CN114478782A - Anti-coagulation factor XI antibodies - Google Patents
Anti-coagulation factor XI antibodies Download PDFInfo
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- CN114478782A CN114478782A CN202111467365.1A CN202111467365A CN114478782A CN 114478782 A CN114478782 A CN 114478782A CN 202111467365 A CN202111467365 A CN 202111467365A CN 114478782 A CN114478782 A CN 114478782A
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- thrombosis
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Abstract
The present technology discloses antibodies that bind to Factor Xi (FXI) and/or its activated form factor xia (FXIa), or fragments of FXI and/or FXIa, and compositions containing the same. Also disclosed are methods of making the antibodies, and uses of the antibodies for making medicaments for treating and/or preventing coagulation-related disorders (e.g., thrombosis and complications or disorders associated with thrombosis).
Description
The application is a divisional application of an invention patent application with the application number of 201880098606.X and the invention name of 'anticoagulant factor XI antibody' in China national stage, which is submitted on 8,9 and 8 in 2018.
Technical Field
The present invention relates to antibodies capable of binding to Factor Xi (FXI) and/or its activated factor xia (FXIa) and FXI and/or FXIa fragments and uses thereof, including use as anticoagulants that can treat thrombi without affecting hemostatic function.
Background
Thrombosis is a condition in which, after coagulation of blood in a blood vessel, the passage of blood flow in the affected area is blocked or impeded. If a blood clot travels along the circulatory system to a critical part of the body, serious complications may result, such as progression to the heart, brain and lungs, which may cause heart attack, stroke and pulmonary embolism. Thrombosis is a major cause of most strokes and myocardial infarctions, Deep Vein Thrombosis (DVT), pulmonary embolism, and other cardiovascular events.1,2Thrombosis may be treated or prevented by anticoagulants (e.g., low molecular weight heparin, warfarin, and direct factor Xa inhibitors). The most common adverse effect of these currently available therapies is impaired hemostatic function.3-5These treatment methods are limited by dose and patient compliance due to the need for close monitoring of the subject after treatment.
Therefore, there is a need for an effective preventive or therapeutic agent for thrombosis with minimal side effects. The present invention satisfies this need in the art.
Disclosure of Invention
Certain embodiments of the invention provide antibodies that bind to Factor Xi (FXI) and/or its activated form factor xia (FXIa), and FXI and/or FXIa fragments. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a recombinant antibody. In certain embodiments, the antibody is a humanized antibody. In certain embodiments, the antibody is an immunologically active portion of an immunoglobulin molecule, such as a Fab, Fvs, or scFv. In certain embodiments, the antibody binds to the a3 domain of FXI and/or FXIa. In some embodiments, the antibody comprises one or more CDR sequences comprising or consisting of 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 the treatment and/or prevention of 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. In certain embodiments, the pharmaceutical composition further comprises one or more pharmaceutically acceptable adjuvants, carriers, excipients, preservatives, or combinations thereof.
The invention provides nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein, or any antibody functional fragment, as well as vectors comprising the nucleic acids, and host cells comprising the vectors. In certain embodiments, the vector is an expression vector capable of producing an antibody or functional fragment thereof encoded by the nucleic acid in a host cell.
The present invention provides a kit comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed herein for use in the treatment and/or prevention of thrombosis and/or complications or disorders associated with thrombosis. Alternatively, the kit comprises a pharmaceutical composition comprising one or more anti-FXI and/or anti-FXIa antibodies disclosed in the specification for the treatment and/or prevention of a thrombus and/or a complication or disorder associated with a thrombus. In certain embodiments, the kit further comprises instructions for use.
The present invention provides methods for treating and/or preventing thrombosis and/or complications or disorders associated with thrombosis. The methods comprise administering to a subject in need thereof an effective dose of one or more anti-FXI and/or anti-FXIa antibodies disclosed herein. Alternatively, the method comprises administering to a subject in need thereof an effective dose of a pharmaceutical composition comprising an anti-FXI antibody, an anti-FXIa antibody or a functional fragment of either antibody.
There is provided the use of an anti-FXI and/or anti-FXIa antibody as disclosed in the specification for the manufacture of a medicament for the treatment and/or prevention of thrombosis and/or a complication or disorder associated with thrombosis.
The present invention provides a method for the preparation of anti-FXI and/or anti-FXIa antibodies as disclosed in the present specification. The method takes steps that include transfecting a host cell with a vector encoding a nucleotide for the antibody and expressing the antibody in the host cell. The method may further comprise purifying the expressed antibody from the host cell. In addition, the purified antibodies can be modified such that the modified recombinant antibodies retain the activity of the corresponding human antibodies. Alternatively, the antibodies disclosed herein can be produced from hybridoma cultures.
Drawings
FIGS. 1A-1E show the anticoagulation effect of five anti-FXI antibodies in human plasma as determined by the APTT assay. 5 different antibodies were added to human plasma at 0 to 400nM for APTT detection as described in example 3. 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.
Fig. 2A-2C show the anticoagulant effect of antibodies 19F6(a), 34F8(B), and 42A5(C) in monkey plasma as measured by APTT. 3 different antibodies were added to monkey plasma at 0 to 400nM for APTT detection as described in example 4.
FIGS. 3A-3F show SPR sensorgrams of FXI binding to immobilized h-19F6(A), h-34F8(B) and h-42A5(C), and SPR sensorgrams of FXIa binding to immobilized h-19F6(D), h-34F8(E) and h-42A5 (F). The data fit to the 1:1 binding model, and curve fitting was performed at the tested concentration range of FXI (0.005-1 ng/mL). Each curve represents a different test concentration of FXI or FXIa.
FIGS. 4A-4C show concentration-response curves for the antibodies h-19F6(A), h-34F8(B), and h-42A5(C) inhibiting hydrolysis of S-2366 by human FXIa.
FIGS. 5A-5B show the inhibitory effect of antibodies h-19F6(A) and h-42A5(B) on FXIa-mediated activation of FIX as the FIXa response. Human FIX (200nM) was placed in a 5mM CaCl-containing solution of FXIa (5nM) and 1. mu. M h-19F6 or h-42A52In PBS, and incubated at room temperature. Samples were collected at designated time intervals and the amount of FIX and FIXa proteins in the samples was determined by western blotting using goat anti-human FIX IgG (Affinity Biologicals). FIGS. 5C-5D illustrate the inhibitory effect of antibodies h-19F6(C) and h-42A5(D) on FXIIa-mediated activation of FXI to FXIa. Human FXI (500nM) was incubated with FXIIa (50nM) in solutions containing 1. mu. M h-19F6 or h-42A 5. Detection of FXI at various time points and representation of FXIa production by Western blotting methodProtein amount of FXIa light chain of (a). Human IgG4 (1. mu.M) was used as a control.
FIGS. 6A-6C show the anticoagulant effect of the antibodies h-34F8, h-19F6 and h-42A5 on APTT in cynomolgus monkeys. Monkeys were administered intravenously at the indicated doses of h-34F8(A), h-19F6(B), and h-42A5 (C). APTT clotting times of ex vivo plasma were determined before dosing (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours post-dosing.
FIGS. 7A-7C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey PT. Monkeys were administered intravenously at the indicated doses of h-34F8(A), h-19F6(B), and h-42A5 (C). The PT clotting time T in ex vivo plasma was determined before dosing (time 0) and at 0.5, 1, 3, 6, 12 and 24 hours after dosing.
FIGS. 8A-8C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey Arteriovenous (AV) shunt thrombosis. Monkey were administered intravenously with increasing doses of h-34F8(A), h-19F6(B) or h-42A5(C) (n-3 for h-34F8 and h-19F6 and n-4 for h-42A 5) and pre-and post-dose weight changes were determined for the AV shunt thromboplastin monkey model. P <0.05, P <0.01 and P <0.001, vs. vehicle control.
FIGS. 9A-9C show the effect of antibodies h-34F8, h-19F6, and h-42A5 on cynomolgus monkey bleeding time. Monkeys were administered intravenously with increasing doses of h-34F8(a), h-19F6(B), or h-42a5(C) (n-3 for the 34F8 and h-19F6 groups and n-4 for the h-42a5 group), and bleeding times were calculated pre-dose and 30 minutes after each dose.
FIGS. 10A-10B show the antithrombotic effects of antibodies h-34F8, h-19F6, and h-42A 5. Four groups of monkeys (n-5) were administered vehicle control, 0.3mg/kg of h-34F8, h-19F6, or h-42A5 intravenously, respectively, with FeCl 2 hours after administration3Was administered to the left femoral artery of each animal to induce thrombosis. The time to reach 80% thrombotic occlusion (a) and 100% thrombotic occlusion (B) was determined by monitoring blood flow velocity. P<0.05 and P<Vehicle control group at 0.01vs.
FIGS. 11A-11D show that treatment with antibodies h-34F8, h-19F6, or h-42A5 did not prolong bleeding time in monkeys. Four groups of monkeys (n-5) were administered vehicle control, 0.3mg/kg of h-34F8, h-19F6, or h-42a5 intravenously, and bleeding time was measured before and 1 hour after dosing. The respective bleeding times of the h-34F8, h-19F6 and h-42A5 treatment groups are shown in FIGS. (A), (B) and (C), respectively. The change in bleeding time between groups of vehicle control, h-34F8, h-19F6, or h-42A5 treated groups is shown in FIG. D.
FIGS. 12A-12B show the effect of antibodies h-34F8, h-19F6, and h-42A5 on monkey plasma clotting time. Four groups of monkeys (n-5) were administered vehicle control, 0.3mg/kg of h-34F8, h-19F6, and h-42a5 intravenously, respectively, and blood was collected before and about 3 hours after dosing for plasma preparation and determination of clotting times APTT and PT. APTT changes and PT changes are shown in FIGS. (A) and (B). P <0.01 and P <0.001vs. vehicle control.
FIG. 13 is the amino acid sequence of human FXI (SEQ ID NO: 203).
FIG. 14 shows the effect of modified antibodies h-19F6 and h-42A5 on cynomolgus monkey APTT. Monkeys were given intravenous doses of the modified h-19F6 and h-42A 5. APTT clotting times of ex vivo plasma were determined before (time 0) and at 0.5, 2, 6, 12, 24, 48, 96, 168, 240 and 336 hours after administration, respectively.
FIG. 15 shows the effect of modified antibodies h-19F6 and h-42A5 on PT in cynomolgus monkeys. Monkeys were given intravenous doses of modified h-19F6 and h-42A 5. Ex vivo plasma PT clotting time measurements were performed before (time 0) and 0.5, 2, 6, 12, 24, 48, 96, 168, 240 and 336 hours after dosing, respectively.
FIGS. 16A-16B show the effect of h-19F6 and h-42A5 on APTT and PT in human plasma. FIG. 16A shows the effect of h-19F6 and h-42A5 on APTT in human plasma. FIG. 16B shows the effect of h-19F6 and h-42A5 on PT in human plasma.
Figure 17 shows the binding specificity of the test antibody to human FXI. In western blot experiments, 10 μ L of human standard plasma, FXI-depleted human plasma were used as FXI positive and FXI negative controls, respectively.
FIG. 18 shows the effect of h-19F6 and h-42A5 on the bleeding times recorded before and 1 hour after dosing in an AV shunt thrombosis model.
FIGS. 19A-19D show the binding properties of h-19F6 and h-42A5 to human FXI. FIG. 19A shows a sensorgram of h-19F6 in mobile phase capturing a specific concentration of FXI on a sensor chip. FIG. 19B shows a sensorgram of h-42A5 in mobile phase capturing a specific concentration of FXI on the sensor chip. FIG. 19C shows the captured antibody when the tested antibody (5. mu.g/mL) was flowed over a sensor chip immobilized with equal amounts of 4 mutant FXIs, the 4 mutations being the replacement of the FXI A1, A2, A3 or A4 domain by the corresponding domain of prekallikrein, respectively. Experiment a known anti-FXI antibody O1a6 was used as a positive control. FIG. 19D shows FXI immobilized on the sensor chip. h-19F6 and h-42A5 (5. mu.g/ml) were injected sequentially into the flow cell at 30. mu.l/min and the response was monitored. Experiments were performed twice and representative results are described.
FIGS. 20A-20B show the binding properties of h-19F6 and h-42A5 to human FXIa. FIG. 20A shows a sensorgram of h-19F6 in mobile phase capturing a specific concentration of FXI on the sensor chip. FIG. 20B shows a sensorgram of h-42A5 in mobile phase capturing a specific concentration of FXI on the sensor chip.
Detailed Description
The following description of the present specification is intended only to illustrate various examples of the present invention. Therefore, the particular modifications discussed should not be construed as limiting the scope of the invention. It will be apparent to those skilled in the art that various equivalent substitutions, changes and modifications can be made to the entities of the present invention without departing from the scope of the invention, and it is to be understood that the invention also includes such equivalent examples.
The intrinsic coagulation cascade mechanism includes the intrinsic pathway and the extrinsic pathway. The intrinsic pathway, also known as the contact activation pathway, is initiated by surface contact and results in activation of FXII. The intrinsic pathways involve FXI, FIX and FVIII. The extrinsic pathway, also known as the Tissue Factor (TF) pathway, is initiated by vascular injury and results in the formation of the TF-FVIIa activation complex. These two pathways can intersect and activate a common pathway, leading to conversion of prothrombin to thrombin and ultimately the formation of a cross-linked fibrin clot. The ideal anticoagulant should be effective in preventing thrombosis without compromising hemostatic action. Evidence suggests that the intrinsic coagulation pathway is important for the expansion phase of coagulation, while the extrinsic pathway is importantAnd the common pathway is more involved in the startup phase and the propagation phase.5-8These findings indicate that the intrinsic pathway plays a secondary role in the normal hemostatic process, while inhibition of the intrinsic pathway may provide antithrombotic benefits with a lower risk of bleeding. FXI is a component of the intrinsic pathway and has recently become an attractive target as it may have the potential to elicit an antithrombotic effect without affecting bleeding.3,5,6
FXI can be activated by factor XIIa through the intrinsic pathway to FXIa, which in turn activates factor IX. Epidemiological studies have shown that lack of FXI in humans is associated with a reduced risk of venous thromboembolism and stroke, while elevated FXI levels are associated with an increased risk of stroke.9-11Furthermore, bleeding tendency is very low in people lacking FXI.12,13Furthermore, FXI deficient mice can be protected from multiple types of thrombosis without increasing the risk of bleeding.14Furthermore, small molecule inhibitors, antibodies and antisense oligonucleotides that inhibit FXI all have antithrombotic properties without the risk of bleeding in many animal models of thrombosis.
The antibodies disclosed herein bind to FXI and/or FXIa and are directed to the intrinsic pathway of coagulation. The structure and role of FXI in coagulation has been previously reported in various documents.33
Animal and clinical studies have shown a close association between FXI and thrombosis.3A number of groups have studied FXI deficient mice and have shown a significant antithrombotic phenotype in several models, including FeCl3Induced arterial and deep vein thrombosis models, pulmonary embolism models, and cerebral arterial occlusion models.14,17,22,23Patients with congenital FXI deficiency are not susceptible to Venous Thromboembolism (VTE) or ischemic stroke in human epidemiological studies, and patients with higher FXI levels are at higher risk of developing VTE and ischemic stroke than patients with lower FXI levels.9-11For physiological hemostasis, the effect of FXI appears to be dispensable. FXI deficient mice did not show excessive bleeding and their tail bleeding times were comparable to wild type animals.23,24Furthermore, patients with severe FXI lack, while possibly exhibiting different bleeding tendencies during surgery, do not exhibit spontaneous bleeding.12,13Combinations of two or more antithrombotic agents are widely used clinically. One previous study showed that aspirin caused similar bleeding tendencies in wild-type and FXI deficient mice, suggesting that targeting FXI may still be safe even in the presence of other antithrombotic therapies.14
All the above findings indicate that FXI/FXIa is a safe drug target for the treatment of thrombosis related diseases without compromising hemostatic function. Therefore, a number of approaches have been applied to the target FXI/FXIa to develop therapies, such as antibodies, oligonucleotides and small molecule inhibitors, for the treatment of thrombosis.5An antibody-type blocker of FXI/FXIa was prepared as described in the specification. The advantages of antibodies include rapid onset of action and low frequency of administration, and the main disadvantage of antibodies is their potential immunogenicity.25At least two of the tested antibodies were humanized prior to in vivo studies. The two humanized antibodies h-19F6 and h-42A5 have high affinity for human FXI/FXIa. Interestingly, they bound different regions of the same domain of FXI (a 3). Without being bound by any theory, these antibodies may both inhibit FXIa activity but have no effect on FXI activation mediated by FXIIa or thrombin.
Various types of FXI/FXIa inhibitors prolong APTT time and show antithrombotic effects in different models. anti-FXI antibody 14E11 increased APTT in the femoral external arteriovenous shunt of baboons by about 1.3-fold and reduced thrombosis.17Antisense oligonucleotides that inhibit FXI expression can reduce baboon plasma FXI levels by about 50% and reduce thrombosis.26,27In addition, the oral bioavailable small molecule FXIa inhibitor ONO-5450598 significantly inhibited thrombosis in a monkey thrombosis model.28Furthermore, antithrombotic effects against the therapeutic target FXI/FXIa have also been demonstrated in a number of non-primate animal models, such as mouse and rabbit thrombosis models.19,29-31A recent clinical trial has shown that this is aimed atThe antisense oligonucleotide of FXI prevents venous thrombosis in knee replacement patients.32As shown in the examples, the antithrombotic effects of h-19F6 and h-42A5 were evaluated in two different primate thrombosis models. In the AV shunt thrombosis model, both antibodies reduced thrombosis in a dose-dependent manner. In FeCl3In the induced thrombosis model, both antibodies extended the time of vessel occlusion by thrombosis. These results provide further evidence of the antithrombotic effect of FXI/FXIa inhibitors. Dose-dependent reduction of thrombosis in the AV shunt thrombosis model of h-19F6 and h-42A5 suggested that thrombosis might be inversely correlated with the extent of FXI inhibition, as shown by the prolongation of APTT. Since h-42A5 is more effective in extending APTT than h-19F6, it can also be deduced that in FeCl3Similar results were obtained when comparing the antithrombotic effects of h-42A5 and h-19F6 in an induced thrombosis model. Thus, the more pronounced the effect of FXI/FXIa inhibitors on prolonging APTT, the more potent the inhibition of FXI/FXIa, possibly leading to better antithrombotic results.
In the development of antithrombotic agents, bleeding risk is the most relevant problem. As previously mentioned, patients lacking FXI may develop a bleeding tendency in the surgical environment. It is not clear to what extent inhibition of plasma FXI activity is still safe in terms of bleeding risk. As shown in the examples, the risk of bleeding was tested that h-19F6 and h-42A5 strongly inhibited the FXI/FXIa action in the same monkey used in the thrombosis experiments. In AV shunt thrombogenic animals, no bleeding tendency was observed with increasing therapeutic doses of h-19F6 or h-42A5, indicating that the risk of bleeding may not be related to the extent of FXI inhibition. In FeCl3Neither h-19F6 nor h-42A5 induced excessive bleeding in thrombosed animals. h-42A5 treatment resulted in an approximately 2-fold increase in plasma APTT, indicating over 99% inhibition of FXI. Previous studies have never assessed the risk of bleeding under such strong conditions of prolonged APTT and high FXI inhibition. The antisense oligonucleotide ISIS416856 only resulted in a 30% increase in APTT when assessing its bleeding risk. Efficient anti-FXI antibody aX in other bleeding risk assessment studies in primatesIMab caused about a 1-fold increase in APTT (from 30.5s to 65.6 s).26Thus, the results described herein indicate that strong inhibition of FXI/FXIa does not increase the bleeding risk in primates. Therefore, FXI can be used as a drug target for thrombosis therapy.
anti-FXI or anti-FXIa antibodies
The present specification provides antibodies that bind to FXI, FXIa and/or fragments of FXI or FXIa and inhibit blood clot formation. These antibodies are able to bind FXI, FXIa and/or fragments of FXI or FXIa (e.g., fragments comprising the a3 domain) and show inhibitory effects at concentrations well below the maximum safe dose. For example, in some embodiments, an antibody at a dose between 0.1mg/kg i.v. and 3mg/kg i.v. exhibits an inhibitory effect on cynomolgus monkey FXI conversion to FXIa. In addition, the antibodies disclosed in the present specification can be used as anticoagulants with excellent safety because they cause minimal risk of bleeding compared to conventional anticoagulants (e.g., heparin).
As shown in the examples, antibodies with anticoagulant properties were determined by preparing a number of anti-human FXI antibodies by immunizing rats with human FXI. Over a dozen such antibodies were identified, some of which were humanized for further development. Humanized rat anti-human FXI antibodies, such as the h-19F6 and h-42A5 antibodies, were characterized in vitro and in vivo. In vitro studies, humanized antibodies inhibited activation of FXI (fxia) -mediated hydrolysis of FXI, but did not inhibit FXI activation induced by factor XIIa. The binding characteristics of the antibody to FXI were determined, with dissociation constants (KD) of 22pM and 35pM for h-19F6 and h-42A5, respectively. These two antibodies bind to different sites in the FXI a3 domain. In vivo studies, two different primate thrombosis models were used to assess the antithrombotic effect and bleeding risk of humanized antibodies. In an Arteriovenous (AV) shunt thrombosis model, both antibodies dose-dependently reduced thrombosis without causing bleeding. In FeCl3In the induced thrombosis model, both antibodies extended the time to thrombosis-mediated vascular occlusion, and neither antibody increased bleeding. Both antibodies showed antithrombotic efficacy without compromising primate hemostasis, further demonstrating that targeting FXI could be usedTreating thrombosis.
In this specification, the term "comprising" with respect to a composition or method means that the composition or method includes at least the recited elements. The term "consisting essentially of means that the composition or method includes the elements described, and may further include one or more additional elements that do not materially affect the novel and basic characteristics of the composition or method. For example, a composition consisting essentially of the elements may include the elements plus one or more residues from the isolation and purification process, a pharmaceutically acceptable carrier such as phosphate buffered saline, and preservatives and the like. The term "consisting of" means that the composition or method includes only the elements recited. Examples defined by each transitional term are within the scope of the present invention.
The term "antibody" as used herein refers to an immunoglobulin molecule or immunologically active part thereof that specifically binds to or has specific immunoreactivity with a specific antigen, which may for example be FXI, FXIa and a specific domain or fragment of FXI or FXIa, such as the a3 domain. In certain embodiments, the antibodies of the present methods, compositions, and kits are full-length immunoglobulin molecules comprising two heavy chains and two light chains, each heavy and light chain comprising three Complementarity Determining Regions (CDRs). In addition to natural antibodies, the term "antibody" also includes genetically engineered or otherwise modified forms of immunoglobulins, such as synthetic antibodies, intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies, peptibodies, and heteroconjugate antibodies (e.g., bispecific antibodies, multispecific antibodies, bispecific antibodies, anti-idiotypic antibodies, diabodies, triabodies, and tetrabodies). The antibodies disclosed herein can be monoclonal or polyclonal. In embodiments where the antibody is an immunologically active portion of an immunoglobulin molecule, the antibody can be Fab, Fab ', Fv, Fab ' F (ab ')2A disulfide-linked Fv, a single-chain Fv antibody (scFv), a single-domain antibody (dAb), or a diabody. Antibodies disclosed in the present specification, comprising immunologically active portions as immunoglobulin molecules, which retain a specific antigen (e.g. FXI or FXIa) binding or specific sheet binding FXI or FXIaThe ability of a segment (e.g., the a3 domain).
In some embodiments, the anti-FXI and/or anti-FXIa antibodies disclosed herein undergo post-translational modifications, such as phosphorylation, methylation, acetylation, ubiquitination, nitrosylation, glycosylation, or lipidation, etc., during protein expression in a mammalian cell line. Techniques for the production of recombinant antibodies and in vitro and in vivo modifications of recombinant antibodies are known in the art. References such as mAbs 6(5):1145 and 1154(2014), the contents of which are incorporated herein by reference.
The specification also discloses polynucleotides or nucleic acids encoding the anti-FXI and/or anti-FXIa antibodies disclosed herein. In some embodiments, the polynucleotide or nucleic acid comprises DNA, mRNA, cDNA, and plasmid DNA. Nucleic acids encoding the antibodies or functional fragments thereof disclosed herein may be cloned into a vector, such as a pTT5 mammalian expression vector, which may further include a promoter and/or other transcriptional or translational control elements, such that the nucleic acids may be expressed into the antibodies or functional fragments thereof.
The present specification Discloses Nucleic Acid (DNA) and/or amino acid (PRT) sequences of some examples of antibodies, which are shown in table 1, including VH, VL and CDR sequences.
Table 1: antibody sequences
In certain embodiments of the invention there are provided humanized anti-FXI and/or anti-FXIa antibodies. There are a variety of techniques known in the art for humanizing non-human antibodies to approximate naturally occurring antibodies in humans. There are 6 CDRs in each antigen binding domain of a natural antibody. These CDRs are short, non-contiguous amino acid sequences that are specifically positioned to form an antigen-binding domain when the antibody assumes its three-dimensional configuration. The remainder of the antigen binding domain is called the "framework" regions, which have less intermolecular variability and form a scaffold for the CDRs to be correctly positioned. In some embodiments, the antibodies or fragments disclosed herein have conserved sequences of the CDR3 regions.
For example, humanization of the antibodies disclosed herein can be accomplished by grafting the CDRs of monoclonal antibodies produced by immunized mice or rats. The CDRs of a mouse monoclonal antibody can be grafted into a human framework, which is then ligated to human constant regions to obtain a humanized antibody. Briefly, a human germline antibody sequence database, a Protein Database (PDB), an inn (international Nonproprietary names) database, and other suitable databases may be searched and the framework most closely approximating the desired antibody found by the search. In addition, some back mutations to the donor amino acid residues were made in the human acceptor framework. In some embodiments, the variable region is linked to a human IgG constant region. For example, the Fc domain of human IgG1, IgG2, IgG3, and IgG4 can be used. Based on the prior art, humanizing monoclonal antibodies produced by non-human species is a fundamental capability of one of ordinary skill in the art.
Table 2 is an example of the variable region sequences of several humanized antibodies.
Table 2: sequences of 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 binding affinity for FXI, FXIa, and/or fragments thereof (e.g., fragments comprising the a3 domain). In some embodiments, the variable region of the antibody comprises an amino acid fragment having an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to, or retains binding affinity for FXI, FXIa and/or a fragment thereof, selected from 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, 209.
The specification also discloses nucleic acid variants comprising antibodies that encode binding to FXI, FXIa, and/or fragments thereof (e.g., a3 domain-containing fragments). In some embodiments, the nucleic acid encoding the antibody variable region comprises a nucleic acid fragment having a nucleic acid sequence at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to a sequence 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.
In some embodiments, the antibodies described above will be subjected to further strategic chemical, production, and control (CMC) developments to enable the novel antibodies disclosed herein (e.g., monoclonal antibodies or humanized monoclonal antibodies) to be marketed from early drug discovery, to clinical trials and subsequent drugs. The modification will further improve the properties of the antibody without compromising the immune function of the antibody. In certain embodiments, the CMC modified antibody is more stable than the unmodified antibody at a variety of temperatures (e.g., 4 ℃, 25 ℃, and 37 ℃), for longer periods of time (e.g., 3 days, 7 days, 14 days, and 28 days), and under repeated freeze/thaw cycles (e.g., -40 ℃/25 ℃ to 5 cycles). In addition, the CMC modified antibody has acceptable solubility. For example, for a given light or heavy chain sequence, certain amino acids may be potential oxidation and glycosylation sites. These amino acid residues at potential oxidation, deamidation or glycosylation sites can be mutated, and other residues in the vicinity can also be mutated and/or optimized to maintain the 3D structure and function of a particular antibody. In some embodiments, one or more amino acid residues in a CDR region with oxidative, deamidation or glycosylation potential are mutated to improve the stability of an antibody or fragment thereof without compromising immune function. In some embodiments, one or more Met residues in the CDR regions with oxidative potential are mutated. In some embodiments, one or more Asn residues in a CDR region with deamidation potential are mutated.
The sequences of the variable regions of several exemplary CMC optimized humanized antibodies are shown in table 3 below.
Table 3: sequences of CMC optimized humanized antibodies
Pharmaceutical composition
The antibodies disclosed in the present specification can be formulated into pharmaceutical compositions. The pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers, excipients, preservatives, or combinations thereof. The pharmaceutical composition can be in various dosage forms, such as injection, lyophilized preparation, liquid preparation, etc. Depending on the dosage form and route of administration, compounds may be selectedSuitable adjuvants, carriers, excipients, preservatives, etc. as additives.34
The pharmaceutical composition may be included in a kit with instructions for use of the composition.
Method of treatment
The present invention provides methods for treating and/or preventing a thrombus in a subject having a thrombus and/or an elevated risk of developing a thrombus. Methods of inhibiting clot formation in a subject are also provided. These methods require administration of an effective dose of an anti-FXI and/or FXIa antibody provided by the present invention to interfere with the intrinsic pathway. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition comprising an anti-FXI and/or anti-FXIa antibody provided by the present invention.
The present specification discloses methods for preventing and/or treating a thrombosis related complication or disorder in a subject in need thereof. Thrombi can lead to a number of complications or disorders, such as embolic stroke, venous thrombosis (e.g., Venous Thromboembolism (VTE), Deep Vein Thrombosis (DVT), and Pulmonary Embolism (PE)), arterial thrombosis (e.g., Acute Coronary Syndrome (ACS), Coronary Artery Disease (CAD), and Peripheral Artery Disease (PAD)). Other conditions associated with thrombosis include: for example, patients who are surgical, bedridden, cancer, heart failure, pregnant or suffering from other medical conditions that may cause thrombosis have a high risk of VTE. The methods disclosed herein relate to prophylactic anticoagulant therapy, i.e., thrombus prevention. These methods entail administering to a subject suffering from a thrombosis related complication as disclosed above an effective dose of an anti-FXI and/or FXIa antibody as disclosed herein or an effective dose of a pharmaceutical composition comprising an anti-FXI and/or FXIa antibody. The antibody or pharmaceutical composition may be administered alone or in combination with any other therapy for treating or preventing a thrombosis related complication or disorder.
The invention also provides methods of treating and/or preventing sepsis in a subject in need thereof. There have been previous attempts to administer anticoagulants to septic patients to improve mortality or morbidity. However, this attempt was not successful because the anticoagulant caused unexpected bleeding. The antibodies disclosed herein can be used as adjunctive therapy to other therapeutic agents (e.g., antibiotics) for sepsis.
The term "subject" as used herein refers to a mammalian subject, preferably a human. By "subject in need thereof is meant a subject who has been diagnosed as having, or at increased risk of developing, a thrombus or a thrombus-related complication or disorder. "subject" and "patient" are used interchangeably in this specification.
The term "treating" in reference to a disorder as used herein refers to partially or completely alleviating the disorder, preventing the disorder, reducing the likelihood of occurrence or recurrence of the disorder, slowing the progression or onset of the disorder, or eliminating, reducing or slowing the occurrence of one or more symptoms associated with the disorder. In the context of a thrombus and/or thrombus-related complication or condition, "treating" can refer to preventing or slowing the growth of an existing blood clot, and/or preventing or slowing the formation of a blood clot. In some embodiments, the term "treating" refers to a reduction in the number or size of blood clots in a subject as compared to a subject not administered the antibody or functional fragment thereof. In some embodiments, the term "treating" or "treatment" refers to a reduction in one or more symptoms of thrombosis and/or a thrombosis-related disorder or complication in a subject following treatment with an antibody or pharmaceutical composition as disclosed herein, as compared to a subject not receiving treatment.
As used herein, an "effective dose" of an antibody or pharmaceutical composition refers to the amount of the antibody or pharmaceutical composition that produces a desired therapeutic effect (e.g., treatment and/or prevention of thrombosis) in a subject. In certain embodiments, an effective dose is the amount of antibody or pharmaceutical composition that produces the greatest therapeutic effect. In other embodiments, the therapeutic effect of the effective dose is less than the maximum therapeutic effect. For example, an effective amount can be an amount that is both therapeutically effective and avoids one or more side effects associated with the maximum therapeutically effective amount. The effective dosage 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 physiological condition of the subject (e.g., age, body weight, sex, type and stage of disease, medical history, general physical condition, response to a given dosage, and other drugs currently used), the nature of any pharmaceutically acceptable carriers, excipients, and preservatives in the composition, and the mode of administration. One skilled in the clinical and pharmacological arts can determine effective dosages by routine experimentation, i.e., by monitoring a subject's response to administration of an antibody or pharmaceutical composition and adjusting the dosage accordingly. For additional guidance, see, e.g., Remington: the Science and Practice of Pharmacy, 22 nd edition, Pharmaceutical Press, London, 2012, and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 12 th edition, McGraw-Hill, New York, 2011, The entire disclosure of which is incorporated herein by reference.
In some embodiments, an effective dose of an antibody disclosed herein is from about 0.01mg/kg to about 30 mg/kg, from about 0.1mg/kg to about 10mg/kg, from about 1mg/kg to about 5 mg/kg.
It is within the ability of one of ordinary skill in the art to select an appropriate route of administration, e.g., subcutaneous, intravenous, intramuscular, intradermal, intrathecal, or intraperitoneal. To provide treatment to a subject in need thereof, the antibody or pharmaceutical composition may be administered continuously or intermittently with immediate, controlled or sustained release. In addition, the antibody or pharmaceutical composition may 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 may be administered over a predetermined period of time. Alternatively, the antibody or pharmaceutical composition may be administered until a specific therapeutic benchmark is reached. In certain embodiments, the methods provided herein comprise the step of evaluating one or more treatment benchmarks to determine whether to continue administration of the antibody or pharmaceutical composition.
Method for producing antibody
The present specification also provides methods of making the anti-FXI and/or anti-FXIa antibodies disclosed herein. In certain embodiments, these methods entail cloning a nucleic acid encoding an anti-FXI and/or anti-FXIa antibody into a vector, transforming a 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. A variety of expression vectors can be used, such as the pTT5 vector and the pcDNA3 vector, as well as a variety of host cell lines, such as CHO cells (e.g., CHO-K1 and ExpicHO) and HEK193T cells.
The present specification also encompasses antibodies produced by the methods disclosed above. The antibody may have undergone one or more post-translational modifications.
The following examples are provided to better illustrate the technical solution and should not be construed as limiting the scope of coverage of any claims. Reference to specific materials is for illustrative purposes only and is not intended to be limiting. Within the scope of the present invention, equivalent means or reactants may be developed by those skilled in the art without the need for creative efforts. It should be understood that many variations of the steps described in this specification may occur while remaining within the scope of the invention. It is the intention of the inventors that such variations are included within the scope of the invention.
Examples
Example 1: materials and methods
A material. Human FXI (Cat No. HFXI 1111), FXIa (Cat No. HFXIa 1111a), FXIIa (HFXIIa 1212a) and FIX (Cat No. HFIX 1009) were purchased from Enzyme Research Laboratory (IN, USA).
And (5) preparing an antibody. Animal immunization and hybridoma screening were performed in Genscript Inc (china, south kyo), and procedures applied to animals in this protocol were approved by the gen-instituted animal care and use committee. Experiments were performed according to approved guidelines. Wistar rats were immunized with human FXI, and spleen cells of animals with good immune response were collected to prepare hybridomas, which were then used for subcloning by limiting dilution. Finally, several monoclonal hybridoma cell clones expressing the desired anti-FXI antibodies were obtained by using ELISA and functional screening, including 19F6, h-34F8, and 42a 5. After determination of the amino acid sequence of their variable regions, 19F6, h-34F8 and 42A5 were humanized to give three humanized antibodies h-19F6, h-34F8 and h-42A5 in the form of IgG 4. These three humanized antibodies were produced in a mammalian transient expression system and purified by protein G chromatography.
Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Standard human plasma purchased from Symens inc. was mixed with equal volumes of each antibody at concentrations from 0 to 400nM for 5 minutes, respectively, and then tested on a CA600 analyzer. In the APTT assay, 50 μ L of plasma-antibody mixture and 25 μ L of APTT reagent (SMN 10445709, Symens Inc.) were mixed for 4 minutes at 37 ℃. Then 25. mu.L of CaCl was added2Solutions (25mM, SMN 10446232, Symen Inc.) and the time to clot formation was determined. In the PT assay, 50 μ L of plasma-antibody mixture was mixed with an equal volume of PT reagent (SMN 10446442, Symens Inc.) at 37 ℃ and the time to clot formation was then determined.
The effect of the antibodies on APTT and PT in monkey plasma was also assessed using the same method as human plasma. In these assays, equal volumes of monkey plasma diluted in Phosphate Buffered Saline (PBS) were used instead of the human plasma-antibody mixture described above.
FXIIa activates FXI. Human FXI (500nM) was preincubated in PBS for 5min at room temperature with 1 μ MIgG4 control or h-19F6 or h-34F8 or h-42A 5. At time zero, FXIIa, HK and kaolin were added to give final concentrations of FXI (250nM), FXIIa (50nM), HK (100nM) and kaolin (0.5 mg/mL). Every 0, 30, 60, 120 minutes, 50 μ Ι _ of sample was collected into dodecyl sulfate sample buffer. The samples were size fractionated on a 10% non-reducing gel and then transferred to a polyvinylidene fluoride membrane. The FXI as well as FXIa light chain levels were determined by western blotting using mouse anti-human FXI IgG (1C5, homemade antibody binding to the FXI C-terminus). Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad).
FXIa mediated FIX activation. Human FIX (200nM) and FXIa (5nM) in a medium containing 5mM CaCl at room temperature in an environment containing 1. mu.M IgG1 control, h-19F6, h-34F8, or h-42A52Was incubated in PBS (g). Every 0, 15, 30, 45 and 60 minutes, 50 μ L of sample was collected into dodecyl sulfate sample buffer. The samples were size fractionated on a 10% non-reducing gel and then transferred to a polyvinylidene fluoride membrane. Use of goat for anti-humanFIX IgG (Affinity Biologicals) was subjected to Western blotting to determine FIX as well as FIXa levels. Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad).
Surface Plasmon Resonance (SPR). The interaction of the antibody with FXI was determined by SPR assay of Biacore T200 system (Biacore, GE Healthcare). Briefly, human IgG capture antibody (Biacore, GE Healthcare) was previously immobilized on a CM5 sensor chip (GE Healthcare), and the test antibody was captured by flowing through the chip. The final amount of captured test antibody was adjusted to 15 Reaction Units (RU) equally by adjusting the capture time. Then, the antigen FXI was flowed over the chip with an association time of 180 seconds and an dissociation time of 1200 seconds. FXI was tested at concentrations of 0.063, 0.313, 0.625, 1.25, 3.125 and 6.25 nM. Data were collected and the affinity between the tested antibody and FXI was analyzed using Biacore Evaluation Software.
To determine the binding site of the test antibody on FXI, four FXI mutants C-terminally labeled with 6xHis were first generated by replacing each Apple domain (a1, a2, A3, and a4) with the corresponding domain of human prekallikrein. Equal amounts of each mutant were immobilized on a CM5 sensor chip, and the antibody to be tested (33.3nM) was passed through the chip with an association time of 180s and an dissociation time of 1200 s. The amount of each antibody captured was recorded using a Biacore Evaluation Software, measured in Reaction Units (RU).
Epitope binding results of the tested antibodies were also analyzed using the Biacore T200 system. Briefly, wild-type FXI with a 6XHis tag was pre-immobilized on a CM5 sensor chip (GE Healthcare), and h-19F6, h-34F8 or h-42A5 (5. mu.g/ml) were sequentially injected into a flow cell on the sensor surface at a flow rate of 30. mu.l/min to monitor changes in response.
Pharmacodynamics of cynomolgus monkey. This animal experiment and subsequent AV thrombosis experiments were performed in Wincon Inc (china, nanning), and the procedures applied to animals in this protocol were approved by the Wincon institutional animal care and use committee. Experiments were performed according to approved guidelines. Animals received varying doses of h-19F6, h-34F8 or h-42A5 as bolus injections. 2mL of blood from the upper limb superficial vein was collected into a collection tube containing 3.2% sodium citrate before dosing (time 0) and at 0.5h, 1h, 3h, 6h, 12h and 24h post-dosing. The tubes were then mixed ten times by gentle inversion at room temperature. Plasma was collected in labeled tubes and stored at-20 ℃ until clotting time analysis. Plasma samples were diluted with equal volumes of phosphate buffered saline (pH 7.4) and then subjected to APTT and PT analysis on an automated analyzer (CA660, Sysmex Inc.).
AV shunt thrombosis and bleeding time test. Cynomolgus monkeys were injected intravenously with antibody and tested 30 minutes later. Then, a tail vein bleeding time test was performed, and thrombosis was induced again. Thrombosis was induced by connecting a shunt device, containing a pre-weighed 10cm long wire, between the femoral artery and the femoral venous cannula. Blood was allowed to flow through the shunt for 10 minutes. The thrombus formed on the wire was weighed. Immediately after the shunt is removed, a blood sample is collected and given the next higher level of antibody to be tested. Four bleeding/thrombosis events were performed in the same animal, with vehicle control and three increasing dose levels of test antibody (0.1, 0.3, 1 mg/kg).
To assess bleeding time, a 2-mL syringe was inserted into the tail vein of the animal. When the amount of blood in the syringe stopped increasing, the elapsed time was manually recorded as the bleeding time.
Ferric chloride (FeCl)3) Resulting thrombosis and bleeding time test. The animal experiments were performed in pharma legacy Laboratories Inc (shanghai, china), and procedures applicable to animals in this protocol were approved by the pharma legacy institutional animal care and use committee. Experiments were performed according to approved guidelines. Cynomolgus monkeys were pre-anesthetized with 1.5mg/kg Zoletil, intubated, and ventilated with a ventilator. Anesthesia was maintained with isoflurane. Blood pressure, heart rate and body temperature were monitored throughout the procedure. In the use of FeCl32 hours before, the administration of either blank vehicle or 0.3mg/kg of h-19F6, h-34F8 or h-42A5 via the limb vein. The left femoral artery was exposed and isolated by blunt dissection. A doppler flow probe is mounted on the artery and the flow is continuously recorded. In the use of FeCl3Previously, blood flow was measured for at least 5 minutes. Then, two sheets were pre-impregnated with 40% FeCl3The filter paper of (a) was applied on the adventitial surface of the blood vessel upstream of the probe for 10 minutes. After removing the filter paper, the application site was washed with brine. Blood flow is measured continuously until blood flow is reduced to 0. The time to 80% occlusion (20% reduction in blood flow to baseline blood flow) and 100% occlusion (0 reduction in blood flow) were recorded. In the same animals, hemostatic effect was assessed using a Surgicutt device and bleeding time was manually recorded before and 1 hour after dosing. At the end of the study (approximately 3 hours post-dose), blood samples were collected.
The binding specificity of the antibody to human FXI in plasma was tested. The test antibodies (h-19F6, h-42A5 and 14E11) were first biotinylated using the EZ-LinkTM Sulfo-NHS-LC-biotinylation kit (Cat No.21435, Thermo Fisher Inc.). These antibodies (25 μ g each) were incubated with 200 μ L of human standard plasma (Siemens Inc.) or FXI-poor human plasma (Hyphen Biomed Inc.) for 1 h. Then 50. mu.L of streptavidin-coated beads (Dynabeads M-280 streptavidin, Thermo Fisher Inc.) were added to the mixture to enrich the biotin-containing antigen-antibody complexes. After 3 washes with PBS, the antigen-antibody complex was eluted and western blotted using mouse anti-human FXI IgG (1C5, home-made antibody binding to FXI C-terminus) as primary antibody. Image results were acquired using a ChemiDocMP imaging system with Image Lab software (Bio-Rad). In western blot, 10 μ L of human standard plasma and FXI-poor human plasma were used as FXI positive and FXI negative controls, respectively.
And (5) carrying out statistical analysis. Numerical data from multiple experiments are presented as mean ± Standard Error of Mean (SEM). Thrombus weight was statistically analyzed in both the AV shunt test and the two bleeding time test using one-way analysis of variance (ANOVA) followed by multiple comparative tests with Dunnett (Dunnett). Kruskal-Wallis grade test was performed to statistically analyze FeCl3Time to occlusion in the resulting thrombosis experiment. P <0.05 was considered statistically significant.
Example 2: generation and sequencing of anti-FXI antibodies.
Respectively immunizing BALB/c mice and Wister rats by using human coagulation Factors XI (FXI), and taking spleen cells of animals with better immune response for preparing hybridoma cells; after the hybridoma cells are subjected to limited dilution, further subcloning is carried out, and 12 monoclonal hybridoma cell lines are successfully obtained through an ELISA capture method and functional screening, wherein the expressed anti-FXI antibodies of the monoclonal hybridoma cell lines are respectively as follows: 3G12, 5B2, 7C9, 7F1, 13F4, 19F6, 21F12, 34F8, 38E4, 42a5, 42F4, and 45H 1.
To determine the amino acid and nucleotide sequences of the light and heavy chain variable regions (VL, VH) of the above antibodies, cDNAs encoding VL and VH were cloned from the corresponding hybridoma cells according to standard PT-PCR protocols. The VL and VH sequences (including CDR sequences) of the corresponding antibodies are detailed in table 1.
Example 3: the anticoagulant activity of the antibody of the present invention in human plasma was evaluated using the Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT) assay indices.
The APTT assay measures the activity of intrinsic and common pathway coagulation; whereas the PT assay measures the activity of extrinsic and common pathway coagulation. The antibodies tested in this experiment were 19F6,34F8, 42a5, 1a6 and 14E 11. In this experiment, antibodies 1a6 and 14E11 were used as positive controls. The sequences of the control antibody variable regions were derived from US patent US 8,388,959 and US patent application 2013/0171144 and reformatted as IgG 4. These antibodies were then expressed using the expihho cell system. APTT and PT assays were performed as described above.
As shown in FIG. 1, all antibodies tested increased the Activated Partial Thromboplastin Time (APTT) and exhibited some concentration dependence in a relatively low concentration range, e.g., as the concentration increased to 100nM (14E11 increased to 200 nM); but all antibodies had no significant effect on Prothrombin Time (PT) (data not provided). These results indicate that all antibodies tested inhibit the intrinsic pathway of coagulation, but not the extrinsic pathway.
Example 4: the anticoagulant activity of the antibody in non-human plasma is evaluated by using an APTT detection index.
Anticoagulant activity of different antibodies (including 19F6,34F8 and 42A5) in plasma of mice, rats and monkeys was tested as in example 3. The results showed that in mouse and rat plasma all antibodies tested had no effect on APTT, but in monkey plasma their APTT values increased and were concentration dependent over a relatively low concentration range (see figure 2 for details). The test shows that the antibody only has cross reaction on monkey FXI/FXIa and has no cross reaction on mouse FXI/FXIa and rat FXI/FXIa.
Example 5: humanization of anti-FXI antibodies
Murine monoclonal antibodies cannot be used directly for therapy due to their short half-life and induction of human anti-mouse antibody responses. One solution to this problem is to humanize murine antibodies. Some antibodies are humanized by CDR grafting. For each murine antibody V was determinedLAnd VHSuitable human acceptor frameworks, and various numbers of back mutations introduced into selected human frameworks to maintain the structure and/or function of the resulting antibody. The humanization of these modified antibodies is considered successful if the affinity and function of these humanized antibodies is substantially comparable to that of the corresponding unmodified antibody. Three humanized V of 19F6,34F8, 42a5LAnd VHThe sequences are shown as h-19F6, h-34F8, h-42A5 and are shown in Table 2.
Example 6: affinity of anti-FXI antibodies to human FXI
The affinity of the anti-FXI antibody to FXI/FXIa was determined by Surface Plasmon Resonance (SPR) technique using BIAcore T200 system. The method comprises the following steps: the humanized antibody is constructed by connecting the variable region of the antibody to be detected with the Fc fragment of human IgG4 and is recombined and expressed in CHO cells; these antibodies were bound to a Biacore CM5 sensor chip coupled to an anti-human IgG capture antibody.
Subsequently purified antigens FXI or FXIa at different concentrations (0.005-1. mu.g/ml) were passed through a CM5 sensor chip with an anti-FXI/FXIa antibody binding time of 180s and dissociation time of 1800 s. The binding data collected was analyzed using Biacore Evaluation Software (supplied from GE Healthcare) to determine the affinity of FXI/FXIa and the detection antibody. The SPR maps of the immobilized h-9F6, h-34F8 and h-42A5 antibodies bound to FXI/FXIa are shown in detail in FIG. 3. As shown in fig. 3, the Response (RU) of each antibody was also enhanced with the gradual increase of the FXI or FXIa concentration. Dissociation constants (KD) were calculated for FXI and FXIa for h-19F6, h-34F8 and h-42A5 and the results are shown in Table 4. Since the difference was less than 10-fold, the affinity of each antibody for FXI and FXIa was considered to be the same.
Table 4: KD values for antibodies against FXI and FXIa
Example 7: determination of the binding site of an anti-FXI antibody on FXI
The binding site of 19F6, 42A5 on FXI was determined using SPR technique. The method comprises the following steps: the human IgG capture antibody is coupled on the surface of a Biacore CM5 sensing chip, the recombinant sample h-19F6 or h-42A5 flows through the surface of the chip to be captured, and the capture quantity of h-19F6 or h-42A5 reaches 15 relative units by adjusting the flow time. Flowing a concentration of wild-type FXI or chimeric FXI (i.e., FXI/PK chimera in which the single apple (A) domain of FXI is replaced by the corresponding domain of human prekallikrein) over the chip surface for h19F6 or h42A5 with a binding time of 180s followed by a dissociation time of 1800 s; data were analyzed using a high performance kinetic model, and only one concentration of wild-type FXI or chimeric FXI was detected in the SPR assay. The results show that both h-19F6 and h-42A5 bind to FXI and FXI/PK chimeras, except for the FXI/PK chimera in which the A3 domain is replaced by the corresponding domain of prekallikrein, suggesting that partial or complete epitopes of h-19F6 and h-42A5 are on the A3 domain.
Example 8: antibody functional neutralization of FXIa
The activity of human FXIa was determined primarily by measuring hydrolysis of a specific and fluorescently labeled substrate (S-2366, Diapharma Inc.). To test the inhibitory activity of the antibodies, the antibodies h-19F6, h-34F8 and h-42A5 to be tested were preincubated with FXIa in PBS buffer at a final concentration of 5nM for 5min at room temperature, then FXIa cleavage was initiated by adding an equal volume of 1mM S-2366 and the change in absorbance at 405nM was read continuously with an M5e microplate reader (Molecular Devices Inc.), and the data were analyzed using GraphPad Prism software, as detailed in FIG. 4. The apparent Ki values of human antibodies h-19F6, h-34F8 and h-42A5 were 0.67, 2.08, 1.43 nM. Therefore, all three antibodies tested showed satisfactory inhibition of FXIa at relatively low concentrations.
Example 9: inhibition of FXIa-mediated FIX activation by antibodies
Experimental procedures for FXIa mediated FIX activation were performed as described above. anti-FXI antibodies can modulate endogenous pathways by inhibiting FXI activation and/or by inhibiting FXIa activity. First, the effect of two antibodies, h-19F6 and h-42A5, on FXIIa-mediated FXI activation was tested and it was found that neither h-19F6 nor h-42A5 prevented FXIIa-mediated conversion of FXI to FXIa (FIGS. 5C and 5D). The effect of these two antibodies on FXIa activity was then tested using FIX as substrate. As shown in FIGS. 5A and 5B, both h-19F6 and h-42A5 reduced FIX activation in a concentration-dependent manner. The inhibitory effect of these two antibodies on FXIa was further confirmed by the use of the chromogenic substrate S-2366 of FXIa. Both antibodies inhibited hydrolysis of S-2366 concentration-dependently (FIG. 4)
Example 10: evaluation of the Effect of anti-FXI antibodies on the clotting time of cynomolgus monkeys
To find animal species suitable for in vivo experiments, cross-reactivity of mouse, rat and monkey FXI antibodies was tested by APTT analysis. Antibodies prolonged APTT in monkey plasma but not in mouse or rat plasma (data not shown). Therefore, prior to in vivo studies of thrombogenic potency, a monkey model was chosen to evaluate the pharmacodynamic effects of the three antibodies on clotting time. The cynomolgus monkey is injected with different antibodies to be detected with specified dosage intravenously, upper limb superficial venous blood is collected before administration and 0.5, 1, 3, 6, 12 and 24 hours after administration, and plasma anticoagulated by citric acid is prepared for determining APTT and PT. In APTT experiments, 50 μ l of diluted plasma samples were taken, 25 μ l of APTT reagent (SMN 10445709, Symen Inc.) was added, mixed well, incubated at 37 ℃ for 4min, CaCl was added2Solutions (25mM, SMN 10446232, Symen Inc.) were mixed in 25. mu.L and the time to thrombosis was recorded. In the PT assay, 50. mu.l of diluted plasma samples were taken,an equal volume of PT reagent (SMN 10446442, Symen Inc) was added, mixed well and incubated at 37 ℃ for thrombogenesis time. The results show that 3 antibodies tested all extended the APTT dose-dependently (see figure 6 for details) without affecting PT (see figure 7 for details).
Both h-19F6 and h-42A5 dose-dependently prolonged APTT (FIGS. 6B and 6C). Notably, at the same dose levels (0.3 and 1mg/kg), h-42A5 was more pronounced than h-19F6 in prolonging APTT, which is consistent with the in vitro effect of the antibody on human APTT (FIG. 16A). In addition, neither antibody affected PT in vivo (fig. 7B and 7C).
Example 11: effect of anti-FXI antibody on cynomolgus monkey arteriovenous shunt thrombosis and tail vein bleeding model
Different doses of the test antibody are administered to the same animal to assess thrombosis and bleeding time. The antibodies detected in this experiment were h-34F8, h-19F6, and h42A 5. Bleeding time and thrombosis were evaluated sequentially before and 30 minutes after each administration of antibody. 4 assessments were made for bleeding/thrombosis for three ascending doses (0.1, 0.3 and 1mg/kg) before and after dosing.
AV shunt thrombus assay: a weighed 10cm long silk thread is put into the tube, the cannula is connected with the femoral artery and the femoral vein, after the blood flow is released for 10min, the silk thread is quickly taken out, the wet weight is weighed, and the thrombus weight is calculated. Thrombus weight is determined by the difference in wire weighing before and after blood flow.
And (3) bleeding time determination: a2 ml syringe was inserted into the tail vein of the animal and timing was started. The timing was stopped when the blood in the syringe no longer increased. This time period is the bleeding time.
The results show that all antibodies of the present invention can inhibit thrombus formation dose-dependently (see fig. 8)) without prolonging the time to tail bleeding (see fig. 9). The effect of h-19F6 and h-42A5 on thrombosis and hemostasis was evaluated in monkey models of AV shunt thrombosis and tail vein hemorrhage. Intravenous injection of h-19F6 resulted in a dose-dependent decrease in clot weight, and a significant decrease was observed at the 1mg/kg dose (fig. 8B). For the h-42a5 treated animals, thrombus weight was significantly reduced in a dose-dependent manner at all dose levels tested (fig. 8C). There was no significant change in bleeding time after treatment with h-19F6 or h-42A5 (FIGS. 9B and 9C).
Example 12: anti-FXI antibody on FeCl3Effect of induced cynomolgus femoral artery thrombosis and Effect on Standard bleeding time
The cynomolgus monkey is anesthetized with 1.5mg/kg Zoletil, trachea intubated, a respirator is started, the anesthetic effect is maintained by isoflurane, and the blood pressure, the heart rate and the body temperature are monitored in the whole process. In the application of FeCl3The first 2 hours of intravenous administration of test antibodies h-34F8, h-19F6, h-42A5 or blank vehicle (control). The left femoral artery was exposed and isolated using a blunt dissection method. Arterial blood flow was continuously detected and recorded with a Doppler flow probe. In the application of FeCl3Previously, blood flow has been detected for at least 5 minutes. Then taking 2 FeCl pieces3Wrapping the surface of the artery above the Doppler flow probe with soaked filter paper, removing the filter paper after 10min, washing the wound with normal saline, and removing the residual FeCl3The solution is washed clean. Blood flow was monitored throughout until the value dropped to 0, and the time to occlusion 80% (20% of blood flow to baseline) and 100% (0 blood flow) were recorded. For the same animal, bleeding time was measured before and 1 hour after dosing using a standard bleeding time assay.
With FeCl3Evaluation of induced arterial thrombosis with antibodies h-34F8, h-19F6 and h-42A5, namely, 4 groups of monkeys were administered with a blank vehicle, h-34F8, h-19F6 and h-42A5, respectively, and after 2 hours, FeCl was applied to the left femoral artery3Causing a thrombus and monitoring the blood flow velocity thereunder. The results show that: the blank control group has 80% blockage time and 100% blockage time of 14.66 +/-1.30 min and 18.5 +/-1.76 min respectively; 0.3mg/kg of h-34F8 and h-42A5, 80% of the occlusion time of the prevention and treatment group is 59.53 +/-16.95 min and 40.80 +/-7.94 min respectively, and 100% of the occlusion time is 70.40 +/-20.76 min and 50.61 +/-9.48 min respectively, which have significant difference compared with a blank control group (see figure 10); although the h-19F6 group showed no significant difference compared with the blank group, it also properly prolonged 80% occlusion (26.43 + -5.72 min) and 100% occlusion time (32.78 + -5.09 min) (see FIG. 10).
(Figure11D) the effect of the h-34F8, h-19F6 and h-42a5 antibodies on hemostasis was evaluated by the standard bleeding time method, and the results showed no significant difference in bleeding status between the groups before and 1 hour after administration (see figures 11A, 11B and 11C), and no significant difference in bleeding time between the h-34F8, h-19F6 and h-42a5 treated groups compared with the blank control group (see Figure11D).
Effect of the h-19F6 and h-42A5 antibodies on hemostasis was evaluated by a skin laceration induced hemorrhage test in which the same FeCl was used3Induced arterial thrombosis (n-5 per group). Bleeding time was recorded before and 1 hour after dosing. No significant difference in bleeding time was observed between pre-and post-antibody administration 1 hour or between the three groups at 1 hour post-administration (fig. 18).
The effect of the antibody on in vitro clotting time of monkey plasma was also determined and the results showed that administration of 0.3mg/ml of the h-34F8, h-19F6, and h-42A5 antibodies significantly prolonged the Activated Partial Thromboplastin Time (APTT) to 3.29. + -. 0.20, 1.67. + -. 0.09, and 2.87. + -. 0.10 fold (FIG. 12A) before administration, but did not affect the Prothrombin Time (PT) (FIG. 12B), compared to the blank control (no APTT prolongation).
Thus, the antibodies disclosed herein are effective in inhibiting the intrinsic pathway of coagulation without unexpectedly increasing bleeding time.
Example 13: evaluation of the Effect of modified anti-FXI antibodies on clotting time of cynomolgus monkeys for extended periods of time
The effect of two additional CMC optimized humanized anti-FXI antibodies ("modified h-19F 6" and "modified h-42a 5", respectively, as shown in figures 14 and 15) whose heavy and light chain sequences are listed in table 3 on clotting time in cynomolgus monkeys over extended periods of time (up to 14 days) was evaluated by APTT and PT assays as described in example 10. Cynomolgus monkeys were injected intravenously with 0.6mg/kg or 2.0mg/kg of the test antibody. Blood was collected from the upper limb superficial vein before administration and at 0.5 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, 96 hours, 168 hours, 240 hours, and 336 hours after administration. As shown in fig. 14, both of the modified antibodies tested showed dose-dependent prolongation of APTT, as shown in fig. 15, neither of which affected PT. Both antibodies show prolonged efficacy over a long period of time, which may be as long as 7 days, as long as 10 days, or as long as 14 days.
Thus, it was unexpectedly found that the modified antibodies disclosed herein effectively inhibit the endogenous coagulation pathway over an extended period of time (up to 14 days) without showing any adverse effects of prolonged bleeding.
Example 14: effect on clotting time of Standard human plasma
Antibodies h-19F6 and h-42a5 were added to normal human plasma, and APTT (fig. 16A) and PT (fig. 16B) were determined (N ═ 3). Both the h-19F6 and h-42A5 antibodies extended the Activated Partial Thromboplastin Time (APTT) of standard human plasma in a concentration-dependent manner (FIG. 16A). Based on established correlation curves between plasma FXI levels and APTT (data not shown), the maximal inhibition levels of H-19F6 and H-42A5 for FXI activity in plasma were about 97% and 99.5%, respectively. Neither antibody affected PT in human plasma (fig. 16B).
Example 15: binding characteristics of h-19F6 and h-42A5 to FXI
First, when h-19F6 and h-42A5 reacted with FXI in standard human plasma, their binding specificity was verified, and no reaction was detected in human FXI-poor plasma (FIG. 17). Biotinylated test antibodies were incubated with human normal plasma or FXI-depleted human plasma. The FXI antibody complex in plasma was eluted and western blotting was performed using mouse anti-human FXI IgG as the primary antibody. In western blot, 10 μ L of human standard plasma or spent FXI plasma was used as FXI positive and FXI negative controls. Previously reported anti-FXI antibody 14E1117Shows the same binding profile as both antibodies (fig. 17).
The affinity of h-19F6 and h-42A5 for FXI was determined using Surface Plasmon Resonance (SPR) techniques. The antibody under test was captured on the sensor chip and then FXI at the indicated concentration was flowed through the chip. Sensorgrams for h-19F6 (FIG. 19A) and h-42A5 (FIG. 19B) were obtained. Dissociation constants for h-19F6 and h-42A5 were 22 and 36pM, respectively (FIGS. 19A and 19B).
The binding sites of these two antibodies on FXI were then determined. FXI is a homodimer consisting of 4 tandem Apple domains (a 1-4) and a catalytic domain. By replacing each Apple domain with the corresponding domain of human prekallikreinFour mutants of FXI were generated and tested for binding properties of h-19F6 or h-42A5 to the four mutants of FXI using SPR. An equal number of 4 mutant FXIs (in which the a1, a2, A3 or a4 domain was replaced with the corresponding domain of prekallikrein) were immobilized on the sensor chip and the antibody to be tested (5 μ g/mL) was run through the chip for binding. The amount of each antibody captured was recorded. Experiments were performed twice and representative results are described. Unexpectedly, both antibodies bound primarily to the A3 domain of FXI, as substitution of the A3 domain resulted in a greater reduction in binding of either antibody compared to substitution of the other 3 Apple domains (fig. 19C). In line with previous studies, another antibody, O1a6, is a known anti-FXI antibody, used as a positive control, that also binds specifically to the A3 domain of FXI.21However, it is speculated that h-19F6 and h-42A5 bind to different sites of FXI, since they have comparable affinity to FXI, but differ greatly in their inhibitory activity against FXI activity (FIG. 16). This hypothesis was tested by epitope binding using the Biacore T200 system. Indeed, binding of h-19F6 to FXI did not prevent further binding of h-42A5 to FXI, indicating that these two antibodies bound to different sites in the A3 domain of FXI (FIG. 19D). Then, the order of flow of the two antibodies was changed and it was found that h-42A5 binding to FXI did not prevent further binding of h-19F6 to FXI (data not shown).
Example 16: binding characteristics of h-19F6 and h-42A5 to FXIa
The antibody bound FXIa with good affinity to FXI (fig. 20A and 20B). The affinity of h-19F6 and h-42A5 for FXIa was determined using Surface Plasmon Resonance (SPR) techniques. Dissociation constants for h-19F6 and h-42A5 were 26 and 81pM, respectively (FIGS. 20A and 20B). The antibody under test was captured on the sensor chip and a specified concentration of FXIa was then flowed through the chip. Sensorgrams for h-19F6 (FIG. 20A) and h-42A5 (FIG. 20B) were obtained.
Reference to the literature
The references listed below, patents and published patent applications, and all references cited in the above specification are hereby incorporated by reference in their entirety as if fully set forth herein.
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Claims (16)
1. An isolated anti-FXI or anti-FXIa antibody that specifically binds human FXI or FXIa, wherein the antibody comprises an amino acid sequence selected from SEQ ID NOs: 155-160, sequences having at least 90% identity, or immunologically active portions thereof.
2. The antibody of claim 1, wherein said antibody specifically binds the a3 domain of human FXI or FXIa.
3. The antibody of claim 1, wherein the antibody comprises a pair of sequences selected from the following pairs of sequences: SEQ ID NO: 153-154, SEQ ID NO: 201-202, and SEQ ID NO: 204, 205, and sequences having at least 90% identity.
4. A pharmaceutical composition comprising the antibody of any one of claims 1-3.
5. Use of the antibody of any one of claims 1-3 in the manufacture of a medicament for inhibiting blood clot formation in a subject.
6. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for inhibiting blood clot formation in a subject.
7. Use of an antibody of any one of claims 1-3 in the manufacture of a medicament for treating or preventing thrombosis or a complication or disorder associated with thrombosis, wherein the amount of the antibody does not impair hemostatic function of the subject.
8. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating or preventing thrombosis or a complication or disorder associated with thrombosis, wherein the amount of the composition does not impair hemostatic function of the subject.
9. Use of an antibody of any one of claims 1-3 in the manufacture of a medicament for treating or preventing sepsis, wherein the amount of the antibody does not impair hemostatic function of the subject.
10. Use of the pharmaceutical composition of claim 4 in the manufacture of a medicament for treating or preventing sepsis, wherein the amount of the composition does not impair hemostatic function of the subject.
11. A method of making the antibody of any one of claims 1-3, comprising expressing in a host cell a nucleic acid encoding the antibody cloned in an expression vector.
12. The method of claim 11, further comprising purifying the expressed antibody from the host cell.
13. The method of claim 11, wherein the expression vector is a pTT5 vector or a pcDNA3 vector.
14. The method of claim 11, wherein the host cell is a CHO cell or a HEK193T cell.
15. An antibody or functional fragment thereof, or an immunologically active portion thereof, prepared by the method of any one of claims 11-14.
16. The antibody of claim 15, wherein the antibody has been post-translationally modified.
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CN202111466510.4A Active CN114478781B (en) | 2018-08-09 | 2018-08-09 | Anticoagulant factor XI antibodies |
CN202410287101.5A Pending CN118496369A (en) | 2018-08-09 | 2018-08-09 | Anticoagulant factor XI antibodies |
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EP (1) | EP3833692A1 (en) |
JP (1) | JP2021534098A (en) |
KR (1) | KR20210042352A (en) |
CN (5) | CN113227150B (en) |
AU (1) | AU2018436195A1 (en) |
BR (1) | BR112021002472A2 (en) |
CA (1) | CA3108708A1 (en) |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
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CN114478781A (en) * | 2018-08-09 | 2022-05-13 | 上海仁会生物制药股份有限公司 | Anti-coagulation factor XI antibodies |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US11958911B2 (en) | 2017-02-10 | 2024-04-16 | Shanghai Benemae Pharmaceutical | Anti-coagulation factor XI antibody |
WO2020154234A1 (en) * | 2019-01-21 | 2020-07-30 | Aronora Inc. | Novel humanized antibodies against factor xi having anti-thrombotic and anti-inflammatory effects and uses thereof |
CN115917812A (en) | 2021-03-31 | 2023-04-04 | 株式会社Lg新能源 | Single-unit manufacturing apparatus having glossmeter and manufacturing method using the same |
CN116410258B (en) * | 2023-04-13 | 2024-07-19 | 上海太阳生物技术有限公司 | Factor XI deficiency plasma protective agent and application thereof |
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- 2018-08-09 CN CN201880098606.XA patent/CN113227150B/en active Active
- 2018-08-09 JP JP2021506623A patent/JP2021534098A/en active Pending
- 2018-08-09 WO PCT/CN2018/099638 patent/WO2020029179A1/en unknown
- 2018-08-09 CA CA3108708A patent/CA3108708A1/en not_active Abandoned
- 2018-08-09 AU AU2018436195A patent/AU2018436195A1/en not_active Abandoned
- 2018-08-09 BR BR112021002472-7A patent/BR112021002472A2/en not_active Application Discontinuation
- 2018-08-09 CN CN202310301532.8A patent/CN116554334A/en not_active Withdrawn
- 2018-08-09 CN CN202111467365.1A patent/CN114478782B/en active Active
- 2018-08-09 EP EP18929685.8A patent/EP3833692A1/en not_active Withdrawn
- 2018-08-09 KR KR1020217006922A patent/KR20210042352A/en unknown
- 2018-08-09 CN CN202111466510.4A patent/CN114478781B/en active Active
- 2018-08-09 CN CN202410287101.5A patent/CN118496369A/en active Pending
- 2018-08-09 MX MX2021001613A patent/MX2021001613A/en unknown
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CN114478781A (en) * | 2018-08-09 | 2022-05-13 | 上海仁会生物制药股份有限公司 | Anti-coagulation factor XI antibodies |
CN114478781B (en) * | 2018-08-09 | 2024-04-02 | 上海仁会生物制药股份有限公司 | Anticoagulant factor XI antibodies |
Also Published As
Publication number | Publication date |
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EP3833692A1 (en) | 2021-06-16 |
CN113227150A (en) | 2021-08-06 |
CN114478782B (en) | 2024-04-02 |
JP2021534098A (en) | 2021-12-09 |
CN113227150B (en) | 2023-07-28 |
WO2020029179A1 (en) | 2020-02-13 |
MX2021001613A (en) | 2021-04-28 |
CN118496369A (en) | 2024-08-16 |
BR112021002472A2 (en) | 2021-07-27 |
KR20210042352A (en) | 2021-04-19 |
AU2018436195A1 (en) | 2021-02-18 |
CN114478781B (en) | 2024-04-02 |
CN116554334A (en) | 2023-08-08 |
CN114478781A (en) | 2022-05-13 |
CA3108708A1 (en) | 2020-02-13 |
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