WO2018145533A1 - 抗凝血因子xi抗体 - Google Patents

抗凝血因子xi抗体 Download PDF

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Publication number
WO2018145533A1
WO2018145533A1 PCT/CN2017/119856 CN2017119856W WO2018145533A1 WO 2018145533 A1 WO2018145533 A1 WO 2018145533A1 CN 2017119856 W CN2017119856 W CN 2017119856W WO 2018145533 A1 WO2018145533 A1 WO 2018145533A1
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Prior art keywords
antibody
seq
fxi
antibodies
fxia
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PCT/CN2017/119856
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English (en)
French (fr)
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王文义
于泉
刘小五
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上海仁会生物制药股份有限公司
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Priority to JP2019543277A priority Critical patent/JP7271430B2/ja
Priority to CA3054322A priority patent/CA3054322A1/en
Priority to EP17895532.4A priority patent/EP3581587A4/en
Priority to BR112019016300-0A priority patent/BR112019016300A2/pt
Priority to MX2019009500A priority patent/MX2019009500A/es
Priority to AU2017397778A priority patent/AU2017397778A1/en
Priority to RU2019128423A priority patent/RU2800719C2/ru
Priority to KR1020197025752A priority patent/KR102690437B1/ko
Publication of WO2018145533A1 publication Critical patent/WO2018145533A1/zh
Priority to US16/537,427 priority patent/US11958911B2/en

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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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • 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
    • C07K2317/56—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
    • C07K2317/70—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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to an antibody capable of binding to Factor XI (FXI) and/or its activating factor XIa (FXIa) and to a fragment of FXI and/or FXIa, and the use thereof, including as an anticoagulant for the preparation of a thrombus which does not affect hemostasis Use of the agent.
  • FXI Factor XI
  • FXIa activating factor XIa
  • a blood clot is a condition in which blood clots in a blood vessel, blocking or hindering the flow of blood through the affected area. If a blood clot travels along the circulatory system to a critical part of the body, it can cause serious complications, such as heart, brain and lungs, which can cause heart attacks, strokes and pulmonary embolism.
  • Anticoagulants such as heparin and warfarin, can treat or prevent blood clots. The most common adverse reaction to current treatments is bleeding. Because patients need to be closely monitored after treatment, these treatments are limited by dose and patient compliance.
  • the present invention satisfies this need in the art.
  • Certain embodiments of the invention provide antibodies that bind to Factor XI (FXI) and/or its activated form Factor XIa (FXIa), as well as FXI and/or FXIa fragments.
  • the antibody is a monoclonal antibody.
  • the antibody is a recombinant antibody.
  • the antibody is a humanized antibody.
  • the antibody is an immunologically active portion of an immunoglobulin molecule, such as a Fab, Fvs or scFv.
  • the antibody binds to the A3 domain of FXI and/or FXIa.
  • the antibody comprises one or more CDR sequences, SEQ ID NOs: 11-16, 27-32, 43-48, 59-64, 75-80, 91-96, 107- 112, 123-128, 139-144, 155-160, 171-176 and 187-192.
  • the present invention provides pharmaceutical compositions for treating and/or preventing thrombosis and/or complications or diseases associated with thrombosis.
  • the pharmaceutical composition comprises one or more anti-FXI and/or anti-FXIa antibodies disclosed herein.
  • the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or combinations thereof.
  • the invention provides nucleic acids encoding anti-FXI and/or anti-FXIa antibodies, or functional fragments of any of the antibodies disclosed herein, as well as vectors comprising the nucleic acids, and host cells comprising the vectors.
  • the vector is an expression vector that produces an antibody or a functional fragment thereof encoded by the nucleic acid in a host cell.
  • the invention provides a kit comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed in the specification for use in the treatment and/or prevention of thrombosis and/or complications or disorders associated with thrombosis .
  • the kit comprises a pharmaceutical composition comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed in the specification for use in the treatment and/or prevention of thrombosis and/or thrombosis-related complications or Illness.
  • the kit further includes instructions for use.
  • the present invention provides methods of treating and/or preventing thrombosis and/or complications or conditions associated with thrombosis.
  • the methods comprise administering to a subject in need thereof a therapeutically effective amount of one or more of the anti-FXI and/or anti-FXIa antibodies disclosed herein.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising an anti-FXI antibody, an anti-FXIa antibody, or a functional fragment of either antibody.
  • the invention provides the use of an anti-FXI and/or anti-FXIa antibody as disclosed herein for the manufacture of a medicament for the treatment and/or prevention of thrombosis and/or thrombosis-related complications or conditions.
  • the invention provides methods of making anti-FXI and/or anti-FXIa antibodies as disclosed herein.
  • the steps taken by the method comprise transforming a host cell using a vector comprising a nucleic acid encoding the antibody and expressing the antibody in the host cell.
  • the method can also include purifying the expressed antibody from the host cell.
  • the purified antibody can be modified, and the modified recombinant antibody retains the activity of the corresponding human antibody.
  • the antibodies disclosed herein can be produced from hybridoma cultures.
  • Figure 1 shows the effect of five anti-FXI antibodies in human plasma as determined by APTT assay. As described in Example 2, after adding 5 different antibodies in a concentration range of 0 to 400 nM in human plasma, APTT detection was performed. The five antibodies tested included 19F6 (A), 34F8 (B), 42A5 (C), 1A6 (D), and 14E11 (E). In this experiment, antibodies 1A6 and 14E11 were used as positive controls.
  • Figure 2 shows the effect of antibodies 19F6 (A), 34F8 (B) and 42A5 (C) on monkey plasma APTT assay.
  • APTT assay was performed after addition of three different antibodies in the human plasma at concentrations ranging from 0 to 400 nM.
  • Figure 3 shows the SPR sensorgram of FXI binding to immobilized h-19F6 (A), h-34F8 (B) and h-42A5 (C), and FXIa with immobilized h-19F6, h-34F8 (E) and h-42A5 (F) combined SPR sensorgram.
  • Data were fitted to a 1:1 binding model and the curve fit at the test concentration of FXI (0.005-1 ng/mL) on the surface plasmon resonance sensorgram showed superposition. Each curve represents a different test concentration for FXI or FXIa.
  • Figure 4 shows concentration-response curves of antibodies h-19F6 (A), h-34F8 (B) and h-42A5 (C) inhibiting human FXIa hydrolysis of S-2366.
  • Figure 5 shows the inhibition of FXIa-mediated FIX activation into FIXa responses by antibodies h-19F6 (A) and h-42A5 (B).
  • Human FIX (200 nM) and FXIa (5 nM) and 1 ⁇ M of control IgG or h-19F6 or h-42A5 were placed in 5 mM CaCl 2 in PBS and incubated at room temperature. At the indicated time intervals, samples were collected and FIX and FIXa were assayed by Western blot using goat anti-human FIX IgG (Affinity Biologicals).
  • Figure 6 shows the effect of antibodies h-34F8, h-19F6 and h-42A5 on cynomolgus monkey APTT.
  • Monkeys were administered intravenously with the indicated doses of h-34F8 (A), h-19F6 (B) and h-42A5 (C).
  • the in vitro clotting time APTT was measured before administration (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours after administration.
  • Figure 7 shows the effect of antibodies h-34F8, h-19F6 and h-42A5 on cynomolgus monkey PT.
  • Monkeys were administered intravenously with the indicated doses of h-34F8 (A), h-19F6 (B) and h-42A5 (C).
  • Ex vivo clotting time PT was measured before administration (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours after administration.
  • Figure 8 shows the effect of antibodies h-34F8, h-19F6 and h-42A5 on arteriovenous (AV) shunt thrombosis in cynomolgus monkeys.
  • Figure 9 shows the effect of antibodies h-34F8, h-19F6 and h-42A5 on the bleeding time of cynomolgus monkeys.
  • the bleeding time was calculated before administration and 30 minutes after each administration.
  • Figure 10 shows the antithrombotic effects of antibodies h-34F8, h-19F6 and h-42A5.
  • the time to reach 80% thromboocclusion (A) and 100% thromboembolism (B) was determined by monitoring blood flow velocity.
  • *P ⁇ 0.05 and **P ⁇ 0.01 vs. vector *P ⁇ 0.05 and **P ⁇ 0.01 vs. vector.
  • Figure 11 illustrates that treatment with antibody h-34F8, h-19F6 or h-42A5 does not prolong the bleeding time in monkeys.
  • the bleeding times of the h-34F8, h-19F6 and h-42A5 treatment groups are shown in Figures (A), (B) and (C), respectively.
  • the changes in bleeding time in the vehicle, h-34F8, h-19F6 or h-42A5 treatment groups are shown in Figure (D).
  • Figure 12 illustrates the effect of antibodies h-34F8, h-19F6 and h-42A5 on monkey plasma clotting time.
  • the APTT changes and PT changes are shown in Figures (A) and (B). **P ⁇ 0.01 and ***P ⁇ 0.001 vs. vector.
  • Figure 13 is the amino acid sequence of human FXI (SEQ ID NO: 203).
  • In vivo coagulation cascade mechanisms include endogenous pathways and exogenous pathways.
  • the endogenous pathway also known as the contact activation pathway, is triggered by the surface contact of blood with a negatively charged foreign body and causes activation of FXII. Endogenous pathways involve FXI, FIX and FVIII.
  • the exogenous pathway also known as the tissue factor (TF) pathway, is triggered by vascular damage and leads to the formation of a TF-FVIIa activation complex. These two pathways satisfy and activate the common pathway leading to the conversion of prothrombin to thrombin and ultimately to the formation of cross-linked fibrin clots.
  • the antibodies disclosed herein bind to FXI and/or FXIa and are directed to the endogenous pathway of coagulation.
  • the structure of FXI and the role of FXI in blood clotting have previously been reported in various publications. See, for example, Emsley et al., Blood 115 (13): 2569-2577 (2010), the contents of which are incorporated herein by reference.
  • the present specification provides antibodies that bind to fragments of FXI, FXIa and/or FXI or FXIa and inhibit clot formation. These antibodies are capable of binding to fragments of FXI, FXIa and/or FXI or FXIa (eg, fragments comprising the A3 domain) and exhibit an inhibitory effect at concentrations well below the maximum safe dose. For example, in some embodiments, an antibody having a dose between 0.1 mg/kg i.v. to 3 mg/kg i.v. exhibits an inhibitory effect on the transformation of cynomolgus FXI to FXIa. Furthermore, the antibodies disclosed in the present specification can be used as anticoagulants with excellent safety because they minimize the risk of bleeding compared to conventional anticoagulants such as heparin.
  • composition or method includes at least the element.
  • consisting essentially of means that the composition or method includes the recited elements and may further comprise one or more novelties and basics that do not materially affect the composition or method.
  • An additional element of the feature For example, a composition consisting essentially of the elements mentioned may include the elements mentioned plus one or more residues from the separation and purification process, a pharmaceutically acceptable carrier such as phosphate buffered saline, and a preservative. Wait.
  • Consisting of means that the composition or method includes only the elements mentioned. Examples defined by each transition term are within the scope of the invention.
  • antibody refers to an immunoglobulin molecule that specifically binds to or has specific antigen immunoreactivity, or an immunologically active portion thereof, such as a specific domain or fragment of FXI, FXIa, and FXI or FXIa, eg, A3 domain.
  • the antibodies of the methods, compositions, and kits are full length immunoglobulin molecules comprising two heavy chains and two light chains, each heavy chain and light chain comprising three complementarity determining regions ( CDR).
  • antibody also includes genetically engineered or other modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies, and heterologous Antibodies (eg, bispecific antibodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, tri-chain antibodies, and four-chain antibodies).
  • synthetic antibodies intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies, and heterologous Antibodies (eg, bispecific antibodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, tri-chain antibodies, and four-chain antibodies).
  • the antibodies disclosed herein may be monoclonal or polyclonal. .
  • the antibody may be Fab, Fab', Fv, Fab'F(ab') 2 , a disulfide-linked Fv, a single-chain Fv antibody (scFv), Single domain antibodies (dAbs) or diabodies.
  • the antibodies disclosed herein, including immunologically active portions of immunoglobulin molecules retain the ability to bind to a specific antigen (e.g., FXI or FXIa) or to bind to a particular fragment of FXI or FXIa (e.g., the A3 domain).
  • the anti-FXI and/or anti-FXIa antibodies disclosed herein are subjected to post-translational modifications associated with expression in a mammalian cell line, including human or non-human host cells, eg, phosphorylation, methylation, acetylation , ubiquitination, nitrosylation, glycosylation or lipidation.
  • post-translational modifications associated with expression in a mammalian cell line including human or non-human host cells, eg, phosphorylation, methylation, acetylation , ubiquitination, nitrosylation, glycosylation or lipidation.
  • Techniques for producing in vitro and in vivo modifications of recombinant antibodies and recombinant antibodies are known in the art. References such as mAbs 6(5): 1145-1154 (2014), the contents of which are incorporated herein by reference.
  • the disclosure also discloses polynucleotides or nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein.
  • the polynucleotide or nucleic acid comprises DNA, mRNA, cDNA, and plasmid DNA.
  • a nucleic acid encoding an antibody or functional fragment thereof disclosed herein can be cloned into a vector, such as a pTT5 mammalian expression vector, which can further include a promoter and/or other transcriptional or translational control elements such that the nucleic acid can be expressed as an antibody or Feature fragment.
  • the present disclosure discloses nucleic acid (DNA) and/or amino acid (PRT) sequences of some antibody examples as shown in Table 1, which includes VH, VL and CDR sequences.
  • Humanized anti-FXI and/or anti-FXIa antibodies are provided in certain embodiments of the invention.
  • a variety of techniques are known in the art for humanizing non-human antibodies to bring them into close proximity to the human body.
  • the remainder of the antigen binding domain is referred to as the "framework" region, which has less intermolecular variability and forms scaffolds to allow proper positioning of the CDRs.
  • humanization of antibodies disclosed herein can be accomplished by transplantation of monoclonal antibody CDRs produced by immunizing mice or rats.
  • the CDRs of the mouse monoclonal antibodies can be grafted into the human framework and subsequently ligated into human constant regions to obtain humanized antibodies.
  • human germline antibody sequence databases, protein database (PDB), INN (International Nonproprietary Names) databases, and other suitable databases can be searched and searched to find the framework most similar to the desired antibody.
  • PDB protein database
  • INN International Nonproprietary Names
  • the variable region is joined to a human IgG constant region.
  • the Fc domains of human IgG1, IgG2, IgG3, and IgG4 can be used. Humanization of monoclonal antibodies produced by non-human species is a fundamental ability of one of ordinary skill in the art based on the prior art.
  • Table 2 is an example of the variable region sequences of several humanized antibodies.
  • the antibodies provided herein include variants of the sequences disclosed herein that contain one or more mutations in the amino acid sequence while retaining the binding affinity for FXI, FXIa and/or fragments thereof (e.g., fragments comprising the A3 domain).
  • the variable region of an antibody comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the following sequence or retained for FXI, FXIa and/or fragments thereof Binding to an affinity amino acid fragment, the sequence is selected from the group consisting of SEQ ID NO: 9, 10, 25, 26, 41, 42, 57, 58, 73, 74, 89, 90, 105, 106, 121, 122, 137, 138, 153, 154, 169, 170, 160, 170, 185, 186, and 197-202.
  • nucleic acid variants comprising an antibody encoding a binding to FXI, FXIa and/or a fragment thereof (eg, comprising an A3 domain fragment).
  • the nucleic acid encoding the variable region of an antibody comprises a nucleic acid sequence having at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the sequence or encoding to FXI, FXIa and/or a fragment thereof having a nucleic acid fragment that binds to an affinity polypeptide selected from the group consisting of SEQ ID NO: 1, 2, 17, 18, 33, 34, 49, 50, 65, 66, 81, 82, 97, 98, 113, 114, 129, 130, 145, 146, 147, 148, 161, 162, 177, and 178.
  • the antibodies disclosed in the present specification can be formulated into pharmaceutical compositions.
  • the pharmaceutical composition may also comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or combinations thereof.
  • the pharmaceutical composition may be in various dosage forms, and may be, for example, an injection preparation, a lyophilized preparation, a liquid preparation, or the like.
  • suitable adjuvants, carriers, excipients, preservatives and the like may be selected as the additive. See, for example, Wang et al., J. Pharm. Sciences 96(1): 1-26 (2007), the contents of which are incorporated herein by reference.
  • the pharmaceutical composition can be included in a kit having instructions for use of the composition.
  • the present invention provides a method of treating and/or preventing thrombosis in a subject having an increased risk of thrombosis and/or thrombosis.
  • Methods of inhibiting clot formation in a subject are also provided. These methods require the administration of a therapeutically effective amount of an anti-FXI and/or FXIa antibody provided herein to interfere with the endogenous pathway.
  • the methods comprise administering to the subject a pharmaceutical composition comprising an anti-FXI and/or anti-FXIa antibody provided herein.
  • thrombosis can lead to many complications or conditions, such as embolic stroke, venous thrombosis (such as venous thromboembolism (VTE), deep vein thrombosis (DVT) and pulmonary embolism (PE)) arterial thrombosis (such as acute coronary syndrome (ACS) , coronary artery disease (CAD) and peripheral arterial disease (PAD).
  • VTE venous thromboembolism
  • DVT deep vein thrombosis
  • PE pulmonary embolism
  • ACS acute coronary syndrome
  • CAD coronary artery disease
  • PAD peripheral arterial disease
  • Other conditions associated with thrombosis include, for example, the following patients having a high risk of VTE, who are surgical patients, bedridden patients, cancer patients, heart failure patients, pregnant patients, or other medical condition patients who may cause thrombosis.
  • the methods disclosed herein relate to prophylactic anticoagulant therapy, i.e., thromboprophylaxis. These methods require administration of a therapeutically effective amount of an anti-FXI and/or FXIa antibody of the present invention or a therapeutically effective amount of a pharmaceutical composition comprising an anti-FXI and/or FXIa antibody to a subject having thrombotic-related complications as disclosed above. .
  • the antibody or pharmaceutical composition can be administered alone or in combination with any other therapies for treating or preventing a thrombosis-related complication or condition.
  • the invention also provides methods of treating and/or preventing sepsis for a subject in need thereof. Attempts have been made to apply anticoagulants to patients with sepsis to improve mortality or morbidity. However, this attempt was unsuccessful because the anticoagulant caused an unexpected bleeding.
  • the antibodies disclosed herein can be used as adjuvant therapy for other therapeutic agents (e.g., antibiotics) of sepsis.
  • subject refers to a mammalian subject, preferably a human.
  • Subject in need refers to a subject who has been diagnosed with a thrombus or thrombosis-related complication or condition, or who has an increased risk of thrombosis or a thrombus-related complication or condition.
  • Subject and patient are used interchangeably throughout this specification.
  • treating means partially or completely alleviating a condition, preventing a condition, reducing the likelihood of occurrence or recurrence of the condition, slowing the progression or occurrence of the condition, or eliminating, reducing or slowing down the condition associated with the condition.
  • the occurrence of one or more symptoms In the case of a thrombus and/or thrombus-related complication or condition, “treating” can mean preventing or slowing the increase in existing blood clots, and/or preventing or slowing the formation of blood clots.
  • the term “treating” refers to a decrease in the number or size of blood clots of a subject compared to a subject not to whom the antibody or functional fragment thereof is administered. In some embodiments, the term “treating” refers to a thrombus and/or thrombosis associated with a subject following treatment with an antibody or pharmaceutical composition as disclosed herein, as compared to a subject not receiving treatment. One or more symptoms of the condition or complication are alleviated.
  • a “therapeutically effective amount" of an antibody or pharmaceutical composition as used herein refers to an amount of an antibody or pharmaceutical composition that produces a desired therapeutic effect (eg, treating and/or preventing a thrombus) in a subject.
  • a therapeutically effective amount is an amount of an antibody or pharmaceutical composition that produces the greatest therapeutic effect.
  • the therapeutically effective amount of the therapeutic effect is less than the maximum therapeutic effect.
  • a therapeutically effective amount can be an amount that has both a therapeutic effect and one or more side effects associated with the maximum therapeutic dose.
  • the therapeutically effective amount of a particular composition will vary based on a variety of factors including, but not limited to, the characteristics of the therapeutic composition (e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiology of the subject. Conditions (eg, age, weight, sex, type and stage of disease, medical history, general physical condition, response to a given dose, and other existing drugs used), any pharmaceutically acceptable carrier, excipient, and The nature of the preservative and the mode of administration. Those skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount by routine experimentation by monitoring the subject's response to administration of the antibody or pharmaceutical composition and adjusting the dosage accordingly.
  • the characteristics of the therapeutic composition e.g., activity, pharmacokinetics, pharmacodynamics, and bioavailability
  • Conditions eg, age, weight, sex, type and stage of disease, medical history, general physical condition, response to a given dose, and other existing drugs used
  • the therapeutically effective amount of an antibody disclosed herein is from about 0.01 mg/kg to about 30 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, from about 1 mg//kg to about 5 mg/kg.
  • an immediate release, controlled release or sustained release antibody or pharmaceutical composition may be administered continuously or intermittently.
  • the antibody or pharmaceutical composition can be administered three times a day, twice a day, or once a day for a period of 3 days, 5 days, 7 days, 10 days, 2 weeks, 3 weeks, or 4 weeks.
  • the antibody or pharmaceutical composition can be administered for a predetermined period of time.
  • the antibody or pharmaceutical composition can be administered until a particular therapeutic basis is reached.
  • the methods provided herein comprise the step of assessing one or more therapeutic criteria to determine whether to continue administering the antibody or pharmaceutical composition.
  • the present specification also provides methods of producing the anti-FXI and/or anti-FXIa antibodies disclosed herein.
  • these methods entail cloning a nucleic acid encoding an anti-FXI and/or anti-FXIa antibody into a vector, transforming the host cell with the vector, and culturing the host cell to express the antibody.
  • the expressed antibody can be purified from the host cell using any known technique.
  • Various expression vectors such as the pTT5 vector and the pcDNA3 vector, as well as various host cell lines, such as CHO cells (e.g., CHO-K1 and ExpiCHO) and HEK193T cells, can be used.
  • the disclosure also encompasses antibodies produced by the methods disclosed above.
  • the antibody may have undergone one or more post-translational modifications.
  • FXI human coagulation factor XI
  • spleen cells of the better immune cells were used for hybridoma cell preparation; hybridoma cells were further subcloned after limited dilution and captured by ELISA.
  • FXI human coagulation factor XI
  • 12 monoclonal hybridoma cell lines were successfully obtained, and the anti-FXI antibodies expressed were: 3G12, 5B2, 7C9, 7F1, 13F4, 19F6, 21F12, 34F8, 38E4, 42A5, 42F4 and 45H1.
  • V L, V H antibody light and heavy chain variable region
  • Example 2 Evaluation of anticoagulant activity of the antibody of the present invention in human plasma using activated partial thromboplastin time (APTT) and prothrombin time (PT) detection indices
  • APTT activated partial thromboplastin time
  • PT prothrombin time
  • the APTT assay measures the activity of endogenous and common pathway coagulation; the PT assay measures the activity of exogenous and common pathway coagulation.
  • the antibodies tested in this experiment were 19F6, 34F8, 42A5, 1A6 and 14E11. In this experiment, antibodies 1A6 and 14E11 were used as positive controls.
  • the sequence of the control antibody variable region is derived from U.S. Patent No. 8,388,959 and U.S. Patent Application No. 2013/0171144, and is reformatted to IgG4. These antibodies were then expressed using the ExpiCHO cell system.
  • Standard human plasma purchased from Symens Inc.
  • various concentrations of the antibody to be tested (0-400 nM) were mixed in equal volumes, incubated for 5 min, and then detected using a CA600 analyzer.
  • APTT assay 50 ⁇ L of the above mixture of plasma and antibody was taken, mixed with 25 ⁇ L of APTT reagent (SMN 10445709, Symens Inc.), and incubated at 37 ° C for 4 min. 25 ⁇ L of CaCl 2 solution (25 mM, SMN 10446232, Symens Inc.) was added and the clotting time was recorded.
  • PT assay 50 ⁇ L of the above mixture of plasma and antibody was taken, and an equal volume of PT reagent (SMN 10446442, Symens Inc.) was added and mixed, and the coagulation time was measured at 37 °C.
  • PT reagent SSN 10446442, Symens Inc.
  • Example 3 Evaluation of the anticoagulant activity of the antibody of the present invention in non-human plasma using the APTT detection index.
  • Some antibodies of the present invention are humanized by CDR grafting (i.e., selecting an appropriate human antibody acceptor framework regions of each murine antibody as a framework of the V L and V H, and subjected to back mutated to ensure that the relevant portion of the antibody structure And the function is not affected too much). If the affinity and function of these humanized antibodies are substantially no less than the corresponding unmodified antibodies, then the modified antibody is considered to be humanized successfully.
  • the affinity of the anti-FXI antibody to FXI/FXIa was determined using a BIAcore T200 system using surface plasmon resonance (SPR) technology.
  • the method is as follows: a humanized antibody is constructed by ligating the variable region of the antibody to be tested with a human IgG4 Fc fragment, and is recombinantly expressed in CHO cells; these antibodies are conjugated to a Biacore CM5 conjugated with an anti-human IgG capture antibody. Sense on the chip.
  • the binding sites of 19F6 and 42A5 on FXI were determined by SPR technique.
  • the method is as follows: a human IgG capture antibody is coupled to the surface of a Biacore CM5 sensor chip, and a recombinant sample h-19F6 or h-42A5 is captured across the surface of the chip, and the amount of capture of h-19F6 or h-42A5 is adjusted by adjusting the flow time. Up to 15 relative units.
  • a certain concentration of wild-type FXI or chimeric FXI flows across the surface of the chip, and h19F6 or h42A5 binding time is For 180 s, the subsequent dissociation time was 1800 s; the data was analyzed using a high-efficiency kinetic model with only one concentration of wild-type FXI or chimeric FXI being tested in the SPR test.
  • the activity of human FXIa is primarily determined by measuring the hydrolysis of a specific and fluorescently labeled substrate (S-2366, Diapharma Inc.). To test the inhibitory activity, the FXIa in 5 ⁇ M final concentration in PBS (Phosphate buffer saline) buffer was pre-incubated with the antibodies h-19F6, h-34F8 and h-42A5 for 5 min at room temperature. After incubation, an equal volume was added. The FXIa cleavage reaction was started at 1 mM S-2366, and the change in absorbance was measured at 405 nm using a M5 e plate reader (Molecular Devices Inc.); the data obtained were analyzed using GraphPad Prism software, as shown in Fig. 4. The apparent Ki values of the human antibodies h-19F6, h-34F8 and h-42A5 were 0.67, 2.08, 1.43 nM. Therefore, all three antibodies tested exhibited satisfactory inhibition of FXIa at relatively low concentrations.
  • FIX Human FIX (200 nM), FXIa (5 nM), and 1 ⁇ M control IgG or h-19F6 or h-42A5 were dissolved in PBS containing 5 mM CaCl 2 and incubated at room temperature. After 0, 15, 30, 45 and 60 minutes, 50 ⁇ L of sample was collected into dodecyl sulfate sample buffer. The sample was placed on a 10% non-reducing gel for size electrophoresis and transferred to a polyvinylidene fluoride membrane. Western blotting was performed using goat anti-human FIX IgG (Affinity Biologicals) to determine FIX and FIXa levels. As shown in Figure 5, both h-19F6 and h-42A5 inhibited FXIa induction induced by FXI compared to the control.
  • APTT test Take 50 ⁇ l of diluted plasma sample, add 25 ⁇ l of APTT reagent (SMN 10445709, Symens Inc.), mix, incubate for 4 min at 37 ° C, add CaCl 2 solution (25 mM, SMN 10446232, Symens Inc.) 25 ⁇ L Evenly, record the setting time.
  • Example 10 Effect of anti-FXI antibody on arteriovenous shunt thrombosis and tail vein hemorrhage in cynomolgus monkeys
  • test antibody Different doses of the test antibody were administered to the same animal to assess thrombus formation and bleeding time.
  • the antibodies tested in this experiment were h-34F8, h-19F6 and h42A5. Briefly, bleeding time and thrombosis were evaluated sequentially before each administration of the antibody and 30 minutes after administration. Four assessments of bleeding/thrombosis were performed for three escalating doses (0.1, 0.3, and 1 mg/kg) before and after administration.
  • AV shunt thrombus measurement put a weighed 10cm long wire in the tube, connect the femoral artery and femoral vein with cannula, release the blood flow for 10min, interrupt the blood flow, quickly take out the silk thread and weigh the weight, calculate the weight of the thrombus .
  • the weight of the thrombus is determined by the difference in the weighing of the threads before and after the blood flow.
  • Bleeding time measurement A 2 ml syringe was inserted into the tail vein of the animal to start timing. Stop timing when the blood in the syringe no longer increases. This period of time is the bleeding time.
  • Example 11 Effect of anti-FXI antibody to cynomolgus femoral artery thrombosis induced by FeCl 3 and the influence on the formation of standard bleeding time
  • the cynomolgus monkeys were anesthetized with 1.5 mg/kg Zoletil, intubated, and ventilated, and blood pressure, heart rate and body temperature were monitored throughout the procedure. In the experiment, isoflurane was used to maintain the anesthetic effect.
  • the test antibody h-34F8, h-19F6, h-42A5 or a blank vector (control) was administered intravenously 2 hours before administration of FeCl 3 .
  • the left femoral artery was exposed and isolated using a blunt dissection method.
  • the blood flow was continuously detected and recorded using a Doppler ultrasound system. 3, before the administration of FeCl, the blood flow has been detected at least 5 minutes.
  • the h-34F8, h-19F6, and h-42A5 antibodies were evaluated by the arterial thrombosis model induced by FeCl 3 , that is, the four groups of cynomolgus monkeys were given blank vector, h-34F8, h-19F6, h-42A5, and left after 2 hours. FeCl 3 was applied to the femoral artery to cause thrombosis, and the blood flow velocity below it was monitored.
  • the antibodies disclosed herein unexpectedly do not prolong the bleeding time while being effective in inhibiting the endogenous pathway of coagulation.

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Abstract

结合凝血因子XI（FXI）和/或其活化形式因子XIa（FXIa），或FXI和/或FXIa的片段的抗体，以及含有该抗体的组合物。还公开了制备该抗体的方法，以及将该抗体用于制备治疗和/或预防凝血相关病症（例如血栓和与血栓相关的并发症或病症）药物方面的用途。

Description

抗凝血因子XI抗体 技术领域
本发明涉及能够结合凝血因子XI(FXI)和/或其活化因子XIa(FXIa)以及结合FXI和/或FXIa片段的抗体及其用途，包括其作为制备可以治疗血栓却不影响止血功能的抗凝剂的用途。
背景技术
血栓是一种血液在血管中凝结后，阻塞或阻碍受影响区域中的血流通过的病症。如果血凝块沿着循环系统行进到身体关键部位，可导致严重的并发症，例如进行到心脏、脑和肺，就可能引起心脏病发作、中风和肺栓塞等疾病。抗凝血剂，如肝素和华法林，可以治疗或者预防血栓。现行治疗手段最常见的不良反应是出血。由于患者治疗后需要受到密切监测，这些治疗方法受剂量和患者依从性的限制。
因此，有必要提供一种副作用最小的有效的预防或治疗血栓形成的药物。本发明满足了本领域的这一需求。
发明内容
本发明的某些实施方案提供了结合凝血因子XI(FXI)和/或其活化形式因子XIa(FXIa)，以及FXI和/或FXIa片段的抗体。在某些实施方案中，该抗体是单克隆抗体。在某些实施方案中，该抗体是重组抗体。在某些实施方案中，该抗体是人源化抗体。在某些实施方案中，该抗体是免疫球蛋白分子的免疫活性部分，例如Fab，Fvs或scFv。在某些实施方案中，该抗体结合FXI和/或FXIa的A3结构域。在一些实施方案中，该抗体包括一个或多个CDR序列，这些CDR序列的序号为SEQ ID NOs:11-16,27-32,43-48,59-64,75-80,91-96,107-112,123-128,139-144,155-160,171-176以及187-192。
本发明提供了用于治疗和/或预防血栓和/或与血栓形成相关的并发症或疾病的药物组合物。该药物组合物包含本发明公开的一种或多种抗FXI和/或抗FXIa抗体。在某些实施方案中，该药物组合物还包含一种或多种药学上可接受的佐剂、载体、赋形剂、防腐剂或其组合。
本发明提供了编码本发明公开的抗FXI和/或抗FXIa抗体，或任一抗体功能片段的核酸，以及包含所述核酸的载体，和包含所述载体的宿主细胞。在某些实施方案中，该载体是在宿主细胞中能产生由该核酸编码的抗体或其功能片段的表达载体。
本发明提供了一种试剂盒，其包含本说明书公开的一种或多种抗FXI和/或抗FXIa抗体，用于治疗和/或预防血栓形成和/或与血栓形成相关的并发症或病症。可选择地，该试剂盒包含药物组合物，其包含本说明书公开的一种或多种抗FXI和/或抗FXIa抗体，用于治疗和/或预防血栓和/或与血栓相关的并发症或病症。在某些实施方案中，试剂盒还包含使用说明书。
本发明提供了治疗和/或预防血栓形成和/或与血栓形成相关的并发症或病症的方法。所述方法包括向有需要的受试者施用治疗有效量的本发明公开的一种或多种抗FXI和/或抗 FXIa抗体。可选择地，所述方法包括向有需要的受试者施用治疗有效量的含有抗FXI抗体，抗FXIa抗体或任一抗体的功能片段的药物组合物。
本发明提供了如本说明书公开的抗FXI和/或抗FXIa抗体用于制备治疗和/或预防血栓和/或与血栓相关的并发症或病症的药物的用途。
本发明提供了制备如本说明书公开的抗FXI和/或抗FXIa抗体的方法。该方法采取的步骤包括使用载体转化宿主细胞，该载体包含编码抗体的核酸，并在宿主细胞中表达抗体。该方法还可以包括从宿主细胞中纯化表达抗体。可选择地，可以对纯化的抗体进行修饰，修饰后的重组抗体保留相应的人抗体的活性。可选择地，本说明书公开的抗体可以从杂交瘤培养产生。
附图说明
图1显示通过APTT检测测定的人血浆中的五种抗FXI抗体的效果。如实施例2所述，人血浆中添加浓度范围为0至400nM的5种不同抗体后，进行APTT检测。测试的五种抗体包括19F6(A)、34F8(B)、42A5(C)、1A6(D)和14E11(E)。在本实验中，抗体1A6和14E11用作阳性对照。
图2显示抗体19F6(A)、34F8(B)和42A5(C)对猴血浆APTT测定的影响。如实施例3所述，人血浆中添加浓度范围为0至400nM的3种不同抗体后，进行APTT检测。
图3显示FXI与固定的h-19F6(A)、h-34F8(B)和h-42A5(C)结合的SPR传感图，以及FXIa与固定的h-19F6、h-34F8(E)和h-42A5(F)结合的SPR传感图。数据和1:1结合模型拟合，并且表面等离子体共振传感图上在FXI(0.005-1ng/mL)的测试浓度下的曲线拟合显示叠加。每条曲线代表FXI或FXIa的不同测试浓度。
图4显示抗体h-19F6(A)、h-34F8(B)和h-42A5(C)抑制人FXIa水解S-2366的浓度-响应曲线。
图5显示抗体h-19F6(A)和h-42A5(B)对FXIa介导的FIX活化为FIXa反应的抑制作用。将人FIX(200nM)与FXIa(5nM)以及1μM对照IgG或h-19F6或h-42A5置于5mM CaCl 2的PBS中，室温下温育。在指定的时间间隔，收集样品，并使用山羊抗人FIX IgG(Affinity Biologicals)通过蛋白质印迹法对FIX以及FIXa进行测定。
图6显示抗体h-34F8，h-19F6和h-42A5对食蟹猴APTT的影响。猴子静脉内施用指定剂量的h-34F8(A)、h-19F6(B)和h-42A5(C)。在给药前(时间0)和给药后0.5、1、3、6、12和24小时测定体外内凝血时间(Ex vivo clotting time)APTT。
图7显示抗体h-34F8、h-19F6和h-42A5对食蟹猴PT的影响。猴子静脉内施用指定剂量的h-34F8(A)、h-19F6(B)和h-42A5(C)。在给药前(时间0)和给药后0.5、1、3、6、12和24小时测定体内外凝血时间(Ex vivo clotting time)PT。
图8显示抗体h-34F8、h-19F6和h-42A5对食蟹猴动静脉(AV)分流血栓形成的影响。对猴子静脉内施用剂量升高的h-34F8(A)、h-19F6(B)或h-42A5(C)(对于h- 34F8和h-19F6组，n＝3；对于h-42A5组，n＝4)，对AV分流血栓猴模型测定凝块重量从给药前发生的变化。*P<0.05，**P<0.01和***P<0.001，vs.载体。
图9显示抗体h-34F8、h-19F6和h-42A5对食蟹猴出血时间的影响。对猴子静脉内施用剂量升高的h-34F8(A)、h-19F6(B)或h-42A5(C)(对于34F8和h-19F6组，n＝3；对于h-42A5组，n＝4)，在给药前和每次给药后30分钟计算出血时间。
图10显示抗体h-34F8、h-19F6和h-42A5的抗血栓效应。四组猴(n＝5)静脉内施用载体、0.3mg/kg的h-34F8、h-19F6或h-42A5，持续2小时，将FeCl 3施用于每只动物的左股动脉，以诱导血栓形成。通过监测血流速度确定达到80％血栓闭塞(A)和100％血栓闭塞(B)的时间。*P<0.05以及**P<0.01 vs.载体。
图11说明使用抗体h-34F8，h-19F6或h-42A5治疗不会延长猴的出血时间。对四组猴(n＝5)静脉内施用载体、0.3mg/kg的h-34F8、h-19F6或h-42A5，并在给药前和给药后1小时测量标准出血时间。h-34F8、h-19F6和h-42A5治疗组各自出血时间分别见图(A)、(B)和(C)所示。载体、h-34F8，h-19F6或h-42A5治疗组出血时间变化见图(D)所示。
图12说明抗体h-34F8、h-19F6和h-42A5对猴血浆凝固时间的影响。四组猴(n＝5)分别静脉内施用载体、0.3mg/kg的h-34F8、h-19F6和h-42A5，并且在给药前和给药后约3小时收集血液，用于血浆制备以及凝血时间APTT和PT的测定。APTT变化和PT变化见图(A)和(B)所示。**P<0.01和***P<0.001 vs.载体。
图13为人FXI(SEQ ID NO：203)的氨基酸序列。
具体实施方式
本说明书的以下描述仅意在说明本发明的各种实例。因此，所讨论的特定变型不应被理解为对本发明范围的限制。对于本领域技术人员，在不脱离本发明的范围的情况下可以对本发明的实体的进行的各种等同替换、改变和修饰，这是显而易见的，并且应当理解为，本发明也包括了这样的等同实例。
体内凝血级联机制包括内源性途径和外源性途径。内源性途径，也称为接触激活途径，通过血液与带负电荷的异物表面接触引发，并导致FXII的激活。内源性途径涉及FXI、FIX和FVIII。外源性途径，也称为组织因子(TF)途径，由血管损伤引发，并导致TF-FVIIa活化复合物的形成。这两个途径满足并激活共同途径，导致凝血酶原转化为凝血酶，并最终形成交联的纤维蛋白凝块。本发明公开的抗体结合FXI和/或FXIa，针对的是凝血的内源性途径。FXI的结构和FXI在凝血中的作用此前已经在各种出版物中被报道过。例如参见Emsley et al.,Blood 115(13):2569-2577(2010)，其内容通过引用并入本说明书。
抗FXI或抗FXIa抗体
本说明书提供结合FXI、FXIa和/或FXI或FXIa的片段并抑制血块形成的抗体。这些抗体能够结合FXI、FXIa和/或FXI或FXIa的片段(例如，包含A3结构域的片段)，并且在远低于最大安全剂量的浓度下显示抑制效果。例如，在一些实施方案中，剂量在0.1mg/kg i.v.至3mg/kg i.v.之间的抗体表现出对食蟹猴FXI向FXIa的转化的抑制效果。此外，本说明书 公开的抗体可以用作具有优异安全性的抗凝剂，因为它们与常规抗凝剂(例如肝素)相比导致出血的风险最小。
本说明书中，关于组合物或方法的术语“包含”是指该组合物或方法至少包括所述的元素。术语“基本上由......组成”是指该组合物或方法包括所提及元素，并可以进一步包括一种或多种不会实质上影响该组合物或方法的新颖性和基本特性的附加元素。例如，基本上由所提及元素组成的组合物可包括所提及元素加上一种或多种来自分离和纯化方法的残留物、药学上可接受的载体如磷酸盐缓冲盐水、和防腐剂等。术语“由...组成”是指组合物或方法仅包括所提及元素。由每个过渡术语定义的实例均在本发明的范围内。
本文所用的术语“抗体”是指与特定抗原特异性结合或具有特定抗原免疫反应性的免疫球蛋白分子或其免疫学活性部分，例如FXI、FXIa以及FXI或FXIa的特定结构域或片段，例如A3结构域。某些实施方案中，本方法、组合物和试剂盒的抗体是全长免疫球蛋白分子，其包含两条重链和两条轻链，每条重链和轻链包含三个互补决定区(CDR)。除天然抗体外，术语“抗体”还包括基因工程或其他修饰形式的免疫球蛋白，例如合成抗体、胞内抗体、嵌合抗体、完全人源抗体、人源化抗体、肽体和异源缀合物抗体(例如双特异性抗体、多特异性抗体、双特异性抗体、抗独特型抗体、双抗体、三链抗体和四链抗体)本发明公开的抗体可以是单克隆抗体或多克隆抗体。在抗体是免疫球蛋白分子的免疫活性部分的实施方案中，抗体可以是Fab、Fab'、Fv，Fab'F(ab') 2、二硫键连接的Fv、单链Fv抗体(scFv)、单结构域抗体(dAb)或双抗体等。本说明书公开的抗体，包括作为免疫球蛋白分子的免疫活性部分，它们保留结合特异性抗原(例如FXI或FXIa)或结合FXI或FXIa的特定片段(例如A3结构域)的能力。
在一些实施方案中，本文公开的抗FXI和/或抗FXIa抗体经过了与哺乳动物细胞系(包括人或非人宿主细胞)中表达相关的翻译后修饰，例如磷酸化、甲基化、乙酰化、泛素化、亚硝基化、糖基化或脂化。用于生产重组抗体和重组抗体体外和体内修饰的技术是本领域已知的。参考文献例如mAbs 6(5):1145-1154(2014)，其内容通过引用并入本说明书。
说明书还公开了编码本文公开的抗FXI和/或抗FXIa抗体的多核苷酸或核酸。在一些实施方案中，该多核苷酸或核酸包括DNA、mRNA、cDNA以及质粒DNA。编码本文公开的抗体或其功能片段的核酸可以克隆到载体中，例如pTT5哺乳动物表达载体，其可以进一步包括启动子和/或其他转录或翻译控制元件，以使核酸可以被表达成抗体或其功能片段。
本说明书公开一些抗体实例的核酸(DNA)和/或氨基酸(PRT)序列见表1，其包括了VH、VL和CDR序列。
表1：抗体序列
Figure PCTCN2017119856-appb-000001
Figure PCTCN2017119856-appb-000002
Figure PCTCN2017119856-appb-000003
Figure PCTCN2017119856-appb-000004
Figure PCTCN2017119856-appb-000005
Figure PCTCN2017119856-appb-000006
Figure PCTCN2017119856-appb-000007
Figure PCTCN2017119856-appb-000008
Figure PCTCN2017119856-appb-000009
Figure PCTCN2017119856-appb-000010
Figure PCTCN2017119856-appb-000011
Figure PCTCN2017119856-appb-000012
Figure PCTCN2017119856-appb-000013
Figure PCTCN2017119856-appb-000014
Figure PCTCN2017119856-appb-000015
Figure PCTCN2017119856-appb-000016
本发明某些实施方案中提供了人源化抗FXI和/或抗FXIa抗体。本领域已知有多种对于非人源抗体进行人源化以使其接近人体天然存在抗体的技术。天然抗体的每个抗原结合结构域中存在6个CDR。这些CDR是短的、非连续的氨基酸序列，当抗体呈现其三维构型时，其被特异性定位以形成抗原结合结构域。抗原结合结构域其余部分称为“框架”区域，这些区域的分子间可变性较小并形成支架使CDR得以正确定位。
例如，本文公开的抗体人源化可以通过移植免疫小鼠或大鼠产生的单克隆抗体CDR来完成。小鼠单克隆抗体的CDR可以移植到人框架中，随后将其连接到人恒定区以获得人源化抗体。简言之，可以搜索人种系抗体序列数据库、蛋白质数据库(PDB)、INN(International Nonproprietary Names))数据库和其他合适的数据库，并且通过搜索找到和所需抗体最近似的框架。此外，一些对供体氨基酸残基所进行的回复突变在人受体框架上进行。在一些实施方案中，可变区与人IgG恒定区连接。例如，可以使用人IgG1、IgG2、IgG3和IgG4的Fc结构域。基于现有技术，将由非人物种产生的单克隆抗体人源化是本领域普通技术人员的基本能力。
表2为几个人源化抗体的可变区序列的示例。
表2：人源化抗体的序列
Figure PCTCN2017119856-appb-000017
本发明提供的抗体包括了本说明书公开序列的变体，其氨基酸序列中含有一个或多个突变，同时保留对FXI、FXIa和/或其片段(例如包含A3结构域的片段)的结合亲和力。在 一些实施方案中，抗体的可变区包括具有与以下序列至少85％，至少90％，至少95％，至少98％或至少99％相同的氨基酸序列或保留对FXI、FXIa和/或其片段结合亲和力的氨基酸片段，该序列选自SEQ ID NO：9、10、25、26、41、42、57、58、73、74、89、90、105、106、121、122、137、138、153、154、169、170、160、170、185、186和197-202。
本说明书还公开了包括编码结合FXI、FXIa和/或其片段(例如，包含A3结构域片段)的抗体的核酸变体。在一些实施方案中，编码抗体可变区的核酸包括具有与以下序列至少85％，至少90％，至少95％，至少98％或至少99％相同的核酸序列或编码对FXI，FXIa和/或其片段具有结合亲和力多肽的核酸片段，该序列选自SEQ ID NO：1、2、17、18、33、34、49、50、65、66、81、82、97、98、113、114、129、130、145、146、147、148、161、162、177和178。
药物组合物
本说明书公开的抗体可以配制成药物组合物。该药物组合物还可包含一种或多种药学上可接受的载体、赋形剂、防腐剂或其组合。该药物组合物可以是各种剂型，例如可以是注射制剂、冻干制剂、液体制剂等。根据剂型和给药途径，可以选择合适的佐剂、载体、赋形剂、防腐剂等作为添加剂。参见例如Wang et al.,J.Pharm.Sciences 96(1):1-26(2007)，其内容通过引用并入本说明书。
该药物组合物可以包括在具有该组合物用法说明的试剂盒中。
治疗方法
本发明对于患有血栓和/或血栓发生风险升高的受试者，提供了治疗和/或预防血栓的方法。还提供了抑制受试者血块形成的方法。这些方法需要施用治疗有效量的本发明提供的抗FXI和/或FXIa抗体，以干预内源性途径。在一些实施方案中，这些方法包括向受试者施用包含本发明提供的抗FXI和/或抗FXIa抗体的药物组合物。
本说明书公开了对于有需要的受试者，预防和/或治疗血栓相关的并发症或病症的方法。血栓会导致许多并发症或病症，例如栓塞性中风、静脉血栓(例如静脉血栓栓塞(VTE)、深静脉血栓(DVT)和肺栓塞(PE))动脉血栓(例如急性冠状动脉综合征(ACS)、冠状动脉疾病(CAD)和外周动脉疾病(PAD))。与血栓形成相关的其它病症包括：例如以下患者具有高VTE风险，他们是外科手术患者、卧床患者、癌症患者、心力衰竭患者、怀孕患者或患有可能引起血栓的其它医学病症患者。本说明书公开的方法涉及预防性抗凝血疗法，即血栓预防。这些方法需要向上文公开的患有血栓相关并发症的受试者施用治疗有效量的本发明公开的抗FXI和/或FXIa抗体或治疗有效量的包含抗FXI和/或FXIa抗体的药物组合物。该抗体或药物组合物可以单独施用或与用于治疗或预防血栓相关并发症或病症的任何其它疗法组合施用。
对于有需要的受试者，本发明还提供了治疗和/或预防败血症的方法。之前已经有向败血症患者施用抗凝剂以改善死亡率或发病率的尝试。然而，由于抗凝剂引起了预期外的出血，该尝试并不成功。本发明公开的抗体可以用作败血症的其它治疗剂(例如抗生素)辅助疗法。
本文所用的术语“受试者”是指哺乳动物受试者，优选人。“有需要的受试者”是指已经诊断出患有血栓或血栓相关的并发症或病症，或发生血栓或与血栓相关的并发症或病症风险增高的受试者。“受试者”和“患者”在本说明书中可互换使用。
本说明书关于病症的术语“治疗”是指部分或完全缓解病症，预防病症，降低病症发生或复发的可能性，减缓所述病症的恶化或发生，或消除、减少或减缓与所述病症相关的一种或多种症状的发生。就血栓和/或血栓相关的并发症或病症而言，“治疗”可以指预防或减缓现有血凝块增大，和/或预防或减缓血块的形成。在一些实施方案中，术语“治疗”是指与未施用抗体或其功能片段的受试者相比，受试者的血块数量或尺寸减少。在一些实施方案中，术语“治疗”是指，与未接受治疗的受试者相比，在接受如本说明书公开的抗体或药物组合物治疗后，受试者血栓和/或血栓形成相关的病症或并发症的一种或多种症状减轻。
本文所用的抗体或药物组合物的“治疗有效量”是指在受试者中产生期望的治疗效果(例如治疗和/或预防血栓)的抗体或药物组合物的量。在某些实施方案中，治疗有效量是产生最大治疗效果的抗体或药物组合物的量。在其它实施方案中，治疗有效量的治疗效果小于最大治疗效果。例如，治疗有效量可以是，既有疗效，又避免最大疗效剂量相关的一种或多种副作用的量。特定组合物的治疗有效量将基于多种因素而变化，所述因素包括但不限于治疗组合物的特征(例如活性、药代动力学、药效学和生物利用度)，受试者的生理状况(例如年龄、体重、性别、疾病类型和阶段、病史、一般身体状况、对给定剂量的反应和其他使用的现有药物)，组合物中任何药学上可接受的载体，赋形剂和防腐剂的性质以及给药方式。临床和药理学领域的技术人员可通过常规实验，即通过监测受试者对施用抗体或药物组合物的反应并相应地调整剂量，来确定治疗有效量。对于附加指导原则，参见例如Remington：The Science and Practice of Pharmacy，第22版，Pharmaceutical Press，London，2012，和Goodman&Gilman's The Pharmacological Basis of Therapeutics，第12版，McGraw-Hill，New York，2011，其全部公开内容通过引用并入本文。
在一些实施方案中，本说明书公开的抗体的治疗有效量在约0.01mg/kg至约30mg/kg，约0.1mg/kg至约10mg/kg，约1mg//kg至约5mg/kg。
选择合适的施用途径，例如皮下施用、静脉内施用、肌内施用、皮内施用、鞘内施用或腹膜内施用，在本领域普通技术人员的能力范围内。为了向有需要的受试者提供治疗，可以连续或间歇地施用速释、控释或缓释的抗体或药物组合物。A另外，该抗体或药物组合物可以一天三次、一天两次、或一天一次给药，持续3天、5天、7天、10天、2周、3周或4周的时间。抗体或药物组合物可以在预定的时间段内施用。可选择地，可施用抗体或药物组合物直至达到特定的治疗基准。在某些实施方案中，本发明提供的方法包括评估一种或多种治疗基准以确定是否继续施用抗体或药物组合物的步骤。
抗体的制备方法
本说明书还提供了生产本文公开的抗FXI和/或抗FXIa抗体的方法。在某些实施方案中，这些方法需要将编码抗FXI和/或抗FXIa抗体的核酸克隆到载体中，用载体转化宿主细胞，并培养宿主细胞以表达抗体。该表达的抗体可以使用任何已知的技术从宿主细胞中纯化。可以使用各种表达载体如pTT5载体和pcDNA3载体，以及各种宿主细胞系，例如CHO细胞(例如CHO-K1和ExpiCHO)和HEK193T细胞。
本公开还涵盖通过上文公开的方法产生的抗体。该抗体可能已经发生一处或多处翻译后修饰。
提供以下实施例是为了更好地说明该技术方案，并不应理解为其限制了任何所要求的实施方案的涵盖范围。对于所提及的具体物质，其仅仅是为了说明目的，并不意在限制本发明。在本发明的范围内，无需创作性劳动，本领域技术人员可能开发出等同的手段或反应物。应当理解为，本说明书描述的步骤可以发生许多变化，这仍在本发明的范围内。发明人的意图是这些变化包括在本发明的范围内。
实施例
实施例1：抗FXI抗体的生成和测序
以人凝血因子XI(FXI)分别免疫BALB/c小鼠、Wister大鼠，取免疫反应较好动物的脾细胞用于杂交瘤细胞制备；杂交瘤细胞有限稀释后进一步亚克隆，并通过ELISA捕获法和功能学筛选，成功获得12个单克隆杂交瘤细胞系，其表达的抗FXI抗体分别为：3G12、5B2、7C9、7F1、13F4、19F6、21F12、34F8、38E4、42A5、42F4和45H1。
为确定上述抗体轻、重链可变区(V L、V H)的氨基酸和核苷酸序列，根据标准PT-PCR操作规程，从相应杂交瘤细胞中克隆编码V L和V H的cDNA。相应抗体VL、VH序列(包括CDR序列)详见表1。
实施例2：使用活化部分凝血活酶时间(APTT)和凝血酶原时间(PT)检测指标评价本发明抗体在人血浆中的抗凝活性
APTT检测方法测量内源性和共同途径凝血的活性；而PT检测方法测量外源性和共同途径凝血的活性。在该实验中测试的抗体是19F6、34F8、42A5、1A6和14E11。在本实验中，抗体1A6和14E11用作阳性对照。对照抗体可变区的序列出自美国专利US 8,388,959和美国专利申请2013/0171144，并重新格式化为IgG4。然后使用ExpiCHO细胞系统表达这些抗体。取标准人血浆(购于Symens Inc.)和不同浓度的待测抗体(0-400nM)等体积混合，孵育5min，随后采用CA600分析仪检测。APTT测定中，取50μL上述血浆和抗体的混合物，加入25μL APTT试剂(SMN 10445709,Symens Inc.)混匀，在37℃孵育4min。加入25μL CaCl 2溶液(25mM,SMN 10446232,Symens Inc.)，记录凝固时间。PT测定中，取50μL上述血浆和抗体的混合物，加入等体积的PT试剂(SMN 10446442,Symens Inc.)混匀，在37℃下测定凝固时间。
如图1所示，所有待测抗体均可增加活化部分凝血活酶时间(APTT)，并在相对低的浓度范围内呈现一定的浓度依赖性，例如随浓度增长至100nM(14E11增长至200nM)；但所有抗体对凝血酶原时间(PT)均无明显影响(未提供数据)。这些结果表明，测试的所有抗体均可抑制凝血的内源性途径，但并不抑制外源性途径。
实施例3：利用APTT检测指标评价本发明抗体在非人血浆中的抗凝活性。
按例2的方法检测抗体(包括19F6,34F8和42A5)在小鼠、大鼠、猴的血浆中的抗凝血活性。结果显示在小鼠、大鼠血浆中所有待测抗体对APTT均无影响，但在猴血浆中其 APTT值显著增加，且在相对低浓度的范围内呈浓度依赖性(详见图2)。试验表明本发明抗体仅对猴FXI/FXIa有交叉反应，对小鼠和大鼠FXI/FXIa没有交叉反应。
实施例4：抗FXI抗体的人源化
由于鼠源抗体半衰期短且在人体内可诱发抗鼠抗体的生成而无法直接用于治疗，因此需要对抗体进行人源化。本发明的一些抗体通过CDR移植人源化(即选择一个合适的人抗体受体框架区作为每一种鼠抗体V L和V H的框架，并对其进行部分回突变以确保相关抗体的结构和功能不受太大的影响)。如果这些人源化抗体的亲和力和功能基本上不逊于相应的未修饰的抗体，则认为该修饰后的抗体人源化成功。19F6、34F8、42A5人源化后表示为h-19F6、h-34F8、h-42A5，其V L和V H序列详见表2。
实施例5：抗FXI抗体对人FXI的亲和力
采用表面等离子体共振(SPR)技术的BIAcore T200系统测定抗FXI抗体与FXI/FXIa的亲和力。方法如下：人源化抗体通过待测抗体可变区与人IgG4 Fc片段的连接而构建，在CHO细胞中进行重组表达；将这些抗体结合到一个已偶联抗人IgG捕获抗体的Biacore CM5传感芯片上。
随后不同浓度(0.005～1μg/ml)的纯化抗原FXI或者FXIa流过CM5传感芯片，抗FXI/FXIa抗体结合时间为180s，解离时间为1800s。收集的结合数据采用Biacore Evaluation Software(提供自GE Healthcare)进行分析以确定FXI/FXIa和检测抗体的亲和力。被固定的h-9F6、h-34F8、h-42A5抗体与FXI/FXIa结合的SPR图谱详见图3。如图3所示，随着FXI或FXIa浓度的逐渐升高，每种抗体的反应(RU)也随之增强。计算h-19F6、h-34F8和h-42A5对FXI和FXIa的解离常数(KD)，结果见表3。由于差异小于10倍，每种抗体对FXI和FXIa的亲和力被认为是相同的。
表3：针对FXI和FXIa的抗体的KD值
Figure PCTCN2017119856-appb-000018
实施例5：确定抗FXI抗体在FXI上的结合位点
采用SPR技术确定19F6、42A5在FXI上的结合位点。方法如下：将人IgG捕获抗体偶联在Biacore CM5传感芯片表面，重组样品h-19F6或h-42A5流过芯片表面而被捕获，通过调整流动时间使h-19F6或h-42A5捕获数量均达到15个相对单位。一定浓度的野生型FXI或嵌合型FXI(即FXI的单个Apple(A)功能域被人前激肽释放酶相应功能域取代的FXI/PK嵌合体)流过芯片表面，h19F6或h42A5结合时间为180s，随后的解离时间为1800s；数据采用一种高效动力学模型进行分析，仅有一种浓度的野生型FXI或嵌合型FXI在SPR测试中被检测。结果显示除了A3功能域被前激肽释放酶相应域取代的FXI/PK嵌合体， h-19F6和h-42A5均可结合FXI及FXI/PK嵌合体，提示h-19F6和h-42A5的部分或完整的抗原表位在A3功能域上。
实施例7：抗体功能性中和FXIa
人FXIa的活性主要通过测量对具有特异性且荧光标记的底物(S-2366，Diapharma Inc.)的水解来确定。为测试抑制活性，将溶于PBS(Phosphate buffer saline)缓冲液中终浓度为5nM的FXIa在室温下预孵育待测抗体h-19F6、h-34F8和h-42A5 5min，孵育后加入等体积的1mM S-2366开始FXIa裂解反应，并用M5 e酶标仪(Molecular Devices Inc.)在405nm下测量吸光度的变化；所得数据采用GraphPad Prism软件进行分析，详见图4。人源抗体h-19F6、h-34F8及h-42A5的表观Ki值为0.67、2.08、1.43nM。因此，测试的所有三种抗体在相对低的浓度下表现出对FXIa的令人满意的抑制作用。
实施例8：抗体对FXIa介导的FIX活化的抑制作用
将人FIX(200nM)、FXIa(5nM)以及1μM对照IgG或h-19F6或h-42A5溶于含有5mM CaCl 2的PBS中在室温下温育。在0、15、30、45和60分钟后，将50μL样品收集到十二烷基硫酸盐样品缓冲液中。将样品置于10％非还原性凝胶上进行大小电泳并转移到聚偏二氟乙烯膜上。使用山羊抗人FIX IgG(Affinity Biologicals)进行Western印迹以测定FIX以及FIXa水平。如图5所示，与对照相比，h-19F6和h-42A5均抑制由FXI诱导的FIXa形成。
实施例9：评估抗FXI抗体对食蟹猴凝血时间的影响
对食蟹猴静脉注射一定剂量的不同待测抗体，于给药前及给药后0.5、1、3、6、12、24小时收集上肢浅静脉血，并制备柠檬酸抗凝的血浆用于测定APTT、PT。1)APTT试验：取50μl稀释后的血浆样品，加入25μl APTT试剂(SMN 10445709,Symens Inc.)，混匀，37℃孵育4min，加入CaCl 2溶液(25mM,SMN 10446232,Symens Inc.)25μL混匀，记录凝固时间。2)PT试验：取50μl稀释后的血浆样品，加入等体积的PT试剂(SMN10446442,Symens Inc)，混匀，37℃下测凝固时间。结果显示，3个待测抗体均可剂量依赖性延长APTT(详见图6)，却不影响PT(详见图7)。
实施例10：抗FXI抗体对食蟹猴动静脉分流血栓形成及尾静脉出血模型的影响
在同一动物体内给予不同剂量的待测抗体评估血栓形成及出血时间。该实验中检测的抗体是h-34F8、h-19F6和h42A5。简言之，在每次施用抗体之前和给药后30分钟，依次评价出血时间和血栓形成。针对给药前和给药后三个递增剂量(0.1、0.3和1mg/kg)，对于出血/血栓形成进行4次评估。
AV分流血栓测定：在管内放入一根已称重的长为10cm的丝线，插管连接股动脉和股静脉，放开血流10min后中断血流，迅速取出丝线称湿重，计算血栓重量。血栓重量由血流前后的丝线称重差值决定。
出血时间测定：取2ml注射器插入动物的尾静脉，开始计时。当注射器内血不再增加时停止计时。该段时间即为出血时间。
结果表明本发明所有抗体可剂量依赖地抑制血栓形成(见图8))，且不延长尾部出血时间(见图9)。
实施例11：抗FXI抗体对FeCl 3诱导的食蟹猴股动脉血栓形成的影响和对标准出血时间的影响
将食蟹猴用1.5mg/kg Zoletil麻醉，气管插管，通呼吸机，全程监测血压、心率和体温。实验中，使用异氟烷维持麻醉效果。在施用FeCl 3前2小时肢静脉给予待测抗体h-34F8、h-19F6、h-42A5或空白载体(对照)。使用钝性分离方法暴露并分离左股动脉。用多普勒超声仪持续探测并记录血流情况。在施用FeCl 3,前，至少已经检测血流5分钟。随后取2片用FeCl 3浸润的滤纸包裹位于多普勒超声仪探针上方的动脉管表面，10min后移除滤纸，用生理盐水冲洗创面，将残留FeCl 3溶液冲洗干净。血流会全程监测直到数值降为0，记录阻塞80％(血流量减少到基线血流20％)时间及100％(血流量为0)的时间。对于同一动物，出血时间于给药前及给药后1小时用标准出血时间测定法测定。
以FeCl 3引起的动脉血栓模型评价h-34F8、h-19F6、h-42A5抗体，即4组食蟹猴分别给予空白载体、h-34F8、h-19F6、h-42A5，2小时后在左股动脉处敷FeCl 3，引起血栓，监测其下方的血流速度。结果显示：空白对照组80％、100％阻塞的时间分别为14.66±1.30min和18.5±1.76min；0.3mg/kg h-34F8、h-42A5预防治疗组80％阻塞时间分别为59.53±16.95min和40.80±7.94min，100％阻塞时间分别为70.40±20.76min和50.61±9.48min，与空白对照组比较有显著性差异(见图10)；h-19F6组虽与空白组比较无显著性差异，但其亦适当延长了80％阻塞(26.43±5.72min)及100％阻塞时间(32.78±5.09min)(见图10)。
以标准出血时间法，评估h-34F8、h-19F6、h-42A5抗体对于止血的影响，结果显示各组给药前及给药后1小时出血状况无明显差异(见图11A、11B、11C)，各治疗组出血时间与空白对照组比较亦无显著差异(见图11D)。
同时测定了3个抗体对猴血浆的体内至体外凝血时间的影响，结果显示，与空白对照组相比较，给予0.3mg/kg h-34F8、h-19F6和h-42A5抗体可显著延长活化部分凝血活酶时间(APTT)至给药前的3.29±0.20、1.67±0.09以及2.87±0.10倍(图12A)，但不影响凝血酶原时间(PT)(图12B)。
因此，本发明公开的抗体在能有效抑制凝血的内源性途径的同时，又出乎意料地不会延长出血时间。

Claims (13)

  1. 一种分离的抗FXI或抗FXIa抗体，其特异性结合人FXI或FXIa，其中，所述抗体包含免疫球蛋白轻链可变结构域，其包含的三个CDR选自SEQ ID NO：11-13、27-29、43-45、59-61、75-77、91-93、107-109、123-125、139-141、155-157、171-173和187-189，或者所述抗体包含免疫球蛋白重链可变结构域，其包含的三个CDR选自SEQ ID NO：14-16、30-32、46-48、62-64、78-80、94-96、110-112、126-128、142-144、158-160、174-176和190-192，或者其免疫活性部分。
  2. 如权利要求1所述的抗体，其中，所述的抗体特异性结合人FXI或FXIa的A3结构域。
  3. 如权利要求1所述的抗体，其中，所述抗体包含的免疫球蛋白轻链可变结构域选自：SEQ ID NO：9、SEQ ID NO：25、SEQ ID NO：41、SEQ ID NO：57、SEQ ID NO：73、SEQ ID NO：89、SEQ ID NO：105、SEQ ID NO：121、SEQ ID NO：137、SEQ ID NO：153、SEQ ID NO：169和SEQ ID NO：185。
  4. 如权利要求1所述的抗体，其中，所述抗体包含的免疫球蛋白重链可变结构域选自：SEQ ID NO：10、SEQ ID NO：26、SEQ ID NO：42、SEQ ID NO：58、SEQ ID NO：74、SEQ ID NO：90、SEQ ID NO：106、SEQ ID NO：122、SEQ ID NO：138、SEQ ID NO：154、SEQ ID NO：170和SEQ ID NO：186。
  5. 一种药物组合物，其包含如权利要求1-4之一所述的抗体。
  6. 如权利要求1-4之一所述的抗体在制备抑制患者血栓形成的药物方面的用途。
  7. 如权利要求1-4之一所述的抗体在制备治疗或预防败血症的药物方面的用途。
  8. 一种制备如权利要求1-4之一所述抗体的方法，其包括将编码抗体的核酸克隆进表达载体，并在宿主细胞中表达。
  9. 如权利要求8所述的方法，其进一步包括从宿主细胞中纯化表达抗体。
  10. 如权利要求8所述的方法，其中，所述表达载体是pTT5载体或pcDNA3载体。
  11. 如权利要求8所述的方法，其中，所述宿主细胞是CHO细胞或HEK193T细胞。
  12. 一种由权利要求8-11之一所述方法产生的抗体或其功能片段或其免疫活性部分。
  13. 如权利要求12所述的抗体，其中，所述抗体已经被翻译后修饰。
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MX2019009500A (es) 2020-01-30
CN108409863A (zh) 2018-08-17
AU2017397778A1 (en) 2019-10-03

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