Patent Application
20240294668
The disclosure belongs to the fields of immunology and pharmacy. In one embodiment, the disclosure relates to a novel antithrombotic antibody, targeting the coagulation factor FIXa-FVIIIa binding site.
Thromboembolic disease is a common clinical disease characterized by arteriovenous and microvascular thrombosis or embolism. Due to the obstruction of blood flow caused by thrombus formation or the interruption of downstream blood flow caused by emboli detachment, ischemia and necrosis of tissues and organs may occur. Globally, approximately 17.9 million people die from cardiovascular diseases, accounting for 31% of the global mortality rate. Thrombosis is a significant factor in triggering various severe cardiovascular and cerebrovascular diseases. Thromboembolic disease has been the primary cause of threats to human health and life in today's society. Main treatment approach for thromboembolic disease is antithrombotic therapy, including anticoagulation, antiplatelet and thrombolysis. Among them, anticoagulant therapy mainly targets different coagulation factors in coagulation cascade signaling pathway, blocking coagulation process by suppressing these factors.
Coagulation factors are various protein components involved in the process of blood clotting. Their physiological function is to be activated during vascular bleeding, adhere to platelets, and seal the leak in the blood vessel. This process is referred to as coagulation. The entire coagulation process can be roughly divided into two stages: the activation of prothrombin and the formation of gelatinous fibrin. For unified naming, the World Health Organization assigns Roman numerals to coagulation factors based on the order of their discovery, such as coagulation factors (F) I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII, XIII, and so on. Some coagulation factors have an “a” added to their number to denote their activated form. For example, the activated form of coagulation factor IX (FIX) is FIXa. and the activated form of coagulation factor VIII (FVIII) is FVIIIa.
Currently, anticoagulants used in clinical include heparin and its derivatives, vitamin K antagonists (such as warfarin), and small molecule inhibitors like rivaroxaban and dabigatran. All these drugs can act on the common coagulation pathway (e.g., factors Ha and Xa) in coagulation cascade. Due to their impacts on the physiological hemostatic function, there is a serious risk of bleeding, especially cerebral hemorrhage. Since the endogenous coagulation pathway is closely related to pathological thrombosis rather than hemostatic function, selective inhibitors of endogenous coagulation factors have become a hot spot in the research of novel anticoagulants.
FIXa is a critical coagulation factor in the endogenous coagulation pathway and is the only soluble form of coagulation protein. FIXa efficiently diffuses from tissue factor-bearing cells to platelets, serving as a crucial link between the initiation and amplification phases of the coagulation cascade. FIX can also be directly activated by FXIa on aggregated platelets. FIXa activates FX by forming a FIXa-FVIIIa complex. However, the binding sites of FVIIIa and FIXa and their binding effects are still unclear in this field.
Embodiments of the present disclosure is to provide a novel antithrombotic antibody targeting the binding site of coagulation factor FIXa-FVIIIa and the use thereof.
In one embodiment of the present disclosure provides an antithrombotic monoclonal antibody or antigen-binding fragment thereof, and the antithrombotic monoclonal antibody or antigen-binding fragment thereof has a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 5; and a light chain CDR1 with the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 7, a light chain CDR3 with the amino acid sequence of SEQ ID NO: 8.
In one embodiment, the monoclonal antibody includes (a) a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region with the amino acid sequence of SEQ ID NO: 2; or (b) a heavy chain variable region with more than 80% (such as more than 85%, 90%, 93%, 95%, 97% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region with more than 80% (such as more than 85%, 90%, 93%, 95%, 97% or 99%) sequence identity to the amino acid sequence of SEQ ID NO: 2, also with the function of (a) antibody.
In another embodiment, the monoclonal antibody includes murine antibody, chimeric antibody or humanized antibody; or, the monoclonal antibody or the antigen-binding fragment thereof includes single-chain antibody (scFV), domain antibody. Fab fragment, Fab′ fragment. Fd fragment. F(ab′)2 fragment.
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof targets the binding site of coagulation factor FIXa and FVIIIa, reduces the formation of a FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and to exert an antithrombotic effect.
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof significantly prolonged the activated partial thromboplastin time (APTT) 2 to 4 times (such as 2.5, 3, 3.5 times; preferably using a normal time of activated partial thromboplastin time in natural body as the control, the normal time is for example 20-40 seconds, more preferably 25-36 seconds); or, the monoclonal antibody or antigen-binding fragment thereof prolonged the activated partial thromboplastin time (APTT) to more than 75 seconds, preferably more than 80 seconds (such as 80-120 seconds, more such as 82, 85, 88, 90, 95, 100, 110 seconds).
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof targets FIXa at the binding sites or adjacent sites of FIXa and FVIIIa, reduces the formation of a FVIIIa-FIXa complex; preferably, in FIXa, the binding sites or adjacent sites of FIXa and FVIIIa include: Asn93, Lys132, Arg165, Thr175; more preferably it also include: Ala95, Lys98, Asp164, Lys173, Tyr177; more preferably it also include: Lys126, Asn129, Asn178, Lys230, Arg233, Asn236.
In another embodiment, the amino acid residues at each site are numbered according to chymotrypsin.
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof does not affect the catalytic activity of FIXa.
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof does not bind to the catalytic site of FIXa, for example, the His57-Asp102-Ser195 site.
In another embodiment, the antithrombotic monoclonal antibody or antigen-binding fragment thereof does not affect prothrombin time (PT).
In another embodiment of the present disclosure provides an isolated polynucleotide or a construct including the polynucleotide, the polynucleotide encodes any of the aforementioned antithrombotic monoclonal antibody or antigen-binding fragment thereof; preferably, the construct is an expression vector.
In another embodiment of the present disclosure provides an antibody expression system, the expression system includes the construct, or includes the exogenous polynucleotide integrated in the genome; preferably, the expression system is a cell expression system.
In another embodiment of the present disclosure provides a method for preparing the antithrombotic monoclonal antibody or antigen-binding fragment thereof, including: expressing the antibody by the antibody expression system under conditions suitable for expression; preferably it also includes purifying and isolating the antibody.
In another embodiment of the present disclosure provides a fusion protein, including the antithrombotic monoclonal antibody or the antigen-binding fragment thereof, and a fusion partner operably linked thereto; preferably, the fusion partner including (but are not limited to): a protein or an active structural domain having an effect of extending half-life in vivo, or a protein or an active structural domain having a synergistic function or binding effect on effectors (carry out another or more functions); more preferably, the protein or active structural domain having an effect of extending half-life in vivo includes (but are not limited to): an immunoglobulin Fc region, preferably a human immunoglobulin Fc region, serum albumin (for example humanized HSA) or a fragment thereof.
In one embodiment, the immunoglobulin is selected from one or a combination of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is selected from one or a combination of IgG1, IgG2, IgG3, or IgG4.
In another embodiment, there is a linker peptide between the antithrombotic monoclonal antibody or the antigen-binding fragment thereof and the fusion partner operably linked thereto; and the linker peptide is preferably selected from a flexible polypeptide chain composed of the group consisting of alanine and/or serine and/or glycine, and the length of the linker peptide is preferably 3-30 amino acids.
In another embodiment of the present disclosure provides an immunoconjugate, and it includes any of the aforementioned antithrombotic monoclonal antibody or the antigen-binding fragment thereof, or the aforementioned fusion protein; and the functional molecules linked (including but are not limited to covalently linked, coupled, attached, adsorbed) thereto; preferably, the functional molecules include (but are not limited to): hydrophilic polymers (such as PEG, PEGylated liposomes, etc.) or detectable markers (for example, including but not limited to: fluorescent markers, chromogenic markers).
In another embodiment of the present disclosure provides a pharmaceutical composition, and the pharmaceutical composition includes the antithrombotic monoclonal antibody or the antigen-binding fragment thereof, the fusion protein, or the immunoconjugate; preferably, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.
In another embodiment of the present disclosure provides a use of the antithrombotic monoclonal antibody or antigen-binding fragment thereof, the fusion protein, or the immunoconjugate, or a pharmaceutical composition including them, in the manufacture of preparations, kits or drug kits for alleviating or treating thromboembolic diseases;
In one embodiment, the thromboembolic diseases include (but are not limited to): venous, arterial or capillary thrombosis, thrombosis in the heart, thrombosis during and/or after contacting blood with an artificial surface, interstitial lung disease (e.g. fibroproliferative and/or idiopathic pulmonary fibrosis), inflammation, neuro-inflammatory diseases, complement activation, fibrinolysis, angiogenesis, coagulation induced by FVIIIa-FIXa complex formation, coagulation induced by FX activation, coagulation induced by FIIa expansion, retinal vascular permeability-related diseases (e.g. embolism); preferably, diseases related to arterial or capillary thrombosis include (but are not limited to): myocardial infarction, stroke, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis, Budd-Chiari syndrome or Paget-Schroetter disease.
In another embodiment of the present disclosure provides a kit or drug kit, including the antithrombotic monoclonal antibody or the antigen-binding fragment thereof, the fusion protein, or the immunoconjugate or pharmaceutical compositions including them.
In another embodiment of the present disclosure provides a method for screening antithrombotic substance (potential substance), including:
In one embodiment, when observing the effect of the candidate substance in binding between FIXa and FVIIIa at the binding sites or adjacent sites of FIXa to determine the functions of the candidate substance, if the candidate substance exerts a strong binding effect (with significance), then it is a antithrombotic substance (including potential substance).
In another embodiment, the “reduction” (also referred to as inhibition, attenuation, etc.) is a statistically significant or significant reduction, such as a reduction of the FVIIIa-FIXa complex by 5%, 10%, 15%, 20%, 30%, 50%, 60%, 80%, 90%, 95% or more.
In another embodiment, a control group is also included, to clearly distinguish the difference between the interaction of FIXa and FVIIIa in testing group and that in the control group.
In another embodiment, the candidate substances include (but are not limited to): regulators designed for FIXa, or upstream or downstream proteins or genes thereof, such as antibodies, interfering molecules (such as interfering RNA), small molecular compounds, gene-modifying constructs or gene-editing constructs, and so on.
After in-depth research, the present inventors have revealed a high-affinity anti-FIXa antibody (FIXa-4) with unique properties targeting FIXa. Although the antibody does not bind to catalytic sites of the substrate of FIXa, it targets the binding site of coagulation factor FIXa and FVIIIa, reduces the formation of a FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and to exert an antithrombotic effect. The antibody of the present disclosure has suitable antithrombotic properties, and has a large effective therapeutic concentration window, but does not increase the risk of bleeding. In addition, the antibody of the present disclosure does not directly bind to substrate catalytic sites of FIXa. Once excessive anticoagulation occurs, exogenous FVIII can be supplied for rescue, to meet the needs for suitable antithrombotic performance in clinical application and for effective avoidance of problems regarding bleeding caused by excessive effects.
As used herein, coagulation factor IX (sequence: GenBnak accession number: 2158) is also known as coagulation factor IX, coagulation factor 9, factor IX, FIX, F9, etc.; FIXa is the activated form of FIX. For example, with the participation of Ca2+, FXIa cleaves FIX and turns it into activated FIX (FIXa). In some embodiments, variants are also included, for example, the variant protein has 1 or more (1-20; more such as 2, 3, 4, 5 or 10) amino acid substitutions, deletions or insertions, and also remains the activity of FIX or FIXa.
As used herein, “antibody” or “immunoglobulin” are used as generic terms herein, including full length antibody, single chain antibody and all parts, domains or fragments thereof (including but are not limited to antigen binding domains or fragments). In addition, the term “sequence” as used herein (e.g. in terms of “immunoglobulin sequence”, “antibody sequence”, “single variable domain sequence”, “VHH sequence” or “protein sequence”, etc.) is to be understood generally including relevant amino acid sequence and the nucleic acid sequence or nucleotide sequence encoding the amino acid sequence, unless a more restrictive interpretation is required herein.
As used herein, “monoclonal antibody” refers to an antibody molecule preparation consisting of a single molecule. Monoclonal antibody exhibits a single-binding specificity and affinity for a specific epitope.
As used herein, “fusion partner”, namely FP, Fusion Partner, refers to another polypeptide that is fused with the target polypeptide. The fusion partner can affect the functional properties of the fusion protein through a variety of different mechanisms, for example, prolonging in vivo half-life of the target polypeptide. For example, the fusion partners include, but are not limited to, a protein or an active structural domain having an effect of extending half-life in vivo, or a protein or an active structural domain having a synergistic function or binding effect on effectors, exerting another or more functions.
As used herein, “conjugate” refers to a product formed by functional molecules (including polypeptides, small molecular compounds, hydrophilic polymers, labels) covalent or non-covalent conjugated to the monoclonal antibody described herein, and the hydrophilic polymer and the polypeptide can be conjugated at any position, such as the N-terminal, C-terminal or a suitable position in the middle of the polypeptide. The hydrophilic polymer is, for example, polysaccharide, polyalkylene glycol, such as polyethylene glycol (PEG), polypropylene glycol (PPG), polyethylene oxide (PEO), copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, and so on.
As used herein, “antithrombotic” can also be interpreted as “increasing vascular permeability” or “anticoagulation”.
“Sequence identity” between two polypeptide sequences indicates the percentage of identical amino acids between the sequences. “Sequence similarity” indicates the percentage of amino acids that are identical or have conservative amino acid substitutions. Methods for evaluating sequence identity between amino acids or nucleotides may be used. For example, amino acid sequence identity is typically measured using sequence analysis software. For example, the program named BLAST of NCBI database can be used to determine identity.
An “effective amount” of an agent refers to the amount necessary to cause a physiological change in a cell or tissue after administration.
A “therapeutically effective amount” of an agent, such as a pharmaceutical composition, refers to an amount that can effectively achieve desired therapeutic or prophylactic results within the required dosage and timeframe. A therapeutically effective amount of an agent, for example, eliminates, reduces, delays, minimizes or prevents the adverse effects of a disease.
An “individual” or “subject” is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In one embodiment, the individual or subject is a human.
The term “pharmaceutical composition” refers to a formulation in a form that allows the biological activity of the active ingredients contained therein is effective, without the inclusion of other components in the formulation that would impart unacceptable toxicity to the subjects receiving the composition.
“Pharmaceutically acceptable carrier” refers to ingredients other than the active ingredient in the pharmaceutical composition that are non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.
The term “treating/preventing” refers to altering natural course of a disease in a treated individual and may be a clinical intervention implemented for prophylaxis or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of diseases, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, improving or mitigating the state of diseases, and preventing or improving prognosis.
The term “humanized antibody” refers to a molecule with antigen-binding sites substantially derived from an immunoglobulin of non-human species, and remaining structure of the immunoglobulin in the molecule is based on the structure and/or sequence of human immunoglobulin. The antigen-binding sites may include an entire variable domain fused to a constant domain, or it may only include complementarity determining regions (CDR) grafted into appropriate framework regions in the variable domain. The antigen-binding site may be wild-type, or modified by one or more amino acid substitutions, e.g., to more closely resemble with human immunoglobulin. Some forms of humanized antibody retain the entire complementarity determining region (CDR) sequences. Other forms have one or more CDRs that are altered relative to the original antibody.
The term “detectable marker” refers to a marker that can be attached to an antibody and is used to determine the presence or absence and the amount of a specific target in the subject. The “detectable marker” can be, but are not limited to: enzyme, fluorescent label, nuclide, quantum dot, colloidal gold, etc. In one embodiment, for example, it may be selected from: horseradish peroxidase (HRP), alkaline phosphatase (AP), glucose oxidase, β-D-galactosidase, urease, catalase, or glucoamylase.
In the present disclosure, a monoclonal antibody was screened with the coagulation factor FIXa as the target, with its antithrombotic effect and mechanisms be studied. After extensive research and screening, an anti-FIXa antibody is provided. It targets the binding site of coagulation factor FIXa and FVIIIa, reduces the formation of FVIIIa-FIXa complex, blocks the conversion of FX to FXa. and exerts an antithrombotic effect. The disclosure also includes antigen-binding fragments of the anti-FIXa antibody.
By hybridoma technology, cell expression and purification for monoclonal antibody, the present disclosure prepared a monoclonal antibody of high purity. The activated partial thromboplastin time (APTT) and prothrombin time (PT) were used to evaluate the antithrombotic effects of the monoclonal antibody. And then chromogenic substrate method was used to detect the effect of monoclonal antibody on the enzyme activity of FIXa. The method of protein-protein docking was adopted to predict the possible binding sites between FIXa and the antibody, and the binding site was verified by competitive assays (indirectly through the chromatic substrate method). The results showed that an anti-FIXa monoclonal antibody named FIXa-4 with high affinity was obtained, and FIXa-4 significantly prolonged APTT in a concentration-gradient dependence. Mechanistic studies found that FIXa-4 did not directly bind to substrate catalytic sites of FIXa, but occupied the binding region of FIXa and FVIIIa. Therefore, the present disclosure provides a novel monoclonal antibody. FIXa-4, the antibody competes with FVIIIa to bind FIXa, reduces the formation of a FVIIIa-FIXa complex, blocks the conversion of FX to FXa, and to exert an antithrombotic effect.
In one embodiment of the present disclosure, the anti-FIXa monoclonal antibody or antigen-binding fragment thereof has a heavy chain CDR1 with the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR2 with the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR3 with the amino acid sequence of SEQ ID NO: 5; and a light chain CDR1 with the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 with the amino acid sequence of SEQ ID NO: 7, a light chain CDR3 with the amino acid sequence of SEQ ID NO: 8.
The anti-FIXa antibody provided by the present disclosure may include framework region FR, such as those listed in Table 1 below. However, the framework region is not limited to the sequences listed in Table 1. Modified antibodies, including partially or entirely altered sequences in the framework regions, are also encompassed within the scope of this disclosure. For example, chimeric antibody or humanized antibody formed by modifications are included.
The antigen-binding properties of an antibody are usually determined by the complementarity determining region CDR. The CDR region is arranged in an order with the FR region, and the FR region does not directly participate in binding. These CDR form a loop structure, and the β-fold created by adjacent FR that are close to each other in the spatial structure, constituting the antigen-binding site of the antibody. The CDR regions are sequences of proteins of interest in immunology, and the CDR regions of the antibody in the present disclosure are entirely novel.
The antibody of the present disclosure can be a complete immunoglobulin molecule or an antigen-binding fragment, including but are not limited to Fab fragment. Fd fragment. Fv fragment. F(ab′)2 fragment, complementarity determining region (CDR) fragment, single chain antibody (scFv), domain antibody, bivalent single-chain antibody, single-chain phage antibody, bispecific diabody, triple-chain antibody, and quadruple-chain antibody.
In one embodiment of the present disclosure, the anti-FIXa antibody has a heavy chain variable region with the amino acid sequence of SEQ ID NO: 1 and a light chain variable region with the amino acid sequence of SEQ ID NO: 2. The present disclosure also includes an antibody having a heavy chain variable region with more than 80% sequence identity to the amino acid sequence of SEQ ID NO: 1 and a light chain variable region with more than 80% sequence identity to the amino acid sequence of SEQ ID NO: 2, and also with the function of the antibody in the example of the present disclosure; preferably, in the heavy/light chain variable region, amino acids in the CDR region are conservative.
Functional variants of the antibody are included in the present disclosure. The variants can compete with the parental antibody for specific binding to FIXa, with an ability to recognize FIXa and a site for recognizing FIXa, and the site is close to those of the specific antibody provided in the examples of the present disclosure (targeting the binding site of blood coagulation factor FIXa-FVIIIa). The functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications may be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may include natural as well as unnatural nucleotides and amino acids. In one embodiment, the sequence modification occurs in regions other than the CDR regions of the antibody.
According to examples of the present disclosure, the antibody or antigen-binding fragment thereof of the present disclosure prolongs the activated partial thromboplastin time (APTT) about 3.5 times, with suitable antithrombotic properties. It has a large effective therapeutic concentration window, but not increase risk of bleeding.
The disclosure also provides a construct including the isolated polynucleotide of the disclosure. Methods for constructing the construct may be used. For example, the construct can be obtained by methods such as in vitro recombinant DNA technology. DNA synthesis technology, and in vivo recombination technology. In one embodiment, it can be constructed by inserting the isolated polynucleotide into the multiple cloning site of the expression vector. Expression vectors in the present disclosure generally refers to various commercially available expression vectors well known in the art, such as bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus or other vectors. The vector may also include one or more regulatory sequences operably linked to the polynucleotide sequence, and the regulatory sequences may include a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the amino acid to be expressed. The promoter can be any nucleotide sequence that shows transcriptional activity in the host cell of choice, including mutated, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides homologous or heterologous to the host cell. The regulatory sequence may also be a suitable transcription termination sequence, a sequence recognized by a host cell to terminate transcription. A terminator sequence is linked to the 3′ terminal of the nucleotide sequence encoding the polypeptide, and any terminator that is functional in the host cell of choice may be used in the present disclosure.
In general, suitable vectors may contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction sites and one or more selectable markers. For example, these promoters can include but are not limited to: the lac or trp promoter of E. coli; λ-phage PL promoter; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the LTR of retroviruses, and several other promoters known to control gene expression in prokaryotic or eukaryotic cells or their viruses. Marker genes may be used to provide phenotypic traits for selection of transformed host cells, for example, it may include but are not limited to dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc. When the polynucleotide is expressed, an enhancer sequence can also be included in the expression vector. If the enhancer sequence is inserted into the vector, the transcription will be enhanced. The enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, with an act on the promoter to enhance gene transcription.
The present disclosure also provides an antibody expression system, and the expression system contains the construct of the present disclosure or the exogenous polynucleotide of the present disclosure integrated in the genome. Any cell suitable for expressing an expression vector can be used as a host cell. For example, the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Specific examples include but are not limited to one or a combination of: bacterial cells of Escherichia coli, Streptomyces, Salmonella typhimurium; fungal cells such as yeast cells, filamentous fungi cells, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, HEK293 cells, or animal cells of Bowes melanoma cells, etc. Methods for constructing the expression system may be used, for example, it may include but not limited to one or a combination of: microinjection, particle bombardment, electroporation, virus-mediated transformation, electroporation, calcium phosphate precipitation, etc.
The present disclosure also includes a fusion protein including the first domain of the antibody according to the disclosure and the second domain for prolonging the half-life in vivo and/or binding to effector molecules or cells.
In the second domain, fragments for prolonging half-life in vivo may include serum albumin or its fragments, domains that bind serum albumin (such as anti-serum albumin antibodies), and so on.
In the second domain, the fragments capable of binding to effector molecules or cells may include immunoglobulin Fc regions, etc., preferably selected from human immunoglobulin Fc regions. The human immunoglobulin Fc region includes mutations for changing Fc-mediated effector functions, and the effector functions include one or more activities of CDC, ADCC and ADCP. The immunoglobulin may be selected from one or a combination of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG may be selected from one or a combination of IgG1, IgG2, IgG3, or IgG4. The immunoglobulin Fc region contained in the antibody fusion protein can enable the fusion protein to form a dimer, also prolong in vivo half-life of the fusion protein and increase Fc-mediated related activities. In a specific embodiment of the present disclosure, the immunoglobulin Fc region can be the Fc region of human IgG1, in one embodiment, it can be the wild-type IgG1 Fc sequence, and the sequence can be introduced with a mutation for changing the effector function mediated by Fc, for example, a) a mutation that alters Fc-mediated CDC activity; b), a mutation that alters Fc-mediated ADCC activity; or c), a mutation that alters Fc-mediated ADCP activity. Such mutations are described in: Leonard G Presta. Current Opinion in Immunology 2008, 20:460-470; Esohe E. Idusogie et al., J Immunol 2000, 164:4178-4184: RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4:443-446; Paul R. Hinton et al., J Immunol. 2006, 176:346-356.
In the fusion protein of the anti-FIXa antibody provided by the present disclosure, a linker peptide may be provided between the first domain and the second domain. The linker peptide is a flexible polypeptide chain composed of alanine (A) and/or serine (S) and/or glycine (G), and the length of the linker peptide may be 3-30 amino acids, preferably 3-9 amino acids, 9-12 amino acids, 12-16 amino acids, 16-20 amino acids. In another specific embodiment of the present disclosure, the length of the linker peptide may be 8 or 15 amino acids.
The present disclosure also provides an isolated polynucleotide encoding the antibody of the present disclosure, or encoding the fusion protein, and the polynucleotide, and the polynucleotide may be RNA, DNA or cDNA, etc. Methods for providing the isolated polynucleotide may be used, for example, it can be prepared by automatic DNA synthesis and/or recombinant DNA technology, or it can be isolated from a suitable natural source.
The present disclosure also provides an immunoconjugate including the antibody of the present disclosure or the fusion protein of the present disclosure. Generally, the immunoconjugate also includes a functional molecule linked (including but not limited to covalently linked, coupled, attached, adsorbed) to the antibody or fusion protein, and the functional molecule may be included but not limited to one or a combination of hydrophilic polymers, detectable markers, radioisotopes, bioactive proteins.
The hydrophilic polymers include but are not limited to: polyethylene glycol. PEGylated liposomes, polysaccharide, polyalkylene glycol, polypropylene glycol (PPG), polyethylene oxide (PEO), copolymers of ethylene glycol and propylene glycol, polyvinyl alcohol, etc., or any combination thereof.
Methods for preparing the immunoconjugate may be used, for example, the antibody and/or fusion protein can be linked directly or through a spacer with suitable length to the functional molecule by chemical cross-linking or genetic engineering fusion expression, to obtain the immunoconjugate.
The immunoconjugate may include an antibody or fusion protein of the disclosure and a detectable marker. The detectable markers include but are not limited to: fluorescent markers, chromogenic markers, protein labels; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals and non-radioactive paramagnetic metal ions. More than one marker may also be included. Markers used to label the antibody for detective and/or analytical and/or diagnostic purposes depends on the particular detective/analytical/diagnostic techniques and/or methods, for example, immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable markers for detective/analytical/diagnostic techniques and/or methods may be used.
Antibodies or fusion proteins of the disclosure can be coupled to a labeling group (labeled polypeptide), which can then be used, for example, for diagnostic purposes. Suitable labeling groups may be selected from radioisotopes (such as those mentioned above) or groups containing radioisotopes, radionuclides, fluorescent groups (such as fluorescent proteins such as GFP, RFP, etc., dyes, rhodamine, fluorescent cyanine and its derivatives such as FITC, cyanine dyes), enzyme groups (such as horseradish peroxidase, alkaline phosphatase, β-galactosidase), chemiluminescent groups, biotin groups, metal particles (e.g. gold particles), magnetic particles (e.g. having a core containing magnetite (Fe3O4)) and/or maghemite (Fe2O3), predetermined polypeptide groups, etc.
The present disclosure also provides a pharmaceutical composition, including the anti-FIXa antibody of the present disclosure, or the fusion protein of the anti-FIXa antibody of the present disclosure, or the immunoconjugate of the present disclosure.
Various pharmaceutically acceptable carriers in the art may also be included in the pharmaceutical composition. The pharmaceutically acceptable carrier is non-toxic to the subject at the dose and concentration used, and may include but not limited to: buffers such as acetate, tris, phosphate, citrate and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as stearyldimethylbenzyl ammonium chloride; hexadimonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; hydrocarbylparabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharide, disaccharide and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; tonicity modifiers, such as trehalose and sodium chloride; sugars, such as sucrose, mannitol, trehalose or sorbitol; surfactants such as polysorbate, salt-forming counterions, such as sodium; metal complexes (such as Zn-protein complexes); and/or nonionic surfactants, such as TWEE®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutical preparations for in vivo administration are generally sterile. Methods for achieving sterility of pharmaceutical preparations may be used, for example, they can be achieved by methods such as filtration through sterile membranes. A suitable pharmaceutically acceptable carrier may be selected based on the formulation requirements of the pharmaceutical composition to prepare it in various forms. For example, the pharmaceutical composition of the present disclosure can include but not limited to tablets, injections, lyophilized preparations and other forms.
In the pharmaceutical composition, the content of the monoclonal antibody, fusion protein or immunoconjugate is usually an effective amount, and the content of the active ingredient corresponding to the effective amount can be determined according to the object to be treated and the specific administration method. For example, based on the total mass of the pharmaceutical composition, the content of the monoclonal antibody, fusion protein and immunoconjugate may be about 0.01-99%, 0.1-70%, 1-30%, 0.01-0.05%, 0.05-0.1%, 0.1-0.3%, 0.3-0.5%, 0.5-1%, 1-3%, 3-5%, 5-10%, 10-20%, 20-30%, 30-50%, 50-70%, or 70-99%.
The monoclonal antibody, fusion protein, and immunoconjugate of the present disclosure can be administered as a single active ingredient, or in combination therapy, that is, in combination with other agents. For example, the combination therapy may be the monoclonal antibody, fusion protein, immunoconjugate combined with at least one other antithrombotic drug. For another example, the combination therapy may be the combined use of the monoclonal antibody, fusion protein, immunoconjugate and antibodies targeting specific antigens on other blood cells.
The present disclosure also provides a detection kit, including the antibody, fusion protein or immunoconjugate of the present disclosure. The kit may also include: containers, controls (negative or positive controls), buffers, auxiliary agents, etc., which can be selected according to specific conditions. Instructions may also be included in the kit, to facilitate the operation.
The present disclosure also provides a use of the antibody, fusion protein, immunoconjugate or pharmaceutical composition of the present disclosure in the manufacture of preparations, kits or drug kits for alleviating or treating thromboembolic diseases.
In the present disclosure, the “thromboembolic diseases” include: venous, arterial or capillary thrombosis, thrombosis in the heart, thrombosis during and/or after contacting blood with an artificial surface, interstitial lung disease (e.g. fibroproliferative and/or idiopathic pulmonary fibrosis), inflammation, neuro-inflammatory diseases, complement activation, fibrinolysis, angiogenesis, coagulation induced by FVIIIa-FIXa complex formation, coagulation induced by FX activation, coagulation induced by FIIa expansion, retinal vascular permeability-related diseases (e.g. embolism), etc.
In one embodiment, diseases related to arterial or capillary thrombosis include (but are not limited to): myocardial infarction, stroke, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, cerebral venous sinus thrombosis. Budd-Chiari syndrome or Paget-Schroetter disease.
A “therapeutically effective amount” of the fusion protein, immunoconjugate, and pharmaceutical composition provided in the present disclosure preferably leads to a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase of the disease, or prevention of damage or disability caused by the disease. For example, for the treatment of thrombotic disorders, a “therapeutically effective amount” preferably reduces thrombus formation (or increases vascular permeability) by at least about 10%, preferably at least about 20%, more preferably at least about 30%, more preferably at least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80% relative to an untreated subject or a non-diseased period in the same subject. The ability to inhibit thrombus formation can be evaluated in in vitro reaction systems, cell models or animal model systems. An appropriate selection of therapeutically effective amount according to the actual situation, for example, the size of the subject, the severity of the subject's symptoms and the selected specific composition or administration route. Prescriptions for treatment (e.g., decisions on dosage, etc.) can be determined by a doctor, considering factors including but not limited to the disease being treated, individual patient conditions, administration site, method of administration, and other factors. A prophylactically effective amount refers to an amount effective at the dosage and time necessary to achieve the desired prophylactic effect. Usually, but not necessarily, a “prophylactically effective amount” is generally lower than a “therapeutically effective amount” because the prophylactic dose is administered to the subject before the onset of the disease or at an early stage of the disease.
The present disclosure further provides a detection method for detecting FIXA antigen using the antibody, including but not limited to qualitative detection, quantitative detection and localization detection. In one embodiment, the detection methods include but are not limited to immunofluorescence, immunohistochemistry, radioimmunoassay and so on.
A method for detecting the presence of FIXa antigen in a sample may include: contacting the sample with the antibody of the present disclosure; observing whether an antibody complex is formed, and the formation of an antibody complex indicates the presence of FIXa antigen in the sample. The sample can be a cell and/or tissue sample: the sample can be fixed or dissolved in a medium; and the level of FIXa antigen in the fixed or dissolved sample can be detected. In some embodiments, the objects for detection may be a cell-containing sample present in a cell preservation solution. In other embodiments, the antibody is also conjugated with markers that can be used for detection or can be detected by other reagents, such as fluorescent dyes, chemicals, polypeptides, enzymes, isotopes, labels, etc.
In the present disclosure, the FIXa-4 antibody is different from the previously reported FIXa inhibitors in that it does not directly act on the catalytic active site of FIXa, but occupies most of the binding region between FIXa and FVIIIa, blocking the formation of the FIXa-FVIIIa complex, then affects the conversion of FX to FXa and triggers anticoagulation. The inventors also observed that the FIXa-4 antibody substantially blocked the production of FXa in vitro, and supplementation with FVIIIa corrected this inhibition. That is, the antithrombotic effect of FIX-4 antibody was partially reversed with the increase of FVIIIa concentration. Compared with the existing antithrombotic drugs, the reversible antithrombotic effect needs to be considered in the development of novel safe and effective antithrombotic drugs. This feature can be used in clinical medication to prevent or treat uncontrollable bleeding side effects caused by excessive anticoagulation. Therefore, this discovery by the present inventors is particularly meaningful. It can be referenced how the FIXa-4 antibody interacts with FIXa to screen or design a class of drugs that can achieve similar competitive binding. These drugs may potentially be drugs with both coagulation-regulating effect and safety.
Therefore, based on the new findings, the present inventors provide a method for screening antithrombotic substance, including: 1) adding candidate substance to the system including FIXa and FVIIIa, and the FIXa and FVIIIa interact with each other (for example, they form a FVIIIa-FIXa complex); (2) detecting the interaction between FIXa and FVIIIa; if the candidate substance competes with FVIIIa to bind FIXa and reduces the formation of the FVIIIa-FIXa complex, it indicates that the candidate substance is a antithrombotic substance; preferably, functions of the candidate substance can be determined by observing the effect of the candidate substance in binding between FIXa and FVIIIa at the binding sites or adjacent sites of FIXa; the binding sites or adjacent sites include: Asn93, Lys132, Arg165, Thr175; more preferably it also include: Ala95, Lys98, Asp164, Lys173, Tyr177; more preferably it also include: Lys126, Asn129, Asn178, Lys230, Arg233, Asn236. The amino acid residues at the sites can form a cluster, occupying the common surface between c170-helix and c131-helix of the FIXa protein.
Further, when observing the effect of the candidate substance in binding between FIXa and FVIIIa at the binding sites or adjacent sites of FIXa to determine the functions of the candidate substance, if the candidate substance exerts a strong binding effect, then it is a antithrombotic substance.
Methods for screening substances acting on a protein or gene or its specific region as a target may be used, and these methods can be used in the present disclosure. The candidate substances can be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and so on. Based on the type of substances to be screened, it may be determined how to select a suitable screening method. In some relatively specific examples, the candidate substances include but are not limited to: regulators designed for FIXa, or upstream or downstream proteins or genes thereof, such as antibodies, interfering molecules (such as interfering RNA), small molecular compounds, gene-modifying constructs or gene-editing constructs, and so on.
After large-scale screening, a class of potential substances that act on the above-mentioned sites of interest with regulatory effects can be obtained.
The disclosure if further illustrated by the specific examples described below. It should be understood that these examples are merely illustrative, and do not limit the scope of the present disclosure. The experimental methods without specifying the specific conditions in the following examples generally used the conventional conditions, such as those described in J. Sambrook, Molecular Cloning: A Laboratory Manual (3rd ed. Science Press, 2002) or followed the manufacturer's recommendation.
When numerical ranges are given in the embodiments, it should be understood that, unless otherwise indicated in the present disclosure, any value between the two endpoints of each numerical range and any value within the range can be selected. Unless otherwise defined, all technical and scientific terms used in the present disclosure have the same meaning as understood in the art. In addition to the specific methods, devices, and materials used in the embodiments, based on their understanding of the prior art and the disclosure of the present disclosure, may also use any methods, devices, and materials similar or equivalent to those described in the embodiments to practice the present disclosure.
4 SPF-level BALB/c male mice (purchased from the Animal Science Department of Shanghai Jiao Tong University School of Medicine) at the age of 6-8 weeks were used for immunization. For the first immunization, purchased full-length 46KD FIXa antigen (Enzyme Research Laboratories) was mixed with complete Freund's adjuvant (Sigma) until the final concentration of FIXa was 100 μg/100 μl. 50 μg/50 μl of the mixture was injected into each of the two foot pads. For the second immunization, complete Freund's adjuvant was replaced by incomplete Freund's adjuvant (Sigma). For the third immunization, the FIXa antigen was diluted to 100 μg/100 μl with PBS and injected intraperitoneally. The fourth immunization, also known as booster immunization, was administered via tail vein injection. A total of 4 immunizations were carried out, with an interval of 2 weeks between each immunization.
On the 3rd day after the booster immunization, splenocytes of the immunized mice were fused with myeloma cells (laboratory cell line SP2/0) under the action of PEG to obtain hybridoma cells.
After culturing hybridoma cells for 7 days, ELISA was used for detecting positive antibodies. 0.1 μg/100 μl of FIXa antigen was coated on an ELISA plate (CORNING), incubated at 37° C. for 1.5 h; then blocked with 2% BSA at 37° C. for 1 h; and then primary antibody, the supernatant of cultured cells, was added and incubated at 37° C. for 1.5 h. After that, mouse secondary antibody was added and incubated at 37° C. for 30 min. Finally, chromogenic substrate TMB (Thermo) was added for 5 min at room temperature in dark, then stop solution 2M H2SO4 was added to terminate the reaction. Antibody-positive cell lines were screened according to the absorbance value measured at 450 nm by a spectrophotometer (Thermo Fisher scientific company). Then the cell lines were cultured for cloning in a semi-solid medium (STEMCELL company), with single clones selected into 96-well plates after 7 days. Following the ELISA method described above, a subsequent screening was conducted, ultimately obtaining antibody-positive single clones.
Sufficient amount of cell pellets was collected for RNA extraction, and then the RNA was reverse transcribed into cDNA. After that, appropriate primers were selected for PCR, and finally the PCR products were sent to the company for sequencing.
4 male nude mice (purchased from the Animal Science Department of Shanghai Jiao Tong University School of Medicine) at the age of 6-8 weeks were selected. Pristane (Sigma company) was injected at a dose of 0.5 ml/mouse 7 days in advance, followed by 0.5-1×106/0.5 ml of hybridoma cells intraperitoneally injected into the mice. After 7-10 days, abdominal distension was observed in the nude mice. A needle was inserted into the left lower abdominal cavity to collect ascites into EP tubes. After centrifuging at 5000 rpm for 10 minutes, the supernatant was collected, with NaN3 added to a final concentration of 0.02%.
Following the protocol outlined in the manual for Protein G Agarose Resin (YEASEN), the ascites collected above was equilibrated, loaded, washed and eluted in sequence. Finally, the eluate was concentrated by ultrafiltration with a 30KD ultrafiltration tube to obtain high-purity monoclonal antibodies.
Following the instructions provided in the Sigma company's subtype detection kit, the antigen was coated, blocked, incubated with primary antibody (0.1 μg/100 μl), incubated with isotype-specific reagent at room temperature for 30 minutes, incubated with secondary antibody (R-antiGoat-HRP) for chromogen development, with stop solution added to terminate the reaction, and finally the absorbance was measured at 450 nm. Affinity detection of the antibody is the same as the above ELISA method.
Antibody coagulation was assessed by activated partial thromboplastin time (APTT) and prothrombin time (PT).
Firstly, instruments and related reagents were preheated to 37° C. Then the antibody was diluted with OVB buffer (SIEMENS), then the APTT program was selected, and the cuvette was placed. 50 μl of APTT reagent, 50 μl of a mixture with plasma and magnetic beads were added in sequence. By pressing the start button, magnetic beads started to oscillate. Then 25 μl of antibody was added, with timing button pressed to start timing. After 180 seconds, 25 μl of 50 mM CaCl2 (SIEMENS) was added, simultaneously the measurement button on the handle was pressed. Magnetic bead oscillation was observed until the plasma coagulates, with the seconds displayed on the instrument recorded. Finally, a curve was drawn according to the log value of the final antibody concentration in plasma and the coagulation time in seconds.
Firstly, instruments and related reagents were preheated to 37° C. Then the antibody was diluted with a mixture of plasma, then the PT program was selected, and the cuvette was placed. Magnetic beads were added for oscillation by pressing the start button, then 50 μl of the mixture of plasma containing antibodies was added, with the timer button pressed to start timing. After 60 s, 100 μl PT reagent was added, simultaneously the measurement button on the handle was pressed. Magnetic bead oscillation was observed until the plasma coagulates, with the seconds displayed on the instrument recorded. A curve was drawn according to the log value of the final antibody concentration in plasma and the seconds.
Firstly, the spectrophotometer was set to 37° C. and the kinetic cycle was selected to a total time of 10 min. an interval of 5 s and 121 cycles. Then, following the instructions of FIXa chromogenic substrate SPECTROZYME (from IMMBIOMED), the following substances were added sequentially to a 96-well plate: 100 μl Tris Buffer (50 mM TRIS, 100 mM NaCl, 5 mM CaCl2, pH7.4, 33% ethylene glycol), 10 μl of 2 μM FIXa protein, 2.5 μl of antibody (with a final concentration of 1000, 10, 1, 0.1, 0.01 μg/ml), and control TBS. Finally, 12.5 μl of 10 mM FIXa chromogenic substrate was added to trigger the reaction. ΔOD/min of the absorbance value was measured at 405 nm.
The three-dimensional model of FIXa was constructed based on the crystal structure of an inhibitor complex of the catalytic domain of FIXa (PDBentry: 3LC3).
By Abody Builder of SabPred (Venkateswarlu D. Structural insights into the interaction of blood coagulation co-factor VIIIa with factor IXa: a computational protein-protein docking and molecular dynamics refinement study. Biochem Biophys Res Commun-2014:452(26) 14.), three-dimensional structure of the antibody variable region was predicted.
The antibody was docked with FIXa through ClusPro. During the docking, the antibody mode was used to shield the non-CDR region of the antibody (Dunbar J et al., SAbPred: a structure-based antibody prediction server. Nucleic Acids Res. 2016 Jul. 8; 44(W1): W474-8).
Diagrams describing the interaction of FIXa with antibodies were made using open-source PyMOL (version 2.5.0).
1.6 FIXa-4 Antibody Competes with FVIIIa for Binding to FIXa
100 μl 10 U/ml thrombin and 25 μl 4 mg/ml FVIII were added to 125 μl Buffer (20 mM Hepes, 300 mM NaCl, 2.5 mM CaCl 2), and incubated at 37° C. for 10 min to obtain FVIIIa (with a final concentration of 0.4 mg/ml). Finally, 1 U Hirudin was added to prevent the disintegration of FVIIIa.
Firstly, 50 ng/2 μl phosphatidylcholine (PC), phosphatidylserine (PS) mixture, 5 μl 20 nM FIXa and 10 μl 3 μM FX were mixed, then the premixed 8 μl FVIIIa at a concentration of 80 μg/ml and 5 μl antibody were added and incubated at 37° C. for 15 min. Subsequently, 20 μl of 20 mM EDTA was added to terminate the reaction. Finally, 150 μl of TBS-Ca2+ buffer containing 0.1% PEG8000 was added to dilute the reaction product.
Then, the spectrophotometer was set to 37° C. and the kinetic cycle was selected to a total time of 5 min, an interval of 10 s and 31 cycles. 40 μl of the reaction product was added to a 96-well plate, and then 40 μl of chromogenic substrate S2765 (1 mM) was added to trigger the reaction. ΔOD/min of the absorbance value was measured at 405 nm.
Appropriate antibody concentration in reaction 1.6.2 was selected. Following the same method, 5 μl of antibody in the above reaction system was replaced with 2.5 μl FVIIIa and 2.5 μl of the antibody (with the final concentration remaining unchanged). ΔOD/min of the absorbance value was measured at 405 nm.
Firstly, 25 μl FX (200 nM), 25 μl Russell Viper Venom (Russell Viper Venom, RVV) (42 nM), 50 μl TBS-Ca2+ buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl2), BSA 0.1%) were mixed. After incubation at 37° C. for 30 min. FX was activated to FXa (with a final concentration of 10 nM).
Then, the spectrophotometer was set to 37° C. and the kinetic cycle was selected to a total time of 5 min, an interval of 10 s and 31 cycles. 40 μl of FXa in a concentration gradient was added to a 96-well plate, and then 40 μl of chromogenic substrate S2765 (1 mM) was added to trigger the reaction. ΔOD/min of the absorbance value was measured at 405 nm. That is the substrate cleavage rate. A standard curve was drawn according to the final concentration of FXa and the corresponding reaction rate.
By mice immunization, hybridoma fusions, antibody sequencing, cell expression and purification, then after a large number of research and screening, the inventors obtained a monoclonal antibody with high affinity to human FIXa.
The half-maximum effect concentration (EC50) of the antibody was determined, and the result is shown in
The present inventors named this monoclonal antibody FIXa-4 antibody.
The amino acid sequence of the heavy chain variable region of the FIXa-4 antibody is as follows (SEQ ID NO: 1):
The amino acid sequence of the light chain variable region of the FIXa-4 antibody is as follows (SEQ ID NO: 2):
Among them, the sequences of the CDR region and surrounding framework regions are listed in Table 1.
The inventors then tested the effects of the monoclonal antibody on activated partial thromboplastin time (APTT) and prothrombin time (PT) to evaluate its role in endogenous and exogenous coagulation pathways. The inventors added different concentrations of the monoclonal antibody (with a final concentration of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 μg/ml) into the APTT or PT reaction system.
The experimental results showed that, compared with the control group, the FIXa-4 antibody significantly prolonged the APTT. Besides, as the concentration of FIXa-4 antibody increased, its prolonging effect on APTT was also enhanced, and the APTT could be prolonged to 88.8 s at most, which was 3.5 times that of the control group at 25.5 s, as shown in
At the same time, the measurement of prothrombin time (PT) showed that the FIXa-4 antibody basically did not affect PT, as shown in
Above experimental data indicate that FIXa-4 antibody inhibits the endogenous coagulation pathway, but does not act on coagulation factors in exogenous coagulation pathways such as FX and FII and coagulation factors in common coagulation pathways.
In the endogenous coagulation pathway, FIXa acts as an enzyme that catalyzes FX to form FXa to mediate the coagulation cascade. Therefore, in order to study the anticoagulant mechanism of the FIXa-4 antibody, the present inventors first examined its effect on the catalytic activity of FIXa. The rate of an enzyme to catalyze substrate cleavage is used to reflect the catalytic activity of the enzyme. The inventors mixed different concentrations of FIXa-4 antibody (with a final concentration of 1000, 10, 1, 0.1, 0.01 μg/ml) and FIXa protein for 3 minutes, and then added it to the FIXa chromogenic substrate reaction system. The OD405 nm was monitored in real time. ΔOD405/min reflects the catalytic activity of the enzyme.
The experimental results showed that the FIXa-4 antibody has no significant effect on the enzymatic activity of FIXa, as shown in
This result indicates that the FIXa-4 antibody does not exert anticoagulant effects by directly binding to the catalytic active site of FIXa.
In order to further study the anticoagulation mechanism of the FIXa-4 antibody, the inventors used protein docking method to predict the binding site of the FIXa-4 antibody and FIXa. Complete structure of full-length FIXa protein without experiments for determination is not available in the Protein Data Bank (PDB). Considering that the present disclosure mainly focuses on the effect of the antibody on the catalytic activity of FIXa, a 3D model of the catalytic domain of FIXa was established based on an inhibitor complex structure (PDBentry: 3LC3) of the catalytic domain of FIXa. Simultaneously, using antibody structure prediction tools, a 3D model of the binding region of the FIXa-4 antibody was established. The optimal ensemble of conformations for their binding was predicted through protein-protein docking. In this ensemble of conformations, a total of 979 docking conformations formed 30 clusters. By observing the representative conformations of each cluster, it was found that most binding conformations of FIXa and FIXa-4 antibody shared a binding surface, and the residues with higher contact frequency were listed in Table 2. In all conformations, the binding of the FIXa-4 antibody to FIXa does not obstruct the entry and exit of substrate to the catalytic site of FIXa (formed by the catalytic triad His57-Asp102-Ser195) (
On the other hand, these contact residues partially overlap with the previously predicted FIXa-FVIIIa binding interface [DeLano W L (2002) The PyMOL molecular graphics system] (
All the residues in the table are numbered according to chymotrypsin. The residues of FIXa within the proximity of 3 Å to the antibody were considered as contact residues. The frequency was calculated as
and the Σ means summation. S* was the number of members of a docking cluster which contained the residue proximate to the antibody and s was the number of members of every cluster. Residues marked with “a” are the residues same to the predicted sites of FIXa binding to FVa. Residues marked with “b” are the residues in the vicinity of those binding residues by less than 2 residues in the sequence.
Example 5 FIXa-4 Antibody Competes with FVIIIa for Binding to FIXa
As mentioned earlier. FIXa-4 competes with FVIIIa for binding to FIXa. and the concentration of FVIIIa increases, the effect of FIXa-4 antibody will be weakened. Firstly, the inventors established an in vitro model that mimics the production of FXa by the FIXa-FVIIIa complex in vivo. In this system, PS/PC, FIXa, FVIIIa, and FX were added to react and produce FXa. Subsequently, FXa chromogenic substrates were added to determine the amount of FXa generated in the reaction.
The inventors found that the addition of different concentrations of FIXa-4 antibody (with a final concentration of 1560, 780, 390, 156, 78, 39, 15.6, 7.8 pM) to this system inhibited the generation of FXa in a dose-dependent manner (
This result proved that FIXa-4 exerts an anticoagulant effect by competing with FVIIIa for binding to FIXa.
In conclusion, the monoclonal antibody FIXa-4 targeting FIXa, obtained by the inventors, exhibits a significant anticoagulant effect. Its mechanism is to inhibit the formation of FVIIIa-FIXa complex by competing with FVIIIa to bind to the site on FIXa, to exert anticoagulant effect.
The research results provide a new target for the treatment of thrombus, and also provide indirect evidence for the binding site of the interaction between FVIIIa and FIXa.
The current anticoagulants have various limitations, including unpredictable pharmacokinetics, lack of reversibility, and, in some cases, immunogenicity. Developing safe and effective antithrombotic drugs remains a key focus in contemporary medical research. When selecting target coagulation factors, two important considerations should be considered. First, this pathway should be blocked before significant amplification of the coagulation cascade occurs. Second, the rate-limiting step should be targeted to provide effective anticoagulation within the broadest therapeutic range. FIXa is a critical coagulation factor in the endogenous coagulation pathway and is the only soluble form of coagulation protein. FIXa efficiently diffuses from tissue factor-bearing cells to platelets, serving as a crucial link between the initiation and amplification phases of the coagulation cascade.
Monoclonal antibodies have the characteristics of strong antigen-binding specificity and long half-life. With continuous development of monoclonal antibody technology, antibody drugs play a crucial role in disease prevention, diagnosis, and treatment. In the present disclosure, a novel anti-FIXa monoclonal antibody was obtained successively through hybridoma, antibody sequencing, cell expression and purification, and the antithrombotic properties of FIXa-4 were described through APTT and PT functional assays. Although the prolongation of APTT by FIXa-4 is dose-dependent, increasing the antibody concentration significantly does not result in uncontrolled prolongation but rather confines the APTT extension to approximately 3.5 times. This result indicates that FIXa-4 has a large effective therapeutic concentration window, but does not increase the risk of bleeding. This can meet the needs in clinical application.
When exploring its antithrombotic mechanism, the inventors found that the FIXa-4 antibody is different from the previously reported FIXa inhibitors in that it does not directly act on the catalytic active site of FIXa, but occupies most of the binding region between FIXa and FVIIIa, blocking the formation of the FIXa-FVIIIa complex, then affects the conversion of FXa to FXa and triggers anticoagulation. More interestingly, the FIXa-4 antibody substantially blocked the production of FXa in vitro, and supplementation with FVIIIa corrected this inhibition. That is, the antithrombotic effect of FIX-4 antibody was partially reversed with the increase of FVIIIa concentration. Compared with the existing antithrombotic drugs, the reversible antithrombotic effect needs to be considered in the development of novel safe and effective antithrombotic drugs. This feature can be used in clinical medication to prevent or treat uncontrollable bleeding side effects caused by excessive anticoagulation.
In summary, the present inventors have prepared a new type of antithrombotic antibody targeting the FIXa-FVIIIa binding site, providing new insights for treating thrombosis and shedding light on the still not entirely clear interaction between FVIIIa and FIXa binding sites.
Each reference provided herein is incorporated by reference to the same extent as if each reference was individually incorporated by reference. In addition, it should be understood that based on the above teaching content of the disclosure, practice of various changes or modifications to the disclosure can be made, and these equivalent forms also fall within the scope of the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110639032.6 | Jun 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/097412 | 6/7/2022 | WO |
