WO2016010014A1 - 新規抗ヒトTie2抗体 - Google Patents
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Definitions
- the present invention relates to a novel anti-human Tie2 antibody.
- Tie2 (Tyrosine kinase with Ig and EGF homology domain 2) is a receptor tyrosine kinase. It is known that Tie2 is mainly expressed in vascular endothelial cells. As its ligand, angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) which are multimeric secreted glycoproteins are known.
- Ang-1 angiopoietin-1
- Ang-2 angiopoietin-2
- Ang-1 functions as an agonist for Tie2.
- Tie2 binds to Ang-1, it is self-phosphorylated by forming a multimer and transmits a signal into the cell to stabilize the blood vessel such as an anti-apoptotic action of vascular endothelial cells, an inhibitory action of vascular permeability, It has been shown to promote maturation and remodeling (Cell, 1996, Vol. 87, p. 1171-1180. Genes Dev., 1994, Vol. 8, p. 1897-1909. Science, 1999, Vol. 286, pages 2511-2514. Nat.Struct.Biol., 2003, Vol.10, pages 38-44).
- Ang-1 is also known to exert vasodilatory and blood flow enhancing effects through nitric oxide production via Tie2 activation (Pharmacol. Res., 2014, Vol. 80, p. 43). -51). Furthermore, Ang-1 is thought to contribute to the stabilization of blood vessels by suppressing internalization of vascular endothelial cadherin through Tie2 activation (Dev. Cell, 2008, Vol. 14, p. 25-36). On the other hand, Ang-2 is said to be able to activate Tie2 on vascular endothelial cells, but its activation is considered to be partial compared to Ang-1 (Mol. Cell Biol., 2009, Vol.29, p.2011-2022).
- Ang-2 binds to the same site of Ang-1 and Tie2 with the same degree of affinity, Ang-2 is endogenous because it replaces the activation of Tie2 by Ang-1 with partial activity. It has been suggested to function as a sex Tie2 antagonist (Science, 1997, Vol. 277, p. 55-60).
- Ang-2 levels have been reported in diseases where vascular vulnerability is considered as one of the etiologies such as diabetes, diabetic retinopathy, sepsis and acute renal failure (Atherosclerosis, 2005, Vol. 180, p.113-118, Br. J. Ophthalmol., 2004, Vol.88, p.1543-1546. Critical Care, 2009, Vol.13, p.207, Intensive Care Med., 2010, Vol. 36, p.462-470).
- Diabetic macular edema with thickened macular region is known as one of the pathological conditions, but there is a report that the thickness of the macular region is decreased in patients whose intraocular Ang-1 concentration has increased by vitreous removal surgery (Br. J. Ophthalmol., 2005, Vol. 89, p. 480-483). Furthermore, retinal edema and hemorrhage were observed in a mouse model of retinal edema in which pericytes of retinal blood vessels were removed, and pathogenesis was inhibited by intravitreal administration of Ang-1 (J. Clin.
- Invest., 2002, Vol.110, p.1619-1628), and administration of an adenovirus containing a gene encoding Ang-1 suppressed vascular endothelial cell damage in the retina in a test using a mouse diabetic retinopathy model suggests that Ang-1 improves the pathological condition.
- Ang-1 improves the pathological condition.
- Ang-2 In relation to severe limb ischemia, plasma Ang-2 is increased in peripheral arterial disease, and Ang-2 expression level is high in ischemic limb muscle or skin tissue of patients with severe limb ischemia (J. Am. Coll Cardial., 2008, Vol. 52, p. 387-393. Int Angiol., 2011, Vol. 30, p. 25-34).
- Angiogenesis 2009, Vol. 12, p. 243-249.
- Patent Document 1 Mouse monoclonal antibody 15B8 (Patent Document 1) has been reported as an antibody exhibiting an agonistic action against human Tie2. It has been reported that 15B8 binds to human Tie2 and induces an anti-apoptotic action of human vascular endothelial cells HUVEC (Patent Document 1).
- An object of the present invention is to provide an anti-human Tie2 antibody capable of preventing or treating diabetic macular edema, diabetic retinopathy or severe limb ischemia by binding to human Tie2 and activating human Tie2.
- the present inventors have found that the heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 from amino acid numbers 1 to 122 and amino acid number 1 of SEQ ID NO: 4
- a tetravalent anti-human Tie2 antibody comprising a light chain variable region consisting of an amino acid sequence from 1 to 113 (Examples 1 to 8), and the anti-human Tie2 antibody binds to human Tie2 (Example 12)
- the anti-apoptotic action of human Tie2-expressing BaF3 cells was induced (Examples 9 and 11), and that the increase in vascular permeability was inhibited (Examples 10 and 13) in a rat vascular permeability enhancement model.
- the anti-human Tie2 antibody was provided and the present invention was completed.
- the present invention may include the following inventions as medically or industrially useful substances or methods.
- An anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions,
- the heavy chain variable region consists of CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2,
- CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 2, and amino acid number 99 of SEQ ID NO: 2.
- CDR3 consisting of up to 111 amino acid sequences
- the light chain variable region is composed of CDR1 consisting of amino acid sequences of amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences of amino acid numbers 55 to 61 of SEQ ID NO: 4, and amino acid number 94 of SEQ ID NO: 4.
- CDR3 consisting of up to 102 amino acid sequences
- One heavy chain variable region and one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment thereof comprises four antigen binding sites; Anti-human Tie2 antibody or antigen-binding fragment thereof.
- the anti-human Tie2 antibody or antigen-binding fragment thereof according to [1], selected from the following (1) or (2): (1) An anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions,
- the heavy chain variable region consists of an amino acid sequence of amino acid numbers 1 to 122 of SEQ ID NO: 2,
- the light chain variable region consists of an amino acid sequence from amino acid numbers 1 to 113 of SEQ ID NO: 4,
- One heavy chain variable region and one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment thereof comprises four antigen binding sites;
- each heavy chain consists of 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 to amino acid numbers 50 to 66, and SEQ ID NO: 2 from amino acid numbers 99 to 111 2 comprising a heavy chain variable region comprising CDR3 consisting of the amino acid sequence and CH1 region, and comprising a CH2 region and a CH3 region, wherein the carboxy terminus (C terminus) of one of the structures is linked via a linker to the other The amino terminus (N terminus) of the structure of Each light chain consists of CDR1 consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid numbers 24 to 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid numbers 55 to 61, and amino acid numbers 94 to 102 of SEQ ID NO: 4
- An anti-human Tie2 antibody which is an antibody produced by post-translational modification of the anti-human Tie2 antibody according to [5].
- the anti-human Tie2 antibody according to [6] wherein the post-translational modification is pyroglutamylation at the heavy chain variable region N-terminus and / or lysine deletion at the heavy chain C-terminus.
- the human Ig ⁇ 1 constant region is a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, and P331S, or a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, P331S, and I253A.
- the described anti-human Tie2 antibody [10] The anti-human Tie2 antibody according to [8], wherein the human Ig ⁇ 4 constant region is a human Ig ⁇ 4 constant region having amino acid mutations of S228P and L235E. [11] The anti-human Tie2 antibody according to any one of [3] to [7], comprising a light chain constant region that is a human Ig ⁇ constant region.
- the anti-human Tie2 according to any one of [3] to [7], comprising a heavy chain constant region that is a human Ig ⁇ 1 constant region or a human Ig ⁇ 4 constant region, and a light chain constant region that is a human Ig ⁇ constant region.
- the human Ig ⁇ 1 constant region is a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, and P331S, or a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, P331S, and I253A.
- the described anti-human Tie2 antibody comprising a heavy chain constant region that is a human Ig ⁇ 1 constant region or a human Ig ⁇ 4 constant region, and a light chain constant region that is a human Ig ⁇ constant region.
- the human Ig ⁇ 1 constant region is a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, and P331S, or a human Ig ⁇ 1 constant region
- the linker comprises an amino acid sequence of the hinge region or a part thereof.
- the anti-human Tie2 antibody according to [16], wherein the linker consists of the amino acid sequence represented by SEQ ID NO: 13.
- the anti-human Tie2 antibody according to [4] comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the anti-human Tie2 antibody according to [4] comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the anti-human Tie2 antibody according to [4] comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- An anti-human Tie2 antibody which is an antibody produced by post-translational modification of the anti-human Tie2 antibody according to any one of [18] to [20].
- the anti-human Tie2 antibody according to [21] wherein the post-translational modification is pyroglutamylation of the heavy chain variable region N-terminus and / or lysine deletion of the heavy chain C-terminus.
- the anti-human according to [21] comprising two heavy chains consisting of amino acid sequences of amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4 Tie2 antibody.
- Human Tie2 epitope is Accession No. The tetravalent anti-human Tie2 antibody or antigen-binding fragment thereof according to [24], which is a human Tie2 epitope comprising amino acids 192, 195 and 197 of NP — 0450.2.
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof according to [2].
- An anti-human Tie2 antibody comprising a step of culturing a host cell selected from the group consisting of the following (a) to (c) and expressing a tetravalent anti-human Tie2 antibody or an antigen-binding fragment thereof:
- Methods for producing the antigen binding fragment (A) a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof according to [2] and a base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof
- a method for producing an anti-human Tie2 antibody comprising culturing a host cell selected from the group consisting of the following (a) to (c) and expressing an anti-human Tie2 antibody: (A) a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody according to any one of [18] to [20] and a polynucleotide comprising a base sequence encoding the light chain of the antibody A host cell transformed with an expression vector; (B) an expression vector comprising a polynucleotide comprising the base sequence encoding the heavy chain of the anti-human Tie2 antibody according to any of [18] to [20] and a poly comprising the base sequence encoding the light chain of the antibody A host cell transformed with an expression vector comprising nucleotide; and (c) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody according
- [37] An anti-human Tie2 antibody produced by the method according to [35].
- a pharmaceutical composition comprising the anti-human Tie2 antibody or antigen-binding fragment thereof according to any one of [1] to [23], [36] and [37] and a pharmaceutically acceptable excipient.
- a pharmaceutical composition comprising the anti-human Tie2 antibody according to [5], the anti-human Tie2 antibody according to [6], and a pharmaceutically acceptable excipient.
- a pharmaceutical composition comprising the anti-human Tie2 antibody according to [18], the anti-human Tie2 antibody according to [23], and a pharmaceutically acceptable excipient.
- Diabetic macular edema, diabetes comprising the step of administering a therapeutically effective amount of the anti-human Tie2 antibody or antigen-binding fragment thereof according to any one of [1] to [23], [36] and [37] A method for preventing or treating retinopathy or severe limb ischemia.
- the anti-human according to any one of [1] to [23], [36] and [37] in the manufacture of a pharmaceutical composition for preventing or treating diabetic macular edema, diabetic retinopathy or severe lower limb ischemia Use of a Tie2 antibody or antigen-binding fragment thereof.
- the anti-human Tie2 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 Tie2 antibody of the present invention can be used as a preventive or therapeutic agent for diabetic macular edema, diabetic retinopathy or severe lower limb ischemia by binding to human Tie2 and activating human Tie2.
- FIG. 5 shows the vascular permeability inhibitory action of fully human type 2-16A2 and TIE-1-Ig ⁇ 1-WT in a rat vascular permeability model.
- the vertical axis shows the amount of Evans Blue dye leakage. (***: p ⁇ 0.0001 vs. Vehicle group).
- Fig. 5 shows the vascular permeability inhibitory action of TIE-1-Ig ⁇ 1-LALA in a rat vascular permeability model.
- the vertical axis shows the amount of Evans Blue dye leakage. (***: p ⁇ 0.0001 vs. Vehicle group).
- FIG. 6 shows retinal edema inhibitory action of TIE-1-Ig ⁇ 1-LALA in a mouse retinal vascular pericyte detachment model.
- the vertical axis represents the sum of the areas of the retinal nerve fiber layer and the retinal ganglion cell layer. (##: p ⁇ 0.005 vs Cont. Group, *: p ⁇ 0.05 vs Veh. Group).
- the blood flow improving action in a hindlimb ischemic mouse model of TIE-1-Ig ⁇ 1-LALA is shown.
- the vertical axis represents blood flow. (*: P ⁇ 0.05 vs control group, **: p ⁇ 0.01 vs control group).
- TIE-1-Ig ⁇ 1-LALA As a part of the epitope analysis of TIE-1-Ig ⁇ 1-LALA, a representative example of the result of the surface plasmon resonance phenomenon is shown.
- the vertical axis represents binding responsiveness (Resonance Unit: RU), and the horizontal axis represents time (seconds).
- TIE-1-Ig ⁇ 1-LALA As part of the epitope analysis of TIE-1-Ig ⁇ 1-LALA, the results of ELISA are shown.
- the vertical axis represents the emission intensity, and the horizontal axis represents the concentration of TIE-1-Ig ⁇ 1-LALA (ng / mL).
- IgG There are five classes of antibodies: IgG, IgM, IgA, IgD, and IgE.
- the basic structure of the antibody molecule is the same for each class, with a heavy chain with a molecular weight of 50,000 to 70,000 and a light chain with 20,000 to 30,000. Consists of chains.
- 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.
- a domain located at the amino terminus (N terminus) of both heavy and light chains is called a variable region because its amino acid sequence is not constant even if it is a specimen from the same class (subclass) of the same species.
- Each domain is called a heavy chain variable region and a light chain variable region.
- the amino acid sequence on the carboxy terminus (C terminus) side of the variable region is almost constant for each class or subclass and is called a constant region.
- the antigen-binding site of an antibody is composed of a heavy chain variable region (VH) and a light chain variable region (VL), and the binding specificity depends on the amino acid sequence of this site.
- VH heavy chain variable region
- VL light chain variable region
- biological activities such as binding to complement and various 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).
- CDR1, CDR2, CDR3 complementarity determining regions
- the remaining part of the variable region is called the framework region (FR) and is relatively constant.
- the heavy chain constant region is composed of three regions, which are called CH1 region, CH2 region and CH3 region in order from the variable region side.
- the light chain constant region is composed of one region.
- a peptide sequence called a hinge region exists between the CH1 region and the CH2 region. The hinge region is involved in the mobility of the structure consisting of the heavy chain variable region and the CH1 region.
- 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 light chain, a heavy chain fragment containing a VH, CH1 region and a part of the hinge region.
- Fab ′ is a monovalent antibody fragment composed of a heavy chain fragment comprising a light chain, a VH, CH1 region, 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.
- An antibody having two or more antigen-binding sites is called a multivalent antibody. Of these, an antibody having four antigen-binding sites is referred to as a tetravalent antibody.
- Various formats (structures) have been reported for tetravalent antibodies (Nat. Rev. Immunol. 2010, Vol. 10, p. 301-316. J. Immunol., 2003, Vol. 170, p. 4854-). 4861. Mol.Immunol., 2000, Vol.37, p.1067-1077. Biochem.J., 2007, Vol.406, p.237-246.J.Immunol.Methods, 2003, Vol.279, p. 219-232).
- a tetravalent antibody two heavy chains, each having a heavy chain variable region and a light chain variable region linked to the N terminus of a heavy chain variable region and a light chain variable region of a bivalent antibody via a linker, respectively.
- a tetravalent antibody comprising four light chains, each heavy chain comprising two structures consisting of a heavy chain variable region and a CH1 region, and a C-terminus of scFv bound to each streptavidin of tetramer streptavidin 4 Tetravalent antibody in which one CF terminus of scFv is bound to each p53 of tetramer p53, and tetravalent in which the N terminus of the CH3 region is linked to the C terminus of dimer scFv via a linker.
- Antibodies etc. have been reported.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention includes an anti-human Tie2 antibody or antigen-binding fragment thereof having the following characteristics.
- An anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions comprising:
- the heavy chain variable region consists of an amino acid sequence of amino acid numbers 1 to 122 of SEQ ID NO: 2
- the light chain variable region consists of an amino acid sequence from amino acid numbers 1 to 113 of SEQ ID NO: 4
- One heavy chain variable region and one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment thereof comprises four antigen binding sites;
- Anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions comprising:
- the heavy chain variable region consists of an amino acid sequence of amino acid numbers 1 to 122 of SEQ ID NO: 2
- the light chain variable region consists of an amino acid sequence from amino acid numbers 1 to 113
- the format of the antibody is not particularly limited, and in the anti-human Tie2 antibody or antigen-binding fragment thereof according to the present invention, for example, Nat. Rev. Immunol. 2010, Vol. 10, p. 301-316, J.A. Immunol. , 2003, Vol. 170, p. 4854-4861, Mol. Immunol. 2000, Vol. 37, p. 1067-1077, Biochem. J. et al. , 2007, Vol. 406, p. 237-246, J. Org. Immunol. Methods, 2003, Vol. 279, p.
- Various tetravalent antibody formats such as those described in 219-232 can be used.
- the anti-human Tie2 antibody of the invention comprises two heavy chains and four light chains,
- Each heavy chain includes two structures consisting of a heavy chain variable region and an CH1 region consisting of the amino acid sequence from amino acid numbers 1 to 122 of SEQ ID NO: 2, and a C2 region and a CH3 region, and the C-terminal of one of the structures Is linked to the other N-terminus of the structure via a linker
- Each light chain includes a light chain variable region and a light chain constant region consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid number 1 to 113,
- Anti-human Tie2 antibody is also referred to as a tandem antibody, and an example thereof is shown in FIG.
- the constant region may be any subclass constant region (for example, the constant region of Ig ⁇ 1, Ig ⁇ 2, Ig ⁇ 3 or Ig ⁇ 4 as the heavy chain constant region, the light chain constant region). Ig ⁇ or Ig ⁇ constant regions) may also be selectable.
- the heavy chain constant region (including the CH1, CH2 and CH3 regions) is a human Ig ⁇ 1 constant region or a human Ig ⁇ 4 constant region.
- the light chain constant region is a human Ig ⁇ constant region.
- examples of the CH1 region, CH2 region, and CH3 region of the human Ig ⁇ 1 constant region include amino acid numbers 350 to 447 of SEQ ID NO: 8.
- a CH1 region consisting of an amino acid sequence, a CH2 region consisting of an amino acid sequence of amino acid numbers 463 to 572 of SEQ ID NO: 8, and a CH3 region consisting of an amino acid sequence of amino acid numbers 573 to 679 of SEQ ID NO: 8 can be mentioned.
- L234A amino acid position 234 according to the EU index of Kabat et al.
- L235A replacement of leucine with alanine at amino acid position 235 according to the EU index of Kabat et al.
- P331S substitution of proline with amino acid position 331 according to the EU index of Kabat et al.
- Etc substitution of proline with amino acid position 331 according to the EU index of Kabat et al.
- the human Ig ⁇ 1 constant region is a human Ig ⁇ 1 constant region having an amino acid mutation of L234A, L235A, and P331S, or L234A, Human Ig ⁇ 1 constant region with amino acid mutations L235A, P331S and I253A.
- Examples of the CH1 region, CH2 region, and CH3 region of the human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, and P331S include, for example, the CH1 region comprising the amino acid sequences of amino acid numbers 350 to 447 of SEQ ID NO: 2, SEQ ID NO: 2 A CH2 region consisting of the amino acid sequence of amino acid numbers 463 to 572, and a CH3 region consisting of the amino acid sequence of amino acid numbers 573 to 679 of SEQ ID NO: 2.
- Examples of the CH1 region, the CH2 region, and the CH3 region of the human Ig ⁇ 1 constant region having amino acid mutations L234A, L235A, P331S, and I253A include, for example, a CH1 region comprising an amino acid sequence of amino acid numbers 350 to 447 of SEQ ID NO: 6, A CH2 region comprising the amino acid sequence from amino acid numbers 463 to 572 of No. 6 and a CH3 region comprising the amino acid sequence from amino acid numbers 573 to 679 of SEQ ID No. 6.
- the human Ig ⁇ 4 constant region is a human Ig ⁇ 4 constant region having amino acid mutations of S228P and L235E.
- the CH1 region, CH2 region, and CH3 region of the human Ig ⁇ 4 constant region having amino acid mutations of S228P and L235E include, for example, the CH1 region consisting of the amino acid sequence of amino acid numbers 350 to 447 of SEQ ID NO: 10, the amino acid number of SEQ ID NO: 10 CH2 region consisting of amino acid sequence from 460 to 569, and CH3 region consisting of amino acid sequence from amino acid number 570 to 676 of SEQ ID NO: 10.
- human Ig ⁇ constant region examples include a human Ig ⁇ constant region consisting of the amino acid sequence of amino acid numbers 114 to 219 of SEQ ID NO: 4.
- the heavy chain constant region is a human Ig ⁇ 1 constant region or a human Ig ⁇ 4 constant region
- the light chain constant region is a human Ig ⁇ constant region.
- the heavy chain constant region is a human Ig ⁇ 1 constant region
- the human Ig ⁇ 1 constant region is a human Ig ⁇ 1 constant region having amino acid mutations of L234A, L235A, and P331S, or amino acid mutations of L234A, L235A, P331S, and I253A. It has a human Ig ⁇ 1 constant region.
- the heavy chain constant region is a human Ig ⁇ 4 constant region
- the human Ig ⁇ 4 constant region is a human Ig ⁇ 4 constant region having amino acid mutations of S228P and L235E.
- any peptide may be used as a linker for linking structures consisting of the heavy chain variable region and the CH1 region as long as the antibody has the function.
- the length and amino acid sequence of the peptide linker can be appropriately selected by those skilled in the art.
- the length of the peptide linker is 5-70 amino acids.
- the peptide linker comprises the amino acid sequence of the hinge region or a portion thereof.
- the hinge region means a region existing between the CH1 region and the CH2 region of the antibody. Examples of the hinge region to be used include an IgG1 or IgG3 hinge region.
- the part of the hinge region means a region in which at least 5 amino acids are contiguous in the hinge region, and preferably means a region in which at least 5 amino acids are contiguous from the N-terminus of the hinge region.
- a part of the hinge region for example, in the case of an IgG1 hinge region, a region that is 5 amino acids continuous from the N-terminus (consisting of amino acid sequences from amino acid numbers 1 to 5 of SEQ ID NO: 13), and an IgG3 hinge region And a region (consisting of amino acid sequences from amino acid numbers 1 to 12 in SEQ ID NO: 14) that is 12 amino acids continuous from the N-terminus.
- the linker comprises an amino acid sequence of a region that is at least 5 amino acids continuous from the N-terminus of the hinge region, and comprises the amino acid sequence GlySer at the C-terminus of the linker.
- Examples of such a linker include a peptide linker consisting of the amino acid sequence shown in any of SEQ ID NOs: 13 to 20, and preferably the linker consists of the amino acid sequence shown in SEQ ID NO: 13.
- the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having any of the following characteristics i) to iv): i) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4. ii) An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 8 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention includes an anti-human Tie2 antibody or antigen-binding fragment thereof subjected to post-translational modification.
- anti-human Tie2 antibodies or antigen-binding fragments thereof of the present invention that have undergone post-translational modification include anti-human Tie2 that has undergone pyroglutamylation at the heavy chain variable region N-terminus and / or lysine deletion at the heavy chain C-terminus
- An antibody or an antigen-binding fragment thereof can be mentioned.
- the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having any of the following features (1) to (4).
- Two heavy chains consisting of an amino acid sequence in which the glutamic acid of amino acid number 1 of SEQ ID NO: 2 is modified with pyroglutamic acid and / or lacks the lysine of amino acid number 679 of SEQ ID NO: 2, and the amino acid shown in SEQ ID NO: 4
- An anti-human Tie2 antibody comprising four light chains consisting of a sequence.
- An anti-human Tie2 antibody comprising four light chains consisting of a sequence.
- An anti-human Tie2 antibody comprising four light chains consisting of a sequence.
- Two heavy chains consisting of an amino acid sequence in which the glutamic acid of amino acid number 1 of SEQ ID NO: 10 is modified with pyroglutamic acid and / or lacks the lysine of amino acid number 676 of SEQ ID NO: 10, and the amino acid shown in SEQ ID NO: 4
- An anti-human Tie2 antibody comprising four light chains consisting of a sequence.
- the anti-human Tie2 antibody of the present invention is an anti-human Tie2 antibody having the following characteristics.
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the present invention also includes an anti-human Tie2 antibody or antigen-binding fragment thereof having the following characteristics.
- An anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions comprising:
- the heavy chain variable region consists of CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 2, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 2, and amino acid number 99 of SEQ ID NO: 2.
- CDR3 consisting of up to 111 amino acid sequences
- the light chain variable region is composed of CDR1 consisting of amino acid sequences of amino acid numbers 24 to 39 of SEQ ID NO: 4, CDR2 consisting of amino acid sequences of amino acid numbers 55 to 61 of SEQ ID NO: 4, and amino acid number 94 of SEQ ID NO: 4.
- CDR3 consisting of up to 102 amino acid sequences
- One heavy chain variable region and one light chain variable region constitute one antigen binding site, and the antibody or antigen binding fragment thereof comprises four antigen binding sites; Anti-human Tie2 antibody or antigen-binding fragment thereof.
- the present invention also includes an anti-human Tie2 antibody having the following characteristics. Comprising two heavy chains and four light chains, Each heavy chain consists of 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 to amino acid numbers 50 to 66, and SEQ ID NO: 2 from amino acid numbers 99 to 111 2 comprising a heavy chain variable region comprising CDR3 consisting of the amino acid sequence of the above and a CH1 region, and a CH2 region and a CH3 region, wherein the carboxy terminus of one of the structures is linked to the other of the structure via a linker.
- Each light chain consists of CDR1 consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid numbers 24 to 39, CDR2 consisting of the amino acid sequence of SEQ ID NO: 4 from amino acid numbers 55 to 61, and amino acid numbers 94 to 102 of SEQ ID NO: 4
- a light chain variable region comprising CDR3 consisting of the amino acid sequence and a light chain constant region, Anti-human Tie2 antibody.
- the anti-human Tie2 antibody of the present invention is an antibody that binds to human Tie2. Whether or not the antibody binds to human Tie2 (Accession No. NP — 0450.2) can be confirmed using a known binding activity measurement method. Examples of the method for measuring the binding activity include a method such as Enzyme-Linked ImmunoSorbent Assay (ELISA).
- ELISA Enzyme-Linked ImmunoSorbent Assay
- a secondary antibody such as an anti-IgG antibody labeled with biotin is reacted, washed, and then reacted with streptavidin to which an enzyme such as alkaline phosphatase is bound. After washing, activity measurement is performed using a reagent that detects the activity (for example, in the case of alkaline phosphatase, Chemiluminescent ultra Sensitive AP Microwell and / or Membrane Substrate (450 nm) (BioFX, APU4-0100-01)). Thus, it can be confirmed whether or not the test antibody binds to human Tie2.
- a specific method for evaluating activity for example, a method as described in Example 12 below can be used.
- the anti-human Tie2 antibody of the present invention includes antibodies that bind to human Tie2 as well as antibodies that bind to other animal-derived Tie2 (eg, monkey Tie2) as long as they bind to human Tie2.
- animal-derived Tie2 eg, monkey Tie2
- the anti-human Tie2 antibody of the present invention binds to human Tie2 and has anti-apoptotic activity against human Tie2-expressing cells.
- a specific method for evaluating that the antibody has anti-apoptotic activity against human Tie2-expressing cells for example, a method as described in Example 4 below can be used.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention comprises two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4 Binds to the same human Tie2 epitope as an antibody or anti-human Tie2 antibody comprising two heavy chains consisting of amino acid sequences from amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of amino acid sequences shown in SEQ ID NO: 4 Also included are tetravalent anti-human Tie2 antibodies or antigen-binding fragments thereof.
- the epitope refers to an antigen site recognized by an antibody.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention binds to a human Tie2 epitope containing at least one amino acid among amino acids 192, 195, and 197 of human Tie2 (Accession No. NP — 0450.2). Also included are tetravalent anti-human Tie2 antibodies or antigen-binding fragments thereof.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention binds to a human Tie2 epitope containing amino acid numbers 192, 195 and 197 of human Tie2 (Accession No. NP — 0450.2), and a tetravalent anti-human Tie2 Also included are antibodies or antigen-binding fragments thereof.
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4 or amino acid sequences from amino acid numbers 1 to 678 of SEQ ID NO: 2
- a tetravalent anti-human Tie2 antibody or antigen-binding fragment thereof that binds to the same human Tie2 epitope as an anti-human Tie2 antibody comprising two heavy chains consisting of 4 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4 , And can be obtained using a known epitope determination method.
- Methods for determining epitopes include, for example, deuterium exchange mass spectrometry, X-ray crystal structure analysis, and ELISA and surface plasmon resonance phenomenon using amino acid substitution mutants of human Tie2 or human Tie2 partial peptides, etc. A method is mentioned.
- human Tie2 substituted with deuterium in the absence of the test antibody and human Tie2 substituted with deuterium in the presence of the test antibody are each decomposed into peptides, and the molecular weight of each peptide is measured. Calculate the percentage of deuterium substitution.
- the human Tie2 epitope of the test antibody can be determined from the difference in the rate of deuterium substitution of human Tie2 depending on the presence or absence of the test antibody.
- ELISA a point mutant of human Tie2 is generated. Mutant human Tie2 is immobilized, and a test antibody is added thereto for reaction.
- a secondary antibody such as a biotin-labeled anti-human kappa light chain antibody is reacted and washed. Thereafter, alkaline phosphatase labeled streptavidin (Thermo Fisher Scientific, 21324) is reacted and washed. Whether or not the test antibody binds to the mutant human Tie2 can be confirmed by performing activity measurement using Chemiluminescent Ultra Sensitive AP Microwell and / or Membrane Substrate (450 nm). By evaluating the binding activity to various mutant human Tie2, the epitope of the test antibody can be determined.
- the test antibody comprises at least one amino acid in the epitope of an anti-human Tie2 antibody comprising two heavy chains consisting of amino acid sequences from 1 to 678 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4 or amino acids from amino acid numbers 1 to 678 of SEQ ID NO: 2 It binds to the same human Tie2 epitope as the anti-human Tie2 antibody comprising a heavy chain consisting of the sequence and a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4. Then it can be determined
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention can be obtained by a method known in the art based on the sequence information of the heavy chain variable region and light chain variable region of the antibody of the present invention disclosed in the present specification. And can be easily made by those skilled in the art.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention is not particularly limited. For example, the following ⁇ Method for producing anti-human Tie2 antibody of the present invention and anti-human Tie2 antibody produced by the method are described below. > Can be produced according to the method described above.
- the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention is further purified if necessary, and then formulated in accordance with a conventional method, followed by diabetic retinopathy, diabetic macular edema, sepsis, acute liver injury, acute kidney injury, acute lung injury It can be used for the prevention or treatment of vascular related diseases such as systemic inflammatory reaction syndrome, peripheral artery occlusion or severe leg ischemia.
- vascular related diseases such as systemic inflammatory reaction syndrome, peripheral artery occlusion or severe leg ischemia.
- the polynucleotide of the present invention includes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention, and the anti-human Tie2 antibody or antigen-binding fragment of the present invention.
- a polynucleotide comprising a base sequence encoding a light chain variable region is included.
- the polynucleotide comprising the base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention has a heavy sequence consisting of the amino acid sequence of amino acid numbers 1 to 122 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 122 examples include, for example, a polynucleotide containing the base sequence of SEQ ID NO: 1 from base No. 1 to 366 Nucleotides are mentioned.
- the polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention is a base sequence encoding a heavy chain consisting of the amino acid sequence shown in SEQ ID NO: 2.
- 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 containing the base sequence which codes the heavy chain which consists of an amino acid sequence shown by sequence number 6 is mentioned, for example.
- the polynucleotide containing the base sequence which codes the heavy chain which consists of an amino acid sequence shown by sequence number 8 the polynucleotide containing the base sequence shown by sequence number 7 is mentioned, for example.
- As a polynucleotide containing the base sequence which codes the heavy chain which consists of an amino acid sequence shown by sequence number 10 the polynucleotide containing the base sequence shown by sequence number 9 is mentioned, for example.
- the polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention is a light chain consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 4.
- polynucleotide containing the base sequence encoding the light chain variable region shown in the amino acid sequence of SEQ ID NO: 4 from amino acid Nos. 1 to 113 examples include, for example, a polynucleotide containing the base sequence of SEQ ID NO: 3 from base No. 1 to 339 Nucleotides are mentioned.
- the polynucleotide comprising the base sequence encoding the light chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention is a base sequence encoding a light chain consisting of the amino acid sequence shown in SEQ ID NO: 4
- 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. As such a 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.
- a gene synthesis method various methods known to those skilled in the art, such as an antibody gene synthesis method described in WO90 / 07861
- the expression vector of the present invention includes a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention and / or the anti-human Tie2 antibody of the present invention or an antigen-binding fragment thereof.
- An expression vector comprising a polynucleotide comprising a base sequence encoding a light chain variable region is included.
- Various formats of tetravalent antibodies and production methods thereof are known in the art, and the expression vector of the present invention can be easily constructed by those skilled in the art according to the format of the tetravalent antibody to be expressed according to these production methods. .
- 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 Tie2 antibody of the present invention, and a base sequence encoding the light chain of the anti-human Tie2 antibody of the present invention. And an expression vector comprising a polynucleotide, or an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody. .
- 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).
- eukaryotic cells eg, animal cells, insect cells, plant cells, yeast
- prokaryotic cells eg, E. coli
- a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention and / or a base encoding the light chain variable region of the anti-human Tie2 antibody of the present invention or an antigen-binding fragment thereof
- the polynucleotide is not particularly limited as long as it can express a polynucleotide containing the sequence and produce a polypeptide encoded by these.
- expression vectors examples include plasmid vectors, virus vectors (eg, adenovirus, adeno-associated virus, retrovirus, Sendai virus), and preferably, pEE6.4 or pEE12.4 (Lonza).
- virus vectors eg, adenovirus, adeno-associated virus, retrovirus, Sendai virus
- pEE6.4 or pEE12.4 e.g. pEE6.4 or pEE12.4 (Lonza).
- an antibody gene can be expressed using an expression vector having a human Ig constant region gene in advance, such as AG- ⁇ 1 and AG- ⁇ (see, for example, 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).
- A a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention and a polynucleotide comprising a base sequence encoding the light chain variable region of the antibody or antigen-binding fragment thereof
- a host cell transformed with an expression vector comprising a nucleotide (B) an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention and a base encoding the light chain variable region of the antibody or antigen-binding fragment thereof
- C a host cell transformed with an expression vector comprising a polynucleotide comprising the sequence
- C a host cell transformed with an expression vector compris
- 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).
- A a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
- B transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody Host cell;
- C a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention; and (d) encoding the light chain of the
- Preferred transformed host cells of the present invention include a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody.
- a host cell transformed with an expression vector, and an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody And a host cell transformed with an expression vector.
- 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, Pichia), and the like, and 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 a tetravalent anti-human Tie2 antibody or its A method of producing an anti-human Tie2 antibody or antigen-binding fragment thereof comprising the step of expressing the antigen-binding fragment is included.
- B an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human Tie2 antibody or antigen-binding fragment thereof of the present invention and a base encoding the light chain variable region of the antibody or antigen-binding fragment thereof
- the method for producing an anti-human Tie2 antibody of the present invention comprises culturing a host cell selected from the group consisting of the following (a) to (c) and expressing the anti-human Tie2 antibody: A method of producing an anti-human Tie2 antibody.
- A a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and a polynucleotide comprising a base sequence encoding the light chain of the antibody;
- B transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding the heavy chain of the anti-human Tie2 antibody of the present invention and an expression vector comprising a polynucleotide comprising the base sequence encoding the light chain of the antibody A host cell; and
- the method for producing the anti-human Tie2 antibody of the present invention is not particularly limited as long as it includes the step of culturing the transformed host cell of the present invention and expressing the anti-human Tie2 antibody.
- 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, pH of the medium 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, antibiotics (eg, tetracycline, neomycin, ampicillin, kanamycin, etc.)) Good.
- the pH of the medium is preferably about 5-8.
- preferred media include LB medium, M9 medium (Mol. Clo., Cold Spring Harbor Laboratory, Vol. 3, A2.2) and the like. 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 and agitation, if necessary.
- the anti-human Tie2 antibody of the present invention or an antigen-binding fragment thereof can be expressed.
- the method of producing the anti-human Tie2 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 Tie2 antibody or antigen-binding fragment thereof, Recovering, preferably isolating or purifying the anti-human Tie2 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 Tie2 antibody of the present invention also includes an anti-human Tie2 antibody or an antigen-binding fragment thereof produced by the method for producing the anti-human Tie2 antibody of the present invention or an antigen-binding fragment thereof.
- the pharmaceutical composition of the present invention includes a pharmaceutical composition comprising the anti-human Tie2 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, intraocular 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 the anti-human Tie2 antibody of the present invention or an antigen-binding fragment 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 present invention containing an anti-human Tie2 antibody or antigen-binding fragment thereof also includes the pharmaceutical composition described below.
- An anti-human Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region consists of an amino acid sequence of SEQ ID NO: 2 from amino acid numbers 1-122;
- the light chain variable region comprises the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 4, one heavy chain variable region and one light chain variable region constitute one antigen-binding site, and the antibody or An anti-human Tie2 antibody or antigen-binding fragment thereof, the antigen-binding fragment of which comprises four antigen-binding sites, as well as an antibody or antigen-binding fragment thereof resulting from post-translational modification of the antibody or antigen-binding fragment thereof
- a pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof.
- the pharmaceutical composition of the present invention containing an anti-human Tie2 antibody also includes the pharmaceutical composition described below.
- Two heavy chains and four light chains each heavy chain comprising two structures consisting of a heavy chain variable region consisting of amino acid sequences from SEQ ID NO: 2 amino acid number 1 to 122 and a CH1 region, and a CH2 region and Including the CH3 region, wherein the C-terminus of one of the structures is linked to the N-terminus of the other structure via a linker, and each light chain is derived from the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 4
- An anti-human Tie2 antibody comprising a light chain variable region and a light chain constant region, and a pharmaceutical composition comprising an anti-human Tie2 antibody which is an antibody produced by post-translational modification of the antibody.
- 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 Tie2 antibody also includes a pharmaceutical composition containing two or more anti-human Tie2 antibodies among the following (1) to (4).
- An anti-human Tie2 antibody comprising two heavy chains consisting of amino acid sequences from amino acid numbers 1 to 678 of SEQ ID NO: 2 and four light chains consisting of the amino acid sequence represented by SEQ ID NO: 4.
- a light chain comprising the amino acid sequence of amino acid numbers 1 to 678 of SEQ ID NO: 2, two heavy chains in which the glutamic acid of amino acid number 1 is modified with pyroglutamic acid, and the amino acid sequence represented by SEQ ID NO: 4
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the pharmaceutical composition of the present invention containing an anti-human Tie2 antibody also includes a pharmaceutical composition containing two or more anti-human Tie2 antibodies among the following (1) to (4).
- An anti-human Tie2 antibody comprising two heavy chains consisting of amino acid sequences from SEQ ID NO: 6 to amino acid numbers 1 to 678 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- a light chain comprising the amino acid sequence of amino acid numbers 1 to 678 of SEQ ID NO: 6, two heavy chains in which glutamic acid of amino acid number 1 is modified with pyroglutamic acid, and an amino acid sequence represented by SEQ ID NO: 4
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 6 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the pharmaceutical composition of the present invention containing an anti-human Tie2 antibody also includes a pharmaceutical composition containing two or more anti-human Tie2 antibodies among the following (1) to (4).
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of amino acid numbers 1 to 678 of SEQ ID NO: 8 and four light chains consisting of the amino acid sequence represented by SEQ ID NO: 4.
- a light chain comprising the amino acid sequence of amino acid numbers 1 to 678 of SEQ ID NO: 8, two heavy chains in which glutamic acid of amino acid number 1 is modified with pyroglutamic acid, and an amino acid sequence represented by SEQ ID NO: 4
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 8 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the pharmaceutical composition of the present invention containing an anti-human Tie2 antibody also includes a pharmaceutical composition containing two or more anti-human Tie2 antibodies among the following (1) to (4).
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence of amino acid numbers 1 to 675 of SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- a light chain comprising the amino acid sequence of amino acid numbers 1 to 675 of SEQ ID NO: 10, two heavy chains in which glutamic acid of amino acid number 1 is modified with pyroglutamic acid, and an amino acid sequence represented by SEQ ID NO: 4
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 10 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4.
- the pharmaceutical composition of the present invention containing an anti-human Tie2 antibody or antigen-binding fragment thereof also includes the pharmaceutical composition described below.
- An anti-human Tie2 antibody comprising two heavy chains consisting of the amino acid sequence shown in SEQ ID NO: 2 and four light chains consisting of the amino acid sequence shown in SEQ ID NO: 4, amino acid sequences from amino acid numbers 1 to 678 of SEQ ID NO: 2
- An anti-human Tie2 antibody comprising 2 heavy chains consisting of 4 and 4 light chains consisting of the amino acid sequence shown in SEQ ID NO: 4, and a pharmaceutically acceptable excipient.
- the amount of the anti-human Tie2 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 used, the binding titer of the antibody, etc. About 001 mg / kg to 100 mg / kg can be used.
- the pharmaceutical composition of the present invention is a blood vessel-related disease such as diabetic retinopathy, diabetic macular edema, sepsis, acute liver injury, acute kidney injury, acute lung injury, systemic inflammatory response syndrome, peripheral arterial occlusion, or severe leg collapse. It can be used as a preventive or therapeutic agent for blood and the like.
- the present invention includes a pharmaceutical composition for the prevention or treatment of diabetic macular edema, diabetic retinopathy or severe leg ischemia comprising the anti-human Tie2 antibody of the present invention.
- the present invention also includes a method for preventing or treating diabetic macular edema, diabetic retinopathy or severe lower limb ischemia, comprising the step of administering a therapeutically effective amount of the anti-human Tie2 antibody of the present invention.
- the present invention also includes the anti-human Tie2 antibody of the present invention for use in the prevention or treatment of diabetic macular edema, diabetic retinopathy or severe lower limb ischemia.
- the present invention includes the use of the anti-human Tie2 antibody of the present invention in the manufacture of a pharmaceutical composition for the prevention or treatment of diabetic macular edema, diabetic retinopathy or severe leg ischemia.
- 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 Tie2 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 Tie2 antibody or antigen-binding fragment thereof comprising four heavy chain variable regions and four light chain variable regions, wherein the heavy chain variable region is derived from the amino acid sequence of amino acid numbers 1 to 122 of SEQ ID NO: 2.
- the light chain variable region comprises an amino acid sequence from amino acid numbers 1 to 113 of SEQ ID NO: 4, one heavy chain variable region and one light chain variable region constitute one antigen binding site,
- the anti-human Tie2 antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof contains four antigen-binding sites, is bound with an anti-human Tie2 antibody or antigen-binding fragment thereof or a modifying agent fused with another peptide or protein.
- anti-human Tie2 antibodies or antigen-binding fragments thereof As long as the antibody of the present invention or the antigen-binding fragment thereof has a binding activity to human Tie2 as a fusion antibody, other peptides or proteins used for fusion are not particularly limited.
- human serum albumin various tag peptides And artificial helix motif peptides, maltose-binding proteins, glutathione S-transferases, various toxins, and other peptides or proteins that can promote multimerization.
- 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 Production of anti-human Tie2 antibody-producing hybridoma
- Human monoclonal antibody development technology “Velocimmune antibody technology: Regeneron (US Pat. No. 6,596,541)” mice were used to produce antibodies. Verosimune mice were immunized with recombinant human Tie2-Fc chimeric protein (R & D, 313-TI-100) together with an adjuvant that elicits an immune response. According to a conventional method, the lymph node of the immunized mouse was removed, lymphocytes were collected, and this was cell-fused with mouse-derived myeloma cells SP2 / 0 (ATCC: CRL-1581) to prepare a hybridoma.
- Hybridomas were monocloned and each clone was cultured in CD hybridoma medium (Invitrogen), which is a serum-free medium.
- the antibody was purified from the obtained culture supernatant using a protein G column (GE Healthcare).
- the antibody obtained by the Verosimune technology is an antibody (also referred to as a chimeric antibody) having a variable region of a human antibody and a constant region of a mouse antibody.
- Example 2 Cell ELISA assay
- human, monkey, rat and mouse Tie2 were analyzed by cell ELISA assay using human Tie2 expressing CHO cells, monkey Tie2 expressing CHO cells, rat Tie2 expressing CHO cells and mouse Tie2 expressing CHO cells. The binding of the antibody to was evaluated.
- Example 3 Evaluation of antagonistic activity using Ang-1 variant
- an Ang-1 variant Proc. Natl. Acad. Sci., 2004, Vol. 101, p. 5547-5552, also referred to as COMP-Ang1
- COMP-Ang1 is an Ang-1 variant in which a site not involved in binding to Tie2 is modified, and retains the binding ability to Tie2 (Proc. Natl. Acad. Sci., 2004, Vol. 101, p. 5547-5552) and Ang-1 and Ang-2 bind to the same site of Tie2 with similar affinity (Science, 1997, Vol. 277, p. 55-60) to COMP-Ang1
- the antagonism to Ang-2 can be evaluated.
- COMP-Ang1 expression vector was introduced into HEK293 cells.
- COMP-Ang1 was purified from the culture supernatant of the HEK293 cells and labeled with biotin.
- the biotin-labeled COMP-Ang1 and the purified antibody obtained in Example 1 were mixed and added to a plate immobilized with a recombinant human Tie2-Fc chimeric protein.
- Streptavidin-labeled HRP was used for detection of bound biotin-labeled COMP-Ang1.
- 2M sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured. This evaluated the antagonistic effect of the antibody on COMP-Ang1.
- Example 4 Evaluation of anti-apoptotic activity using human Tie2-expressing BaF3 cells
- a mouse pro B cell line BaF3 cell stably expressing human Tie2 (hereinafter referred to as human Tie2 expressing BaF3 cell) is described in Immunity, 1998, Vol. 9, p.
- a plasmid containing the human Tie2 gene represented by SEQ ID NO: 21 was introduced into the cells by electroporation. Thereafter, the anti-apoptotic activity of the antibody was evaluated using the same cells.
- Human Tie2-expressing BaF3 cells are suspended in 2 ⁇ 10 5 cells / mL in RPMI 1640 medium (Life Technologies) containing 0.05% fetal bovine serum and per well in a 96-well plate for suspension cells (Sumitomo Bakelite, MS-8096R). 80 ⁇ L was seeded. Thereafter, 20 ⁇ L of the purified antibody or Ang-1 obtained in Example 1 was added. After culturing in a CO 2 incubator set at 37 ° C. for 72 hours, 50 ⁇ L of the cell suspension was transferred to a white 96-well plate (Nunc, 236108).
- cell viability was measured by adding 50 ⁇ L of the substrate solution diluted with the attached buffer to the cell suspension. Anti-apoptotic activity was assessed by measuring viability.
- Example 5 Analysis of purified antibody solution using size exclusion chromatography and electrophoresis
- the purified antibody solutions identified in Examples 2-4 above were analyzed by size exclusion chromatography. As a result, three fractions were detected from each purified antibody solution. As a result of analyzing each fraction solution by electrophoresis, it became clear that each fraction contained an antibody monomer, a dimer, and a multimer more than a trimer.
- Example 4 the anti-apoptotic activity of each fraction solution was evaluated by the method shown in Example 4. As a result, a strong anti-apoptotic activity was observed in the fraction containing a dimer and the fraction containing a trimer or higher multimer. On the other hand, almost no anti-apoptotic activity was observed in the fraction containing the monomer. 15B8 was analyzed by size exclusion chromatography in the same manner as described above, and as a result, a fraction showing a dimer or higher multimer was detected, but a monomer fraction was hardly detected.
- the fraction containing a dimerized or more multimerized antibody contained a strong anti-apoptotic activity. Since the antibody that formed the dimer becomes a tetravalent antibody, it was suggested that an antibody having a valence of 4 or more has a strong anti-apoptotic activity through activation of human Tie2.
- Example 6 Evaluation of anti-apoptotic activity by cross-linked antibody
- the anti-human Tie2 antibody having a valence of 4 or more is important for inducing anti-apoptotic activity via Tie2. Therefore, the anti-apoptotic activity of the anti-human Tie2 antibody increased in quantity by crosslinking with an anti-mouse IgG antibody was evaluated.
- cells human Tie2-expressing BaF3 cells and human vascular endothelial cells HUVEC that endogenously express human Tie2 were used.
- Human Tie2-expressing BaF3 cells were cultured in RPMI 1640 medium and HUVEC were cultured in EBM-2 serum-free medium (Lonza), respectively, and a purified antibody solution containing the anti-human Tie2 antibody identified in Examples 2 to 4 was added. Further anti-mouse IgG antibody was added to crosslink the antibody. Cell viability was measured using CellTiter Glo Luminescent Cell Viability Assay. Anti-apoptotic activity was assessed by measuring viability.
- Example 7 Sequencing of a bivalent anti-human Tie2 antibody
- the genes encoding the heavy and light chains of the antibody were cloned from the hybridoma producing anti-human Tie2 antibody 2-16 and sequenced.
- the framework regions (FR) of 2-16 light chain and heavy chain are replaced with FRs of other human antibodies, and the modified anti-human Tie2 antibody A variable region of 2-16A2 was prepared.
- a gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, p. 499-505) on the 5 ′ side of the heavy chain variable region gene of 2-16A2, and human Ig ⁇ 1 constant on the 3 ′ side
- the region genes (consisting of the base sequences from base numbers 367 to 1356 of SEQ ID NO: 11) were respectively connected, and this heavy chain gene was inserted into the GS vector pEE6.4.
- a gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, p. 499-505) is located on the 5 ′ side of the light chain variable region gene, and the human ⁇ chain constant is located on the 3 ′ side.
- the region genes (consisting of base sequences from base numbers 340 to 657 of SEQ ID NO: 3) were connected to each other, and this light chain gene was inserted into the GS vector pEE12.4.
- the heavy chain gene sequence and light chain gene sequence of the prepared antibody were analyzed with a sequencer.
- the base sequence of the heavy chain of the prepared 2-16A2 fully human antibody (fully human type 2-16A2) is shown in SEQ ID NO: 11, the amino acid sequence encoded thereby is SEQ ID NO: 12, and the light chain base 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 shown in SEQ ID NO: 12 consists of the amino acid sequence from amino acid numbers 1 to 122 of SEQ ID NO: 12, and the variable region of the light chain shown in SEQ ID NO: 4 from amino acid number 1 of SEQ ID NO: 4 It consists of up to 113 amino acid sequences.
- Antibody expression was carried out by two methods, transient expression and constitutive expression, using the aforementioned GS vector into which the heavy chain and light chain genes of fully human type 2-16A2 were inserted.
- transient expression both the heavy and light chain expression vectors described above were introduced into FreeStyle 293 cells (Invitrogen) cultured at about 1 million cells / mL in FreeStyle 293 Expression medium (Invitrogen). Transfected with reagent 293fectin (Invitrogen) and cultured for 5 days.
- Expi293 cells (Invitrogen) cultured at about 3 million cells / mL in Expi293 Expression medium (Invitrogen)
- the above-described both heavy chain and light chain expression vectors are introduced into the gene introduction kit Expifectamine293 Transfection Kit (Invitrogen).
- Expifectamine293 Transfection Kit (Invitrogen).
- both the above heavy chain and light chain expression vectors can be electroporated. And transfected for 7 days.
- Each culture supernatant was purified using a protein A column or protein G column (GE Healthcare) to obtain a purified antibody of a fully human antibody.
- the above-described GS vector into which the antibody heavy chain and light chain genes were inserted was digested with NotI and PvuI, and ligation kit Ligation-Convenience Kit (NIPPONGENE) or ligation reagent Ligation high Ver. 2 (TOYOBO) was used for ligation to construct a GS vector in which both heavy chain and light chain genes were inserted.
- the antibody was expressed by transfection of this expression vector into CHO-K1SV cells.
- the culture supernatant was purified with a protein A column, protein G column or MabSelect SuRe (GE Healthcare, 17-5438-02) to obtain a purified antibody of a fully human antibody.
- Example 8 Production of tetravalent anti-human Tie2 antibody
- the tetravalent antibody produced in this example includes two heavy chains and four light chains.
- Each heavy chain includes two structures consisting of a heavy chain variable region and a CH1 region, and a CH2 region and a CH3 region, and the C terminus of the structure consisting of one of the heavy chain variable region and the CH1 region is already connected via a linker.
- One of the structures is linked to the N-terminus.
- Each light chain includes a light chain variable region and a light chain constant region. The format of this tetravalent antibody is shown in FIG.
- a linker comprising the amino acid sequence shown in SEQ ID NO: 13 at the C-terminus of the structure consisting of the heavy chain variable region and CH1 region of fully human type 2-16A2 (consisting of amino acid sequences from amino acid numbers 1 to 220 of SEQ ID NO: 12)
- a gene encoding a tetravalent anti-human Tie2 antibody heavy chain linked to the N-terminus of the fully human type 2-16A2 heavy chain was prepared.
- a gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, p. 499-505) was ligated to the 5 'side of the heavy chain gene and inserted into the GS vector pEE6.4.
- a method similar to the antibody expression and purification method described in Example 7 by combining the heavy chain vector and the GS vector pEE12.4 in which the light chain gene of fully human type 2-16A2 prepared in Example 7 is inserted was used to prepare a tetravalent anti-human Tie2 antibody.
- the anti-human Tie2 antibody is referred to as TIE-1-Ig ⁇ 1-WT.
- the constant region in the heavy chain of TIE-1-Ig ⁇ 1-WT is a human Ig ⁇ 4 constant region into which amino acid mutations of S228P and L235E have been introduced (the amino acid sequence from amino acid numbers 123 to 220 of SEQ ID NO: 10 and the amino acid of SEQ ID NO: 10)
- a gene encoding a tetravalent anti-human Tie2 antibody heavy chain replaced with the amino acid sequence of numbers 350 to 676 was prepared.
- a gene encoding a signal sequence (Protein Engineering, 1987, Vol. 1, No. 6, p. 499-505) was ligated to the 5 'side of the heavy chain gene and inserted into the GS vector pEE6.4.
- a method similar to the antibody expression and purification method described in Example 7 by combining the heavy chain vector and the GS vector pEE12.4 in which the light chain gene of fully human type 2-16A2 prepared in Example 7 is inserted was used to prepare a tetravalent anti-human Tie2 antibody.
- the anti-human Tie2 antibody is referred to as TIE-1-Ig ⁇ 4-PE.
- the base sequence of the heavy chain of TIE-1-Ig ⁇ 1-WT is shown in SEQ ID NO: 7, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 8, respectively.
- the heavy chain base sequence of TIE-1-Ig ⁇ 4-PE is shown in SEQ ID NO: 9, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 10, respectively.
- the light chain of both antibodies is the same as the light chain of fully human type 2-16A2, and the base sequence of the light chain of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 4, respectively.
- TIE-1-Ig ⁇ 1-LALA tetravalent anti-human Tie2 antibody
- TIE-1-Ig ⁇ 1-I253A tetravalent anti-human Tie2 antibody
- the base sequence of the heavy chain of TIE-1-Ig ⁇ 1-LALA is shown in SEQ ID NO: 1, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2, respectively.
- the base sequence of the heavy chain of TIE-1-Ig ⁇ 1-I253A is shown in SEQ ID NO: 5, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 6, respectively.
- the light chain of both antibodies is the same as the light chain of fully human type 2-16A2, and the base sequence of the light chain of the antibody is shown in SEQ ID NO: 3, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 4, respectively.
- variable regions of the four types of tetravalent anti-human Tie2 antibody heavy chains represented by SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 are common, and amino acids 1 to 120 of SEQ ID NO: 2 CDR1, CDR2, and CDR3 of the heavy chain variable region are amino acid sequences from amino acid numbers 31 to 35, 50 to 66, and 99 to 111 of SEQ ID NO: 2, respectively.
- variable region of the light chain of the four types of tetravalent anti-human Tie2 antibodies represented by SEQ ID NO: 4 consists of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 4, and CDR1, CDR2, CDR3 consists of amino acid sequences from amino acid numbers 24 to 39, 55 to 61, and 94 to 102 of SEQ ID NO: 4, respectively.
- the linker (consisting of the amino acid sequence shown in SEQ ID NO: 13) is replaced with another linker (TIE-1-Ig ⁇ 1- 4 types of linkers consisting of amino acid sequences shown in SEQ ID NOs: 17 to 20 for WT, and 7 types of linkers consisting of amino acid sequences shown to SEQ ID NOs: 14 to 20 for TIE-1-Ig ⁇ 4-PE, respectively.
- Tetravalent anti-human Tie2 antibodies (11 types in total) were prepared.
- TIE-1-Ig ⁇ 1-WT TIE-1-Ig ⁇ 4-PE
- eleven types of antibodies in which the linker was replaced were evaluated.
- all anti-human Tie2 antibodies showed similar anti-apoptotic activity. It became clear to show.
- Example 9 Evaluation of anti-apoptotic action of divalent anti-human Tie2 antibody and tetravalent anti-human Tie2 antibody
- an antibody having a valence of 4 or more has a strong anti-apoptotic activity through activation of human Tie2. Therefore, the efficacy of the bivalent anti-human Tie2 antibody and the tetravalent anti-human Tie2 antibody was compared using the anti-apoptotic effect on human Tie2-expressing BaF3 cells as an index.
- the anti-apoptotic effect on human Tie2-expressing BaF3 cells was evaluated using the bivalent antibody fully human type 2-16A2 and the tetravalent antibody TIE-1-Ig ⁇ 1-WT.
- the test antibodies, fully human type 2-16A2 and TIE-1-Ig ⁇ 1-WT were purified by MabSelect SuRe, and fractionated into monomer fractions by size exclusion chromatography, which were 99.98% and 99.98%, respectively. Obtained as 74% monomer purity.
- Each antibody was diluted with phosphate buffered saline (PBS) from 5 ng / mL to 5000 ng / mL at a 3-fold common ratio in 7 steps, and 20 ⁇ L was added per well.
- PBS phosphate buffered saline
- Ang-1 diluted with PBS (R & D, 923-AN-025 / CF. Seven levels with a final common ratio of 1 ng / mL to 1000 ng / mL at approximately three times the common ratio.
- Each well to which (dilution) was added was prepared.
- the measured value of the well added with PBS instead of the test antibody was 0%, and Ang-1 was 300 ng / mL and 1000 ng / mL instead of the test antibody, respectively.
- the average value of the measured values of the added wells was set as 100%.
- the calculated anti-apoptotic activity was analyzed, and the EC 50 value of the test antibody was calculated by sigmoid-Emax model nonlinear regression analysis.
- Table 1 Anti-apoptotic activity of divalent anti-human Tie2 antibody and tetravalent anti-human Tie2 antibody
- the tetravalent antibody TIE-1-Ig ⁇ 1-WT has a strong anti-apoptotic action. From the above, it has been clarified that the tetravalent antibody has superior anti-apoptotic activity than the divalent antibody.
- Example 10 Evaluation of rat blood vessel permeability inhibitory action of divalent anti-human Tie2 antibody and tetravalent anti-human Tie2 antibody
- the mustard oil-induced vascular permeability model is a modification of Miles assay (J. Physiol., 1952, Vol. 118, p. 228-257) widely used as a plasma leakage evaluation system. It has been reported that Ang-1 inhibits vascular permeability enhancement (Nature Medicine, 2000, Vol. 6, p. 460-463).
- Evans Blue dye 45 mg / kg, Sigma-Aldrich, E2129 dissolved in physiological saline was intravenously administered, and immediately after that it was diluted to 5% with mineral oil (Sigma-Aldrich, M8410). Allyl acid (also referred to as mustard oil; Nacalai Tesque, 01415-92) was applied to one ear and 20 ⁇ L of mineral oil was applied to the opposite ear. After 30 minutes, both ears were sampled, weighed and then immersed in 1 mL of formamide and incubated at 70 ° C. overnight to extract Evans blue dye in the ear tissue.
- the concentration of Evans blue dye was determined from the absorbance of the extract (measurement wavelength: 620 nm, control wavelength: 740 nm), and the amount of Evan Blue dye in the extract was calculated. Thereafter, the amount of dye leakage per ear weight was calculated by dividing the amount of Evans blue dye by the ear weight. A value obtained by subtracting the Evans blue dye leakage amount of the mineral oil application ear from the Evans blue dye leakage amount of the same individual mustard oil application ear was calculated as the final Evans blue dye leakage amount of each individual. The amount of Evans Blue dye leakage was used as an indicator of vascular permeability. The results are shown in FIG.
- the fully human type 2-16A2 which is a bivalent antibody did not inhibit the dye leakage, whereas the tetravalent antibody TIE-1-Ig ⁇ 1-WT has a dye leakage.
- the tetravalent antibody TIE-1-Ig ⁇ 1-WT inhibits enhanced vascular permeability. From the above, it has been clarified that the tetravalent antibody has a superior vascular permeability enhancement action than the divalent antibody.
- Example 11 Evaluation of anti-apoptotic effect of tetravalent anti-human Tie2 antibody (2)
- TIE-1-Ig ⁇ 1-LALA and TIE-1-Ig ⁇ 1-I253A the anti-apoptotic activity of the antibody against human Tie2-expressing BaF3 cells was evaluated according to the method of Example 9.
- Each tetravalent anti-human Tie2 antibody was evaluated in the same antibody concentration range as in Example 9.
- the EC 50 value of each antibody and the maximum activity of the anti-apoptotic activity were evaluated with the average value of the measured values of wells added with 100, 300 and 1000 ng / mL of Ang-1 being 100%.
- Table 2 Anti-apoptotic activity of each tetravalent anti-human Tie2 antibody
- TIE-1-Ig ⁇ 1-LALA and TIE-1-Ig ⁇ 1-I253A exhibit the same level of anti-apoptotic activity as Ang-1.
- TIE-1-Ig ⁇ 1-LALA exhibits an anti-apoptotic activity comparable to Ang-1, whereas 15B8 has only a partial anti-apoptotic activity similar to Ang-2. It became clear not to show.
- TIE-1-Ig ⁇ 1-LALA was evaluated for its binding activity to various Tie2 proteins.
- Recombinant human Tie2-Fc chimeric protein R & D, 313-TI-100
- recombinant monkey Tie2-Fc chimeric protein (Sino Biological, 90292-C02H)
- recombinant rat Tie2-Fc chimeric protein R & D, 3874
- recombinant mouse Tie2-Fc chimeric protein R & D, 762-T2-100
- PBS 1 ⁇ g / mL
- 1 well in a white maxisoap 384 well plate Nunc, 460372
- TBS-T Tris buffered saline
- BlockingOne Nacalai Tesque, 03953-95
- Tween Wako, 310-7375
- TBS-T Tris buffered saline
- TIE-1-Ig ⁇ 1-LALA was diluted 8 times from 0.03 ng / mL to 100 ng / mL with a TBS-T solution containing 5% BlockingOne at a 3-fold common ratio. 20 ⁇ L was added.
- a well to which a TBS-T solution was added instead of the test antibody was prepared. After incubating at room temperature for 1.5 hours, it was washed with a TBS-T solution.
- biotin-labeled anti-human kappa light chain antibody (Immunobiological Laboratories, 17249) was diluted to 0.1 ⁇ g / mL with a TBS-T solution containing 5% BlockingOne, and 20 ⁇ L was added per well.
- TIE-1-Ig ⁇ 1-LALA has the same high binding activity to human, monkey, rat and mouse Tie2.
- 15B8 was found to have binding activity to human and monkey Tie2, but the binding activity to rat and mouse Tie2 was found to be extremely weak.
- TIE-1-Ig ⁇ 1-LALA showed high binding activity to humans, monkeys, rats, and mouse Tie2 without any species difference.
- 15B8 showed a species difference in binding activity. From the above, it was suggested that the human Tie2 epitope of TIE-1-Ig ⁇ 1-LALA is different from the epitope of 15B8.
- Example 13 Evaluation of rat blood vessel permeability inhibitory action of TIE-1-Ig ⁇ 1-LALA
- Example 10 Evaluation of rat blood vessel permeability inhibitory action of TIE-1-Ig ⁇ 1-LALA
- TIE-1-Ig ⁇ 1-LALA was used as a test antibody, and antibody administration doses were 0.1 and 0.3 mg / kg. The results are shown in FIG.
- TIE-1-Ig ⁇ 1-LALA significantly inhibited pigment leakage as compared to the Vehicle group. From the above, it was revealed that TIE-1-Ig ⁇ 1-LALA inhibits the increase in vascular permeability.
- Example 14 Inhibitory effect on retinal edema of mice with pericyte shed
- pericyte loss is one of the characteristic lesions (Retina, 2013, Fifth edition, p. 925-939).
- Streptozotocin-induced diabetic rat models are widely used in diabetic retinopathy research, but it takes several months before pericyte detachment is observed, and pericyte detachment is the cause. No capillary aneurysm is observed, the ratio of pericytes and endothelial cells is different from that of humans (Retina, 2013, Fifth edition, p.
- evaluating the inhibitory effect on retinal edema using a pathologic model exhibiting pericyte detachment, a characteristic lesion in diabetic retinopathy patients is to evaluate the effectiveness against diabetic retinopathy and diabetic macular edema Useful for.
- Pericyte detachment-induced retinal edema is described in J. Org. Clin. Invest. , 2002, Vol. 110, p. The method reported in 1619-1628 was derived with some modifications. That is, 25 mg / kg of anti-PDGFR ⁇ monoclonal antibody 1B3 (International Publication No. 2008/130704) diluted with PBS was subcutaneously administered to 2-day-old C57BL / 6J mice (Charles River Japan Co., Ltd.). Caused dropout.
- Control group also referred to as Cont. Group
- Vehicle group also referred to as Veh.
- mice administered with anti-PDGFR ⁇ antibody TIE-1-Ig ⁇ 1- Group TIE-1-Ig ⁇ 1-LALA group administered with PBS instead of LALA (0.1, 0.3 and 1 mg / kg): 23, 21 and 21 anti-PDGFR ⁇ antibodies were administered respectively, and TIE-1-Ig ⁇ 1 -Groups administered each dose of LALA
- TIE-1-Ig ⁇ 1-LALA group (1 mg / kg) was found to significantly inhibit retinal edema with retinal blood vessels from which pericytes had fallen.
- TIE-1-Ig ⁇ 1-LALA inhibits retinal edema caused by retinal blood vessels from which pericytes are lost, suggesting that it is effective against diabetic macular edema and diabetic retinopathy.
- the hind limb ischemia model is a model that causes ischemia of hind limb tissues by ligating and removing blood vessels in one hind limb, and is a representative model for evaluating improvement of ischemic symptoms (J. Vasc. Surg., 2012, Vol. 56, pp. 1669-1679).
- TIE-1-Ig ⁇ 1-LALA diluted with PBS was subcutaneously administered at 1 mg / kg, and skin blood flow in normal and ischemic limbs was measured one week after antibody administration.
- pentobarbital sodium 60 mg / kg was intraperitoneally administered, placed on a heat-retaining table, and skin blood flow in the foot was measured 15 minutes after anesthetic administration.
- FIG. 5 shows the results of measuring blood flow with the plantar region as the region of interest (Region of interest: ROI).
- TIE-1-Ig ⁇ 1-LALA As shown in FIG. 5, it was revealed that the TIE-1-Ig ⁇ 1-LALA group significantly improved blood flow in normal limbs and ischemic limbs compared to the control group. Therefore, it was suggested that TIE-1-Ig ⁇ 1-LALA is effective for peripheral arterial diseases such as severe limb ischemia.
- Example 16 Epitope evaluation of TIE-1-Ig ⁇ 1-LALA: hydrogen deuterium exchange mass spectrometry
- a fully human 2-16A2 Fab prepared in Example 7 (hereinafter referred to as a fully human 2-16A2-Fab) was prepared. Because fully human 2-16A2-Fab has the same variable region as TIE-1-Ig ⁇ 1-LALA, these antibodies recognize the same epitope.
- Accession No. A human Tie2 protein hereinafter referred to as human Tie2 (1-452)
- human Tie2 (1-452) consisting of amino acids 1 to 452 of NP — 0450.2 was produced. This amino acid sequence is the site used to identify the Tie2 binding site of Ang-2 (Nat. Struct. Mol. Biol., Vol. 13, p. 524-532).
- fully human 2-16A2-Fab is a structure composed of the heavy chain variable region and CH1 region of fully human 2-16A2 (consisting of amino acid sequences 1 to 221 of SEQ ID NO: 12). Expression of the antibody described in Example 7 by combining the GS vector pEE6.4 inserted with a heavy chain gene encoding GS and the GS vector pEE12.4 inserted with a fully human 2-16A2 light chain gene Using a method similar to the method and purification method, fully human 2-16A2-Fab was produced.
- human Fie (amino acid represented by SEQ ID NO: 23) was first linked to human Tie2 (1-452) with a thrombin recognition sequence (consisting of the amino acid sequence represented by SEQ ID NO: 22) as a linker.
- a protein fused with the sequence (hereinafter referred to as human Tie2 (1-452) -Fc chimeric protein) was prepared.
- a gene encoding the human Tie2 (1-452) -Fc chimeric protein is inserted into the GS vector pEE12.4, and the expression method and purification method described in Example 7 are used.
- a Tie2 (1-452) -Fc chimeric protein was prepared.
- the prepared human Tie2 (1-452) -Fc chimeric protein was incubated with thrombin (GE Healthcare, 27-0846-01) at 22 ° C. for 16 hours, and Benzamidine Sepharose 4 Fast Flow (high sub) ( Human Tie2 (1-452) was prepared by removing thrombin and human Fc using GE Healthcare) and MabSelect SuRe and cleaving the Fc part.
- H / D exchange mass spectrometry For the purpose of identifying the epitope site, deuterium deuterium exchange mass spectrometry (hereinafter referred to as H / D exchange mass spectrometry, Anal.Bional.Chem., 2010, Vol.397) using the NanoAQUITY UPLC HDX system (Waters). P. 967-979).
- a fully human 2-16A2-Fab and human Tie2 (1-452) mixed solution (final concentrations are 50 ⁇ M and 25 ⁇ M, respectively) is prepared. And left at 37 ° C. overnight.
- Example 17 Epitope evaluation of TIE-1-Ig ⁇ 1-LALA: surface plasmon resonance phenomenon analysis and ELISA
- an epitope candidate for human Tie2 of TIE-1-Ig ⁇ 1-LALA was identified.
- an amino acid variant of human Tie2 (1-452) -Fc chimeric protein was prepared, and the binding activity using surface plasmon resonance (SPR analysis) and ELISA was analyzed. evaluated.
- Biacore T200 (GE Healthcare) was used for SPR analysis.
- Anti-human IgG (Fc) antibody Human Antibody Capture Kit, GE Healthcare
- the binding amount of the antibody per unit solid-phased antigen (hereinafter referred to as the binding ratio) was calculated by dividing the binding amount by the capture amount.
- Table 7 shows the arithmetic average of three trials and the relative values of the binding ratios of each mutant when the binding ratio of the human Tie2 (1-452) -Fc chimeric protein is 100%. Typical measurement data is shown in FIG. A method for relatively comparing the amount of binding in Biacore is described in, for example, Analytical Biochemistry, 2003, Vol. 312, p. 113-124.
- the fully human type 2-16A2-Fab was compared with the human Tie2 (1-452) -Fc chimeric protein, human Tie2 (1-452) g1-Fc, human Tie2 (1-452) g2-Fc, human Tie2 (1-452) g3-Fc, human Tie2 (1-452) g4-Fc, human Tie2 (1-452) g5-Fc, human Tie2 (1-452) m3-Fc, human Tie2 (1-452) It was revealed that the binding was reduced in A1-Fc, human Tie2 (1-452) A2-Fc, human Tie2 (1-452) A3-Fc and human Tie2 (1-452) A4-Fc. Table 7: Results of SPR analysis
- Human Tie2 (1-452) -Fc chimeric protein and 23 mutants were diluted to 1 ⁇ g / mL with PBS, 20 ⁇ L per well was added to a white maxisoap 384 well plate, and overnight at 4 ° C. Was immobilized by incubating with a. On the next day, the solid phase solution was removed, the plate was washed with a TBS-T solution, and 50 ⁇ L of Blocker TM Casein in TBS (Thermo Fisher Scientific, 37532) was added, followed by incubation for 60 minutes for blocking.
- TBS-T solution 50 ⁇ L of Blocker TM Casein in TBS (Thermo Fisher Scientific, 37532) was added, followed by incubation for 60 minutes for blocking.
- TIE-1 washed with TBS-T solution and diluted in 8 steps from 0.03 ng / mL to 100 ng / mL using Blocker TM Casein in TBS containing 0.05% Tween 20 (Nacalai Tesque, 28353-85) -20 ⁇ L of Ig ⁇ 1-LALA was added per well. After incubating at room temperature for 90 minutes, the biotin-labeled anti-human kappa light chain antibody was washed 3 times with a TBS-T solution and diluted to 0.1 ⁇ g / mL using 0.05% Tween20-containing Blocker TM Casein in TBS. 20 ⁇ L was added.
- the plate was washed three times with a TBS-T solution, and 20 ⁇ L of alkaline phosphatase-labeled streptavidin diluted to 0.1 ⁇ g / mL using Blocker TM Casein in TBS containing 0.05% Tween 20 was added. .
- the substrate was washed with a TBS-T solution three times and diluted 5-fold with 20 mM TBS (pH 9.8) containing 1 mM MgCl 2. As a result, 50 ⁇ L was added. After incubating for 40 minutes at light-shielded room temperature, the luminescence intensity was measured with an EnVision TM multilabel counter.
- TIE-1-Ig ⁇ 1-LALA for the human Tie2 (1-452) -Fc chimeric protein and 23 variants were calculated.
- the convergence value when TIE-1-Ig ⁇ 1-LALA binds to human Tie2 (1-452) -Fc chimeric protein is defined as 100%
- the maximum applied concentration is 100 ng / mL TIE-1-Ig ⁇ 1-
- the relative value of the luminescence intensity when LALA bound to the human Tie2 (1-452) -Fc chimeric protein and each of the 23 mutants was calculated (Tables 8 and 9).
- the EC 50 value and the convergence value were calculated by sigmoid-Emax model nonlinear regression analysis. A graph is shown in FIG.
- TIE-1-Ig ⁇ 1-LALA is compared with the human Tie2 (1-452) -Fc chimeric protein in terms of Tie2 (1-452) g1-Fc, Tie2 (1-452) g2-Fc, Tie2 ( 1-45) It was revealed that the binding activity to g4-Fc and Tie2 (1-452) g5-Fc was decreased.
- Tie2 (1-452) A1-Fc although a decrease in the relative value of the luminescence intensity was observed, no difference was observed in the EC 50 value, so it was determined that there was no change in the binding activity. did.
- Table 8 ELISA results
- Table 9 ELISA results
- Tie2 (1-452) g1-Fc having amino acid mutations of I194A, N197A and L198A was found to have decreased binding by ELISA, whereas Tie2 (1-452) g3-Fc having an amino acid mutation of I194A was No decrease in binding was observed in the ELISA method.
- Tie2 (1-452) g6-Fc having an amino acid mutation of L198A did not show decreased binding in both the ELISA method and the SPR analysis method. From these results, it was considered that amino acid number 197 contributes to the decrease in binding of Tie2 (1-452) g1-Fc.
- TIE-1-Ig ⁇ 1-LALA is an Accession No. It was revealed that amino acids 192, 195 and 197 of NP_000450.2 were bound as epitopes.
- the anti-human Tie2 antibody of the present invention is useful for the prevention or treatment of various blood vessel-related diseases.
- the polynucleotide, expression vector, transformed host cell, and method for producing an antibody of the present invention are useful for producing the anti-human Tie2 antibody.
- the description of “Artificial Sequence” is described in the numerical heading ⁇ 223> of the following sequence listing.
- the base sequence shown in SEQ ID NO: 1 in the sequence listing is the heavy chain base sequence of TIE-1-Ig ⁇ 1-LALA
- the amino acid sequence shown in SEQ ID NO: 2 is encoded by SEQ ID NO: 1.
- the nucleotide sequence represented by SEQ ID NO: 3 in the sequence listing includes TIE-1-Ig ⁇ 1-LALA, TIE-1-Ig ⁇ 1-I253A, TIE-1-Ig ⁇ 1-WT, TIE-1-Ig ⁇ 4-PE, and fully human type 2.
- the base sequence of the light chain of ⁇ 16A2 and the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of the light chain encoded by SEQ ID NO: 3.
- the base sequence shown in SEQ ID NO: 5 in the sequence listing is the heavy chain base sequence of TIE-1-Ig ⁇ 1-I253A
- the amino acid sequence shown in SEQ ID NO: 6 is the heavy chain amino acid encoded by SEQ ID NO: 5.
- the base sequence represented by SEQ ID NO: 7 in the sequence listing is the heavy chain base sequence of TIE-1-Ig ⁇ 1-WT
- the amino acid sequence represented by SEQ ID NO: 8 is the heavy chain amino acid encoded by SEQ ID NO: 7. Is an array.
- the base sequence shown in SEQ ID NO: 9 in the sequence listing is the heavy chain base sequence of TIE-1-Ig ⁇ 4-PE, and the amino acid sequence shown in SEQ ID NO: 10 is the heavy chain amino acid encoded by SEQ ID NO: 9. Is an array.
- the nucleotide sequence represented by SEQ ID NO: 11 in the sequence listing is the heavy chain nucleotide sequence of fully human type 2-16A2, and the amino acid sequence represented by SEQ ID NO: 12 is the heavy chain amino acid encoded by SEQ ID NO: 11, respectively. Is an array.
- the amino acid sequences shown in SEQ ID NOs: 13 to 20 are linker amino acid sequences.
- the amino acid sequence shown in SEQ ID NO: 22 is a thrombin recognition site.
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Abstract
Description
[1]4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は、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111までのアミノ酸配列からなるCDR3とを含み、
該軽鎖可変領域は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含み、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2抗体又はその抗原結合フラグメント。
[2]以下の(1)又は(2)から選択される、[1]に記載の抗ヒトTie2抗体又はその抗原結合フラグメント:
(1)4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は配列番号2のアミノ酸番号1から122までのアミノ酸配列からなり、
該軽鎖可変領域は配列番号4のアミノ酸番号1から113までのアミノ酸配列からなり、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2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Tie2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Tie2抗体又はその抗原結合フラグメント。
[3][1]に記載の抗ヒトTie2抗体であって、該抗体は2つの重鎖及び4つの軽鎖を含み、
各重鎖は、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111までのアミノ酸配列からなるCDR3とを含む重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のカルボキシ末端(C末端)がリンカーを介してもう一方の該構造のアミノ末端(N末端)と連結されており、
各軽鎖は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体。
[4]以下の(1)又は(2)から選択される、[3]に記載の抗ヒトTie2抗体:
(1)2つの重鎖及び4つの軽鎖を含み、
各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体;並びに
(2)(1)の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2抗体。
[5][4]に記載の抗ヒトTie2抗体であって、該抗ヒトTie2抗体は、2つの重鎖及び4つの軽鎖を含み、
各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体。
[6][5]に記載の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2抗体。
[7]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、[6]に記載の抗ヒトTie2抗体。
[8]ヒトIgγ1定常領域又はヒトIgγ4定常領域である重鎖定常領域を含む、[3]~[7]のいずれかに記載の抗ヒトTie2抗体。
[9]ヒトIgγ1定常領域がL234A、L235A、及びP331Sのアミノ酸変異を有するヒトIgγ1定常領域、又は、L234A、L235A、P331S、及びI253Aのアミノ酸変異を有するヒトIgγ1定常領域である、[8]に記載の抗ヒトTie2抗体。
[10]ヒトIgγ4定常領域がS228P及びL235Eのアミノ酸変異を有するヒトIgγ4定常領域である、[8]に記載の抗ヒトTie2抗体。
[11]ヒトIgκ定常領域である軽鎖定常領域を含む、[3]~[7]のいずれかに記載の抗ヒトTie2抗体。
[12]ヒトIgγ1定常領域又はヒトIgγ4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、[3]~[7]のいずれかに記載の抗ヒトTie2抗体。
[13]ヒトIgγ1定常領域がL234A、L235A、及びP331Sのアミノ酸変異を有するヒトIgγ1定常領域、又は、L234A、L235A、P331S、及びI253Aのアミノ酸変異を有するヒトIgγ1定常領域である、[12]に記載の抗ヒトTie2抗体。
[14]ヒトIgγ4定常領域がS228P及びL235Eのアミノ酸変異を有するヒトIgγ4定常領域である、[12]に記載の抗ヒトTie2抗体。
[15]リンカーが5~70アミノ酸からなるペプチドリンカーである、[3]~[7]のいずれかに記載の抗ヒトTie2抗体。
[16]リンカーがヒンジ領域又はその一部分のアミノ酸配列を含む、[15]に記載の抗ヒトTie2抗体。
[17]リンカーが配列番号13に示されるアミノ酸配列からなる、[16]に記載の抗ヒトTie2抗体。
[18]配列番号2に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、[4]に記載の抗ヒトTie2抗体。
[19]配列番号6に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、[4]に記載の抗ヒトTie2抗体。
[20]配列番号10に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、[4]に記載の抗ヒトTie2抗体。
[21][18]~[20]のいずれかに記載の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2抗体。
[22]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、[21]に記載の抗ヒトTie2抗体。
[23]配列番号2のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、[21]に記載の抗ヒトTie2抗体。
[24][18]又は[23]に記載の抗ヒトTie2抗体と同じヒトTie2エピトープに結合する、4価の抗ヒトTie2抗体又はその抗原結合フラグメント。
[25]ヒトTie2エピトープが、Accession No.NP_000450.2のアミノ酸番号192、195及び197のアミノ酸を含むヒトTie2エピトープである、[24]に記載の4価の抗ヒトTie2抗体又はその抗原結合フラグメント。
[26][2]に記載の抗ヒトTie2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。
[27][2]に記載の抗ヒトTie2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。
[28][18]~[20]のいずれかに記載の抗ヒトTie2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。
[29][18]~[20]のいずれかに記載の抗ヒトTie2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。
[30][26]及び/又は[27]に記載のポリヌクレオチドを含む発現ベクター。
[31][28]及び/又は[29]に記載のポリヌクレオチドを含む発現ベクター。
[32]以下の(a)~(d)からなる群より選択される、[30]に記載の発現ベクターで形質転換された宿主細胞。
(a)[2]に記載の抗ヒトTie2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)[2]に記載の抗ヒトTie2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)[2]に記載の抗ヒトTie2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)[2]に記載の抗ヒトTie2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。
[33]以下の(a)~(d)からなる群より選択される、[31]に記載の発現ベクターで形質転換された宿主細胞。
(a)[18]~[20]のいずれかに記載の抗ヒトTie2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)[18]~[20]のいずれかに記載の抗ヒトTie2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)[18]~[20]のいずれかに記載の抗ヒトTie2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)[18]~[20]のいずれかに記載の抗ヒトTie2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。
[34]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、4価の抗ヒトTie2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Tie2抗体又はその抗原結合フラグメントを生産する方法:
(a)[2]に記載の抗ヒトTie2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)[2]に記載の抗ヒトTie2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)[2]に記載の抗ヒトTie2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。
[35]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Tie2抗体を発現させる工程を包含する、抗ヒトTie2抗体を生産する方法:
(a)[18]~[20]のいずれかに記載の抗ヒトTie2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)[18]~[20]のいずれかに記載の抗ヒトTie2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)[18]~[20]のいずれかに記載の抗ヒトTie2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Tie2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。
[36][34]に記載の方法で生産された抗ヒトTie2抗体又はその抗原結合フラグメント。
[37][35]に記載の方法で生産された抗ヒトTie2抗体。
[38][1]~[23]、[36]及び[37]のいずれかに記載の抗ヒトTie2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。
[39][5]に記載の抗ヒトTie2抗体、[6]に記載の抗ヒトTie2抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
[40][18]に記載の抗ヒトTie2抗体、[23]に記載の抗ヒトTie2抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
[41]糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療用医薬組成物である、[38]~[40]のいずれかに記載の医薬組成物。
[42][1]~[23]、[36]及び[37]のいずれかに記載の抗ヒトTie2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、糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血を予防又は治療する方法。
[43]糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療に使用するための、[1]~[23]、[36]及び[37]のいずれかに記載の抗ヒトTie2抗体又はその抗原結合フラグメント。
[44]糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療用医薬組成物の製造における、[1]~[23]、[36]及び[37]のいずれかに記載の抗ヒトTie2抗体又はその抗原結合フラグメントの使用。
本発明の抗ヒトTie2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Tie2抗体又はその抗原結合フラグメントが含まれる。
4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は配列番号2のアミノ酸番号1から122までのアミノ酸配列からなり、
該軽鎖可変領域は配列番号4のアミノ酸番号1から113までのアミノ酸配列からなり、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2抗体又はその抗原結合フラグメント。
各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体である。
以下、当該フォーマットの4価抗体をタンデム抗体とも称し、その一例を図1に示す。
i)配列番号2に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
ii)配列番号6に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
iii)配列番号8に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
iv)配列番号10に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(1)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679のリジンを欠くアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(2)配列番号6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6のアミノ酸番号679のリジンを欠くアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(3)配列番号8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8のアミノ酸番号679のリジンを欠くアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(4)配列番号10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10のアミノ酸番号676のリジンを欠くアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
配列番号2のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は、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111までのアミノ酸配列からなるCDR3とを含み、
該軽鎖可変領域は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含み、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2抗体又はその抗原結合フラグメント。
2つの重鎖及び4つの軽鎖を含み、
各重鎖は、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111までのアミノ酸配列からなるCDR3とを含む重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のカルボキシ末端がリンカーを介してもう一方の該構造のアミノ末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体。
本発明のポリヌクレオチドには、本発明の抗ヒトTie2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Tie2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Tie2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Tie2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。当該分野において種々のフォーマットの4価抗体及びその生産方法が公知であり、本発明の発現ベクターは、これらの生産方法に従って、発現させる4価抗体のフォーマットに応じて当業者に容易に構築され得る。
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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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、4価の抗ヒトTie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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Tie2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、眼内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。
4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、該重鎖可変領域は配列番号2のアミノ酸番号1から122までのアミノ酸配列からなり、該軽鎖可変領域は配列番号4のアミノ酸番号1から113までのアミノ酸配列からなり、1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む抗ヒトTie2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Tie2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。
2つの重鎖及び4つの軽鎖を含み、各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、抗ヒトTie2抗体、並びに、該抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2抗体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678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2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を2つ、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4つ含む、抗ヒトTie2抗体。
(3)配列番号2のアミノ酸番号1から678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を2つ、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4つ含む、抗ヒトTie2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を2つ、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4つ含む、抗ヒトTie2抗体。
(1)配列番号6のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(2)配列番号6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を2つ、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4つ含む、抗ヒトTie2抗体。
(3)配列番号6のアミノ酸番号1から678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を2つ、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4つ含む、抗ヒトTie2抗体。
(4)配列番号6に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(1)配列番号8のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(2)配列番号8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を2つ、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4つ含む、抗ヒトTie2抗体。
(3)配列番号8のアミノ酸番号1から678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を2つ、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4つ含む、抗ヒトTie2抗体。
(4)配列番号8に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(1)配列番号10のアミノ酸番号1から675までのアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
(2)配列番号10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を2つ、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4つ含む、抗ヒトTie2抗体。
(3)配列番号10のアミノ酸番号1から675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を2つ、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4つ含む、抗ヒトTie2抗体。
(4)配列番号10に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、抗ヒトTie2抗体。
配列番号2に示されるアミノ酸配列からなる重鎖を2つ及び配列番号4に示されるアミノ酸配列からなる軽鎖を4つ含む抗ヒトTie2抗体、配列番号2のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ及び配列番号4に示されるアミノ酸配列からなる軽鎖を4つ含む抗ヒトTie2抗体、並びに、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Tie2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、4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、該重鎖可変領域は配列番号2のアミノ酸番号1から122までのアミノ酸配列からなり、該軽鎖可変領域は配列番号4のアミノ酸番号1から113までのアミノ酸配列からなり、1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、抗ヒトTie2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Tie2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Tie2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ヒトTie2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ヒトTie2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。
ヒトモノクローナル抗体開発技術「ベロシミューン」(VelocImmune antibody technology:Regeneron社(米国特許6596541号))マウスを用いて抗体を作製した。ベロシミューンマウスに、免疫反応を惹起するアジュバントと共に、組換えヒトTie2-Fcキメラ蛋白質(R&D社、313-TI-100)を免疫した。常法に従い、免疫したマウスのリンパ節を摘出しリンパ球を回収し、これをマウス由来ミエローマ細胞SP2/0(ATCC:CRL-1581)と細胞融合することでハイブリドーマを作製した。ハイブリドーマのモノクローン化を行い、各クローンについて、無血清培地であるCDハイブリドーマメディウム(Invitrogen社)で培養した。得られた培養上清からプロテインGカラム(GEヘルスケア社)を用いて抗体を精製した。ベロシミューン技術により得られた抗体は、ヒト抗体の可変領域とマウス抗体の定常領域を有する抗体(キメラ抗体とも称する)である。
抗体の抗原結合活性を測定するために、ヒトTie2発現CHO細胞、サルTie2発現CHO細胞、ラットTie2発現CHO細胞及びマウスTie2発現CHO細胞を用いた細胞ELISAアッセイにより、ヒト、サル、ラット及びマウスTie2に対する抗体の結合を評価した。
抗体のAng-2拮抗活性を評価するために、Ang-1改変体(Proc.Natl.Acad.Sci.、2004、Vol.101、p.5547-5552。COMP-Ang1とも称する。)とTie2との結合阻害を評価した。COMP-Ang1はTie2への結合に関与しない部位を改変したAng-1改変体であり、Tie2への結合能を保持していること(Proc.Natl.Acad.Sci.、2004、Vol.101、p.5547-5552)及びAng-1とAng-2はTie2の同じ部位へ同程度の親和性で結合すること(Science、1997、Vol.277、p.55-60)から、COMP-Ang1への拮抗作用を評価することで、Ang-2への拮抗作用を評価することができる。
ヒトTie2を安定的に発現するマウスプロB細胞株BaF3細胞(以下、ヒトTie2発現BaF3細胞と称する)は、Immunity、1998、Vol.9、p.677-686に記載の方法に従い、配列番号21で示されるヒトTie2遺伝子を含むプラスミドを当該細胞にエレクトロポレーションにて導入することで作製した。以後、同細胞を用いて、抗体の抗アポトーシス活性を評価した。
前述の実施例2~4で同定した精製抗体溶液をサイズ排除クロマトグラフィーで解析した。その結果、各精製抗体溶液から3つのフラクションが検出された。各フラクション溶液を電気泳動で解析した結果、各フラクションには抗体の単量体、2量体、及び3量体以上の多量体がそれぞれ含まれていることが明らかとなった。
実施例5の検討から、抗ヒトTie2抗体の価数が4価以上であることがTie2を介した抗アポトーシス活性を誘導するのに重要であると考えられた。そこで、抗マウスIgG抗体で架橋することで多量化した抗ヒトTie2抗体の抗アポトーシス活性を評価した。細胞として、ヒトTie2発現BaF3細胞及び内在的にヒトTie2を発現しているヒト血管内皮細胞HUVECを用いた。
抗ヒトTie2抗体2-16を産生するハイブリドーマから抗体の重鎖及び軽鎖をコードする遺伝子をクローニングし、配列決定した。
4価の抗ヒトTie2抗体を作製した。本実施例で作製した4価抗体は、2つの重鎖及び4つの軽鎖を含む。各重鎖は、重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該重鎖可変領域及びCH1領域からなる構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されている。各軽鎖は、軽鎖可変領域及び軽鎖定常領域を含む。本4価抗体のフォーマットを図1に示す。
実施例5の結果から、4価以上の価数をもつ抗体がヒトTie2活性化を介した強力な抗アポトーシス活性を有することが示唆された。そこで、ヒトTie2発現BaF3細胞に対する抗アポトーシス作用を指標として、2価の抗ヒトTie2抗体及び4価の抗ヒトTie2抗体の薬効を比較した。
表1:2価の抗ヒトTie2抗体及び4価の抗ヒトTie2抗体の抗アポトーシス活性
マスタードオイル誘発血管透過性モデルは、血漿漏出評価系として広く使用されているMiles assay(J.Physiol.、1952、Vol.118、p.228-257)に変更を加えたもので、本モデルにおいてAng-1が血管透過性亢進を阻害することが報告されている(Nature Medicine、2000、Vol.6、p.460-463)。そこで、2価の抗ヒトTie2抗体及び4価の抗ヒトTie2抗体の血管透過性阻害作用を比較するために、本モデルを用いて、完全ヒト型2-16A2及びTIE-1-Igγ1-WTを評価した。
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TIE-1-Igγ1-LALA及びTIE-1-Igγ1-I253Aについて、実施例9の方法に従い、ヒトTie2発現BaF3細胞に対する抗体の抗アポトーシス活性を評価した。実施例9と同じ抗体濃度範囲で各4価抗ヒトTie2抗体の評価を行った。但し、Ang-1の100、300及び1000ng/mLをそれぞれ添加したウェルの測定値の平均値を100%として、各抗体のEC50値及び抗アポトーシス活性の最大活性を評価した。
表2:各4価抗ヒトTie2抗体の抗アポトーシス活性
15B8について、実施例9の方法に従い、ヒトTie2発現BaF3細胞に対する抗アポトーシス活性を評価した。実施例9と同じ抗体濃度範囲で15B8(特許文献1)の評価を行った。Ang-2(R&D社、623-AN-025)はAng-1と同様に評価を行った。但し、Ang-1の1000ng/mLを添加したウェルの測定値の平均値を100%として、EC50値及び抗アポトーシス活性の最大活性を評価した。
表3:15B8の抗アポトーシス活性
TIE-1-Igγ1-LALAについて、各種Tie2蛋白質に対する結合活性を評価した。組換えヒトTie2-Fcキメラ蛋白質(R&D社、313-TI-100)、組換えサルTie2-Fcキメラ蛋白質(Sino Biological社、90292-C02H)、組換えラットTie2-Fcキメラ蛋白質(R&D社、3874-T2-100)又は組換えマウスTie2-Fcキメラ蛋白質(R&D社、762-T2-100)をPBSにて1μg/mLに調製し、白色マキシソープ384ウェルプレート(Nunc社、460372)に1ウェルあたり20μL添加し、一晩4℃でインキュベートすることで、固相化した。翌日固相液を除き、20%のBlockingOne(ナカライテスク社、03953-95)を含むトリス緩衝生理食塩水(TBS)-0.05%Tween(Wako社、310-7375)(以下、TBS-T溶液と称する)を1ウェルあたり50μL添加し、室温で1時間静置した。被験抗体として、TIE-1-Igγ1-LALAを、5%のBlockingOneを含むTBS-T溶液を用いて0.03ng/mLから100ng/mLまで約3倍公比で8段階希釈し、1ウェルあたり20μL添加した。コントロールとして、被験抗体の代わりにTBS-T溶液を添加したウェルを準備した。室温で1.5時間インキュベートした後、TBS-T溶液で洗浄した。2次抗体として、ビオチン標識した抗ヒトカッパー軽鎖抗体(免疫生物研究所、17249)を5%のBlockingOneを含むTBS-T溶液で0.1μg/mLに希釈し、1ウェルあたり20μL添加した。室温で1時間インキュベートした後、TBS-T溶液で洗浄し、5%のBlockingOneを含むTBS-T溶液で0.1μg/mLに希釈したアルカリフォスファターゼ標識ストレプトアビジン(Thermo Fisher Scientific社、21324)を検出試薬として、1ウェルあたり20μL添加した。室温で1時間インキュベートした後、TBS-T溶液で洗浄し、1mM MgCl2含有20mM TBS(pH9.8)で5倍希釈したChemiluminescent Ultra Sensitive AP Microwell and/or Membrane Substrate(450nm)(BioFX社、APU4-0100-01)を基質として20μL添加した。室温で30分間インキュベートした後、EnVisionTM マルチラベルカウンター(PerkinElmer社)にて発光強度を測定した。算出された結合活性を解析し、Sigmoid-Emaxモデル非線形回帰分析により被験抗体のEC50値を算出した。
表4:TIE-1-Igγ1-LALAの結合活性
実施例12の方法に従い、15B8の各種Tie2蛋白質に対する結合活性を評価した。但し、2次抗体としてHRP標識抗マウスカッパー軽鎖抗体(SouthernBiotech社、1050-05)を使用し、基質としてTMB発色試薬(を使用し、測定器としてARVOマルチラベルリーダー(PerkinElmer社)を用いて450nmの吸光度を測定した。また、被験抗体として15B8を使用し、抗体濃度は1000ng/mLから0.3ng/mLまで約3倍公比(8段階希釈)で実施した。算出された結合活性を解析し、Sigmoid-Emaxモデル非線形回帰分析により被験抗体のEC50値を算出した(表5)。
表5:15B8の結合活性
実施例10の方法に従って、TIE-1-Igγ1-LALAのラット血管透過性阻害作用を評価した。但し、被験抗体としてTIE-1-Igγ1-LALAを使用し、抗体投与用量を0.1及び0.3mg/kgで実施した。結果を図3に示す。
糖尿病性網膜症患者の網膜血管において、周皮細胞の脱落は特徴的な病変の1つである(Retina、2013、Fifth edition、p.925-939)。糖尿病網膜症研究においては、ストレプトゾトシン誘発糖尿病ラットモデルが広く使用されているが、周皮細胞の脱落が観察されるまでには数か月の期間を要することや、周皮細胞の脱落がその成因の1つとされている毛細血管瘤は観察されないこと、周皮細胞と内皮細胞の割合がヒトとは異なること(Retina、2013、Fifth edition、p.925-939)、及び、明らかな網膜浮腫が観察されないことなどから、このモデルの有用性には一定の限界がある(Diabetes Metab.J.、2013、Vol.37、p.217-224)。一方、抗PDGF受容体β(PDGFRβ)抗体投与により周皮細胞を脱落した網膜血管を有するマウスでは、高血糖ではないが網膜血管の拡張や網膜の浮腫、出血など、糖尿病網膜症及び糖尿病黄斑浮腫に類似の病変が観察されたことから、周皮細胞の脱落と糖尿病網膜症及び糖尿病黄斑浮腫における血管脆弱化との関連が報告されている(J.Clin.Invest.、2002、Vol.110、p.1619-1628)。したがって、糖尿病網膜症患者に特徴的な病変である周皮細胞の脱落を呈する病態モデルを用いて網膜浮腫に対する阻害作用を評価することは、糖尿病網膜症及び糖尿病黄斑浮腫に対する有効性を評価するために有用である。
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後肢虚血モデルは、片側後肢の血管を結紮及び摘出することにより後肢組織の虚血を引き起こすモデルであり、虚血症状の改善を評価する代表的なモデルである(J.Vasc.Surg.、2012、Vol.56、p.1669-1679)。
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TIE-1-Igγ1-LALAの認識エピトープを同定するために、実施例7で作製した完全ヒト型2-16A2のFab(以下、完全ヒト型2-16A2-Fabと称する)を作製した。完全ヒト型2-16A2-FabはTIE-1-Igγ1-LALAと同じ可変領域を持つため、これらの抗体は同じエピトープを認識する。抗原として、Accession No.NP_000450.2のアミノ酸番号1から452からなるヒトTie2蛋白質(以下、ヒトTie2(1-452)と称する)を作製した。当該アミノ酸配列はAng-2のTie2結合部位を同定するときに用いられた部位である(Nat.Struct.Mol.Biol.、Vol.13、p.524-532)。
実施例16のH/D交換質量分析からTIE-1-Igγ1-LALAのヒトTie2に対するエピトープ候補が同定された。さらに詳細にエピトープ部分を推定するため、ヒトTie2(1-452)-Fcキメラ蛋白質のアミノ酸変異体を作製し、表面プラズモン共鳴現象解析(surface plasmon resonance:SPR解析)及びELISAを用いた結合活性を評価した。
表6:変異体ヒトTie2(1-452)-Fcキメラ蛋白質
表7:SPR解析結果
表8:ELISA結果
Claims (34)
- 4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は、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111までのアミノ酸配列からなるCDR3とを含み、
該軽鎖可変領域は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含み、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2抗体又はその抗原結合フラグメント。 - 以下の(1)又は(2)から選択される、請求項1に記載の抗ヒトTie2抗体又はその抗原結合フラグメント:
(1)4つの重鎖可変領域及び4つの軽鎖可変領域を含む抗ヒトTie2抗体又はその抗原結合フラグメントであって、
該重鎖可変領域は配列番号2のアミノ酸番号1から122までのアミノ酸配列からなり、
該軽鎖可変領域は配列番号4のアミノ酸番号1から113までのアミノ酸配列からなり、
1つの該重鎖可変領域と1つの該軽鎖可変領域が1つの抗原結合部位を構成し、該抗体又はその抗原結合フラグメントが4つの抗原結合部位を含む、
抗ヒトTie2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Tie2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Tie2抗体又はその抗原結合フラグメント。 - 請求項1に記載の抗ヒトTie2抗体であって、該抗体は2つの重鎖及び4つの軽鎖を含み、
各重鎖は、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111までのアミノ酸配列からなるCDR3とを含む重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のカルボキシ末端(C末端)がリンカーを介してもう一方の該構造のアミノ末端(N末端)と連結されており、
各軽鎖は、配列番号4のアミノ酸番号24から39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55から61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94から102までのアミノ酸配列からなるCDR3とを含む軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体。 - 以下の(1)又は(2)から選択される、請求項3に記載の抗ヒトTie2抗体:
(1)2つの重鎖及び4つの軽鎖を含み、
各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体;並びに
(2)(1)の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2抗体。 - 請求項4に記載の抗ヒトTie2抗体であって、該抗ヒトTie2抗体は、2つの重鎖及び4つの軽鎖を含み、
各重鎖は、配列番号2のアミノ酸番号1から122までのアミノ酸配列からなる重鎖可変領域及びCH1領域からなる構造を2つ、並びにCH2領域及びCH3領域を含み、一方の該構造のC末端がリンカーを介してもう一方の該構造のN末端と連結されており、
各軽鎖は、配列番号4のアミノ酸番号1から113までのアミノ酸配列からなる軽鎖可変領域及び軽鎖定常領域を含む、
抗ヒトTie2抗体。 - 請求項5に記載の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2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6に記載の抗ヒトTie2抗体。
- 配列番号2に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、請求項4に記載の抗ヒトTie2抗体。
- 配列番号6に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、請求項4に記載の抗ヒトTie2抗体。
- 配列番号10に示されるアミノ酸配列からなる重鎖を2つ、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4つ含む、請求項4に記載の抗ヒトTie2抗体。
- 請求項8~10のいずれか1項に記載の抗ヒトTie2抗体の翻訳後修飾により生じた抗体である、抗ヒトTie2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Tie2抗体。
- 配列番号2のアミノ酸番号1から678までのアミノ酸配列からなる重鎖を2つ、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4つ含む、請求項11に記載の抗ヒトTie2抗体。
- 請求項8又は13に記載の抗ヒトTie2抗体と同じヒトTie2エピトープに結合する、4価の抗ヒトTie2抗体又はその抗原結合フラグメント。
- ヒトTie2エピトープが、Accession No.NP_000450.2のアミノ酸番号192、195及び197のアミノ酸を含むヒトTie2エピトープである、請求項14に記載の4価の抗ヒトTie2抗体又はその抗原結合フラグメント。
- 請求項2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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。
- 請求項8~10のいずれか1項に記載の抗ヒトTie2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。
- 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。
- 請求項18及び/又は19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20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2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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21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8~10のいずれか1項に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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、4価の抗ヒトTie2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Tie2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2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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2に記載の抗ヒトTie2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Tie2抗体を発現させる工程を包含する、抗ヒトTie2抗体を生産する方法:
(a)請求項8~10のいずれか1項に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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8~10のいずれか1項に記載の抗ヒトTie2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Tie2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24に記載の方法で生産された抗ヒトTie2抗体又はその抗原結合フラグメント。
- 請求項25に記載の方法で生産された抗ヒトTie2抗体。
- 請求項1~13、26及び27のいずれか1項に記載の抗ヒトTie2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5に記載の抗ヒトTie2抗体、請求項6に記載の抗ヒトTie2抗体、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8に記載の抗ヒトTie2抗体、請求項13に記載の抗ヒトTie2抗体、及び、薬学的に許容される賦形剤を含む、医薬組成物。
- 糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療用医薬組成物である、請求項28~30のいずれか1項に記載の医薬組成物。
- 請求項1~13、26及び27のいずれか1項に記載の抗ヒトTie2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、糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血を予防又は治療する方法。
- 糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療に使用するための、請求項1~13、26及び27のいずれか1項に記載の抗ヒトTie2抗体又はその抗原結合フラグメント。
- 糖尿病黄斑浮腫、糖尿病網膜症又は重症下肢虚血の予防又は治療用医薬組成物の製造における、請求項1~13、26及び27のいずれか1項に記載の抗ヒトTie2抗体又はその抗原結合フラグメントの使用。
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KR1020177001003A KR102455680B1 (ko) | 2014-07-15 | 2015-07-14 | 신규 항인간 Tie2 항체 |
CN201580038358.6A CN106687478B (zh) | 2014-07-15 | 2015-07-14 | 新的抗人Tie-2抗体 |
MYPI2017700117A MY185811A (en) | 2014-07-15 | 2015-07-14 | Novel anti-human tie-2 antibody |
CA2954754A CA2954754C (en) | 2014-07-15 | 2015-07-14 | Novel anti-human tie-2 antibody |
EP15822476.6A EP3170841A4 (en) | 2014-07-15 | 2015-07-14 | NOVEL ANTI-HUMAN Tie2 ANTIBODY |
RU2017104642A RU2702553C2 (ru) | 2014-07-15 | 2015-07-14 | Новое антитело против tie-2 человека |
JP2016534433A JP6699551B2 (ja) | 2014-07-15 | 2015-07-14 | 新規抗ヒトTie2抗体 |
SG11201700271RA SG11201700271RA (en) | 2014-07-15 | 2015-07-14 | NOVEL ANTI-HUMAN Tie2 ANTIBODY |
MX2017000615A MX2017000615A (es) | 2014-07-15 | 2015-07-14 | Anticuerpo anti-tie 2 humano novedoso. |
AU2015290673A AU2015290673B2 (en) | 2014-07-15 | 2015-07-14 | Novel anti-human Tie2 antibody |
US15/134,803 US9683051B2 (en) | 2014-07-15 | 2016-04-21 | Anti-human tie-2 antibody |
IL249964A IL249964B (en) | 2014-07-15 | 2017-01-08 | A new anti-tie2 antibody of human origin |
PH12017500094A PH12017500094A1 (en) | 2014-07-15 | 2017-01-13 | Novel anti-human tie-2 antibody |
US15/585,374 US9902782B2 (en) | 2014-07-15 | 2017-05-03 | Anti-human tie-2 antibody |
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WO2019235856A1 (ko) * | 2018-06-07 | 2019-12-12 | 기초과학연구원 | Tie2에 결합하는 항체 및 이의 용도 |
JP2023502725A (ja) * | 2019-11-21 | 2023-01-25 | ユニティ バイオテクノロジー インコーポレイテッド | Tie-2に対する抗体および使用方法 |
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TW201617366A (zh) | 2016-05-16 |
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PH12017500094A1 (en) | 2017-05-22 |
JP2020103302A (ja) | 2020-07-09 |
US9902782B2 (en) | 2018-02-27 |
KR20170029508A (ko) | 2017-03-15 |
US20170253669A1 (en) | 2017-09-07 |
MA40182A (fr) | 2017-05-24 |
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MX2017000615A (es) | 2017-05-01 |
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