WO2015125904A1 - 新規抗ヒトpai-1抗体 - Google Patents
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- WO2015125904A1 WO2015125904A1 PCT/JP2015/054704 JP2015054704W WO2015125904A1 WO 2015125904 A1 WO2015125904 A1 WO 2015125904A1 JP 2015054704 W JP2015054704 W JP 2015054704W WO 2015125904 A1 WO2015125904 A1 WO 2015125904A1
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- C07K16/38—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against protease inhibitors of peptide structure
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A61K2039/507—Comprising a combination of two or more separate antibodies
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- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the present invention relates to a novel anti-human PAI-1 antibody.
- Plasminogen activator inhibitor-1 is one of serine protease inhibitors produced from vascular endothelium. PAI-1 binds to a plasminogen activator (PA), which is a serine protease, and deactivates the enzyme activity of PA (Mol. Cell. Endocrinol., 1990, Vol. 68, p. 1-). 19). PAI-1 includes active PAI-1, latent PAI-1, and PAI-1 that forms a complex by binding to PA. The properties of PAI-1 that inhibit this PA are as follows. Only PAI-1 has (Blood, 1987, Vol. 70, p. 1090-1098).
- tissue plasminogen activator tissue plasminogen activator (tissue) plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA, also called urokinase), both of which are plasminogen.
- tissue tissue plasminogen activator
- uPA urokinase-type plasminogen activator
- PAI-1 binds to tPA and uPA to form a complex.
- the biological properties that inactivate PA are well elucidated, and as a result of the binding of active PAI-1 to PA, it inhibits the fibrinolytic system and promotes thrombus formation.
- a feature of the three-dimensional structure of active PAI-1 is that the active central loop, which is a binding site with PA, is extended and exposed to the outside of the protein (J. Biol. Chem., 2001, Vol. 276, p. .. 44912-44918).
- active PAI-1 can bind to its cofactor, vitronectin, to form a complex (J. Biol. Chem., 1988, Vol. 263, p. 15454-15461).
- Vitronectin suppresses the conformational change from the active form to the latent form of PAI-1 (J. Biol. Chem., 1990, Vol. 265, p. 18490-18498).
- Latent PAI-1 is a stable conformation in which the active center loop is folded inward, and once latent, PAI-1 cannot bind to PA (J. Biol. Chem., 2008, Vol.283, p.18147-18157. Nature, 1992, Vol.355, p.270-273).
- active PAI-1 includes active PAI-1 present as a monomer and active PAI-1 present as a complex of vitronectin, which bind to PA and deactivate PA. It has a function to make it.
- pulmonary fibrosis such as idiopathic pulmonary fibrosis, stroma Pneumonia, systemic lupus erythematosus, scleroderma, diabetic nephropathy, lupus nephritis, graft-versus-host disease, glomerulonephritis, nephrotic syndrome, renal fibrosis, chronic obstructive pulmonary disease, acute kidney injury, acute lung injury, Acute respiratory failure, age-related macular degeneration, disseminated intravascular coagulation syndrome, postoperative adhesion, symptomatic vitreous macular adhesion, diabetic retinopathy, arteriosclerosis, myocardial infarction, cerebral infarction, lung infarction, conjunctival scar after glaucoma surgery It is considered to be involved in diseases such as peritoneal
- active PAI-1 can be used in various diseases related to pathogenesis. It is expected to be useful for prevention and treatment.
- Examples of antibodies exhibiting a function inhibitory action against active human PAI-1 include mouse monoclonal antibodies MA-33B8 (Patent Document 1), MA-33H1F7 (Non-Patent Document 1), MA-55F4C12 (Non-Patent Document 1), MA-56A7C10 (patent document 2), MA-33B8 humanized antibody CT140 (patent document 1), and fully human antibody MEDI-579 (CAT-1001 or PICK167_A01-fgl IgG1) produced by phages. Patent Document 3) and the like have been reported.
- MA-56A7C10 and MEDI-579 are about 4.0-fold and about 20-fold higher binding to active human PAI-1, respectively, compared to human PAI-1 having a three-dimensional structure other than the active form.
- the activity is shown (patent document 2 and patent document 3).
- MEDI-579 also has an inhibitory effect on mouse PAI-1, and experimental results using pathological model mice of lupus nephritis, scleroderma, diabetic nephropathy and thrombosis are expected to have a medicinal effect on these diseases. (Patent Document 3).
- PAI-1 having other three-dimensional structure means latent PAI-1 and PAI-1 complexed with tPA or uPA.
- More than 90% of total PAI-1 in human plasma is stored in platelets. In plasma excluding platelets, about two-thirds of total PAI-1 is active (Br. J. Haematol., 1988, Vol. 70, p. 327-333. Blood, 1988, Vol. 71, p.220-225). Not only PAI-1 activity in plasma (active PAI-1 activity shown by the ability to bind to tPA), but also total PAI-1 level in plasma (by anti-PAI-1 antibody that detects all PAI-1) The total amount of PAI-1 detected) and the amount of complex of PAI-1 and tPA in plasma (the amount of complex detected by the combination of anti-PAI-1 antibody and anti-tPA antibody) vary. (Thromb. Res., 2008, Vol.
- tPA-PAI-1 complex is present in about one third of the amount of active PAI-1. (Blood, 1990, Vol. 76, p. 930-937). In tissues, not only tPA but also uPA exists, and a complex of PAI-1 and uPA also exists. In conclusion, PAI-1 other than active PAI-1 is present in the human body in a non-negligible proportion of total PAI-1.
- An object of the present invention is to provide an anti-human PAI-1 antibody that prevents or treats pulmonary fibrosis by selectively inhibiting active human PAI-1 as compared with conventional anti-human PAI-1 antibodies. There is.
- an anti-human PAI-1 antibody having high selectivity for active human PAI-1 As a result of considerable inventive studies in the production of an anti-human PAI-1 antibody having high selectivity for active human PAI-1, the present inventors consisted of amino acid sequences from amino acid numbers 1 to 118 of SEQ ID NO: 2.
- An anti-human PAI-1 antibody comprising a heavy chain variable region and a light chain variable region consisting of amino acid sequences from amino acid numbers 1 to 108 of SEQ ID NO: 4 was prepared (Examples 1 to 3), and the anti-human PAI- 1 antibody found to inhibit active human PAI-1 in plasma (Example 5) and to have high selectivity for active human PAI-1 complexed with vitronectin (Example 6) It was.
- the anti-human PAI-1 antibody was provided and the present invention was completed.
- this invention includes the following invention as a medically or industrially useful substance or method.
- CDR1 consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid numbers 31 to 35
- CDR2 consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid numbers 50 to 66
- amino acids from amino acid numbers 99 to 107 of SEQ ID NO: 2
- a heavy chain variable region comprising CDR3 consisting of a sequence, CDR1 consisting of an amino acid sequence of amino acid numbers 24 to 34 of SEQ ID NO: 4, CDR2 consisting of an amino acid sequence of amino acid numbers 50 to 56 of SEQ ID NO: 4, and a sequence An anti-human PAI-1 antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising CDR3 consisting of the amino acid sequence of amino acid numbers 89 to 97 of number 4.
- An anti-human PAI-1 antibody or an antigen-binding fragment thereof selected from any of the following 1) to 2): 1) an anti-human PAI-1 comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4 An antibody or an antigen-binding fragment thereof; and 2) an anti-human PAI- described in (1), which is an antibody or an antigen-binding fragment thereof produced by post-translational modification of the anti-human PAI-1 antibody or the antigen-binding fragment thereof of 1) 1 antibody or antigen-binding fragment thereof.
- an anti-human PAI-1 antibody comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4
- the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (2) which is an antibody or antigen-binding fragment thereof produced by post-translational modification of the antigen-binding fragment thereof.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof according to any one of (1) to (5) comprising a heavy chain constant region that is a human Ig ⁇ 1 constant region.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof according to any one of (1) to (5) comprising a light chain constant region that is a human Ig ⁇ constant region.
- the anti-human PAI-1 antibody according to any one of (1) to (5) which comprises a heavy chain constant region which is a human Ig ⁇ 1 constant region and a light chain constant region which is a human Ig ⁇ constant region Antigen binding fragment.
- the anti-human PAI-1 antibody according to (3) comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the antigen-binding fragment according to any one of (1) to (8) which is a single-chain variable region fragment, Fab, Fab ′, or F (ab ′) 2 .
- An anti-human PAI-1 antibody which is an antibody produced by post-translational modification of the anti-human PAI-1 antibody according to (9).
- a polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (3).
- An expression vector comprising the polynucleotide according to (16) and / or (17).
- (A) A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (3) and the light chain variable region of the antibody or antigen-binding fragment thereof A host cell transformed with an expression vector comprising a polynucleotide comprising the base sequence;
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (3) and the light chain variable of the antibody or antigen-binding fragment thereof
- An anti-human PAI-1 antibody comprising a step of culturing a host cell selected from the group consisting of the following (a) to (c) to express an anti-human PAI-1 antibody or an antigen-binding fragment thereof: Or a method of producing an antigen-binding fragment thereof.
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (3) and the light chain variable region of the antibody or antigen-binding fragment thereof A host cell transformed with an expression vector comprising a polynucleotide comprising the base sequence;
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to (3) and the light chain variable of the antibody or antigen-binding fragment thereof
- a method for producing an anti-human PAI-1 antibody comprising culturing a host cell selected from the group consisting of the following (a) to (c) to express an anti-human PAI-1 antibody.
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody according to (9) and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody
- a pharmaceutical composition comprising: (27) A pharmaceutical composition comprising the anti-human PAI-1 antibody according to (9), the anti-human PAI-1 antibody according to (13), and a pharmaceutically acceptable excipient. (28) The pharmaceutical composition according to any one of (25) to (27), which is a pharmaceutical composition for preventing or treating pulmonary fibrosis. (29) The pharmaceutical composition according to (28), wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.
- (30) Lung fiber comprising the step of administering a therapeutically effective amount of the anti-human PAI-1 antibody or antigen-binding fragment thereof according to any one of (1) to (15), (23), and (24) Of preventing or treating symptom.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof includes a fusion antibody with another peptide and protein, and a modified antibody to which a modifying agent is bound.
- the anti-human PAI-1 antibody of the present invention prevents a disease in which active human PAI-1 is involved in pathogenesis by inhibiting the function of active human PAI-1, for example, prevention of pulmonary fibrosis such as idiopathic pulmonary fibrosis Or it can be used as a therapeutic agent.
- FIG. 2 shows the activity of fully human HOT-1 in the inhibition of monkey blood activated PAI-1.
- the vertical axis represents plasma active PAI-1 concentration.
- IgG There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE.
- the basic structure of an antibody molecule is common to each class, and is composed of a heavy chain having a molecular weight of 50,000 to 70,000 and a light chain having a molecular weight of 20,000 to 30,000.
- the heavy chain is usually composed of a polypeptide chain containing about 440 amino acids, has a characteristic structure for each class, and corresponds to IgG, IgM, IgA, IgD, IgE, Ig ⁇ , Ig ⁇ , Ig ⁇ , Ig ⁇ . It is called.
- IgG has IgG1, IgG2, IgG3, and IgG4 subclasses, and the corresponding heavy chains are called Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, and Ig ⁇ 4.
- the light chain is usually composed of a polypeptide chain containing about 220 amino acids, and two types of L-type and K-type are known, and are called Ig ⁇ and Ig ⁇ , respectively.
- the peptide structure of the basic structure of an antibody molecule has a molecular weight of 150,000 to 190,000, in which two heavy chains and two light chains that are homologous are linked by a disulfide bond (SS bond) and a non-covalent bond, respectively. .
- the two light chains can be paired with any heavy chain.
- Each antibody molecule always consists of two identical light chains and two identical heavy chains.
- the antigen-determining site of an antibody is composed of VH and VL, and the binding specificity depends on the amino acid sequence of this site.
- biological activities such as binding to complement and various Fc receptor-expressing cells reflect differences in the structure of the constant region of each class Ig. It has been found that the variability of the light chain and heavy chain variable regions is almost limited to the three small hypervariable regions present in both chains, these regions being complementarity determining regions (CDRs; each N-terminal). From the side, it is called CDR1, CDR2, CDR3). The remaining part of the variable region is called the framework region (FR) and is relatively constant.
- CDRs complementarity determining regions
- antigen-binding fragments including antibody VH and VL also have antigen-binding activity.
- antigen-binding fragments include single-chain variable region fragments (scFv), Fab, Fab ′, and F (ab ′). 2 is mentioned.
- Fab is a monovalent antibody fragment composed of a heavy chain fragment comprising a light chain, VH, CH1 domain and part of the hinge region.
- Fab ′ is a monovalent antibody fragment composed of a heavy chain fragment comprising a light chain, a VH, CH1 domain, and a part of the hinge region. The cysteine residues that made up the bond are included.
- An F (ab ′) 2 fragment is a bivalent antibody fragment in which two Fab ′ fragments are joined by an inter-heavy chain SS bond in the hinge region.
- scFv is a monovalent antibody fragment composed of VH and VL linked by a linker.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes an anti-human PAI-1 antibody having the following characteristics.
- An anti-human PAI-1 antibody comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4 Its antigen-binding fragment.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention is preferably an antibody having the above characteristics and further comprising a heavy chain constant region and a light chain constant region.
- a heavy chain constant region any subclass constant region (eg, Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3, or Ig ⁇ 4 as the heavy chain and Ig ⁇ or Ig ⁇ as the light chain) can be selected, but the heavy chain constant region is human.
- the Ig ⁇ 1 constant region is preferred.
- human Ig ⁇ 1 constant region examples include a human Ig ⁇ 1 constant region consisting of an amino acid sequence from amino acid numbers 119 to 448 of SEQ ID NO: 2.
- the light chain constant region is preferably a human Ig ⁇ constant region.
- human Ig ⁇ constant region examples include a human Ig ⁇ constant region consisting of the amino acid sequence of amino acid numbers 109 to 214 of SEQ ID NO: 4.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes the above heavy chain variable region and light chain variable region, the heavy chain constant region is a human Ig ⁇ 1 constant region, and the light chain constant region is human Ig ⁇ . More preferred is an anti-human PAI-1 antibody which is a constant region.
- the antigen-binding fragment of the invention is scFv, Fab, Fab ′, or F (ab ′) 2 .
- the anti-human PAI-1 antibody of the present invention comprises an anti-human PAI-1 comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. It is an antibody.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes an anti-human PAI-1 antibody or antigen-binding fragment thereof that has undergone post-translational modification.
- anti-human PAI-1 antibodies or antigen-binding fragments thereof of the invention that have undergone post-translational modification include anti-humans that have undergone pyroglutamylation at the heavy chain variable region N-terminus and / or lysine deletion at the heavy chain C-terminus And human PAI-1 antibody or antigen-binding fragment thereof.
- the anti-human PAI-1 antibody of the present invention is an anti-human PAI-1 antibody having the following characteristics.
- An anti-human PAI-1 antibody comprising a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.
- the anti-human PAI-1 antibody of the present invention is an anti-human PAI-1 antibody having the following characteristics.
- An anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers from 1 to 447 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4.
- the present invention also includes an anti-human PAI-1 antibody or antigen-binding fragment thereof having the following characteristics.
- CDR1 consisting of amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2
- CDR2 consisting of amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 2
- a heavy chain variable region comprising CDR3, CDR1 comprising an amino acid sequence of SEQ ID NO: 4 from amino acid numbers 24 to 34, CDR2 comprising an amino acid sequence of SEQ ID NO: 4 from amino acid numbers 50 to 56, and SEQ ID NO: 4
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention is an antibody that binds to active human PAI-1 complexed with vitronectin (also referred to as VTN-PAI-1 complex).
- Active human PAI-1 present in vivo includes active human PAI-1 present as a monomer and a VTN-PAI-1 complex.
- the conformational change from the active form of human PAI-1 to the latent form is suppressed by binding to vitronectin, and the active form of human PAI-1 present as a monomer is suppressed.
- the structure of active human PAI-1 is more stably maintained.
- the active center loop structure of active human PAI-1 is not affected (Nat. Struct. Biol., 2003, Vol. 10, p. 541-544). Therefore, by evaluating the binding activity of an anti-human PAI-1 antibody to the VTN-PAI-1 complex, the selectivity of the antibody against active human PAI-1 can be more accurately evaluated.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes not only a VTN-PAI-1 complex but also an antibody that binds to active human PAI-1 present as a monomer. .
- Whether or not it binds to the active PAI-1 present as a VTN-PAI-1 complex or monomer can be confirmed using a known binding activity measurement method.
- the method for measuring the binding activity include methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and surface plasmon resonance (SPR).
- ELISA Enzyme-Linked Immunosorbent Assay
- SPR surface plasmon resonance
- vitronectin BD Biosciences, 354238
- recombinant activated human PAI-1 Molecular Innovation, PAI-A
- a test antibody is added and reacted.
- a secondary antibody such as an anti-IgG antibody labeled with horseradish peroxidase (HRP) or the like is reacted, washed, and a reagent for detecting its activity (for example, in the case of HRP labeling, a color kit for peroxidase ( Whether or not the test antibody binds to the VTN-PAI-1 complex can be confirmed by measuring the activity using Sumitomo Bakelite Co., Ltd.).
- HRP horseradish peroxidase
- a reagent for detecting its activity for example, in the case of HRP labeling, a color kit for peroxidase ( Whether or not the test antibody binds to the VTN-PAI-1 complex can be confirmed by measuring the activity using Sumitomo Bakelite Co., Ltd.).
- SPR for example, Biacore (registered trademark) T200 (GE Healthcare Japan) can be used.
- a test antibody is immobilized on the surface of a sensor chip, and recombinant active human PAI-1 (Molecular Innovation, PAI-A) is added to the flow path.
- active human PAI-1 Molecular Innovation, PAI-A
- association rate constant (ka) association rate constant
- kd dissociation rate constant
- KD dissociation constant
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention is an antibody that binds to the VTN-PAI-1 complex and has an inhibitory activity against active human PAI-1 in plasma. More preferably, the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention binds to the VTN-PAI-1 complex, has inhibitory activity against active human PAI-1 in plasma, and is latent This antibody does not bind to human PAI-1 or human PAI-1 complexed with PA.
- a method for specifically evaluating the inhibitory activity against active human PAI-1 in plasma a method as described in Example 5 below can be used.
- “Does not bind to latent human PAI-1” means that when the binding activity of an antibody to latent human PAI-1 is evaluated, the EC50 value (ng / mL) is> 10,000, and the EC50 value (Ng / mL)> 10000 means that the antibody concentration showing 50% of the maximum activity is higher than 10000 ng / mL.
- the method used for this evaluation is as follows. (Evaluation of binding activity to latent human PAI-1) Recombinant latent human PAI-1 (Molecular Innovation, PAI-L) was diluted to 1000 ng / mL with phosphate buffered saline (PBS) and added to a Nunkumaxisorp transparent 96 plate (Nunc).
- Latent human PAI-1 is immobilized by adding 100 ⁇ L per well and allowing the plate to stand at 4 ° C. overnight.
- the solid phase solution is removed by reverse centrifugation, 200 ⁇ L of blocking agent (Thermo SCIENTIFIC, 37532) is added per well, and the mixture is allowed to stand at room temperature for 30 minutes.
- Wash with a washing solution Tris buffered saline (TBS) containing 0.05% Tween-20), and in 8 steps from 100000 ng / mL to 0.01 ng / mL with PBS containing 0.1% bovine serum-derived albumin (BSA).
- TBS Tris buffered saline
- BSA bovine serum-derived albumin
- MEDI-579 Patent Document 3
- MA-56A7C10 Hycult Biotech, HM2182
- a mouse Fc region Dilute in 7 steps from 10000 ng / mL to 0.01 ng / mL with PBS containing% BSA and add to wells.
- a well to which PBS containing 0.1% BSA is added instead of the test antibody is prepared. After washing 3 times with the washing solution, 100 ⁇ L of detection antibody diluted 2000 times using a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20 times with PBS is added and reacted.
- a blocking agent Nacalai Tesque, 03953-95
- an HRP-labeled goat anti-mouse Ig antibody (Southern Biotech, 1010-05) is used for detection of a test antibody having an Fc region of a mouse antibody, and an HRP is used for detection of a test antibody having an Fc region of a human antibody.
- a labeled rabbit anti-human Ig antibody (Southern Biotech, 6145-05) is used. Incubate for 30 minutes at room temperature, then wash 3 times with wash solution. Add 100 ⁇ L of the coloring solution contained in the peroxidase coloring kit (Sumitomo Bakelite Co., Ltd.) per well and let stand at room temperature for about 15 minutes, then add 100 ⁇ L of the reaction stopping solution contained in the coloring kit, and obtain the OD450 value.
- test antibody When the test antibody has a mouse Fc region, if the test antibody does not reach the maximum concentration (10000 ng / mL) measured in the MA-56A7C10 evaluation system even when the maximum concentration (100,000 ng / mL) is added, MA-56A7C10 The measurement value of the maximum density in the evaluation system is set to 100%. The calculated PAI-1 binding rate is analyzed, and the EC50 value of the test antibody is calculated by 4-parameter logistic curve regression.
- Does not bind to human PAI-1 complexed with plasminogen activator means human PAI-1 complexed with plasminogen activator (PA) uPA or tPA (respectively uPA ⁇ Mean that the EC50 value (ng / mL) for each conjugate is> 10,000 when the binding activity of the antibody to the PAI-1 conjugate and tPA-PAI-1 conjugate) is evaluated.
- EC50 value (ng / mL)> 10000 means that the antibody concentration showing 50% of maximum activity is greater than 10000 ng / mL. The method used for this evaluation is as follows.
- uPA 60,000 units for intravenous urokinase “Benesis (registered trademark)”; Mitsubishi Tanabe Pharma Co., Ltd.
- PBS urokinase
- Nunkumaxisorp transparent 96 plates Add 100 ⁇ L and allow the plate to stand at 4 ° C. overnight to immobilize uPA.
- the solid phase solution is removed by reverse centrifugation, 200 ⁇ L of blocking agent (Thermo SCIENTIFIC, 37532) is added per well, and the mixture is allowed to stand at room temperature for 30 minutes.
- Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) diluted to 1000 ng / mL with PBS containing 0.1% BSA is added at 100 ⁇ L per well and captured by uPA. Wash with a washing solution (0.05% Tween-20-containing TBS), add 100 ⁇ L of test antibody diluted in 8 steps from 0.1000 ng / mL to 0.01 ng / mL with PBS containing 0.1% BSA, and add room temperature Let stand for 30 minutes.
- a washing solution 0.05% Tween-20-containing TBS
- MEDI-579 Patent Document 3
- MA-56A7C10 Hycult Biotech, HM2182
- a mouse Fc region Dilute in 7 steps from 10000 ng / mL to 0.01 ng / mL with PBS containing% BSA and add to wells.
- a well to which PBS containing 0.1% BSA is added instead of the test antibody is prepared. After washing 3 times with the washing solution, 100 ⁇ L of detection antibody diluted 2000 times using a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20 times with PBS is added and reacted.
- a blocking agent Nacalai Tesque, 03953-95
- an HRP-labeled goat anti-mouse Ig antibody (Southern Biotech, 1010-05) is used for detection of a test antibody having an Fc region of a mouse antibody, and an HRP for detection of a test antibody having an Fc region of a human antibody.
- a labeled rabbit anti-human Ig antibody (Southern Biotech, 6145-05) is used. Incubate for 30 minutes at room temperature, then wash 3 times with wash solution. Add 100 ⁇ L of the coloring solution contained in the peroxidase coloring kit (Sumitomo Bakelite Co., Ltd.) per well and let stand at room temperature for about 15 minutes, then add 100 ⁇ L of the reaction stopping solution contained in the coloring kit, and obtain the OD450 value.
- test antibody When the test antibody has a mouse Fc region, if the test antibody does not reach the maximum concentration (10000 ng / mL) measured in the MA-56A7C10 evaluation system even when the maximum concentration (100,000 ng / mL) is added, MA-56A7C10 The measurement value of the maximum density in the evaluation system is set to 100%. The calculated PAI-1 binding rate is analyzed, and the EC50 value of the test antibody is calculated by 4-parameter logistic curve regression.
- tPA (Activacin (Registered Trademark) Note 6 million; Kyowa Hakko Kirin Co., Ltd.) was diluted with PBS to 1000 units / mL, and 100 ⁇ L per well was added to Nunkumaxi Soap Clear 96 Plate (Nunc Co.) at 4 ° C. The tPA is immobilized on the plate by allowing the plate to stand overnight. The solid phase solution is removed by reverse centrifugation, 200 ⁇ L of blocking agent (Thermo SCIENTIFIC, 37532) is added per well, and the mixture is allowed to stand at room temperature for 30 minutes.
- blocking agent Thermo SCIENTIFIC, 37532
- Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) diluted to 1000 ng / mL with PBS containing 0.1% BSA is added at 100 ⁇ L per well and captured by tPA. Wash with a washing solution (0.05% Tween-20-containing TBS), add 100 ⁇ L of test antibody diluted in 8 steps from 0.1000 ng / mL to 0.01 ng / mL with PBS containing 0.1% BSA, and add room temperature Let stand for 30 minutes.
- a washing solution 0.05% Tween-20-containing TBS
- MEDI-579 Patent Document 3
- MA-56A7C10 Hycult Biotech, HM2182
- a mouse Fc region Dilute in 7 steps from 10000 ng / mL to 0.01 ng / mL with PBS containing% BSA and add to wells.
- a well to which PBS containing 0.1% BSA is added instead of the test antibody is prepared. After washing 3 times with the washing solution, 100 ⁇ L of detection antibody diluted 2000 times using a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20 times with PBS is added and reacted.
- a blocking agent Nacalai Tesque, 03953-95
- an HRP-labeled goat anti-mouse Ig antibody (Southern Biotech, 1010-05) is used for detection of a test antibody having an Fc region of a mouse antibody, and an HRP for detection of a test antibody having an Fc region of a human antibody.
- a labeled rabbit anti-human Ig antibody (Southern Biotech, 6145-05) is used. Incubate for 30 minutes at room temperature, then wash 3 times with wash solution. Add 100 ⁇ L of the coloring solution contained in the peroxidase coloring kit (Sumitomo Bakelite Co., Ltd.) per well and let stand at room temperature for about 15 minutes, then add 100 ⁇ L of the reaction stopping solution contained in the coloring kit, and obtain the OD450 value.
- test antibody When the test antibody has a mouse Fc region, if the test antibody does not reach the maximum concentration (10000 ng / mL) measured in the MA-56A7C10 evaluation system even when the maximum concentration (100,000 ng / mL) is added, MA-56A7C10 The measurement value of the maximum density in the evaluation system is set to 100%. The calculated PAI-1 binding rate is analyzed, and the EC50 value of the test antibody is calculated by 4-parameter logistic curve regression.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention comprises an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4
- binding of an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 to active human PAI-1 Inhibits and binds to active human PAI-1 complexed with vitronectin and does not bind to latent human PAI-1 nor to human PAI-1 complexed with plasminogen activator
- anti-human PAI-1 antibodies or antigen-binding fragments thereof are also included.
- an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 labeled with HRP or the like and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 or from amino acid number 1 of SEQ ID NO: 2
- a reagent for detecting the activity of the anti-human PAI-1 antibody comprising a heavy chain consisting of up to 447 amino acids and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 after reacting and washing (for example, In the case of HRP labeling, the activity is measured using a peroxidase coloring kit (Sumitomo Bakelite Co., Ltd.).
- an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 or amino acid numbers 1 to 447 of SEQ ID NO: 2
- the test antibody is determined from the amino acid sequence shown in SEQ ID NO: 2.
- An anti-human PAI-1 antibody comprising a heavy chain comprising and a light chain comprising the amino acid sequence represented by SEQ ID NO: 4 or a heavy chain comprising the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 2 and the amino acid represented by SEQ ID NO: 4 It can be determined that the binding between an anti-human PAI-1 antibody containing a light chain consisting of a sequence and active human PAI-1 is inhibited.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention comprises an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 Alternatively, it binds to the same human PAI-1 epitope as an anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid number 1 to 447 and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 4. Also included are anti-human PAI-1 antibodies or antigen-binding fragments thereof.
- the epitope refers to an antigen site recognized by an antibody.
- human PAI-1 partial peptide is immobilized, and a test antibody is added thereto for reaction.
- a secondary antibody such as an anti-IgG antibody labeled with HRP or the like is reacted, washed, and a reagent for detecting its activity (for example, in the case of HRP labeling, a color kit for peroxidase (Sumitomo Bakelite)) etc.
- a reagent for detecting its activity for example, in the case of HRP labeling, a color kit for peroxidase (Sumitomo Bakelite)
- the anti-human PAI-1 antibody in which the epitope of the test antibody comprises a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 or amino acid numbers 1 to 447 of SEQ ID NO: 2
- the epitope of the anti-human PAI-1 antibody comprising the heavy chain consisting of the amino acid sequence of FIG.
- test antibody is a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2
- an anti-human PAI-1 antibody comprising a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 or a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 2 and the amino acid sequence shown in SEQ ID NO: 4 It can be determined that it binds to the same human PAI-1 epitope as the anti-human PAI-1 antibody comprising the light chain.
- the anti-human PAI-1 antibody of the present invention includes a complex formed with vitronectin in addition to binding to the VTN-PAI-1 complex as long as the antibody binds to the VTN-PAI-1 complex.
- Antibodies that bind to active PAI-1 derived from these animals for example, active monkey PAI-1 complexed with vitronectin are also included.
- the anti-human PAI-1 antibody of the present invention can be obtained by using a method known in the art based on the sequence information of the heavy chain variable region and the light chain variable region of the antibody of the present invention disclosed in the present specification. Can be easily made by those skilled in the art.
- the anti-human PAI-1 antibody of the present invention is not particularly limited. For example, the following ⁇ Method for producing anti-human PAI-1 antibody of the present invention and anti-human PAI-1 produced by the method are described below. It can be produced according to the method described in ⁇ Antibodies>.
- the anti-human PAI-1 antibody of the present invention is further purified if necessary and then formulated according to a conventional method, and is pulmonary fibrosis such as idiopathic pulmonary fibrosis, interstitial pneumonia, systemic lupus erythematosus, scleroderma, diabetes Nephropathy, lupus nephritis, graft-versus-host disease, glomerulonephritis, nephrotic syndrome, renal fibrosis, chronic obstructive pulmonary disease, acute kidney injury, acute lung injury, acute respiratory failure, age-related macular degeneration, disseminated blood vessel Internal coagulation syndrome, postoperative adhesion, symptomatic vitreous macular adhesion, diabetic retinopathy, arteriosclerosis, myocardial infarction, cerebral infarction, pulmonary infarction, diseases with eye fibrosis such as conjunctival scarring after glaucoma surgery, peritoneal sclerosis It can be used for
- the polynucleotide of the present invention includes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof, and the anti-human PAI-1 antibody of the present invention or the same
- a polynucleotide comprising a base sequence encoding the light chain variable region of an antigen-binding fragment is included.
- the polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention is an amino acid sequence from amino acid numbers 1 to 118 of SEQ ID NO: 2.
- polynucleotide containing the base sequence encoding the heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 2 from amino acid Nos. 1 to 118 include, for example, a polynucleotide containing the base sequence of SEQ ID NO: 1 from base No. 1 to 354 Nucleotides are mentioned.
- the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody of the present invention is a polynucleotide comprising a base sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2. It is a nucleotide.
- Examples of the polynucleotide containing the base sequence encoding the heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 include the polynucleotide containing the base sequence shown in SEQ ID NO: 1.
- the polynucleotide comprising a base sequence encoding the light chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention is an amino acid sequence from amino acid numbers 1 to 108 of SEQ ID NO: 4.
- polynucleotide containing the base sequence encoding the light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid No. 1 to 108 include, for example, the polynucleotide comprising the base sequence of SEQ ID NO: 3 from base No. 1 to 324 Is mentioned.
- the polynucleotide comprising a nucleotide sequence encoding the light chain variable region of the anti-human PAI-1 antibody of the present invention comprises a polynucleotide comprising a nucleotide sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. It is a nucleotide.
- Examples of the polynucleotide containing the base sequence encoding the light chain consisting of the amino acid sequence shown in SEQ ID NO: 4 include the polynucleotide containing the base sequence shown in SEQ ID NO: 3.
- the polynucleotide of the present invention can be easily prepared by those skilled in the art using a method known in the art based on the base sequence.
- the polynucleotide of the present invention can be synthesized using gene synthesis methods known in the art.
- gene synthesis method various methods known to those skilled in the art, such as an antibody gene synthesis method described in WO90 / 07861, can be used.
- the expression vector of the present invention includes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof and / or the anti-human PAI-1 antibody of the present invention or the An expression vector comprising a polynucleotide comprising a base sequence encoding the light chain variable region of an antigen-binding fragment is included.
- Preferred expression vectors of the present invention include an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody of the present invention, and encodes the light chain of the anti-human PAI-1 antibody of the present invention.
- An expression vector comprising a polynucleotide comprising a base sequence, or a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody And an expression vector containing.
- Expression vectors used for expressing the polynucleotide of the present invention include various host cells such as eukaryotic cells (eg, animal cells, insect cells, plant cells, yeast) and / or prokaryotic cells (eg, E. coli).
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention and / or the light chain variable region of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention
- the polynucleotide is not particularly limited as long as it can express a polynucleotide containing a nucleotide sequence encoding and can produce a polypeptide encoded by these.
- expression vectors examples include plasmid vectors, virus vectors (eg, adenovirus, retrovirus) and the like.
- virus vectors eg, adenovirus, retrovirus
- pEE6.4 or pEE12.4 can be used.
- an antibody gene can be expressed by introducing a variable region gene fragment into an expression vector having a human Ig constant region gene in advance, such as AG- ⁇ 1 and AG- ⁇ (for example, see WO94 / 20632).
- the expression vector of the present invention may contain a promoter operably linked to the polynucleotide of the present invention.
- promoters for expressing the polynucleotide of the present invention in animal cells include virus-derived promoters such as CMV, RSV, SV40, actin promoter, EF (longation factor) 1 ⁇ promoter, heat shock promoter, and the like.
- the promoter for expression in bacteria include trp promoter, lac promoter, ⁇ PL promoter, tac promoter, and the like.
- promoters for expression in yeast include GAL1 promoter, GAL10 promoter, PH05 promoter, PGK promoter, GAP promoter, and ADH promoter.
- the expression vector of the present invention may contain a start codon and a stop codon.
- the expression vector of the present invention comprises an enhancer sequence, the antibody of the present invention or the 5 ′ and 3 ′ untranslated regions of the gene encoding the heavy chain variable region or light chain variable region thereof, secretory signal sequence, splicing It may contain a junction, a polyadenylation site, or a replicable unit.
- the expression vector of the present invention may contain a start codon, a stop codon, a terminator region, and a replicable unit.
- the expression vector of the present invention may contain a selection marker (for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene) that is usually used depending on the purpose. .
- a selection marker for example, tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, neomycin resistance gene, dihydrofolate reductase gene
- the transformed host cell of the present invention includes a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
- the transformed host cell of the present invention is a host cell transformed with the expression vector of the present invention selected from the group consisting of the following (a) to (d).
- C a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody of the present invention; and (d) an anti-human PAI-1 antibody of
- Preferred transformed host cells of the present invention include a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
- the host cell to be transformed is not particularly limited as long as it is compatible with the expression vector to be used and can be transformed with the expression vector to express the antibody.
- Examples of host cells to be transformed include various cells (for example, animal cells (for example, CHO-K1SV cells), insect cells, etc., such as natural cells or artificially established cells usually used in the technical field of the present invention. (For example, Sf9), bacteria (such as Escherichia), yeast (such as Saccharomyces and Pichia), and the like, preferably CHO cells (CHO-K1SV cells, CHO-DG44 cells, etc.), 293 cells, Cultured cells such as NS0 cells can be used.
- the method for transforming the host cell is not particularly limited, and for example, a calcium phosphate method, an electroporation method, or the like can be used.
- a host cell selected from the group consisting of the following (a) to (c) is cultured, and the anti-human PAI-1 antibody or A method of producing an anti-human PAI-1 antibody or antigen-binding fragment thereof comprising the step of expressing the antigen-binding fragment is included.
- the method for producing an anti-human PAI-1 antibody of the present invention comprises culturing a host cell selected from the group consisting of the following (a) to (c), and anti-human PAI-1 antibody:
- a method of producing an anti-human PAI-1 antibody comprising the step of expressing (A) A host transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human PAI-1 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody cell;
- the method of producing the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes the step of culturing the transformed host cell of the present invention and expressing the anti-human PAI-1 antibody or antigen-binding fragment thereof. As long as it is, it is not particularly limited.
- Preferred host cells used in the method include the above-described preferred transformed host cells of the present invention.
- the cultured host cells can be cultured by a known method.
- the culture conditions such as temperature, medium pH, and culture time are appropriately selected.
- the medium include MEM medium containing about 5 to 20% fetal bovine serum (Science, 1959, Vol. 130, No. 3373, p. 432-7), DMEM medium ( Virology, 1959, Vol. 8, p. 396), RPMI 1640 medium (J. Am. Med. Assoc., 1967, Vol. 199, p. 519), 199 medium (Exp. Biol. Med., 1950, Vol. 73, p.1-8) and the like can be used.
- the pH of the medium is preferably about 6 to 8, and the culture is usually carried out at about 30 to 40 ° C. for about 15 to 72 hours with aeration and stirring as necessary.
- the host cell is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, 1985, Vol. 82, p. 8404) can be used as the medium. .
- the pH of the medium is preferably about 5 to 8, and the culture is usually carried out at about 20 to 40 ° C. for about 15 to 100 hours with aeration and agitation as necessary.
- a liquid medium containing a nutrient source is appropriate.
- the nutrient medium preferably contains a carbon source, inorganic nitrogen source or organic nitrogen source necessary for the growth of the transformed host cell.
- the carbon source include glucose, dextran, soluble starch, and sucrose.
- the inorganic nitrogen source or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, and meat extract. , Soybean meal, potato extract and the like.
- other nutrients eg, inorganic salts (eg, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, etc.), antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.) Good.
- the pH of the medium is preferably about 5-8.
- Escherichia coli for example, LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, 2001, Vol. 3, A2.2) can be used as a preferable medium. Cultivation is usually carried out at about 14 to 39 ° C. for about 3 to 24 hours with aeration and agitation as necessary.
- yeast for example, a Burkholder minimum medium (Proc. Natl. Acad. Sci. USA, 1980, Vol. 77, p. 4505) can be used as the medium. Cultivation is usually carried out at about 20 to 35 ° C. for about 14 to 144 hours with aeration or agitation as necessary.
- the method of producing the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes the step of culturing the transformed host cell of the present invention and expressing the anti-human PAI-1 antibody or antigen-binding fragment thereof. In addition, it may further comprise a step of recovering, preferably isolating or purifying the anti-human PAI-1 antibody or antigen-binding fragment thereof from the transformed host cell. Isolation or purification methods include, for example, methods using solubility such as salting out and solvent precipitation, methods using differences in molecular weight such as dialysis, ultrafiltration and gel filtration, ion exchange chromatography and hydroxylapatite chromatography.
- the antibody accumulated in the culture supernatant can be purified by various chromatography, for example, column chromatography using a protein A column or a protein G column.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes an anti-human PAI-1 antibody or antigen-binding fragment thereof produced by the method for producing the anti-human PAI-1 antibody or antigen-binding fragment of the present invention. Is also included.
- the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof and a pharmaceutically acceptable excipient.
- the pharmaceutical composition of the present invention can be prepared by an ordinarily used method using an excipient usually used in the art, that is, a pharmaceutical excipient or a pharmaceutical carrier.
- Examples of dosage forms of these pharmaceutical compositions include parenteral agents such as injections and infusions, and can be administered by intravenous administration, subcutaneous administration, or the like.
- excipients, carriers, additives and the like corresponding to these dosage forms can be used within a pharmaceutically acceptable range.
- the pharmaceutical composition of the present invention may contain a plurality of types of anti-human PAI-1 antibodies of the present invention or antigen-binding fragments thereof.
- the present invention also includes an antibody that has not undergone post-translational modification or an antigen-binding fragment thereof, and a pharmaceutical composition containing an antibody or antigen-binding fragment thereof that is generated by post-translational modification of the antibody or antigen-binding fragment thereof. .
- the pharmaceutical composition of the invention containing an anti-human PAI-1 antibody or antigen-binding fragment thereof also includes the pharmaceutical composition described below.
- An anti-human PAI-1 antibody comprising the heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2 and the light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4 or an antigen thereof
- a pharmaceutical composition comprising an anti-human PAI-1 antibody or antigen-binding fragment thereof which is a binding fragment and an antibody or antigen-binding fragment thereof produced by post-translational modification of the antibody or antigen-binding fragment thereof.
- the pharmaceutical composition of the present invention comprises a heavy chain C-terminal lysine-deleted antibody, an antibody that has undergone N-terminal post-translational modification or an antigen-binding fragment thereof, a heavy chain C-terminal lysine that has been deleted, and N-terminal post-translational modification. And / or a pharmaceutical composition containing a heavy chain C-terminal lysine and an antibody that has not undergone N-terminal post-translational modification.
- the pharmaceutical composition of the present invention containing an anti-human PAI-1 antibody includes a pharmaceutical composition containing two or more anti-human PAI-1 antibodies of the following (1) to (4): Is also included.
- An anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 447 of SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4.
- an anti-human PAI-1 antibody comprising an amino acid sequence represented by SEQ ID NO: 2 and comprising a heavy chain in which glutamic acid of amino acid number 1 is modified with pyroglutamic acid and a light chain comprising the amino acid sequence represented by SEQ ID NO: 4 .
- the pharmaceutical composition of the present invention containing an anti-human PAI-1 antibody or an antigen-binding fragment thereof also includes the pharmaceutical composition described below.
- An anti-human PAI-1 antibody comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2 and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4; a heavy consisting of the amino acid sequence from amino acid numbers 1 to 447 of SEQ ID NO: 2
- a pharmaceutical composition comprising an anti-human PAI-1 antibody comprising a chain and a light chain comprising the amino acid sequence shown in SEQ ID NO: 4, and a pharmaceutically acceptable excipient.
- the amount of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention in the formulation varies depending on the degree and age of the patient's symptoms, the dosage form of the formulation to be used, the binding titer of the antibody, etc. For example, about 0.001 mg / kg to 100 mg / kg can be used.
- the pharmaceutical composition of the present invention can be used as a prophylactic or therapeutic agent for diseases in which active human PAI-1 is involved in pathogenesis, for example, pulmonary fibrosis such as idiopathic pulmonary fibrosis.
- the present invention includes a pharmaceutical composition for preventing or treating pulmonary fibrosis such as idiopathic pulmonary fibrosis, comprising the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof.
- the present invention also includes a method for treating or preventing pulmonary fibrosis such as idiopathic pulmonary fibrosis, comprising the step of administering a therapeutically effective amount of the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention. included.
- the present invention also includes the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof for use in the prevention or treatment of pulmonary fibrosis such as idiopathic pulmonary fibrosis.
- the present invention includes the use of the anti-human PAI-1 antibody of the present invention or an antigen-binding fragment thereof in the manufacture of a pharmaceutical composition for preventing or treating pulmonary fibrosis such as idiopathic pulmonary fibrosis.
- a person skilled in the art can prepare an antibody or antigen-binding fragment thereof as a fusion antibody obtained by fusing another peptide or protein using a method known in the art or a modified antibody to which a modifying agent is bound. It is also possible to do.
- the anti-human PAI-1 antibody or antigen-binding fragment thereof of the present invention includes such a fusion or modified form of antibody or antigen-binding fragment.
- an anti-human PAI-1 comprising a heavy chain variable region comprising the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4
- the antibody or antigen-binding fragment thereof also includes an anti-human PAI-1 antibody or antigen-binding fragment thereof fused with another peptide or protein, or an anti-human PAI-1 antibody or antigen-binding fragment thereof bound with a modifying agent.
- the antibody or antigen-binding fragment thereof of the present invention has a binding activity with the VTN-PAI-1 complex as a fusion antibody, other peptides or proteins used for fusion are not particularly limited.
- the modifying agent used for the modification is not particularly limited.
- concentration mol / L is expressed as M.
- 1M sodium hydroxide aqueous solution means 1 mol / L sodium hydroxide aqueous solution.
- Example 1 Examination of immune antigen and production of anti-human PAI-1 antibody-producing hybridoma
- an optimal human PAI-1 peptide antigen was examined.
- a multiple antigen peptide 8 (MAP8) peptide J. Biol. Chem., was added to the C-terminus of the peptide antigen STAVIVSARMAPEEII (SEQ ID NO: 5) of 16 amino acid residues in human PAI-1. 1988, Vol. 263, No. 4, p. 1719-1725), it was revealed that an antibody that neutralizes active human PAI-1 in plasma can be obtained.
- MAP8 multiple antigen peptide 8
- STAVIVSARMAPEEII SEQ ID NO: 5
- mice Human monoclonal antibody development technology “Velosimune” (Velocimmune technology: Regeneron (US Pat. No. 6,596,541)) mice were used to produce antibodies. Specifically, a peptide antigen in which a KLH protein is linked to the C-terminus of a peptide amino acid STAVIVSARMAPEEII (SEQ ID NO: 5) of 16 amino acid residues in human PAI-1 together with an adjuvant that elicits an immune response, in a Verosimune mouse Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) was immunized. Mice were immunized several times and the increase in antibody titer in plasma was confirmed.
- STAVIVSARMAPEEII SEQ ID NO: 5
- the resulting antibodies are human antibody variable regions and mouse antibody constant regions.
- An antibody having a region also referred to as a chimeric antibody.
- Example 2 An ELISA assay was used to measure the antigen-specific binding activity of the antibody.
- a plate prepared by immobilizing vitronectin and adding active human PAI-1, and a latent type instead of active human PAI-1 The test was performed using a plate prepared by adding human PAI-1.
- Vitronectin (BD Biosciences, 354238) was diluted to 1000 ng / mL with phosphate buffered saline (PBS), and 100 ⁇ L per well was added to Nunkumaxisorp transparent 96 plates (Nunc) at 4 ° C. By keeping the plate overnight, vitronectin was immobilized.
- Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) or recombinant latent human PAI-1 (Molecular Innovation) diluted with PBS to a concentration of 1000 ng / mL with vitronectin. PAI-L) was added at 100 ⁇ L per well and allowed to stand at 4 ° C. for 1 hour.
- the solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Nacalai Tesque, 03953-95) solution diluted 3-fold with PBS was added per well. After leaving still at room temperature for 1 hour, the blocking agent was removed by reverse centrifugation.
- a blocking agent solution Nacalai Tesque, 03953-95
- purified antibody samples were added at 1000 ng / mL to 0.1 ng / mL for plates with activated human PAI-1.
- the plate to which latent human PAI-1 was added was diluted in stages from 3000 ng / mL to 0.3 ng / mL, and 100 ⁇ L was added per well.
- a well to which a blocking agent solution diluted 20 times with PBS instead of an antibody was added was prepared. After incubation at room temperature for 1 hour, the plate was washed 3 times with a washing solution (Tris buffered saline (TBS) containing 0.05% Tween-20), and diluted 2000 times with a blocking agent solution diluted 20 times with PBS. 100 ⁇ L of HRP-labeled rabbit anti-mouse Ig antibody (DAKO, P260) was added. After incubating at room temperature for 30 minutes, the plate was washed 3 times with a washing solution.
- TBS Tris buffered saline
- DAKO HRP-labeled rabbit anti-mouse Ig antibody
- the measured value of the well to which a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20-fold with PBS instead of the antibody was added was 0%.
- the maximum measured value of each antibody was set as 100%.
- the PAI-1 binding rate calculated from the measured value was analyzed, and the EC50 value of the antibody was calculated by four parameter logistic curve regression.
- HOT-1 chimeric antibody
- Example 3 Production of fully human antibody
- the aforementioned antibody is an antibody in which the variable region is derived from human and the constant region is derived from mouse. Therefore, the present inventors constructed an expression vector containing both heavy chain and light chain genes using a GS vector (Lonza), which is a mammalian cell expression vector, and produced a fully human antibody.
- GS vector Longza
- RNA was extracted from the hybridoma, and cDNA was prepared using a cDNA amplification kit (SMARTER RACE cDNA Amplification kit; Clontech). Polymerase chain reaction (PCR) was then used to extend and amplify the heavy and light chain variable regions.
- a gene encoding a signal sequence (Nigel White et al., Protein Engineering, 1987, Vol. 1, No. 6, p. 499-505) 5 ′ of the heavy chain variable region gene of HOT-1 and 3 ′
- the human Ig ⁇ 1 constant region gene (consisting of the nucleotide sequence from nucleotide numbers 355 to 1344 of SEQ ID NO: 1) was ligated to each, and this heavy chain gene was inserted into the GS vector pEE6.4.
- a gene encoding a signal sequence is located 5 ′ of the light chain variable region gene of HOT-1, and a constant region gene of human ⁇ chain (amino acid of SEQ ID NO: 3) is located 3 ′.
- the light chain genes were inserted into the GS vector pEE12.4.
- the heavy chain gene sequence of the antibody inserted into pEE6.4 and the light chain gene sequence of the antibody inserted into pEE12.4 were analyzed with a sequencer, and the resulting amino acid sequence was used to determine the database of Kabat et al. (“Sequences of Proteins of The CDR sequence was determined with reference to "Immunological Interest", US Department of Health and Human Services, US Government Printing Office).
- the base sequence of the heavy chain of the fully human antibody of HOT-1 thus prepared (fully human HOT-1) is shown in SEQ ID NO: 1, the amino acid sequence encoded thereby is shown in SEQ ID NO: 2, and the base of the light chain of the antibody The sequence is shown in SEQ ID NO: 3, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 4, respectively.
- the variable region of the heavy chain represented by SEQ ID NO: 2 consists of the amino acid sequence of amino acid numbers 1 to 118 of SEQ ID NO: 2, and the CDR1, CDR2, and CDR3 of the heavy chain are amino acid numbers 31 to 35 of SEQ ID NO: 2, respectively. It consists of an amino acid sequence from 50 to 66, 99 to 107.
- variable region of the light chain represented by SEQ ID NO: 4 consists of the amino acid sequence of amino acid numbers 1 to 108 of SEQ ID NO: 4, and the CDR1, CDR2, and CDR3 of the light chain are amino acid numbers 24 to 34 of SEQ ID NO: 4, respectively. It consists of an amino acid sequence from 50 to 56, 89 to 97.
- the above-mentioned GS vector in which the heavy chain and light chain genes of fully human HOT-1 are inserted is digested with NotI and PvuI, and ligated using a DNA Ligation Kit (Takara Bio Inc.).
- a GS vector in which both the light chain and light chain genes were inserted was constructed.
- antibody expression was performed by two methods, transient expression and constitutive expression.
- FreeStyleMAX Invitrogen
- FreeStyleMAX was used as a gene transfer reagent for FreeStyle 293 cells (Invitrogen) cultured at approximately 1 million cells / mL in FreeStyle 293 Expression medium (Invitrogen). And cultured for 7 days.
- a purified human G antibody was obtained from the culture supernatant using a protein G column (GE Healthcare).
- the antibody was expressed by transfecting the GS vector into CHO-K1SV cells (Lonza) using electroporation.
- a fully human antibody was purified from the culture supernatant using a protein A column (GE Healthcare).
- Example 5 Measurement of active PAI-1 inhibitory activity in human plasma
- HOT-1 chimeric antibody
- Example 3 the plasma of obese patients was used.
- An active PAI-1 inhibition assay was performed.
- the concentration of active PAI-1 is low and it is difficult to construct an assay system. Therefore, the plasma of obese patients whose plasma active PAI-1 concentration is known to be high is used. (Nature Medicine, 1996, Vol. 2, p. 800-803).
- uPA Urokinase for intravenous injection 60,000 units “Benesis” (registered trademark); Mitsubishi Tanabe Pharma Co., Ltd.) was diluted with PBS to 50 units / mL, and assay plate Numkumaxisorp white 96 plate (Nunc) 100 ⁇ L per well was added to the plate, and the plate was allowed to stand at 4 ° C. overnight to immobilize uPA.
- the solid phase solution was removed by reverse centrifugation, 200 ⁇ L of a blocking agent (Nacalai Tesque, 03953-95) solution diluted 3-fold with PBS was added per well, and the mixture was allowed to stand at room temperature for 1 hour.
- a mixed solution of plasma of the obese patient and the antibody sample (plasma-antibody mixed solution) was prepared.
- the plasma was diluted with PBS containing 0.1% bovine serum-derived albumin (BSA) so that the plasma of the obese patient was diluted 35-fold when the plasma-antibody mixture was prepared (plasma-antibody
- BSA bovine serum-derived albumin
- the concentration of active PAI-1 derived from human plasma contained in the mixture is about 360 pg / mL).
- the antibody sample was diluted with 0.1% BSA-containing PBS so that the final antibody concentration was 1000 ng / mL to 1 ng / mL in a 7-step dilution series. Thereafter, the plasma and the antibody sample were mixed to prepare a plasma-antibody mixed solution.
- a calibration curve sample recombinant active human PAI-1 (Molecular Innovation, PAI-A) diluted in 7 stages from 10000 pg / mL to 10 pg / mL with PBS containing 0.1% BSA was prepared.
- a control sample a sample prepared by mixing PBS containing 0.1% BSA with obese patient plasma instead of the antibody was prepared. These plasma-antibody mixed solution, calibration curve sample, and control sample were allowed to stand at room temperature for 30 minutes and pre-incubated.
- the plate was washed 3 times with a washing solution, and a biotinylated anti-mouse Ig antibody (2000-fold diluted with a blocking agent solution (Nacalai Tesque, 03953-95) diluted 20-fold with PBS) ( Ancell, 234010) was added at 100 ⁇ L per well.
- a biotinylated anti-mouse Ig antibody 2000-fold diluted with a blocking agent solution (Nacalai Tesque, 03953-95) diluted 20-fold with PBS) ( Ancell, 234010) was added at 100 ⁇ L per well.
- the plate was washed 3 times with a washing solution, and alkaline phosphatase-labeled streptavidin (Thermo) diluted 1000-fold with a blocking agent (Nacalai Tesque, 03953-95) diluted 20-fold with PBS.
- SCIENTIFIC was added at 100 ⁇ L per well.
- alkaline phosphatase substrate (Chemiluminescent AP; SurModics, APU4-0100-01) diluted 5-fold with 0.1 mM magnesium chloride aqueous solution containing 2 mM Tris (pH 9.8) was added per well, and the signal value was measured with an EnVision counter ( Perkin Elmer).
- the active human PAI-1 concentration was calculated using a calibration curve. Each antibody was tested in duplicate, and the average value was calculated. Well in which 0.1% BSA-containing PBS mixed with obesity patient plasma instead of antibody was added and active human PAI-1 concentration was set as 100%, and antibody 1000 ng / mL was mixed with obesity patient plasma The active human PAI-1 concentration was set at 0%. The calculated inhibition rate of active human PAI-1 was analyzed, and the IC50 value of the antibody was calculated by 4-parameter logistic curve regression.
- the IC50 value of HOT-1 (chimeric antibody) was 11.3 ng / mL, and the IC50 of fully human HOT-1 was 6.0 ng / mL. Both antibodies were active in human plasma. It was found to inhibit the uPA binding activity of PAI-1.
- Example 6 Evaluation of binding selectivity for VTN-PAI-1 complex
- ELISA assay the binding selectivity of the fully human HOT-1 prepared in Example 3 to the VTN-PAI-1 complex was evaluated.
- the following four types of human PAI-1 solid phase plates (1) to (4) were prepared and tested using each plate.
- VTN-PAI-1 complex-immobilized plate Vitronectin (BD Biosciences, 354238) was diluted to 1000 ng / mL with PBS, and 100 ⁇ L per well was added to Nunkumaxisorp transparent 96 plates (Nunc). Vitronectin was immobilized by adding and allowing the plate to stand overnight at 4 ° C.
- the solid phase solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Thermo SCIENTIFIC, 37532) was added per well and allowed to stand at room temperature for 30 minutes.
- a blocking agent Thermo SCIENTIFIC, 37532
- Recombinant active human PAI-1 (Molecular Innovation, PAI-A) diluted to 1000 ng / mL with PBS containing 0.1% BSA was added at 100 ⁇ L per well and captured by vitronectin.
- the binding activity of the antibody to VTN-PAI-1 was evaluated by the test on this plate.
- Latent human PAI-1 solid-phase plate Recombinant latent human PAI-1 (Molecular Innovation, PAI-L) was diluted to 1000 ng / mL with PBS, and a Nunkumaxisorp transparent 96 plate ( The latent human PAI-1 was immobilized by adding 100 ⁇ L per well to Nunc) and allowing the plate to stand at 4 ° C. overnight. The solid phase solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Thermo SCIENTIFIC, 37532) was added per well and allowed to stand at room temperature for 30 minutes. The binding activity of the antibody against latent human PAI-1 was evaluated by the test on this plate.
- a blocking agent Thermo SCIENTIFIC, 37532
- uPA-PAI-1 complex-immobilized plate uPA (60,000 units for intravenous injection of urokinase “Benesis” (registered trademark); Mitsubishi Tanabe Pharma Co., Ltd.) was diluted with PBS to 100 units / mL, UPA was solid-phased by adding 100 ⁇ L per well to a Nunkumaxisorp transparent 96 plate (Nunc) and allowing the plate to stand at 4 ° C. overnight. The solid phase solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Thermo SCIENTIFIC, 37532) was added per well and allowed to stand at room temperature for 30 minutes.
- a blocking agent Thermo SCIENTIFIC, 37532
- tPA-PAI-1 complex-immobilized plate tPA (Activacin (registered trademark) Note 6 million; Kyowa Hakko Kirin Co., Ltd.) was diluted with PBS to 1000 units / mL, and Nunkumaxisorp transparent 96 plate (Nunc) was added with 100 ⁇ L per well, and the plate was allowed to stand at 4 ° C. overnight to immobilize tPA.
- the solid phase solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Thermo SCIENTIFIC, 37532) was added per well and allowed to stand at room temperature for 30 minutes.
- Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) diluted to 1000 ng / mL with PBS containing 0.1% BSA was added at 100 ⁇ L per well and captured by tPA. The binding activity of the antibody to the tPA-PAI-1 complex was evaluated by the test on this plate.
- Each prepared human PAI-1 solid phase plate was washed with a washing solution (0.05% Tween-20-containing TBS), and diluted in 8 steps from 100000 ng / mL to 0.01 ng / mL with PBS containing 0.1% BSA. 100 ⁇ L / well of fully human HOT-1 was added and allowed to stand at room temperature for 30 minutes.
- a washing solution 0.05% Tween-20-containing TBS
- MEDI-579 Patent Document 3 which is a fully human anti-human PAI-1 antibody, MA-33B8 (Progen Biotechnik), which is a mouse anti-human PAI-1 antibody, and MA-33H1F7 (Progen Biotechnik) MA-55F4C12 (Hycult Biotech, HM2180) and MA-56A7C10 (Hycult Biotech, HM2182) were diluted in 7 steps from PBS ng / mL to 0.01 ng / mL using 0.1% BSA-containing PBS. 100 ⁇ L was added per well and allowed to stand at room temperature for 30 minutes. As a control, wells were prepared in which PBS containing 0.1% BSA was added instead of the antibody.
- a detection antibody diluted 2000 times using a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20 times with PBS was added and allowed to react.
- HRP-labeled goat anti-mouse Ig antibody (Southern Biotech, 1010-05) is used for detection of a test antibody having an Fc region of a mouse antibody, and HRP is used for detection of a test antibody having an Fc region of a human antibody.
- a labeled rabbit anti-human Ig antibody (Southern Biotech, 6145-05) was used. After incubating at room temperature for 30 minutes, the plate was washed 3 times with a washing solution.
- Example 7 Evaluation of binding to monomeric active human PAI-1
- SPR analysis was performed.
- analysis was performed using Biacore (registered trademark) T200 (GE Healthcare Japan) and recombinant active human PAI-1 (Molecular Innovation, PAI-A).
- Biacore registered trademark
- T200 GE Healthcare Japan
- PAI-1 Molecular Innovation, PAI-A
- Example 8 Evaluation of binding selectivity for monkey PAI-1
- An ELISA assay was used to assess the binding selectivity of fully human HOT-1 to activated monkey PAI-1 complexed with vitronectin (also referred to as VTN-monkey PAI-1 complex).
- the following four monkey PAI-1 solid phase plates (1) to (4) were prepared and tested using each plate.
- MEDI-579 was used as a comparative antibody.
- VTN-monkey PAI-1 complex-immobilized plate According to the method described in Example 6 (1), the binding activity of the antibody to VTN-monkey PAI-1 was evaluated. However, recombinant activated monkey PAI-1 (Molecular Innovation, CYPAI) was used instead of recombinant activated human PAI-1. In addition, fully human HOT-1 and MEDI-579 were used after diluting them in 7 steps from 10000 ng / mL to 0.01 ng / mL.
- Latent monkey PAI-1 immobilized plate According to the method described in Example 6 (2), the binding activity of the antibody to latent monkey PAI-1 was evaluated.
- recombinant latent monkey PAI-1 (Molecular Innovation, CYPAI-L) was used instead of recombinant latent human PAI-1.
- fully human HOT-1 and MEDI-579 were used after diluting them in 7 steps from 10000 ng / mL to 0.01 ng / mL.
- (3) uPA-monkey PAI-1 complex-immobilized plate According to the method described in Example 6 (3), the binding activity of the antibody to the uPA-monkey PAI-1 complex was evaluated.
- recombinant activated monkey PAI-1 (Molecular Innovation, CYPAI) was used instead of recombinant activated human PAI-1.
- Example 9 Measurement of active PAI-1 inhibitory activity in human and monkey plasma
- Evaluation of active PAI-1 inhibitory activity in human and monkey plasma of fully human HOT-1 and MEDI-579 was performed.
- human plasma the plasma of obese patients was used as in Example 5.
- monkey plasma LPS was intravenously administered to cynomolgus monkeys, and the collected plasma was used.
- Example 5 For the evaluation of active PAI-1 inhibitory activity in human plasma, the method of Example 5 was followed. Regarding the evaluation of active PAI-1 inhibitory activity in monkey plasma, monkey plasma was used in place of human plasma in the method described in Example 5. For monkey plasma, the plasma was diluted with PBS containing 0.1% BSA so that the monkey plasma was diluted 120-fold when the plasma-antibody mixture was prepared. In both evaluation systems, the final concentration of each antibody was 300 ng / mL to 0.3 ng / mL (7 step dilution).
- Each active PAI-1 concentration was calculated using a calibration curve. Each antibody was tested in duplicate, and the average value was calculated.
- the active type PAI-1 concentration in a well containing a sample prepared by mixing PBS containing 0.1% BSA with obesity patient plasma or monkey plasma instead of the antibody was set as 100%, and MEDI-579 antibody 300 ng / mL was set as obesity patient
- Each active PAI-1 concentration in wells mixed with plasma or monkey plasma was set as 0%.
- the calculated inhibition rate of each active PAI-1 was analyzed, and the IC50 value of the antibody was calculated by 4-parameter logistic curve regression.
- the IC50 value of fully human HOT-1 was 4.2 ng / mL and the IC50 value of MEDI-579 was 0.73 ng / mL with respect to the uPA binding activity of active PAI-1 in human plasma.
- the IC50 value of fully human HOT-1 was 37 ng / mL and the IC50 value of MEDI-579 was 0.83 ng / mL for the uPA binding activity of monkey plasma active PAI-1.
- Example 10 Measurement of PAI-1 inhibitory activity in an in vitro intravascular reproduction system
- uPA and tPA are produced from the vascular endothelium in the vascular wall and bound to the vascular endothelium (Ba Amsterdam Clin. Haematol., 1993, Vol. 6, No. 3, p. 559-576). Blood, 2011, Vol.118, No.11, p.3182-3185). That is, uPA and tPA are present not only in the blood but also in the blood vessel wall.
- Example 9 the inhibitory activity of active PAI-1 in plasma by an anti-PAI-1 antibody was measured. Since this experimental system extracts only plasma components, the influence of the blood vessel wall is taken into consideration. Absent.
- an evaluation system in which active PAI-1, uPA and uPA substrate peptide are mixed with an assay plate on which a uPA-PAI-1 complex is immobilized is used as a fully human.
- the activity of type HOT-1 and MEDI-579 was measured using the activity of uPA as an index.
- Recombinant activated human PAI-1 (Molecular Innovation, PAI-A) was diluted with PBS to a concentration of 10 ⁇ g / mL, added to Numkumaxisope white 96 plate (Nunc) at 100 ⁇ L per well, and at room temperature.
- the active PAI-1 was immobilized by allowing the plate to stand for 20 minutes.
- the solid phase solution was removed by reverse centrifugation, 200 ⁇ L of a blocking agent (Nacalai Tesque, 03953-95) solution was added per well, and the mixture was allowed to stand at room temperature for 15 minutes.
- uPA 60,000 units for intravenous injection of urokinase “Benesis” (registered trademark); Mitsubishi Tanabe Pharma Corporation) is adjusted to 50 Unit / mL with a blocking agent diluted 3 times with PBS. Dilute and add 100 ⁇ L per well. After standing at room temperature for 20 minutes, 200 ⁇ L of bicarbonate buffer (Sigma, C3041-50CAP) was added per well and allowed to stand at 37 ° C. overnight.
- each antibody was tested in duplicate and the average value was calculated.
- the average value of wells added with PBS containing 0.1% BSA instead of the antibody was taken as 0%, and the antibody and recombinant human active PAI-1
- the average value of the measured values of wells to which PBS containing PBS containing 0.1% BSA and PBS was added was set to 100%.
- the calculated uPA activity rate was analyzed, and the EC50 value of the antibody was calculated by 4-parameter logistic curve regression. Since PAI-1 inhibits uPA activity, the higher the human PAI-1 inhibitory activity of the antibody, the higher the uPA activity.
- the EC50 value of fully human HOT-1 was 12.7 ng / mL and the EC50 value of MEDI-579 was 42.1 ng / mL using the uPA activity in this evaluation system as an index. It was revealed that fully human HOT-1 has higher uPA activation ability. Therefore, in this evaluation system, it was revealed that fully human HOT-1 has a higher PAI-1 inhibitory activity.
- Example 11 Measurement of antibody concentration in monkey plasma
- Example 6 From the results of Example 6, it was revealed that fully human HOT-1 showed a strong binding activity to the VTN-PAI-1 complex and had a very high selective binding activity.
- antibodies in blood are known to be metabolized as antigen-antibody complexes by binding to antigens in addition to being metabolized as they are (Journal of Pharmaceutical Sciences, 2012, Vol. 101, No. 12). P4367-4382).
- PAI-1 in plasma exists in various complex states such as latent PAI-1 and tPA-PAI-1 complex (Blood, 1990, Vol. 76, p.
- anti-PAI-1 antibodies with low selectivity are metabolized by forming antigen-antibody complexes with various PAI-1.
- fully human HOT-1 binds selectively to VTN-PAI-1 and is not easily metabolized as a complex with latent PAI-1 or PA-PAI-1. There was a possibility of being. In fact, it is known that the persistence of antibody concentration in blood greatly depends on the metabolic rate of this antigen-antibody complex (Bioanalysis, 2011, Vol. 3, No. 6, p. 659-675). From the results of Example 8, since fully human HOT-1 showed high selective binding activity to the VTN-monkey PAI-1 complex, the antibody concentration in plasma when the antibody was administered to monkeys was measured. MEDI-579 was used as a comparative antibody.
- Cynomolgus monkeys were intravenously administered with fully human HOT-1 or MEDI-579 diluted in PBS. Treatment groups were set up as follows. [Treatment group] HOT-1 administration group (3 animals): Group (2.0 mg / kg) administered with fully human HOT-1 MEDI-579 administration group (2 animals): Group administered with MEDI-579 (2.0 mg / kg) Blood was collected before antibody administration and 0.25, 1, 2, 4, 8, 24, 48, 96, 168, 336, and 504 hours after antibody administration to obtain plasma. For fully human HOT-1, additional blood was collected after 672, 936, 1200, and 1440 hours to obtain plasma.
- ECL electrochemiluminescence
- the ECL assay for fully human HOT-1 is shown below.
- Vitronectin was diluted with TBS to 100 ng / mL, and 25 ⁇ L was added per well using Multi-array 96-well Plate (Meso Scale Discovery, L15XA-1). By leaving the plate at 4 ° C. overnight, vitronectin was immobilized on the plate. The vitronectin solid phase solution was removed by reverse centrifugation, washed with a washing solution (0.05% Tween-20-containing TBS) three times, and diluted with TBS to 100 ng / mL. Recombinant activated human PAI-1 (Molecular Innovation, Inc., PAI-A) was added at 25 ⁇ L per well and allowed to stand at 4 ° C.
- PAI-1 Molecular Innovation, Inc., PAI-A
- Antibody-treated monkey plasma is diluted 100-fold with 0.05% Tween-20-containing blocking agent 1 solution, and further 0.05% Tween-20-containing blocking agent 1 solution containing 1% of antibody-untreated monkey plasma ( (Hereinafter referred to as plasma-Tween-containing blocking agent 1 solution) and diluted to be within the range of the calibration curve, and 25 ⁇ L was added per well.
- plasma-Tween-containing blocking agent 1 solution 1% of antibody-untreated monkey plasma
- 25 ⁇ L was added per well.
- wells containing fully human HOT-1 diluted in 11 stages from 100 ng / mL to 0.0017 ng / mL using a plasma-Tween-containing blocking agent 1 solution were prepared.
- As a control a well to which a plasma-Tween-containing blocking agent 1 solution was added instead of an antibody was prepared.
- the plasma concentration of MEDI-579 was measured according to the method of ECL assay of fully human HOT-1. However, the concentrations of vitronectin and recombinant active human PAI-1 were used at 75 ng / mL. Further, as a blocking step after solidifying the recombinant active PAI-1, the following blocking treatment was performed instead of the blocking agent 1 treatment.
- Blocking agent 2 (Meso Scale Discovery, R93BA-1) dissolved in TBS at 5 w / v% was added at 150 ⁇ L per well and allowed to stand at room temperature for 1 hour. After removing the blocking agent and washing three times with the washing solution, 50 ⁇ L of blocking agent 3 (Meso Scale Discovery, R51BB-3) diluted 4-fold with blocking agent 1 was added per well.
- a calibration curve was prepared. The regression equation was analyzed using a 5-parameter logistic model. Weighting was 1 / y 2. The antibody concentration at each blood collection point was calculated from the calibration curve. Each plasma test was performed in triplicate, and the average value of the calculated concentrations was determined. For each test substance and each test monkey, the obtained antibody concentration was analyzed by BioBook (IDBS), and the vertical axis represents the logarithmic value of the antibody concentration, and the horizontal axis represents the blood sampling time from the slope of the ⁇ layer. Half-life was calculated.
- IDBS BioBook
- Example 12 Measurement of active PAI-1 inhibitory activity in monkey blood
- MEDI-579 was used as a comparative antibody.
- Example 11 the inhibitory activity of blood active PAI-1 was measured using plasma obtained from monkeys administered with fully human HOT-1 or MEDI-579.
- uPA 60,000 units for intravenous injection of urokinase “Benesis (registered trademark)”; Mitsubishi Tanabe Pharma Co., Ltd.
- PBS 50 units / mL
- Nunkumaxisorp white 96 plate 100 ⁇ L was added, and the plate was allowed to stand at 4 ° C. overnight to immobilize uPA.
- the solid phase solution was removed by reverse centrifugation, and 200 ⁇ L of a blocking agent (Thermo SCIENTIFIC, 37532) was added per well and allowed to stand at room temperature for 1 hour.
- the plate was washed with a washing solution (TBS containing 0.05% Tween-20), diluted 10-fold with 0.1% BSA-containing PBS, 100 ⁇ L per well, and allowed to stand at room temperature for 30 minutes.
- a washing solution TBS containing 0.05% Tween-20
- human active PAI-1 (Molecular Innovation, PAI-A) was diluted in 7 steps from 300 ng / mL to 0.003 ng / mL using PBS containing 0.1% BSA. 100 ⁇ L was added per well and allowed to stand at the same time as plasma for 30 minutes at room temperature.
- wells were prepared in which PBS containing 0.1% BSA was added instead of the antibody.
- Biotinylation (Dojindo Laboratories, LK03) anti-PAI-1 diluted 10,000 times using a blocking agent (Nacalai Tesque, 03953-95) solution diluted 20 times with PBS
- the antibody (Progen Biotechnik, MA-33H1F7) was added at 100 ⁇ L per well for reaction.
- MA-33H1F7 was used as an anti-PAI-1 antibody that recognizes the uPA-PAI-1 complex on the plate. Biotinylation was performed by the method specified in the kit of Dojindo Laboratories.
- the active PAI-1 concentration was calculated using a calibration curve. The results of the inhibitory action of each antibody on the active PAI-1 in monkey plasma are shown in FIG. Each plasma was tested in duplicate, and the average value was calculated. After that, the monkey plasma values of each group used in the test were averaged. Calculate the inhibition ratio from the active PAI-1 concentration in each plasma, assuming that the active PAI-1 concentration before antibody administration is 100% and the active PAI-1 concentration for calibration curve is 0 ng / mL. The number of days in which the plasma active PAI-1 concentration was suppressed by 50% or more compared to the pre-dose was determined.
- the anti-human PAI-1 antibody of the present invention is useful for prevention or treatment of various diseases in which active human PAI-1 is involved in pathogenesis.
- the polynucleotide, expression vector, transformed host cell, and method for producing an antibody of the present invention are useful for producing the anti-human PAI-1 antibody.
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Abstract
Description
(1)配列番号2のアミノ酸番号31から35までのアミノ酸配列からなるCDR1、配列番号2のアミノ酸番号50から66までのアミノ酸配列からなるCDR2、及び配列番号2のアミノ酸番号99から107までのアミノ酸配列からなるCDR3とを含む重鎖可変領域、並びに、配列番号4のアミノ酸番号24から34までのアミノ酸配列からなるCDR1、配列番号4のアミノ酸番号50から56までのアミノ酸配列からなるCDR2、及び配列番号4のアミノ酸番号89から97までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(2)以下の1)~2)のいずれかから選択される、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
1)配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント;並びに
2)1)の抗ヒトPAI-1抗体又はその抗原結合フラグメントの翻訳後修飾により生じた抗体又はその抗原結合フラグメントである、(1)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(3)配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、(2)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(4)配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む抗ヒトPAI-1抗体又はその抗原結合フラグメントの翻訳後修飾により生じた抗体又はその抗原結合フラグメントである、(2)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(5)翻訳後修飾が、重鎖可変領域N末端のピログルタミル化及び/又は重鎖C末端のリジン欠失である、(2)又は(4)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(6)ヒトIgγ1定常領域である重鎖定常領域を含む、(1)~(5)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(7)ヒトIgκ定常領域である軽鎖定常領域を含む、(1)~(5)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(8)ヒトIgγ1定常領域である重鎖定常領域を含み、ヒトIgκ定常領域である軽鎖定常領域を含む、(1)~(5)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(9)配列番号2に示されるアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、(3)に記載の抗ヒトPAI-1抗体。
(10)一本鎖可変領域フラグメント、Fab、Fab’、又はF(ab’)2である、(1)~(8)のいずれかに記載の抗原結合フラグメント。
(11)(9)に記載の抗ヒトPAI-1抗体の翻訳後修飾により生じた抗体である、抗ヒトPAI-1抗体。
(12)翻訳後修飾が、重鎖可変領域N末端のピログルタミル化及び/又は重鎖C末端のリジン欠失である、(11)に記載の抗ヒトPAI-1抗体。
(13)配列番号2のアミノ酸番号1から447までのアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、(11)に記載の抗ヒトPAI-1抗体。
(14)(9)又は(13)に記載の抗ヒトPAI-1抗体と活性型ヒトPAI-1との結合を阻害し、ビトロネクチンと複合体を形成した活性型ヒトPAI-1に結合し、かつ、潜在型ヒトPAI-1にも、プラスミノーゲンアクチベーターと複合体を形成したヒトPAI-1にも結合しない、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(15)(9)又は(13)に記載の抗ヒトPAI-1抗体と同じヒトPAI-1エピトープに結合する、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(16)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチド。
(17)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチド。
(18)(16)及び/又は(17)に記載のポリヌクレオチドを含む発現ベクター。
(19)以下の(a)~(d)からなる群より選択される、(18)に記載の発現ベクターで形質転換された宿主細胞。
(a)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(20)以下の(a)~(d)からなる群より選択される、(18)に記載の発現ベクターで形質転換された宿主細胞。
(a)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)(9)に記載の抗ヒトPAI-1抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(21)以下の(a)~(c)からなる群より選択される宿主細胞を培養し、抗ヒトPAI-1抗体又はその抗原結合フラグメントを発現させる工程を包含する、抗ヒトPAI-1抗体又はその抗原結合フラグメントを生産する方法。
(a)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(22)以下の(a)~(c)からなる群より選択される宿主細胞を培養し、抗ヒトPAI-1抗体を発現させる工程を包含する、抗ヒトPAI-1抗体を生産する方法。
(a)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)(9)に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗ヒトPAI-1抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(23)(21)に記載の方法で生産された抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(24)(22)に記載の方法で生産された抗ヒトPAI-1抗体。
(25)(1)~(15)、(23)、及び(24)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む、医薬組成物。
(26)(3)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント、(4)に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント、及び、薬学的に許容される賦形剤を含む、医薬組成物。
(27)(9)に記載の抗ヒトPAI-1抗体、(13)に記載の抗ヒトPAI-1抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
(28)肺線維症の予防又は治療用医薬組成物である、(25)~(27)のいずれかに記載の医薬組成物。
(29)肺線維症が特発性肺線維症である、(28)に記載の医薬組成物。
(30)(1)~(15)、(23)、及び(24)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの治療有効量を投与する工程を包含する、肺線維症を予防又は治療する方法。
(31)肺線維症が特発性肺線維症である、(30)に記載の方法。
(32)肺線維症の予防又は治療に使用するための、(1)~(15)、(23)、及び(24)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(33)肺線維症が特発性肺線維症である、(32)に記載の使用。
(34)肺線維症の予防又は治療用医薬組成物の製造における、(1)~(15)、(23)、及び(24)のいずれかに記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの使用。
(35)肺線維症が特発性肺線維症である、(34)に記載の使用。
本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントには、以下の特徴を有する抗ヒトPAI-1抗体が含まれる。
配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
配列番号2に示されるアミノ酸配列からなる重鎖であって、ただし、配列番号2のアミノ酸番号1のグルタミン酸がピログルタミン酸に修飾され及び/又は配列番号2のアミノ酸番号448のリジンを欠く重鎖、並びに配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
配列番号2のアミノ酸番号1から447までのアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
配列番号2のアミノ酸番号31から35までのアミノ酸配列からなるCDR1、配列番号2のアミノ酸番号50から66までのアミノ酸配列からなるCDR2、及び配列番号2のアミノ酸番号99から107までのアミノ酸配列からなるCDR3とを含む重鎖可変領域、並びに、配列番号4のアミノ酸番号24から34までのアミノ酸配列からなるCDR1、配列番号4のアミノ酸番号50から56までのアミノ酸配列からなるCDR2、及び配列番号4のアミノ酸番号89から97までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(潜在型ヒトPAI-1に対する結合活性評価)
組換え潜在型ヒトPAI-1(Molecular Innovation社、PAI-L)をリン酸緩衝生理食塩水(PBS)で1000ng/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、潜在型ヒトPAI-1を固相化する。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置する。洗浄液(0.05% Tween-20含有トリス緩衝生理食塩水(TBS))で洗浄し、0.1%ウシ血清由来アルブミン(BSA)含有PBSで100000ng/mLから0.01ng/mLまで8段階に希釈した被験抗体を1ウェルあたり100μL添加し、室温で30分間静置する。比較抗体として、被験抗体がヒトFc領域を有する場合はMEDI-579(特許文献3)を、被験抗体がマウスFc領域を有する場合はMA-56A7C10(Hycult Biotech社、HM2182)を、それぞれ0.1%BSA含有PBSで10000ng/mLから0.01ng/mLまで7段階に希釈し、ウェルに添加する。コントロールとして、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルを準備する。洗浄液で3回洗浄した後、PBSで20倍に希釈したブロッキング剤(ナカライテスク社、03953-95)溶液を使用して2000倍に希釈した検出抗体を1ウェルあたり100μL添加して反応させる。検出抗体としては、マウス抗体のFc領域を有する被験抗体の検出用にHRP標識ヤギ抗マウスIg抗体(Southern Biotech社、1010-05)を、ヒト抗体のFc領域を有する被験抗体の検出用にHRP標識ウサギ抗ヒトIg抗体(Southern Biotech社、6145-05)を用いる。室温にて30分間インキュベートした後、洗浄液で3回洗浄する。ペルオキシダーゼ用発色キット(住友ベークライト社)に含まれる発色液を1ウェルあたり100μL添加し、室温で15分間程度静置した後、前記発色キットに含まれる反応停止液を100μL添加して、そのOD450値をEnVisionカウンター(パーキンエルマー社)で測定する。被験抗体の各濃度におけるPAI-1結合率を算出するにあたり、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルの測定値を0%とし、被験抗体の最大濃度における測定値を100%と設定する。ただし、被験抗体がヒトFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMEDI-579の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MEDI-579の評価系における最大濃度の測定値を100%と設定する。被験抗体がマウスFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMA-56A7C10の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MA-56A7C10の評価系における最大濃度の測定値を100%と設定する。算出されたPAI-1結合率を解析し、4パラメータロジスティック曲線回帰により被験抗体のEC50値を算出する。
(uPA-PAI-1複合体に対する結合活性評価)
uPA(ウロキナーゼ静注用6万単位「ベネシス(登録商標)」;田辺三菱製薬株式会社)をPBSで100unit/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、uPAを固相化する。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置する。0.1%BSA含有PBSで1000ng/mLに希釈した組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を1ウェルあたり100μL添加し、uPAに捕捉させる。洗浄液(0.05% Tween-20含有TBS)で洗浄し、0.1%BSA含有PBSで100000ng/mLから0.01ng/mLまで8段階に希釈した被験抗体を1ウェルあたり100μL添加し、室温で30分間静置する。比較抗体として、被験抗体がヒトFc領域を有する場合はMEDI-579(特許文献3)を、被験抗体がマウスFc領域を有する場合はMA-56A7C10(Hycult Biotech社、HM2182)を、それぞれ0.1%BSA含有PBSで10000ng/mLから0.01ng/mLまで7段階に希釈し、ウェルに添加する。コントロールとして、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルを準備する。洗浄液で3回洗浄した後、PBSで20倍に希釈したブロッキング剤(ナカライテスク社、03953-95)溶液を使用して2000倍に希釈した検出抗体を1ウェルあたり100μL添加して反応させる。検出抗体としては、マウス抗体のFc領域を有する被験抗体の検出用にHRP標識ヤギ抗マウスIg抗体(Southern Biotech社、1010-05)を、ヒト抗体のFc領域を有する被験抗体の検出用にHRP標識ウサギ抗ヒトIg抗体(Southern Biotech社、6145-05)を用いる。室温にて30分間インキュベートした後、洗浄液で3回洗浄する。ペルオキシダーゼ用発色キット(住友ベークライト社)に含まれる発色液を1ウェルあたり100μL添加し、室温で15分間程度静置した後、前記発色キットに含まれる反応停止液を100μL添加して、そのOD450値をEnVisionカウンター(パーキンエルマー社)で測定する。被験抗体の各濃度におけるPAI-1結合率を算出するにあたり、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルの測定値を0%とし、被験抗体の最大濃度における測定値を100%と設定する。ただし、被験抗体がヒトFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMEDI-579の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MEDI-579の評価系における最大濃度の測定値を100%と設定する。被験抗体がマウスFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMA-56A7C10の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MA-56A7C10の評価系における最大濃度の測定値を100%と設定する。算出されたPAI-1結合率を解析し、4パラメータロジスティック曲線回帰により被験抗体のEC50値を算出する。
(tPA-PAI-1複合体に対する結合活性評価)
tPA(アクチバシン(登録商標)注600万;協和発酵キリン株式会社)をPBSで1000unit/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、tPAを固相化する。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置する。0.1%BSA含有PBSで1000ng/mLに希釈した組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を1ウェルあたり100μL添加し、tPAに捕捉させる。洗浄液(0.05% Tween-20含有TBS)で洗浄し、0.1%BSA含有PBSで100000ng/mLから0.01ng/mLまで8段階に希釈した被験抗体を1ウェルあたり100μL添加し、室温で30分間静置する。比較抗体として、被験抗体がヒトFc領域を有する場合はMEDI-579(特許文献3)を、被験抗体がマウスFc領域を有する場合はMA-56A7C10(Hycult Biotech社、HM2182)を、それぞれ0.1%BSA含有PBSで10000ng/mLから0.01ng/mLまで7段階に希釈し、ウェルに添加する。コントロールとして、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルを準備する。洗浄液で3回洗浄した後、PBSで20倍に希釈したブロッキング剤(ナカライテスク社、03953-95)溶液を使用して2000倍に希釈した検出抗体を1ウェルあたり100μL添加して反応させる。検出抗体としては、マウス抗体のFc領域を有する被験抗体の検出用にHRP標識ヤギ抗マウスIg抗体(Southern Biotech社、1010-05)を、ヒト抗体のFc領域を有する被験抗体の検出用にHRP標識ウサギ抗ヒトIg抗体(Southern Biotech社、6145-05)を用いる。室温にて30分間インキュベートした後、洗浄液で3回洗浄する。ペルオキシダーゼ用発色キット(住友ベークライト社)に含まれる発色液を1ウェルあたり100μL添加し、室温で15分間程度静置した後、前記発色キットに含まれる反応停止液を100μL添加して、そのOD450値をEnVisionカウンター(パーキンエルマー社)で測定する。被験抗体の各濃度におけるPAI-1結合率を算出するにあたり、被験抗体の代わりに0.1%BSA含有PBSを添加したウェルの測定値を0%とし、被験抗体の最大濃度における測定値を100%と設定する。ただし、被験抗体がヒトFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMEDI-579の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MEDI-579の評価系における最大濃度の測定値を100%と設定する。被験抗体がマウスFc領域を有する場合、被験抗体を最大濃度(100000ng/mL)添加してもMA-56A7C10の評価系における最大濃度(10000ng/mL)の測定値に達しない時は、MA-56A7C10の評価系における最大濃度の測定値を100%と設定する。算出されたPAI-1結合率を解析し、4パラメータロジスティック曲線回帰により被験抗体のEC50値を算出する。
本発明のポリヌクレオチドには、本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチド、及び、本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドが含まれる。
本発明の発現ベクターには、本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチド及び/又は本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターが含まれる。
本発明の形質転換された宿主細胞には、以下の(a)~(d)からなる群より選択される、本発明の発現ベクターで形質転換された宿主細胞が含まれる。
(a)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(a)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)本発明の抗ヒトPAI-1抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントを生産する方法には、以下の(a)~(c)からなる群より選択される宿主細胞を培養し、抗ヒトPAI-1抗体又はその抗原結合フラグメントを発現させる工程を包含する、抗ヒトPAI-1抗体又はその抗原結合フラグメントを生産する方法が含まれる。
(a)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
(a)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)本発明の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。
本発明の医薬組成物には、本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む医薬組成物が含まれる。本発明の医薬組成物は、当該分野において通常用いられている賦形剤、即ち、薬剤用賦形剤や薬剤用担体等を用いて、通常使用される方法によって調製することができる。これら医薬組成物の剤型の例としては、例えば、注射剤、点滴用剤等の非経口剤が挙げられ、静脈内投与、皮下投与等により投与することができる。製剤化にあたっては、薬学的に許容される範囲で、これら剤型に応じた賦形剤、担体、添加剤等を使用することができる。
配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む抗ヒトPAI-1抗体又はその抗原結合フラグメント、並びに、該抗体又はその抗原結合フラグメントの翻訳後修飾により生じた抗体又はその抗原結合フラグメントである、抗ヒトPAI-1抗体又はその抗原結合フラグメントを含有する医薬組成物。
(1)配列番号2のアミノ酸番号1から447までのアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
(2)配列番号2に示されるアミノ酸配列からなり、アミノ酸番号1のグルタミン酸がピログルタミン酸に修飾された重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
(3)配列番号2のアミノ酸番号1から447までのアミノ酸配列からなり、アミノ酸番号1のグルタミン酸がピログルタミン酸に修飾された重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
(4)配列番号2に示されるアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、抗ヒトPAI-1抗体。
配列番号2に示されるアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む抗ヒトPAI-1抗体、配列番号2のアミノ酸番号1から447までのアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む抗ヒトPAI-1抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
当業者であれば、当該分野で公知の方法を用いて、抗体又はその抗原結合フラグメントを、他のペプチド又は蛋白質を融合した融合抗体として作製することや、修飾剤を結合させた修飾抗体として作製することも可能である。本発明の抗ヒトPAI-1抗体又はその抗原結合フラグメントには、このような融合体や修飾体の形態の抗体又は抗原結合フラグメントも含まれる。例えば、配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメントには、他のペプチド又は蛋白質と融合した抗ヒトPAI-1抗体又はその抗原結合フラグメント、或いは修飾剤を結合させた抗ヒトPAI-1抗体又はその抗原結合フラグメントも含まれる。本発明の抗体又はその抗原結合フラグメントは融合抗体としてVTN-PAI-1複合体との結合活性を有している限り、融合に用いられる他のペプチド又は蛋白質は特に限定されず、例えば、ヒト血清アルブミン、各種タグペプチド、人工ヘリックスモチーフペプチド、マルトース結合蛋白質、グルタチオンSトランスフェラーゼ、各種毒素、その他多量体化を促進しうるペプチド又は蛋白質等が挙げられる。本発明の抗体又はその抗原結合フラグメントは修飾抗体としてVTN-PAI-1複合体との結合活性を有している限り、修飾に用いられる修飾剤は特に限定されず、例えば、ポリエチレングリコール、糖鎖、リン脂質、リポソーム、低分子化合物等が挙げられる。
活性型ヒトPAI-1に選択性の高い抗体を取得するために、最適なヒトPAI-1ペプチド抗原を検討した。ペプチドの長さやアミノ酸配列を検討した結果、ヒトPAI-1中の16アミノ酸残基のペプチド抗原STAVIVSARMAPEEII(配列番号5)のC末端にMultiple Antigen Peptide 8(MAP8)ペプチド(J.Biol.Chem.、1988、Vol.263、No.4、p.1719-1725)をつなげたものを免疫した場合に、血漿中の活性型ヒトPAI-1を中和する抗体を取得できることが明らかになった。さらに抗原につなげる蛋白質や免疫方法等を創意検討した結果、このペプチド抗原のC末端にKeyhole Limpet Hemocyanin(KLH)蛋白質をつなげたものと組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を混合して同時に免疫した場合に、血漿中の活性型ヒトPAI-1を強力に中和し、かつビトロネクチンと複合体を形成した活性型ヒトPAI-1(VTN-PAI-1複合体)に選択性の高い抗体を取得できることが明らかになった。
抗体の抗原特異的結合活性を測定するために、ELISAアッセイを使用した。ここで、活性型ヒトPAI-1に対する選択性を評価するために、ビトロネクチンを固相化し、活性型ヒトPAI-1を添加して作製したプレートと、活性型ヒトPAI-1の代わりに潜在型ヒトPAI-1を添加して作製したプレートを用いて試験した。
前述の抗体は、可変領域がヒト由来であり、定常領域がマウス由来の抗体である。そこで、本発明者らは、哺乳細胞発現用ベクターであるGSベクター(Lonza社)を用いて、重鎖及び軽鎖の両遺伝子を含む発現ベクターを構築し、完全ヒト型抗体を作製した。具体的には、実施例2で同定されたHOT-1について、ハイブリドーマからRNAを抽出し、cDNA増幅キット(SMARTer RACE cDNA Amplification kit;Clontech社)を用いて、cDNAを作製した。次いで、ポリメラーゼ連鎖反応(PCR)を用いて、重鎖及び軽鎖の可変領域を伸長及び増幅した。HOT-1の重鎖可変領域遺伝子の5’側にシグナル配列(Nigel Whittleら、Protein Engineering、1987、Vol.1、No.6、p.499-505)をコードする遺伝子を、そして3’側にヒトIgγ1の定常領域遺伝子(配列番号1の塩基番号355から1344までの塩基配列からなる)をそれぞれ繋げ、この重鎖遺伝子をGSベクターpEE6.4に挿入した。また、HOT-1の軽鎖可変領域遺伝子の5’側にシグナル配列(Nigel Whittleら、前出)をコードする遺伝子を、そして3’側にヒトκ鎖の定常領域遺伝子(配列番号3のアミノ酸番号325から642までの塩基配列からなる)をそれぞれ繋げ、この軽鎖遺伝子をGSベクターpEE12.4に挿入した。
精製した完全ヒト型HOT-1のアミノ酸修飾を分析した結果、精製抗体の大部分において、重鎖C末端のリジン欠失が生じていた。
実施例2で同定したHOT-1(キメラ抗体)及び実施例3で作製した完全ヒト型HOT-1についてヒト血漿中の活性型PAI-1阻害活性を評価するために、肥満患者の血漿を用いて、活性型PAI-1阻害アッセイを実施した。健常人の血漿を用いた場合、活性型PAI-1の濃度が低くアッセイ系の構築が困難であるため、血漿中活性型PAI-1濃度が高いことが知られている肥満患者の血漿を用いた(Nature Medicine、1996、Vol.2、p.800-803)。
ELISAアッセイを用いて、実施例3で作製した完全ヒト型HOT-1のVTN-PAI-1複合体に対する結合選択性を評価した。次の(1)~(4)の4通りのヒトPAI-1固相化プレートを作製し、各プレートを用いて試験した。
(1)VTN-PAI-1複合体固相化プレート
ビトロネクチン(BD Biosciences社、354238)をPBSで1000ng/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、ビトロネクチンを固相化した。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置した。0.1%BSA含有PBSで1000ng/mLに希釈した組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を1ウェルあたり100μL添加し、ビトロネクチンに捕捉させた。本プレートでの試験により、VTN-PAI-1に対する抗体の結合活性を評価した。
(2)潜在型ヒトPAI-1固相化プレート
組換え潜在型ヒトPAI-1(Molecular Innovation社、PAI-L)をPBSで1000ng/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、潜在型ヒトPAI-1を固相化した。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置した。本プレートでの試験により、潜在型ヒトPAI-1に対する抗体の結合活性を評価した。
(3)uPA-PAI-1複合体固相化プレート
uPA(ウロキナーゼ静注用6万単位「ベネシス」(登録商標);田辺三菱製薬株式会社)をPBSで100unit/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、uPAを固相化した。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置した。0.1%BSA含有PBSで1000ng/mLに希釈した組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を1ウェルあたり100μL添加し、uPAに捕捉させた。本プレートでの試験により、uPA-PAI-1複合体に対する抗体の結合活性を評価した。
(4)tPA-PAI-1複合体固相化プレート
tPA(アクチバシン(登録商標)注600万;協和発酵キリン株式会社)をPBSで1000unit/mLとなるように希釈し、ヌンクマキシソープ透明96プレート(Nunc社)に1ウェルあたり100μL添加し、4℃で一晩プレートを静置することで、tPAを固相化した。逆遠心により固相液を除去し、ブロッキング剤(Thermo SCIENTIFIC社、37532)を1ウェルあたり200μL添加し、室温にて30分間静置した。0.1%BSA含有PBSで1000ng/mLに希釈した組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を1ウェルあたり100μL添加し、tPAに捕捉させた。本プレートでの試験により、tPA-PAI-1複合体に対する抗体の結合活性を評価した。
表1:抗ヒトPAI-1抗体の各種ヒトPAI-1に対する結合活性
完全ヒト型HOT-1の単量体活性型ヒトPAI-1への結合活性を測定するために、SPR解析を行った。SPR解析においては、Biacore(登録商標)T200(GEヘルスケアジャパン)及び組換え活性型ヒトPAI-1(Molecular Innovation社、PAI-A)を用いて解析を行った。その結果、完全ヒト型HOT-1が単量体として存在する活性型ヒトPAI-1に結合することが明らかとなった。
ELISAアッセイを用いて、ビトロネクチンと複合体を形成した活性型サルPAI-1(VTN-サルPAI-1複合体とも称する)に対する完全ヒト型HOT-1の結合選択性を評価した。次の(1)~(4)の4通りのサルPAI-1固相化プレートを作製し、各プレートを用いて試験した。比較抗体としてMEDI-579を使用した。
実施例6(1)に記載の方法に従って、VTN-サルPAI-1に対する抗体の結合活性を評価した。但し、組換え活性型ヒトPAI-1の代わりに、組換え活性型サルPAI-1(Molecular Innovation社、CYPAI)を使用した。また、完全ヒト型HOT-1及びMEDI-579を10000ng/mLから0.01ng/mLまで7段階に希釈して使用した。
(2)潜在型サルPAI-1固相化プレート
実施例6(2)に記載の方法に従って、潜在型サルPAI-1に対する抗体の結合活性を評価した。但し、組換え潜在型ヒトPAI-1の代わりに、組換え潜在型サルPAI-1(Molecular Innovation社、CYPAI-L)を使用した。また、完全ヒト型HOT-1及びMEDI-579を10000ng/mLから0.01ng/mLまで7段階に希釈して使用した。
(3)uPA-サルPAI-1複合体固相化プレート
実施例6(3)に記載の方法に従って、uPA-サルPAI-1複合体に対する抗体の結合活性を評価した。但し、組換え活性型ヒトPAI-1の代わりに、組換え活性型サルPAI-1(Molecular Innovation社、CYPAI)を使用した。また、完全ヒト型HOT-1及びMEDI-579を10000ng/mLから0.01ng/mLまで7段階に希釈して使用した。
(4)tPA-サルPAI-1複合体固相化プレート
実施例6(4)に記載の方法に従って、tPA-サルPAI-1複合体に対する抗体の結合活性を評価した。但し、組換え活性型ヒトPAI-1の代わりに、組換え活性型サルPAI-1(Molecular Innovation社、CYPAI)を使用した。また、完全ヒト型HOT-1及びMEDI-579を10000ng/mLから0.01ng/mLまで7段階に希釈して使用した。
表2:抗PAI-1抗体の各種サルPAI-1に対する結合活性
完全ヒト型HOT-1及びMEDI-579のヒト及びサル血漿中の活性型PAI-1阻害活性の評価を実施した。ヒト血漿については、実施例5と同様に肥満患者の血漿を用いた。サル血漿については、カニクイザルにLPSを静脈内投与し、採取した血漿を用いた。
血管壁においてuPA及びtPAが血管内皮から産生されて、血管内皮に結合していることが知られている(Baillieres Clin.Haematol.、1993、Vol.6、No.3、p.559-576。Blood、2011、Vol.118、No.11、p.3182-3185)。つまり、uPA及びtPAは血中だけでなく血管壁にも存在する。実施例9では、抗PAI-1抗体による血漿中の活性型PAI-1の阻害活性を測定したが、この実験系は血漿中成分のみを抽出していることから血管壁の影響が考慮されていない。そのため、実際の血管中における生体反応を反映しているとは言い難いと考えられる。そこで、実際の血管内状態を模した系として、uPA-PAI-1複合体を固相化したアッセイプレートに活性型PAI-1、uPA及びuPA基質ペプチドを混合した評価系を用いて、完全ヒト型HOT-1及びMEDI-579の活性をuPAの活性を指標に測定した。
実施例6の結果から、完全ヒト型HOT-1はVTN-PAI-1複合体に対して強い結合活性を示し、かつ極めて高い選択的結合活性を有することが明らかになった。一般的に、血中における抗体はそのまま代謝される以外に抗原に結合して抗原-抗体複合体として代謝されることが知られている(Journal of Pharmaceutical Sciences、2012、Vol.101、No.12、p4367-4382)。血漿中におけるPAI-1はVTN-PAI-1以外にも、潜在型PAI-1及びtPA-PAI-1複合体等の様々な複合体の状態で存在する(Blood、1990、Vol.76、p.930-937)ことから、選択性の低い抗PAI-1抗体は様々なPAI-1と抗原-抗体複合体を形成して代謝される。一方で完全ヒト型HOT-1はVTN-PAI-1選択的に結合するため潜在型PAI-1やPA-PAI-1との複合体としては代謝されにくく、そのため血中における濃度が長期間維持される可能性が考えられた。実際抗体の血中濃度持続はこの抗原-抗体複合体の代謝速度に大きく依存することが知られている(Bioanalysis、2011、Vol.3、No.6、p.659-675)。実施例8の結果から、完全ヒト型HOT-1はVTN-サルPAI-1複合体に対する高い選択的結合活性を示したことから、サルに抗体を投与した際の血漿中抗体濃度を測定した。比較抗体として、MEDI-579を使用した。
[処置群]
HOT-1投与群(3匹):
完全ヒト型HOT-1を投与した群(2.0mg/kg)
MEDI-579投与群(2匹):
MEDI-579を投与した群(2.0mg/kg)
抗体投与前、及び、抗体投与0.25、1、2、4、8、24、48、96、168、336、504時間後に採血を行い、血漿を得た。完全ヒト型HOT-1に関しては追加で、672、936、1200、1440時間後にも採血を行い、血漿を得た。
サルの血中で長期間かつ強力にPAI-1活性を阻害することが、肺線維症等のPAI-1が病態に関与する疾患の予防又は治療に望ましい。そこで、完全ヒト型HOT-1のサル血中における活性型PAI-1阻害活性を評価した。比較抗体としてMEDI-579を使用した。
Claims (31)
- 配列番号2のアミノ酸番号31から35までのアミノ酸配列からなるCDR1、配列番号2のアミノ酸番号50から66までのアミノ酸配列からなるCDR2、及び配列番号2のアミノ酸番号99から107までのアミノ酸配列からなるCDR3とを含む重鎖可変領域、並びに、配列番号4のアミノ酸番号24から34までのアミノ酸配列からなるCDR1、配列番号4のアミノ酸番号50から56までのアミノ酸配列からなるCDR2、及び配列番号4のアミノ酸番号89から97までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 以下の(1)~(2)のいずれかから選択される、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
(1)配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、抗ヒトPAI-1抗体又はその抗原結合フラグメント;並びに
(2)上記(1)の抗ヒトPAI-1抗体又はその抗原結合フラグメントの翻訳後修飾により生じた抗体又はその抗原結合フラグメントである、請求項1に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。 - 配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域、及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む、請求項2に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 配列番号2のアミノ酸番号1から118までのアミノ酸配列からなる重鎖可変領域及び配列番号4のアミノ酸番号1から108までのアミノ酸配列からなる軽鎖可変領域を含む抗ヒトPAI-1抗体又はその抗原結合フラグメントの翻訳後修飾により生じた抗体又はその抗原結合フラグメントである、請求項2に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 翻訳後修飾が、重鎖可変領域N末端のピログルタミル化及び/又は重鎖C末端のリジン欠失である、請求項2又は4に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- ヒトIgγ1定常領域である重鎖定常領域を含む、請求項1~5のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- ヒトIgκ定常領域である軽鎖定常領域を含む、請求項1~5のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- ヒトIgγ1定常領域である重鎖定常領域を含み、ヒトIgκ定常領域である軽鎖定常領域を含む、請求項1~5のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 配列番号2に示されるアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、請求項3に記載の抗ヒトPAI-1抗体。
- 一本鎖可変領域フラグメント、Fab、Fab’、又はF(ab’)2である、請求項1~8のいずれか1項に記載の抗原結合フラグメント。
- 請求項9に記載の抗ヒトPAI-1抗体の翻訳後修飾により生じた抗体である、抗ヒトPAI-1抗体。
- 翻訳後修飾が、重鎖可変領域N末端のピログルタミル化及び/又は重鎖C末端のリジン欠失である、請求項11に記載の抗ヒトPAI-1抗体。
- 配列番号2のアミノ酸番号1から447までのアミノ酸配列からなる重鎖及び配列番号4に示されるアミノ酸配列からなる軽鎖を含む、請求項11に記載の抗ヒトPAI-1抗体。
- 請求項9又は13に記載の抗ヒトPAI-1抗体と活性型ヒトPAI-1との結合を阻害し、ビトロネクチンと複合体を形成した活性型ヒトPAI-1に結合し、かつ、潜在型ヒトPAI-1にも、プラスミノーゲンアクチベーターと複合体を形成したヒトPAI-1にも結合しない、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 請求項9又は13に記載の抗ヒトPAI-1抗体と同じヒトPAI-1エピトープに結合する、抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチド。
- 請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチド。
- 請求項16及び/又は17に記載のポリヌクレオチドを含む発現ベクター。
- 以下の(a)~(d)からなる群より選択される、請求項18に記載の発現ベクターで形質転換された宿主細胞。
(a)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 以下の(a)~(d)からなる群より選択される、請求項18に記載の発現ベクターで形質転換された宿主細胞。
(a)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;
(c)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;及び
(d)請求項9に記載の抗ヒトPAI-1抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 以下の(a)~(c)からなる群より選択される宿主細胞を培養し、抗ヒトPAI-1抗体又はその抗原結合フラグメントを発現させる工程を包含する、抗ヒトPAI-1抗体又はその抗原結合フラグメントを生産する方法。
(a)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの重鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗体又はその抗原結合フラグメントの軽鎖可変領域をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 以下の(a)~(c)からなる群より選択される宿主細胞を培養し、抗ヒトPAI-1抗体を発現させる工程を包含する、抗ヒトPAI-1抗体を生産する方法。
(a)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドとを含む発現ベクターで形質転換された宿主細胞;
(b)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターと該抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞;並びに
(c)請求項9に記載の抗ヒトPAI-1抗体の重鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞、及び、該抗ヒトPAI-1抗体の軽鎖をコードする塩基配列を含むポリヌクレオチドを含む発現ベクターで形質転換された宿主細胞。 - 請求項21に記載の方法で生産された抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 請求項22に記載の方法で生産された抗ヒトPAI-1抗体。
- 請求項1~15、23、及び24のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント及び薬学的に許容される賦形剤を含む、医薬組成物。
- 請求項3に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント、請求項4に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント、及び、薬学的に許容される賦形剤を含む、医薬組成物。
- 請求項9に記載の抗ヒトPAI-1抗体、請求項13に記載の抗ヒトPAI-1抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
- 肺線維症の予防又は治療用医薬組成物である、請求項25~27のいずれか1項に記載の医薬組成物。
- 請求項1~15、23、及び24のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの治療有効量を投与する工程を包含する、肺線維症を予防又は治療する方法。
- 肺線維症の予防又は治療に使用するための、請求項1~15、23、及び24のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメント。
- 肺線維症の予防又は治療用医薬組成物の製造における、請求項1~15、23、及び24のいずれか1項に記載の抗ヒトPAI-1抗体又はその抗原結合フラグメントの使用。
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CA2939897A CA2939897A1 (en) | 2014-02-21 | 2015-02-20 | New anti-human pai-1 antibody |
RU2016137486A RU2016137486A (ru) | 2014-02-21 | 2015-02-20 | Новое антитело против pai-1 человека |
KR1020167025778A KR20160124840A (ko) | 2014-02-21 | 2015-02-20 | 신규 항인간 pai-1 항체 |
MX2016010852A MX2016010852A (es) | 2014-02-21 | 2015-02-20 | Anticuerpo anti-inhibidor de activador de plasminogeno 1 (pai-1) humano novedoso. |
EP15751458.9A EP3109320B1 (en) | 2014-02-21 | 2015-02-20 | New anti-human pai-1 antibody |
US15/120,257 US9803024B2 (en) | 2014-02-21 | 2015-02-20 | Anti-human PAI-1 antibody |
PL15751458T PL3109320T3 (pl) | 2014-02-21 | 2015-02-20 | Nowe przeciwciało anty-ludzki PAI-1 |
CN201580009780.9A CN106029884B (zh) | 2014-02-21 | 2015-02-20 | 新型抗人pai-1抗体 |
ES15751458T ES2726915T3 (es) | 2014-02-21 | 2015-02-20 | Nuevo anticuerpo anti-PAI-1 humano |
BR112016019332A BR112016019332A2 (pt) | 2014-02-21 | 2015-02-20 | anticorpo pai-1 anti-humano inovador |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002034776A2 (en) * | 2000-10-26 | 2002-05-02 | K.U.Leuven Research And Development | Epitopes of pai-1 |
WO2011139973A2 (en) * | 2010-05-03 | 2011-11-10 | Abbott Laboratories | Methods of inhibiting fibrosis using anti-pai-1 antibodies |
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JP2006507297A (ja) * | 2002-05-13 | 2006-03-02 | チルドレンズ・ホスピタル・ロサンジェルス | ケロイドおよび他の皮膚または内部創傷または病変における異常瘢痕形成の処置および予防 |
WO2009033095A2 (en) * | 2007-09-07 | 2009-03-12 | Cisthera, Incorporated | Humanized pai-1 antibodies |
CN101654405A (zh) * | 2008-08-19 | 2010-02-24 | 信谊药厂 | 抗凝化合物、组合物及其用途 |
US20100254979A1 (en) | 2009-03-06 | 2010-10-07 | Cisthera, Incorporated | Humanized PAI-1 Antibodies and Uses Thereof |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2002034776A2 (en) * | 2000-10-26 | 2002-05-02 | K.U.Leuven Research And Development | Epitopes of pai-1 |
WO2011139973A2 (en) * | 2010-05-03 | 2011-11-10 | Abbott Laboratories | Methods of inhibiting fibrosis using anti-pai-1 antibodies |
Non-Patent Citations (2)
Title |
---|
ANN-PASCALE BIJNENS. ET AL.: "Importance of the Hinge Region between alpha-Helix F and the MainPart of Serpins, Based upon Identification of the Epitope of Plasminogen Activator Inhibitor Type 1 Neutralizing Antibodies", JBC, vol. 275, 2000, pages 6375 - 6380, XP055221556 * |
HIROYOSHI OBA: "Jinko Kotai Library ni yoru Kotai Iyaku Kaihatsu", KOTAI IYAKU NO SAIZENSEN, 2007, pages 157 - 169, XP008184908 * |
Cited By (1)
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