpiCHO细胞。按照1μg DNA/mL转染细胞的量比，使用ExpiCHO Expression System(Cat.no.A29133)进行转染，按试剂说明书进行。采用标准方法，放置于37℃摇床震荡培养(8％CO 2)，第8天收取细胞培养液，4000rpm离心，取上清并使用0.45uM的滤膜过滤。经检测，获得目的抗体。
Biacore测定
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一系列浓度梯度的人FXI/FXIa。利用Biacore仪器(Biacore T200，GE)实时检测反应信号，从而获得结合和解离曲线。在每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液或pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54，GE)将生物芯片洗净再生。实验中用到的缓冲液为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用双蒸水稀释至1×(pH 7.4)。
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，得出亲和力数值，部分抗体测试结果见下图1A，图1B及表6。
表6.抗FXI/FXIa抗体的亲和力测定
抗体 抗原 ka(1/Ms) kd(1/s) K D(M)
3871 FXI 1.32E+06 2.66E-05 2.00E-11
3875 FXI 7.99E+05 3.02E-05 3.78E-11
3882 FXI 1.973E+6 5.846E-5 2.963E-11
0012 FXIa 5.43E+05 5.27E-04 1.06E-09
1209 FXIa 6.74E+05 4.73E-05 7.03E-11
1267 FXIa 8.73E+05 1.79E-05 2.22E-11
3807 FXIa 1.59E+06 2.35E-04 1.48E-10
3871 FXIa 1.24E+06 7.08E-04 5.70E-10
3875 FXIa 1.53E+06 3.36E-04 2.20E-10
3882 FXIa 1.06E+06 3.87E-04 3.64E-10
实施例10.抗FXI/FXIa抗体的体外FXIa酶活抑制试验
为测试抗FXI/FXIa抗体的功能，检测抗FXI/FXIa抗体与胰蛋白酶FXIa结合并抑制FXIa酶切特异性底物的能力，以及抗FXI/FXIa抗体与胰蛋白酶原FXI结合并阻断FXIIa对FXI酶切的能力。
预先设置SpectraMax M5酶标仪到37℃，冰上预冷384孔板(thermo，Cat.94410153)，加入分别用1×PBS稀释的20μL待测抗体(10ug/mL)和10μL FXIa胰蛋白酶(5μg/mL)，在37℃孵育5min，然后放冰上孵育5min，加入10μL S-2366(2mM)(Chromogenix，S821090)后，立即用SpectraMax M5酶标仪，在37℃读取405nm的动力学曲线(一分钟读一次，60min)。
部分抗体测试结果见图2A，其中，使用人IgG同种型作为阴性对照(NC)。
检测抗FXI/FXIa抗体阻断FXIIa对FXI酶切的能力则是，冰上预冷384孔板后，加入分别用缓冲液(20mM HEPES，pH 7.4，150mM NaCl和0.1％BSA)稀释的10μl FXI(12μg/ml)、10μl FXIIa(7.8μg/ml)、10μl的FXI/FXIa待测抗体(300μg/ml)、10μl的Dextran(100μg/ml)，在37℃孵育60min，然后放冰上孵育5min，加入10μl S-2366(8mM)(Chromogenix，S821090)后，酶标仪检测。
部分抗体测试结果见图2B，其中，使用人IgG同种型作为阴性对照(NC)。
实施例11.人血/猴血的aPTT、PT抗凝血活性检测
用枸橼酸钠管新鲜采集人血/猴血，3000rpm离心15min，取上层血浆。
用Sysmex试剂盒测定在不同浓度的候选抗体(PBS稀释)的存在下的活化部分凝血活酶时间(aPTT)，将所要测试的候选抗体用血浆在37℃下孵育3分钟，然后通过加入25mM氯化钙试剂启动凝血，测定发生凝血时的时间。测定使aPTT相对于PBS延长50％(即aPTT1.5)的候选抗体的浓度，部分抗体测试结果见图3A，图4A和表7。
用Sysmex试剂盒测定在不同浓度的候选抗体(PBS稀释)存在下的凝血酶原时间(PT)，将所要测试的候选抗体用血浆在37℃下孵育3分钟，然后通过加入促凝血酶原激酶启动凝血，测定发生凝血时的时间。部分抗体测试结果见图3B和图4B。
结果显示，3807、3871、3875、3882比Bay1213790在更低浓度就能达到aPTT1.5倍的延长，对FXI的抑制作用更强，能更有效抑制凝血，并且上述所有抗体均无延长PT的作用。
表7.抗FXI/FXIa抗体的aPTT1.5测定
抗体 血浆 aPTT1.5(ug/mL*)
0012 人血 14.59
1209 人血 4.68
3807 人血 1.33
3871 人血 0.88
3875 人血 0.87
3882 人血 0.63
Bay1213790 人血 2.15
0012 猴血 >8.33
1209 猴血 4.08
3807 猴血 1.00
3871 猴血 1.12
3882 猴血 1.14
Bay123790 猴血 2.73
*所述浓度为***终浓度。
实施例12.食蟹猴体内药代动力学(PK)/药效学(PD)试验
正常成年雄性食蟹猴(体重在4.0-4.7kg范围)，按照体重分配入组(体重第1、4、5入一组，体重第2、3、6入另一组)，每组3只，观察14天。
给药前一天以枸橼酸钠采血管采集血样，作为给药前样本，测定aPTT、PT、血浆药物浓度、血浆活性XI因子(Factor XI,FXI)比例(FXI:C％，即有凝血活性的FXI的比例)和血浆游离FXI浓度；同时测定每只动物的出血时间。两组动物中，其中一组空白不给药；另一组给药3882，5毫克/千克体重(mg/kg,mpk)。药物按照5毫克/毫升(mg/mL)浓度溶于磷酸盐缓冲液(PBS)，静脉推注给药。
空白对照组于给药后15分钟(min)、3小时(h)分别观测出血时间；于给药后15min、3h、6h和1天(d)分别按照上述方法采集血浆并观测aPTT和PT。给药组于给药后15min、3h、2d、4d、1周(w)、2w和3w分别观测出血时间；于给药后15min、3h、6h、1d、2d、4d、1w、2w、3w、4w、5w和6w分别按照上述方法采集血浆并观测aPTT、PT、血浆药物浓度、血浆FXI:C％和血浆游离FXI浓度。两组动物均于给药1d后进行AV-shunt造栓，造栓时间10min，造栓后称取栓净重。
其中aPTT、PT和血浆FXI:C％以凝血仪和相应试剂盒测定，血浆游离FXI浓度和血浆药物浓度以ELISA方法测定。血栓重量以给药组与对照组进行对比，以t检验进行统计分析。
抗体测试结果参见图5A-图5D，其中，阴性对照(NC)为未给药。结果显示，5mpk良好的抑制了血栓生成，同时，延长了内源性凝血时间，但对动物出血时间和外源性凝血时间没有显著影响，PK结果显示半衰期为20天左右。其中，图5B中的“**”代表P值<0.01，为显著性差异。
实施例13.食蟹猴体内药效学(PD)试验
正常成年雄性食蟹猴(体重在7-9kg范围)6只，随机分为3组，分别为：
BAY1213790静脉给药1毫克/千克体重(mg/kg)组；
3882静脉给药1毫克/千克体重(mg/kg)组；
3882皮下给药1毫克/千克体重(mg/kg)组。
静脉给药组动物在给药前及给药后5min、1h、1d、2d、3d、5d、1w、2w、3w、4w，皮下给药组在给药前及给药后1h、1d、2d、3d、5d、1w、2w、3w、4w，以枸橼酸钠采血管采集血样，以凝血仪和相应试剂盒测定aPTT和血浆FXI:C％。
测试结果参见图6A，图6B。结果显示，3882皮下给药和静脉给药均能显著延长内源性凝血时间，抑制FXI的活性。在等剂量给药条件下，3882和BAY1213790相比，3882对内源性凝血时间的延长维持时间更长，对FXI的抑制作用更强。
>BAY1213790的重链：
Figure PCTCN2021104253-appb-000056
Figure PCTCN2021104253-appb-000057
>BAY1213790的轻链：
Figure PCTCN2021104253-appb-000058
虽然以上描述了本公开的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本公开的保护范围由所附权利要求书限定。

Claims (18)

  1. 一种抗FXI/FXIa抗体或其抗原结合片段，包含：
    重链HCDR1，其包含SEQ ID NO：63所示序列；
    重链HCDR2，其包含SEQ ID NO：64所示序列；
    重链HCDR3，其包含SEQ ID NO：9或SEQ ID NO：39所示序列；
    轻链LCDR1，其包含SEQ ID NO：10或SEQ ID NO：40所示序列；
    轻链LCDR2，其包含SEQ ID NO：65所示序列；和
    轻链LCDR3，其包含SEQ ID NO：66所示序列。
  2. 如权利要求1所述的抗FXI/FXIa抗体或其抗原结合片段，包含：
    (a)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：37、38、39所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：40、41、42所示序列；
    (b)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：7、8、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、11、12所示序列；
    (c)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：22、23、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
    (d)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：24、25、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；
    (e)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：26、27、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列；或
    (f)重链HCDR1、HCDR2、HCDR3分别包含SEQ ID NO：28、25、9所示序列，轻链LCDR1、LCDR2、LCDR3分别包含SEQ ID NO：10、29、12所示序列。
  3. 如权利要求1-2任一项所述的抗FXI/FXIa抗体或其抗原结合片段，其为鼠源抗体、嵌合抗体、人源化抗体、人抗体或其片段；
    优选地，其为人源化抗体或其片段；
    更优选地，人源化抗体或其片段的轻链模板为IGKV3-11*01，重链模板为IGHV1-69-2*01。
  4. 如权利要求3所述的抗FXI/FXIa抗体或其抗原结合片段，其VH具有Y27F、T28N、F29I、T30K、A93L、R94Y、E73T、R66K、V67A、T75A、T76N中任一或任意组合的回复突变，和/或VL具有R45K、L46R、L47W、I58V、F71Y中任一或任意组合的回复突变。
  5. 如权利要求1-4任一项所述的抗FXI/FXIa抗体或其抗原结合片段，包含：
    如SEQ ID NO：5、17-20、30-33、35、43、45-49、53-58之一所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：6、21、34、36、44、50-52之一所示或与之具有至少90％同一性的VL。
  6. 如权利要求5所述的抗FXI/FXIa抗体或其抗原结合片段，包含：
    如SEQ ID NO：58所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：51所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：5所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：6所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：17所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：21所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：18所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：21所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：19所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：21所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：20所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：21所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：30所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：34所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：31所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：34所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：32所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：34所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：35所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：36所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：43所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：44所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：45所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：51所示或与之具有至少90％同一性的VL；
    如SEQ ID NO：49所示或与之具有至少90％同一性的VH，和
    如SEQ ID NO：51所示或与之具有至少90％同一性的VL。
  7. 如权利要求1-6任一项所述的抗FXI/FXIa抗体或其抗原结合片段，其VH与人源或小鼠CH1连接，VL与人源或小鼠CL或Cκ连接，
    优选地，所述人源CH1如SEQ ID NO：13或59所示，所述Cκ的序列如SEQ ID NO：14所示。
  8. 如权利要求1-7任一项所述的抗FXI/FXIa抗体或其抗原结合片段，其包含 恒定区Fc，
    优选地，所述Fc为IgG1的Fc、IgG4的Fc、或IgG4P的Fc；
    更优选地，所述IgG1的Fc序列如SEQ ID NO：67所示，所述IgG4P的Fc的序列如SEQ ID NO：60所示。
  9. 如权利要求1-8任一项所述的抗FXI/FXIa抗体或其抗原结合片段，其中：
    重链如SEQ ID NO：61所示或与之具有至少80％同一性，
    轻链如SEQ ID NO：62所示或与之具有至少80％同一性；或
    重链如SEQ ID NO：15所示或与之具有至少80％同一性，
    轻链如SEQ ID NO：16所示或与之具有至少80％同一性。
  10. 如权利要求1-9任一项所述的抗FXI/FXIa抗体或其抗原结合片段，所述抗原结合片段选自：scFv、Fv、Fab或Fab’片段。
  11. 如权利要求1-10任一项所述的抗FXI/FXIa抗体或其抗原结合片段，其具有以下任一或多项的特性：
    预防或阻止凝血途径的活化；
    阻断FXI/FXIa结合至FIX、FXIIa、凝血酶中的一个或多个；
    阻断FIX、FXI、FXIa中的一个或多个结合至血小板受体；
    以小于或等于10 -9K D的亲和力结合至人FXI和/或FXIa蛋白；
    结合至FXI/FXIa时，阻止其催化结构域呈现活性构象；
    可用于皮下给药或静脉给药。
  12. 一种多核苷酸，其编码权利要求1-11任一项所述的抗FXI/FXIa抗体或其抗原结合片段。
  13. 一种载体，其含有权利要求12所述的多核苷酸。
  14. 一种宿主细胞，其包含权利要求13所述的载体。
  15. 一种制备抗FXI/FXIa抗体或其抗原结合片段的方法，包括：
    在权利要求14所述的宿主细胞中表达抗FXI/FXIa抗体或其抗原结合片段，以及
    从所述宿主细胞中分离所述抗FXI/FXIa抗体或其抗原结合片段。
  16. 一种药物组合物，其含有：
    -可药用的赋形剂、稀释剂或载体；以及
    -选自以下的任一项或其任意组合：权利要求1-11任一项所述的抗FXI/FXIa抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的载体；
    优选地，所述药物组合物为皮下注射剂或静脉注射剂。
  17. 选自以下的任一项或其任意组合在制备药物或药物组合物中的用途：
    权利要求1-11任一项所述的抗FXI/FXIa抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的载体；
    其中，所述药物或药物组合物用于治疗和/或预防选自以下的任一项：血栓形成性或血栓栓塞性疾病、血栓形成性或血栓栓塞性并发症、心律失常、心源性血栓栓塞、弥漫性血管内凝血；
    优选地，所述血栓形成性或血栓栓塞性疾病或其并发症选自：心脏冠状动脉疾病、有ST段升高的心肌梗死(STEMI)、无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄，导致外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成、短暂性脑缺血发作、血栓形成性脑卒中和血栓栓塞性脑卒中、由慢性血栓栓塞(CTEPH)引起的肺病、由CTEPH引起的肺动脉高压、或其组合；
    优选地，所述心脏冠状动脉疾病是急性冠状动脉综合征。
  18. 一种治疗和/或预防疾病的方法，所述疾病选自以下任一项：
    血栓形成性或血栓栓塞性疾病、血栓形成性或血栓栓塞性并发症、心律失常、心源性血栓栓塞、弥漫性血管内凝血；
    所述方法包括步骤：
    向受试者施用治疗或预防疾病有效量的权利要求1-11任一项所述的抗FXI/FXIa抗体或其抗原结合片段、权利要求12所述的多核苷酸、权利要求13所述的载体、或其任意组合；
    优选地，所述血栓形成性或血栓栓塞性疾病或其并发症选自：心脏冠状动脉疾病、有ST段升高的心肌梗死(STEMI)、无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄，导致外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成、短暂性脑缺血发作、血栓形成性脑卒中和血栓栓塞性脑卒中、由慢性血栓栓塞(CTEPH)引起的肺病、由CTEPH引起的肺动脉高压、或其组合；
    优选地，所述心脏冠状动脉疾病是急性冠状动脉综合征；
    优选地，所述受试者患有与心房纤维性颤动有关的缺血性中风和/或深层静脉血栓形成。
PCT/CN2021/104253 2020-07-02 2021-07-02 抗FXI/FXIa抗体、其抗原结合片段及医药用途 WO2022002249A1 (zh)

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KR1020237003848A KR20230034361A (ko) 2020-07-02 2021-07-02 항-fxi/fxia 항체, 이의 항원-결합 단편, 및 이의 약제학적 용도
AU2021302202A AU2021302202A1 (en) 2020-07-02 2021-07-02 Anti-FXI/FXIa antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
CA3184718A CA3184718A1 (en) 2020-07-02 2021-07-02 Anti-fxi/fxia antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
US18/013,524 US20230220111A1 (en) 2020-07-02 2021-07-02 Anti-fxi/fxia antibody, antigen-binding fragment thereof, and pharmaceutical use thereof
BR112022026533A BR112022026533A2 (pt) 2020-07-02 2021-07-02 Anticorpo anti-fxi/fxia, fragmento de ligação do mesmo, e antígenos e uso farmacêutico do mesmo
CN202180004913.9A CN114269790B (zh) 2020-07-02 2021-07-02 抗FXI/FXIa抗体、其抗原结合片段及医药用途
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