WO2023032955A1 - 抗成長ホルモン抗体 - Google Patents
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- C12N5/10—Cells modified by introduction of foreign genetic material
Definitions
- the present invention provides antibodies or antigen-binding fragments thereof that specifically bind to human growth hormone, nucleic acids encoding them, vectors comprising said nucleic acids, cells comprising said nucleic acids or vectors, said antibodies or antigens thereof
- a medicament comprising a binding fragment or a method for reducing insulin-like growth factor 1 (IGF-1) levels in the blood comprising administering a therapeutically effective amount of said antibody or antigen-binding fragment thereof.
- IGF-1 insulin-like growth factor 1
- GH Growth Hormone
- GH has isoforms with molecular weights of 22 kDa and 20 kDa (GH-22K, GH-20K), and the major molecular species in human serum is GH-22K. is known to exist in multiple isoforms (Non-Patent Document 1).
- GH-V-22K, GH-V-20K GH-V-20K with molecular weights of 22 kDa and 20 kDa are secreted from the placenta
- GH acts directly on tissues, and acts on growth hormone receptors (growth hormone receptors, hereinafter referred to as "GHR") expressed in the liver and other tissues to act as insulin-like growth factor 1 (GHR).
- GHR growth hormone receptors
- IGF-1 insulin-like growth factor 1
- release stimulation such as growth-related effects such as bone elongation and muscle growth, metabolism-related effects such as metabolism promotion, blood sugar increase, and maintenance of homeostasis.
- GH hypersecretion and hyposecretion are associated with many diseases.
- GH hypersecretion is caused by benign pituitary adenomas, the typical manifestations of which are known as acromegaly and gigantism.
- Acromegaly also known as acromegaly, is an enlargement of the tip of the body, such as the forehead, nose, chin, limbs, and tongue, which causes excessive sweating and discomfort due to bristling of body hair, thickened skin, and enlarged sebaceous and sweat glands. This disease is accompanied by unpleasant body odor and a deepened voice due to proliferation of oral cartilage. If GH hypersecretion develops by puberty, it results in gigantism.
- acromegaly The prevalence of acromegaly is reported to be 4-24 per 100,000 population, and the number is not large, making it a rare disease. is accompanied by symptoms such as diabetes, hypertension, hyperlipidemia and other metabolic disorders, sleep apnea syndrome, and cardiac hypertrophy, and is also at risk of angina pectoris, myocardial infarction, and cerebrovascular disease, resulting in excessive GH secretion. are known to cause high mortality if left untreated. In addition, acromegaly is known to be more likely to be complicated by colon cancer, thyroid cancer, etc., and early detection and treatment are important.
- Non-Patent Document 4 describes that GH expression in cancer cells is involved in the progression of endometrial cancer, breast cancer, etc.
- Non-Patent Document 5 describes a tumor model mouse of pegvisomant, a GH receptor antagonist. has been described as a tumor suppressor in Non-Patent Document 6 describes the relationship between GH and diabetic nephropathy
- Non-Patent Document 7 describes the relationship between GH and arthralgia and arthritis
- Non-Patent Document 8 describes IGF- An association between 1 and pulmonary inflammation has been described.
- Treatments for acromegaly and gigantism include surgery to remove pituitary adenomas that oversecrete GH, regression by radiotherapy, and drug therapy.
- Pituitary adenomectomy like other surgical procedures, carries the risk of complications, including death, and requires a high degree of skill.
- Radiation therapy also carries similar risks and can take years to be effective.
- Somatostatin analogues octreotide acetate sustained-release formulation, lanreotide acetate sustained-release formulation, etc.
- dopamine agonists bromocriptine mesylate
- pegvisomant are currently available on the market. However, there is still no drug that satisfies efficacy, safety and convenience at the same time.
- Patent Document 1 discloses anti-human GH mouse monoclonal antibodies (mAbs) (hGH-25, hGH-26) that bind to GH-22K but do not substantially bind to GH-20K, and binds to GH-20K.
- mAbs anti-human GH mouse monoclonal antibodies
- an anti-human GH mouse mAb hGH-33 that does not substantially bind to GH-22K
- an anti-human GH mouse mAb hGH-12
- Non-Patent Document 9 8 types of anti-human GH mouse mAbs were obtained, and among them, clones EB1, EB2, It has been described that NA71 inhibited the growth of human GH- and hCS-dependent Nb2 rat lymphoma cell lines, but the strength of the inhibitory activity ( IC50 ) was not specified.
- Non-Patent Document 10 shows that the anti-human GH mouse mAbs EB1 and EB2 described in Non-Patent Document 1 reversely promoted chondrocyte proliferation in dwarf mice and mammary gland stimulation in pigeons of human GH and hCS. ing.
- Non-Patent Document 11 describes multiple anti-human GH mouse mAbs, clones EB1 and EB2 described in Non-Patent Documents 10 and 11 inhibit the binding of human GH to human GHR, and 3T3-F422A adipocytes It has been shown to inhibit the inhibitory activity of human GH on glucose uptake into the strain, but the strength of inhibition ( IC50 ) was not specified.
- Non-Patent Document 12 is related to Patent Document 1.
- Non-Patent Document 12 in a GH-dependent proliferation test of a Ba/F3 cell line forcibly expressing human GHR/G-CSFR, hGH-25 and hGH-26 only inhibited GH-22K-dependent cell proliferation.
- -33 inhibited only GH-20K-dependent cell proliferation
- hGH-12 inhibited both GH- 22K and GH-20K-dependent cell proliferation. Not specified.
- Non-Patent Document 13 describes three anti-human GH mouse mAbs, of which two clones (AC8, F11) that strongly inhibit the binding of human GH to the Nb2 rat lymphoma cell line are human GH-dependent Nb2. It has been shown to inhibit cell proliferation, but the strength of inhibition ( IC50 ) is not specified.
- Non-Patent Document 14 describes 10 types of human GH-specific mouse mAbs, which suppress mitogen-stimulated (PHA)-induced T cell proliferation, but no inhibitory activity against human GH was shown. not
- Non-Patent Document 15 describes three types of anti-human GH mouse mAbs and shows that only the clone (B-2) that cross-reacts with hPL at high concentrations inhibited human GH-human GHR binding. However, the strength of inhibition (IC 50 ) has not been specified.
- Non-Patent Document 16 describes a novel method for measuring human GH activity in serum using a Ba/F3 cell line (Ba/F3-hGHR) in which human GHR is forcibly expressed.
- Anti-human GH mouse mAb (clone 5801) has been used as a positive control antibody to demonstrate that is GH-dependent.
- Non-Patent Document 17 describes an anti-human GH mouse mAb (6J33) with neutralizing activity.
- the 6J33 antibody has been shown to have neutralizing activity, but the potency of inhibition ( IC50 ) has not been specified.
- Growth hormone isoforms and segments/fragments Molecular structure and laboratory measurement (Palo E.F.D. et al., Clinica Chimica Acta 2006, 364, 67-76) Cloning of two novel growth hormone transcripts expressed in human placenta (Boguszewski C.L. et al., J Clin Endocrinol Metab 1998, 83, 2878-2885) Targeting growth hormone function: strategies and therapeutic applications (Lu M. et al., Signal Transduction and Targeted Therapy 2019, 4:3) Tumor-Derived Human Growth Hormone As a Therapeutic Target in Oncology (Jo K Perry et al. Trends Endocrinol Metab.
- the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human GH.
- the present invention includes the following embodiments.
- an antibody or antigen-binding fragment thereof that specifically binds to human growth hormone comprising a heavy chain variable region (VH) and a light chain variable region (VL);
- VH comprises VH complementarity determining region (CDR) 1 (VHCDR1), VHCDR2, and VHCDR3;
- VHCDR1 comprises the amino acid sequence of SEQ ID NO: 5, or an amino acid sequence in which 1 or 2 amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 5
- VHCDR2 comprises the amino acid sequence of SEQ ID NO: 6, or an amino acid sequence in which 1 or 2 amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 6
- VHCDR3 comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which 1 or 2 amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 7
- VL comprises VL Complementarity Determining Region (C
- VH comprises the amino acid sequence of SEQ ID NO: 5 as VH complementarity determining region (CDR) 1 (VHCDR1), the amino acid sequence of SEQ ID NO: 6 as VHCDR2, and the amino acid sequence of SEQ ID NO: 7 as VHCDR3;
- CDR complementarity determining region
- VLCDR1 amino acid sequence of SEQ ID NO: 8 as VL complementarity determining region
- SEQ ID NO: 9 amino acid sequence of SEQ ID NO: 9 as VLCDR2
- amino acid sequence of SEQ ID NO: 10 as VLCDR3.
- a VH comprising an amino acid sequence having 90% or more identity to the amino acid sequence of 11, 12, or 19, and/or a region other than CDR1 to CDR3 in the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20 contains an amino acid sequence in which one or several amino acids are substituted, added, or deleted, or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20 VL
- the antibody or antigen-binding fragment thereof according to any one of (1) to (4), comprising (6) the antibody of (5), comprising VH having the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19 and/or VL having the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20; or an antigen-binding fragment thereof.
- the antibody is a heavy chain constant region containing an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 17, or 90% of the amino acid sequence of SEQ ID NO: 17
- a heavy chain constant region comprising an amino acid sequence having at least the same identity, and/or a light chain constant region comprising an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO: 18, or a light chain constant region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 18;
- the antibody or antigen-binding fragment thereof according to any one of (1) to (8), comprising (10) The antibody according to any one of (1) to (9), wherein the antibody comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO: 17 and/or a light chain constant region having the amino acid sequence of SEQ ID NO: 18.
- Antibodies or antigen-binding fragments thereof (11) The antibody or antigen-binding fragment thereof according to any one of (1) to (10), wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or scFv. (12) A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of (1) to (11). (13) A vector comprising the nucleic acid of (12). (14) A cell containing the nucleic acid of (12) or the vector of (13). (15) A pharmaceutical comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (11). (16) The medicament according to (15), for reducing the level of insulin-like growth factor 1 (IGF-1) in blood.
- IGF-1 insulin-like growth factor 1
- (17) for treating and/or preventing a disease selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, and pulmonary inflammation medicine.
- a disease selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, and pulmonary inflammation medicine.
- a method for reducing levels of insulin-like growth factor 1 (IGF-1) in the blood, comprising a therapeutically effective amount of the antibody of any one of (1) to (11) or antigen-binding thereof A method comprising administering a fragment.
- IGF-1 insulin-like growth factor 1
- the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human GH.
- FIG. 1 shows serum IGF-1 levels two days after administration of anti-GH mouse mAb (13H02) to hypophysectomized/GH supplemented rats.
- the normal control group (Normal) was hypophysectomized/GH non-supplemented rats, and the negative control group was hypophysectomized/GH supplemented rats administered control mouse IgG1.
- FIG. 2 shows serum IGF-1 levels two days after administration of anti-GH mouse-human chimeric mAb (Ch-13H02) in hypophysectomized/GH supplemented rats.
- the normal control group was hypophysectomized/GH non-supplemented rats, and the negative control group was hypophysectomized/GH supplemented rats to which control human IgG1 was administered.
- FIG. 1 shows serum IGF-1 levels two days after administration of anti-GH mouse mAb (13H02) to hypophysectomized/GH supplemented rats.
- the normal control group (Normal) was hypophysectomized/GH non-suppl
- FIG 3 shows serum IGF-1 levels two days after administration of anti-GH humanized antibody Fc variant (Hu-13H02m) to hypophysectomized/GH supplemented rats.
- the normal control group (Normal) was hypophysectomized/GH non-supplemented rats, and the negative control group was hypophysectomized/GH supplemented rats to which control human IgG1 was administered.
- the present invention provides antibodies or antigen-binding fragments thereof (hereinafter also referred to as “anti-GH antibodies”) that specifically bind to human growth hormone (GH). .
- Growth hormone is a hormone secreted from cells in the anterior lobe of the pituitary gland, and is involved in individual growth and metabolism. GH promotes bone elongation by both direct effects on tissues and stimulation of insulin-like growth factor 1 (IGF-1) release through its action on GH receptors (GHR) expressed in the liver and other tissues. It shows growth-related effects such as muscle growth, metabolism-related effects such as metabolism promotion, blood sugar increase, and maintenance of homeostasis. IGF-1 is a peptide hormone with a structure similar to that of insulin, and is secreted in various tissues including the liver. IGF-1 has a wide range of physiological functions, such as acting on various organs, promoting cell proliferation and differentiation protein synthesis, and suppressing cell death.
- sequence information for GH of other mammals such as mice and monkeys can be obtained from publicly accessible databases.
- GH refers to the GH protein.
- the gene encoding the GH protein may simply be called GH. It will be clear from the context to those skilled in the art when “GH” refers to the gene encoding the GH protein.
- GH is typically human GH, but may be non-human mammalian GH (eg, mouse, rat, monkey, etc.).
- the term "antibody” refers to a glycoprotein comprising at least two heavy chains and two light chains interconnected by disulfide bonds, and in the case of IgM, a J chain. is.
- a heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region.
- Heavy chain constant regions include ⁇ , ⁇ , ⁇ , ⁇ and ⁇ chains, and isotypes such as IgG, IgM, IgA, IgD and IgE exist depending on their differences.
- Heavy chain constant regions comprise three domains, CH1, CH2 and CH3, and in the case of IgE and IgM, CH4.
- a light chain comprises a light chain variable region (VL) and a light chain constant region, the light chain constant region comprising one domain, CL.
- VL light chain variable region
- ⁇ light chain constant regions
- the VH and VL regions comprise four regions (FR1, FR2, FR3, FR4) that are relatively conserved among variable regions, further referred to as framework regions (FR), and complementarity determining regions (CDRs). It is subdivided into three hypervariable regions (CDR1, CDR2, CDR3) which are called and contribute to antigen binding.
- the VH contains 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 (CDRH1), FR2, CDR2 (CDRH2), FR3, CDR3 (CDRH3), FR4.
- VL contains 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1 (CDRL1), FR2, CDR2 (CDRL2), FR3, CDR3 (CDRL3), FR4.
- CDR can be determined, for example, according to the method of Kabat et al. (Kabat E.A. et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.).
- Antibodies as used herein include all classes and subclasses of intact immunoglobulins.
- the antibodies described herein are, for example, IgG, and more specifically, the constant region thereof is human IgG1, IgG1 effector-deficient variants, IgG2, IgG4, or IgG4 (S228P). More specifically, it may be IgG1, or a variant in which the effector function of IgG1 is eliminated.
- isotypes with weak effector functions or variants in which effector functions are eliminated from the viewpoint of safety but the antibodies described herein are Since it targets soluble GH, there is little need for it.
- antigen-binding fragments of antibodies include separated heavy chains, light chains, Fab, Fab', F(ab')2, Fv, scFv, and the like.
- antibodies or antigen-binding fragments thereof include derivatives thereof.
- Derivatives of antibodies or antigen-binding fragments thereof include antibodies in which amino acid mutations are artificially introduced into the constant region, antibodies in which the configuration of the domain of the constant region is altered, antibodies with two or more Fc molecules per molecule, and sugar chain alterations.
- the antibodies described in this specification may be either polyclonal antibodies or monoclonal antibodies, but monoclonal antibodies are particularly preferred.
- a “monoclonal antibody” (also referred to as “mAb”) is an antibody obtained from a clone, typically derived from a single antibody-producing cell. That is, mAbs are identical in amino acid sequence to individual antibodies that make up a homogeneous or substantially homogeneous population, except for possible naturally occurring mutations that may occur in minor amounts. A mAb binds to a single antigenic determinant (epitope) on an antigen and has high specificity.
- Polyclonal antibodies are mixtures of mAbs that differ in one or more amino acid sequences.
- mAbs include fully human antibodies and non-human antibodies.
- Non-human antibodies are those other than fully human antibodies, and include, for example, animal antibodies such as mice, rats, rabbits, and guinea pigs, chimeric antibodies, humanized antibodies, and veneered antibodies.
- a "chimeric antibody” is an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a non-human animal antibody and the constant region sequences are human antibodies. Refers to antibodies derived from
- Humanized antibody means that only the CDR sequences and some amino acid residues of the framework in the variable region are derived from non-human animal antibodies, and the rest of the sequences, that is, most of the framework sequences and all constant It refers to an antibody whose region sequence is derived from a human antibody.
- a “veneered antibody” retains some and usually all of the CDRs of a non-human animal antibody and some of the non-human animal variable region framework residues, but does not contribute to B or T cell epitopes.
- Other variable region framework residues obtained, e.g., one of a humanized antibody, by replacing exposed residues (Padlan, Mol. Immunol. 28:489, 1991) with residues at corresponding positions in the human antibody sequence. There are two types.
- Fully human antibody refers to an antibody that is entirely derived from a human antibody, including CDR sequences.
- the term "specifically binds” means that an antibody binds to a certain antigen with a significantly higher affinity than the affinity (nonspecific interaction) to a non-antigen substance. Affinity can be measured by conventional methods. Examples of such methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and the like. In such affinity measurement, the antibody and/or antigen can be labeled with an enzyme, fluorescent substance, or the like, and the labeling substance can be measured to detect the affinity.
- ELISA enzyme-linked immunosorbent assay
- SPR surface plasmon resonance
- K D equilibrium dissociation constant
- k on binding rate constant
- k off dissociation rate constant
- k on refers to the rate constant for antibody binding to antigen and k off refers to the rate constant for antibody dissociation from the antigen-antibody complex.
- K D refers to the equilibrium dissociation constant of an antigen-antibody interaction and is calculated as k off /k on .
- the antibodies described herein have a K D of, for example, 1 ⁇ 10 ⁇ 8 mol/L or less, 1 ⁇ 10 ⁇ 8 mol/L or less, when measured according to the method described in Example 12 herein 9 mol/L or less, 1 ⁇ 10 -10 mol/L or less, 5 ⁇ 10 -11 mol/L or less, 2 ⁇ 10 -11 mol/L or less and/or 1.0 ⁇ 10 -13 or more, 1.0 ⁇ 10 -12 or more, or 1.0 ⁇ 10 ⁇ 11 or more, to human GH.
- the antibodies described herein have a high affinity for GH-22K among isoforms of human GH (GH-22K, GH-20K).
- cross-reactivity refers to other proteins, such as non-human Reactivity to mammalian GH, proteins showing structural similarity to human GH (e.g., human placental lactogen (PL), human prolactin (PRL)), and/or other isoforms of human GH (GH-20K) Say.
- PL placental lactogen
- PRL human prolactin
- GH-20K isoforms of human GH
- Cross-reactivity of anti-GH antibodies can be measured in vitro, for example, by competitive ELISA described in Examples 3, 4 and 14 and SPR described in Example 13.
- an antibody or antigen-binding fragment thereof described herein comprises VH and VL, wherein VH comprises VH complementarity determining region (CDR) 1 (VHCDR1), VHCDR2, and VHCDR3, and VL comprises Contains VL Complementarity Determining Region (CDR) 1 (VLCDR1), VLCDR2, and VLCDR3.
- VHCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:5.
- VHCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:6, or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:6.
- VHCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:7 or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:7.
- VLCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:8, or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:8.
- VLCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:9 or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:9.
- VLCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:10 or an amino acid sequence in which 1 or 2 amino acids are substituted, added or deleted in the amino acid sequence of SEQ ID NO:10.
- VHCDR1 comprises or consists of an amino acid sequence in which one amino acid is substituted, added, or deleted in the amino acid sequence of SEQ ID NO:5, and VHCDR2 has one amino acid in the amino acid sequence of SEQ ID NO:6.
- VHCDR3 comprises or consists of an amino acid sequence in which one amino acid is substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 7, or or consists of
- VLCDR1 comprises or consists of an amino acid sequence in which one amino acid is substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 8
- VLCDR2 consists of one in the amino acid sequence of SEQ ID NO: 9 comprising or consisting of an amino acid sequence in which amino acids are substituted, added, or deleted
- VLCDR3 comprises or consists of an amino acid sequence in which one amino acid is substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 10, or consists of
- the antibody or antigen-binding fragment thereof described herein has the amino acid sequence of SEQ ID NO: 5 as VH complementarity determining region (CDR) 1, the amino acid sequence of SEQ ID NO: 6 as VHCDR2, and the amino acid sequence of SEQ ID NO: 6 as VHCDR3. It contains the amino acid sequence of SEQ ID NO: 7, VL contains the amino acid sequence of SEQ ID NO: 8 as VL complementarity determining region (CDR) 1, the amino acid sequence of SEQ ID NO: 9 as VLCDR2, and the amino acid sequence of SEQ ID NO: 10 as VLCDR3.
- VHCDR1-CDR3 may consist of the amino acid sequences of SEQ ID NOs: 5-7, respectively, and VLCDR1-CDR3 may consist of the amino acid sequences of SEQ ID NOs: 8-10, respectively.
- the framework region sequences of the antibodies or antigen-binding fragments thereof described herein are not limited as long as the antibody or antigen-binding fragment thereof can specifically bind to GH.
- the antibodies or antigen-binding fragments thereof described herein are An amino acid sequence in which one or several amino acids are substituted, added, or deleted in a region other than CDR1 to CDR3 in the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, or SEQ ID NO: 3, 11, 12 , or 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of the 19 amino acid sequences VH comprising identical amino acid sequences, wherein the regions of CDR1 to CDR3 consist of SEQ ID NOs: 5 to 7, respectively, and/or in the amino acid sequences of SEQ ID NOs: 4, 13, 14, or 20, in regions other than CDR1 to CDR3 , an amino acid sequence in which one
- the antibodies or antigen-binding fragments thereof described herein are An amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, or to the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19 contains amino acid sequences with 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity
- An amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of VH and/or SEQ ID NO: 4, 13, 14, or 20, or SEQ ID NO: 4, 13, 14, or 20 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence of A VL containing an amino acid sequence with
- “several” may be, for example, 2 to 10, 2 to 8, 2 to 5, for example 2 or 3.
- identity means the ratio of matching amino acid residues.
- the identity of amino acid sequences can be determined, for example, by BLAST (Basic Local Alignment Search Tool) analysis.
- the antibody or antigen-binding fragment thereof described herein is a VH having the amino acid sequence of SEQ ID NO:3, 11, 12, or 19 and/or Contains VL with amino acid sequence.
- the antibodies or antigen-binding fragments thereof described herein are A heavy chain constant region comprising an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 17, or 90% or more, 91% or more of the amino acid sequence of SEQ ID NO: 17,
- a light chain constant region comprising an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of number 18, or 90% or more, 91% or more, or 92 of the amino acid sequence of SEQ ID NO: 18 % or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater identity.
- the C-terminal amino acid sequence of SEQ ID NO: 17 the C
- the antibody or antigen-binding fragment thereof described herein comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:17 and/or a light chain constant region having the amino acid sequence of SEQ ID NO:18.
- the C-terminal lysine residue may or may not be present.
- amino acid sequence of the constant region may contain substitutions that extend blood half-life (see, for example, Hinton P.R. et al., J. Biol. Chem. 279:6213-6216, 2004).
- An antibody or antigen-binding fragment thereof comprising a VH and/or a VL comprising may have biological activity equivalent to an antibody or antigen-binding fragment thereof having the original amino acid sequence.
- the "equivalent biological activity” includes (i) specific binding activity to GH, (ii) GH neutralizing activity, (iii) IGF-1 production inhibitory activity when bound to GH, or (iv) any two or more or all of these.
- the antibodies described herein neutralize GH action.
- neutralizing refers to binding to a target protein and neutralizing or attenuating its action
- neutralizing activity refers to the degree of neutralizing or attenuating action.
- neutralizing activity refers to the degree of neutralization or attenuation of GH, unless otherwise stated or the context makes it clear to other proteins. say.
- the antibodies described herein reduce IGF-1 levels in plasma and/or tissues by specifically binding GH and blocking its interaction with GHR.
- the neutralizing activity of the antibodies described herein can be determined, for example, in vitro (eg, in the GH-GHR binding inhibition assay or the GH-dependent Nb2 cell growth inhibition assay described in Example 2 herein). can be measured.
- the antibodies described herein are 10,000 pmol/L or less, 1,000 pmol/L or less, or 100 pmol/L or less, and/or 1 pmol/L in the GH-GHR binding inhibition assay described in Example 2. /L or higher, 0.1 pmol/L or higher, or 0.01 pmol/L or higher.
- the antibodies described herein are 1,000 pmol/L or less, 100 pmol/L or less , or 10 pmol/L or less, and/or Or has an IC 50 of 1 pmol/L or higher, 0.1 pmol/L or higher, or 0.01 pmol/L or higher.
- the antibodies described herein reduce serum IGF-1 levels, may be used to alleviate and treat symptoms of GH hypersecretion, and may be used in humans who will develop some symptoms in the future due to GH excess. It may be used as a prophylactic agent against The antibodies described herein may be used in patients who have failed conventional therapeutic agents for GH hypersecretion. In one embodiment, the antibodies described herein are used to treat disorders associated with GH hypersecretion, including, for example, acromegaly, gigantism, certain cancers, diabetic nephropathy, arthritis, and pulmonary inflammation. and/or in prophylaxis.
- the antibodies described herein may have excellent efficacy (e.g., equal to or greater than pegvisomant), safety, and/or good blood kinetics.
- the antibodies described herein reduce serum IGF-1 levels in vivo.
- the antibodies described herein dose-dependently reduce elevated serum IGF-1 levels caused by continuous administration of exogenous human GH in rats lacking endogenous GH due to hypophysectomy.
- the antibodies described herein neutralize endogenous GH and persistently lower serum IGF-1 levels by administration to a mammal, eg, a monkey.
- the antibodies described herein have little or no toxicity to mammals.
- the antibodies described herein exhibit good physical properties (eg, at least one of water solubility, acid stability, heat stability, hydrophobic interactions).
- the antibody described herein is known in the art, for example, from B cells obtained by immunizing an animal with an antigen, using hybridoma methods, various display methods, single-cell screening methods, and the like. can be selected and acquired by a wide variety of methods.
- Fully human monoclonal antibodies can also be produced by using genetically modified animals in which human antibody genes are integrated into antigen-immunized animals, or by using antibody display libraries constructed from human antibody gene pools.
- Hybridoma technology selects and acquires target antibody-producing strains from immortalized monoclonal hybridoma cell lines by fusing antibody-producing B cells obtained by immunizing animals such as mice and rats with antigens and myeloma cells. It is a technique to obtain monoclonal antibodies by Although the technique is not limited, it can be implemented as follows, for example.
- Human GH or a fragment thereof can be used as an antigen for producing the antibody described in this specification.
- Animals such as mice or rats are immunized multiple times with these antigens along with various adjuvants, and the animals with elevated serum antibody titers are given the final immunization with the antigen.
- Collect lymphocytes containing The lymphocytes (B cells) are fused with a myeloma cell line using polyethylene glycol or an electric cell fusion device, and a selective medium (for example, HAT (hypoxanthine-aminopterin-thymidine) in which only the fused cells of both can survive) (selective medium containing ) to establish hybridomas.
- a selective medium for example, HAT (hypoxanthine-aminopterin-thymidine) in which only the fused cells of both can survive
- the monoclonal hybridomas thus obtained can be appropriately cultured, and the human GH-binding activity and human GH-neutralizing activity of the culture supernatant can be measured to select human GH-specific neutralizing antibody-producing hybridomas.
- Details of hybridoma production techniques are known, for example, see Koehler, G. et al., Nature 256: 495-497, 1975, Hnasko R.M. et al., Methods Mol. Biol. 1318: 15-28, 2015.
- Antibody production methods using display technology include, for example, phage display, yeast display, cDNA display, ribosome display, and animal cell display. Specifically, antibody fragments (scFv, Fab, etc.) encoded by genes isolated from B cell pools isolated from immunized animals, non-immunized animals, diseased patients, or healthy individuals described in the above hybridoma technology are displayed in various display systems. to build a library. From this library, a target antibody can be obtained by concentrating, amplifying, and isolating antibodies with affinity to the antigen and their genes by a selection procedure called biopanning. Details of display technology are known, for example, US Pat. No. 5,223,409, US Pat. No. 5,885793, US Pat. Patent No.
- B cells isolated from the above-mentioned immunized animals or diseased patients are seeded, for example, in very small cells at a rate of 1 cell/cell, and the antibody activity secreted by each cell is detected by fluorescence.
- FACS fluorescence-activated cell sorting
- the antibody of interest can be obtained by isolating the antibody gene from the B cells collected in this way. Details of single-cell screening techniques for B cells are known, see, for example, Love J.C. et al., Nat.
- a chimeric antibody can be produced, for example, by genetically linking a non-human antibody variable region sequence and a human antibody constant region sequence and producing it by the method described below. Details of chimeric antibody production techniques are known, see, for example, US Pat. No. 5,482,856, Morrison S.L. et al., Proc. Natl. Acad. Sci.
- Humanized antibodies for example, combine some and usually all of the CDRs of a non-human antibody and some of the non-human variable region framework residues into human antibody variable regions that are highly homologous to the non-human antibody variable region sequences. It can be produced by transplanting. Details of humanized antibody production techniques are known, for example, US Pat. No. 5,530,101, US Pat. No. 5,585,089, US Pat. See Queen C.L. et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989; Tsurushita N. et al., Methods 36: 69-83, 2005; can do.
- a veneered antibody retains, for example, some and usually all of the CDRs of the non-human antibody and some of the non-human variable region framework residues, and has B-cell or T-cell epitopes (e.g., exposed residues) ( Padlan, Mol. Immunol. 28:489, 1991) by substituting residues from corresponding positions in the human antibody sequence for other variable region framework residues.
- B-cell or T-cell epitopes e.g., exposed residues
- a fully human antibody can be produced, for example, from B cells obtained by antigen-immunizing an animal into which a human antibody gene has been introduced, using the above-mentioned monoclonal antibody production technology. It can also be produced from various antibody display libraries prepared from B cell pools isolated from diseased patients or healthy individuals. Details of techniques for producing fully human antibodies are known, see, for example, Green L.L. et al., Nat. Genet. 7: 13-21, 1994, Lonberg N. et al., Nature 368: 856-859, 1994, Mendez M.J. et al., Nat. Genet. 15: 146-156, 1997, Ishida I.
- the heavy and light chain variable regions of chimeric, humanized, veneered, or fully human antibodies can be linked, for example, to any human heavy and light chain constant regions, respectively.
- the choice of heavy chain constant region isotype depends on whether complement dependent cytotoxicity or antibody dependent cytotoxicity is desired as the target and mechanism of action of the antibody.
- human IgG1 and IgG3 generally have strong complement-dependent cytotoxicity and antibody-dependent cytotoxicity
- human IgG2 and IgG4 have weak effector functions.
- the light chain constant region can be selected to be either lambda or kappa.
- Human constant regions exhibit allotypic variation among different individuals, but include constant regions with any of the natural allotypes or any substitutions of residues occupying polymorphic positions of the natural allotypes.
- One or more amino- or carboxy-terminal amino acids of the light and/or heavy chains of the antibody e.g., the C-terminal lysine of the heavy chain
- Antibodies described herein can have increased serum half-life by substituting amino acid residues in the heavy chain constant region.
- Typical substitutions for prolonging blood half-life include, for example, a method of substituting glutamine for threonine at position 250 of the heavy chain, and a method of substituting leucine for methionine at position 428 of heavy chain, according to EU numbering as in Kabat. , there is a method of substituting asparagine at position 434 of the heavy chain with serine, and multiple combinations of the above amino acid substitutions can be made (see US Pat. Nos. 7,083,784, 7,217,797 and 8,088,376). 3. Production of Antibodies Described Herein In one embodiment, the invention provides a method of producing an antibody or antigen-binding fragment described herein.
- the invention provides an isolated nucleic acid encoding an antibody or antigen-binding fragment described herein.
- a nucleic acid molecule may be RNA or DNA.
- the nucleic acids described herein can be used to produce the antibodies or antigen-binding fragments described herein.
- Nucleic acids encoding the antibodies or antigen-binding fragments described herein can be, for example, B cells that produce the antibodies described herein, hybridomas, isolated clones of various antibody displays (phage, yeast, animal cells, cDNA, etc.). ) and sequenced.
- the genes encoding the antibody heavy and/or light chain variable region amino acid sequences can be isolated and sequenced using oligonucleotide probes that specifically bind to these constant region sequences.
- antibodies whose amino acid sequences are known should be prepared using the nucleic acid encoding the amino acid sequence.
- Antibodies or antigen-binding fragments produced by genetic recombination techniques are also referred to as recombinant antibodies or recombinant antigen-binding fragments.
- the invention provides a vector comprising a nucleic acid encoding an antibody or antigen-binding fragment described herein.
- a vector comprising a nucleic acid encoding an antibody or antigen-binding fragment described herein can be transfected into a eukaryotic host cell, e.g., operably linked to a nucleic acid sequence encoding the antibody or antibody-binding fragment.
- Expression control sequences that can be converted or transfected e.g., origins of replication, promoters, enhancers (Queen C. et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites (e.g., ribosome binding sites). , an RNA splice site, a polyadenylation site and a transcription terminator sequence)), and can be easily constructed by known genetic recombination techniques.
- the present invention provides a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment described herein, or a vector comprising said nucleic acid.
- a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment described herein, or a vector comprising said nucleic acid.
- a cell expressing a recombinant antibody or a recombinant antigen-binding fragment is obtained by introducing a vector containing a nucleic acid encoding the antibody or antigen-binding fragment into a host cell and transiently It can be obtained by direct or stable expression.
- host cells include E. coli cells, yeast cells, insect cells, mammalian cells, etc.
- Preferred hosts include mammalian cells, such as human HEK293 embryonic kidney-derived cells, monkey COS cells, Chinese hamster ovary ( CHO) cells, mouse myeloma cells (Sp2/0, NS0) and the like. Details of techniques for producing cells expressing recombinant antibodies or recombinant antigen-binding fragments are known.
- Recombinant antibodies or recombinant antigen-binding fragments are purified from cell extracts or culture supernatants by culturing the aforementioned recombinant cells and by standard protein purification techniques, including various column chromatography and ultraconcentration. It can be manufactured by Details of techniques for purifying recombinant antibodies or recombinant antigen-binding fragments are known.
- the above-described method for producing an antibody with a known amino acid sequence, method for constructing a vector containing a nucleic acid encoding an antibody or an antigen-binding fragment, recombinant cell production technology, and technology for purifying a recombinant antibody or a recombinant antigen-binding fragment include, for example: , Ossipow V. et al., Monoclonal Antibodies, Methods Mol. Biol. 1131 (2nd ed.), 2014.
- recombinant antibodies or recombinant antigen-binding fragments are transgenic plants (e.g., tobacco, duckweed) or transgenic animals (e.g., cattle, It can also be expressed in goat's milk, purified according to the same method as described above, and produced. See, for example, Perdigones A.S. et al., Cell Engineering 7: 143-164, 2011, Pollock D.P. et al., J. Immunol. Methods 231: 147-157, 1999 for methods of producing antibody transgenic plants and antibody transgenic animals. sea bream.
- Antigen-binding fragments can also be produced by enzymatic digestion of antibodies or (for short polypeptides of, eg, about 50 amino acids or less) by chemical synthesis, in addition to the methods for producing antibodies described above.
- Methods for enzymatic digestion and chemical synthesis of antibodies are known.
- enzymatic digestion for example, the details of techniques for preparing Fab by partial digestion of IgG with papain or the like are known; for example, see Zao Y. et al., Protein Expression and Purification 67: 182-189, 2009. can.
- Chemical syntheses can also be produced, for example, by automated polypeptide synthesizers using solid phase methods, see, eg, US Pat. No. 5,807,715; US Pat. No. 4,816,567; and US Pat. No. 6,331,415. can do.
- the invention relates to pharmaceuticals comprising the antibodies or antigen-binding fragments described herein.
- the antibodies or antigen-binding fragments or medicaments described herein can be used to reduce levels of IGF-1 in blood. In one embodiment, the antibodies or antigen-binding fragments or medicaments described herein can be used to prevent and/or treat symptoms associated with excess GH and/or excess IGF-1 in an individual. can.
- Conditions associated with excess GH and/or excess IGF-1 include acromegaly, gigantism, pituitary gigantism, glucose intolerance, diabetic nephropathy, sleep apnea, decreased energy, menstruation. Irregularity, cancer, increased incidence of tumors, malocclusion, excessive sweating, numbness in hands and feet, arthritis, joint pain due to osteoarthritis, lung inflammation, and the like. Symptoms associated with excess GH and/or excess IGF-1 are preferably acromegaly, gigantism, glucose intolerance, diabetic nephropathy, sleep apnea, more preferably acromegaly, gigantism is.
- the antibody or antigen-binding fragment or medicament described herein reduces blood IGF-1 levels.
- Blood IGF-1 level is, for example, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% before administration % or less.
- therapeutic administration of an antibody or antigen-binding fragment or medicament described herein results in a reduction or amelioration of one or more symptoms of acromegaly and/or a reduced incidence of acromegaly.
- Symptoms of acromegaly include, but are not limited to, excessive IGF-1, profuse sweating, unpleasant body odor, thickened skin, darkened skin, oily skin, skin tags, fatigue, Muscle weakness, deepening of voice due to enlarged vocal cords and sinuses and/or cartilage in the pharynx, severe snoring, sleep apnea, visual disturbances, headache, enlarged tongue, back pain, joint pain, joint pain restricted range of motion, irregular menstrual cycle, decreased libido, erectile dysfunction, enlarged hands, enlarged legs, widened facial features, protruding lower teeth than upper teeth (opposite occlusion), Enlarged liver, enlarged heart, enlarged kidneys, enlarged spleen, increased chest circumference (
- the clinical criteria and prognostic indicators of acromegaly are well known in the art, and the diagnosis or evaluation of acromegaly is established.
- Individuals suitable for treatment and/or prophylaxis with the antibodies or antigen-binding fragments or pharmaceuticals described herein can be selected according to the clinical criteria, evaluation, and the like.
- Assessment of acromegaly severity includes, for example, measurement of blood IGF-1 levels, measurement of GH before and after oral glucose testing, and magnetic resonance imaging (MRI) of the head to detect pituitary tumors. It can be carried out based on known tests. Relief, amelioration, modulation, reduction in incidence, or delay in onset or progression of acromegaly symptoms can also be measured by testing blood levels of IGF-1.
- Symptoms of gigantism include, but are not limited to, excessive IGF-1, excessive height growth, excessive muscle growth, excessive organ growth, delayed puberty, diplopia, difficulty in peripheral vision, and frontal protuberance. , protruding chin, headaches, increased sweating, irregular menstruation, large hands, large feet, thick fingers, thick toes, milk production, facial thickening, weakness, adrenal insufficiency, diabetes insipidus, hypogonadism, and thyroid gland Hypofunctional disorder and the like can be mentioned.
- the clinical criteria and prognostic indicators of gigantism are well known in the art, and the diagnosis or evaluation of gigantism has been established.
- Individuals suitable for treatment and/or prophylaxis with the antibodies or antigen-binding fragments or pharmaceuticals described herein can be selected according to the clinical criteria, evaluation, and the like.
- Evaluation of gigantism severity e.g., computed tomography (CT) or MRI of the head to detect pituitary tumors, measurement of GH before and after oral glucose test, measurement of blood prolactin, blood IGF -1 measurement, blood cortisol measurement, blood estradiol measurement, blood testosterone measurement, and thyroid hormone measurement, etc., can be performed based on known tests.
- Relief, amelioration, modulation, reduction in incidence, or delay in onset or progression of gigantism symptoms can also be measured by measuring blood IGF-1 levels.
- treatment means improvement of symptoms associated with excess GH, continued improvement of symptoms, prevention of recurrence, reduction of other treatments, and other medical treatments related to excess GH. It includes all treatments, including improving quality of life for those with symptoms of GH excess.
- prevention includes preventing or reducing the risk of symptoms associated with excess GH.
- the medicaments described herein further contain pharmaceutically acceptable carriers (excipients, fillers, binders, lubricants, etc.) and/or known additives (buffers, etc.) tonicity agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.).
- pharmaceutically acceptable carriers excipients, fillers, binders, lubricants, etc.
- known additives buffers, etc. tonicity agents, chelating agents, coloring agents, preservatives, fragrances, flavoring agents, sweetening agents, etc.
- compositions described herein can also contain one or more other agents.
- the medicaments described herein are usually administered systemically or locally, orally or parenterally.
- the medicament described herein can be administered to a living body by, for example, intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, external preparation, or the like.
- the dosage of the pharmaceuticals described herein varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., and varies according to various conditions. For example, it can be administered to an individual as a medicament or as an active ingredient at 0.0001 to 100 mg/kg, preferably 0.01 to 100 mg/kg per day.
- the medicament described herein can be a pharmaceutically acceptable formulation.
- the above preparations can be made into sterile solutions, suspensions, injections such as freeze-dried preparations, tablets, granules, powders, capsules, emulsions, suspensions, syrups, etc., according to commonly used means. can be done.
- the medicaments described herein can be used alone or in combination with other conventional treatment methods.
- the timing of administration and the timing of treatment of the drug and the other drug described herein are not limited, and they may be administered to the subject at the same time or at different times.
- the invention provides a method for reducing levels of IGF-1 in blood comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof described herein.
- the method may be for treating and/or preventing symptoms associated with excess GH and/or excess IGF-1 as described above.
- the method includes identifying an individual at risk for a disease associated with excess GH and/or excess IGF-1, evaluating the individual for risk factors, and/or evaluating or diagnosing the individual for symptoms. may further include
- GH binding activity measurement method Recombinant human GH (Pharmaceuticals and Medical Devices Regulatory Science Foundation) diluted to 5 ⁇ g/mL with Dulbecco's phosphate buffer (PBS) was immobilized on a 96 half-well immunoplate (Corning) at 50 ⁇ L/well, followed by 1% bovine serum. Blocking was performed with PBS containing albumin (BSA). After that, 50 ⁇ L/well of positive control (anti-GH mouse mAb (MAB1067, R&D Systems)) diluted with assay buffer (0.2% BSA, 0.05% Tween 20-containing PBS) or sample containing anti-GH mouse mAb was added and incubated at room temperature.
- PBS Dulbecco's phosphate buffer
- Example 2 Method for measuring GH neutralizing activity> We established (a) GH-GH receptor (GHR) binding inhibition assay and (b) GH-dependent Nb2 cell growth inhibition assay to evaluate the GH-neutralizing activity of anti-GH antibodies.
- GHR GH-GH receptor
- GH-dependent Nb2 cell growth inhibition assay to evaluate the GH-neutralizing activity of anti-GH antibodies.
- GH-GHR binding inhibition activity was measured using biotin-labeled GH against GHR extracellular region protein-human Fc fusion (GHR-hFc) immobilized on an ELISA plate via anti-human Fc ⁇ mouse antibody. was measured by calculating the inhibition rate of the binding signal of Biotin-labeled GH was prepared by labeling recombinant GH using the EZ-Link Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). A specific method is as follows.
- Anti-human Fc ⁇ mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted in 1 ⁇ g/mL PBS was immobilized on a 96 half-well immunoplate at 50 ⁇ L/well, and blocked with 1% BSA-containing PBS. After that, 50 ⁇ L/well of GHR-hFc (R&D Systems) diluted to 0.1 ⁇ g/mL with assay buffer (0.2% BSA, 0.05% Tween 20-containing PBS) was added to the plate and captured on the plate.
- biotin-labeled GH diluted to a final concentration of 5.5 ng/mL with assay buffer and a specimen (sample containing anti-GH mouse mAb) diluted with assay buffer were added.
- a mixture of equal volumes was added at 50 ⁇ L/well and allowed to stand at room temperature for 2 hours.
- non-labeled GH or anti-GH mouse mAb was used instead of the sample.
- the GH-GHR binding inhibition rate of each sample was calculated assuming that the absorbance when only biotin-labeled GH was added was 0% inhibition and the absorbance when biotin-labeled GH was not added was 100% inhibition, and IC50 was calculated.
- the IC50 of the positive control antibody MAB1067 under the above conditions was 6,860 pmol/L.
- Nb2 rat lymphoma cell line proliferation assay known as a standard GH activity assay (Omae Y et al., Endocrinol Japan 36, 9-13, 1989).
- a specific method is as follows. Nb2-11 cell line (EC90741101, ECACC) adapted to Fischer's Medium containing 10% fetal bovine serum and 10% horse serum (HS) was placed in Fischer's Medium containing 1% HS in a CO 2 incubator (37°C, 5% CO 2 ) was cultured for about 24 hours and placed in a serum-starved state.
- IC 50 was calculated by setting the absorbance when GH was not added and anti-GH antibody was not added as 100% inhibition, and the absorbance when GH was added and anti-GH antibody was not added as 0% inhibition.
- the IC50 of the positive control antibody MAB1067 under the above conditions was 21,500 pmol/L.
- Example 3 Cross-reactivity of anti-GH mouse mAb to experimental animal-derived GH> Cross-reactivity of anti-GH mouse mAb to monkey-, mouse-, and rat-derived GH was determined by competitive ELISA. A specific method is described below.
- a mixture of anti-GH mouse mAb diluted to ng/mL was added at 50 ⁇ L/well and allowed to stand at room temperature for 2 hours. After washing the plate with a washing buffer (0.05% Tween20-containing PBS), HRP-labeled anti-mouse IgG goat antibody (Jackson ImmunoResearch Laboratories, Inc) diluted to 0.1 ⁇ g/mL in assay buffer was added at 50 ⁇ L/well. It was allowed to stand for 2 hours. After washing the plate with a washing buffer, 50 ⁇ L/well of TMB solution was added as an HRP substrate, and enzymatic reaction was allowed to occur at room temperature.
- a washing buffer 0.05% Tween20-containing PBS
- HRP-labeled anti-mouse IgG goat antibody Jackson ImmunoResearch Laboratories, Inc
- IC 20 (nmol/L) of the anti-GH mouse mAb was obtained from the concentration-dependent curves of human GH and experimental animal-derived GH, and cross-reactivity to each experimental animal-derived GH was calculated by the following formula 1.
- Example 4 Cross-reactivity test of anti-GH mouse mAb against GH-like proteins (proteins having structural similarity to GH)> Cross-reactivity of anti-GH mouse mAb to proteins having structural similarity to GH (placental lactogen (PL), prolactin (PRL)) was measured by competitive ELISA. A specific method is described below.
- IC 20 (nmol/L) of the anti-GH mouse mAb was determined from the concentration-dependent curves of human GH and GH-like proteins, and cross-reactivity to GH-like proteins was calculated by the following formula 2.
- Cross-reactivity to GH-like proteins (%) IC20 (human GH)/ IC20 (GH-like proteins) x 100 ... (formula 2)
- Example 5 Preparation of anti-GH mouse mAb> (1) Preparation of anti-GH mouse mAb Female mice (7 weeks old) were given 5 ⁇ g or 10 ⁇ g of recombinant human GH (Fujifilm Wako Pure Chemical Industries, Ltd., Pharmaceuticals and Medical Devices Regulatory Science Foundation) twice a week with various adjuvants. , subcutaneously immunized 7-13 times. Blood was collected from these mice over time, and plasma antibody titers were measured using the ELISA described in Example 1.
- mice with sufficiently elevated plasma antibody titers were immunized intraperitoneally or tail vein with 10 ⁇ g of recombinant human GH for the final immunization, euthanized 3 to 4 days later, and lymph nodes and spleens were removed. Electrofusion of isolated lymphocytes and mouse myeloma cell line P3U1 (JCRB0708, National Institutes of Biomedical Innovation, Health and Nutrition) to create a semi-solid selection medium containing hypoxanthine-aminopterin-thymidine (HAT) (STEMCELL Technologies) in a CO 2 incubator for 8-12 days.
- HAT hypoxanthine-aminopterin-thymidine
- Proliferated monoclonal hybridomas were picked by a colony picker, transferred to 96-well culture plates containing hypoxanthine-thymidine (HT)-containing liquid medium (STEMCELL Technologies), and cultured in a CO 2 incubator for 3-4 days.
- HT hypoxanthine-thymidine
- a 10-fold dilution of the hybridoma culture supernatant thus obtained was subjected to ELISA described in Example 1 to measure GH binding activity.
- a two-fold dilution of the culture supernatant was also subjected to a GH-GHR binding inhibition assay according to the method described in Example 2(a) to measure GH neutralizing activity.
- Anti-GH mouse mAb-producing hybridomas that showed an inhibitory activity of 70% or more in the GH-GHR binding inhibition assay were expanded in a 24-well plate, and the culture supernatant reproduced GH binding activity and GH-GHR binding inhibitory activity. Those that produced a single IgG subtype were selected as neutralizing mAb-producing hybridomas and cryopreserved.
- the anti-GH mouse mAb-producing hybridomas selected above were expanded and cultured in DMEM medium containing 10% Ultra Law IgG, the mAb was purified from the culture supernatant using rProtein A Sepharose FF (GE Healthcare), and the anti-GH mouse mAb was obtained. Obtained. Also, the purity was confirmed by SDS-PAGE, and the antibody concentration was quantified based on the absorbance at 280 nm with a molecular extinction coefficient (1 mg/mL) of 1.38.
- Mouse mAb preparations thus prepared were tested in the GH-GHR binding inhibition assay described in Example 2, the GH-dependent Nb2 cell proliferation inhibition assay, cross-reactivity to experimental animal-derived GH described in Example 3, Cross-reactivity to the GH-like protein described in Example 4 was examined, and an anti-GH mouse mAb, 13H02 (IgG2a/ ⁇ ), which was comprehensively superior in terms of GH-binding properties and GH-neutralizing activity, was selected.
- Example 6 Efficacy of anti-GH mouse mAb (13H02) in hypophysectomized/GH supplemented rats> The efficacy of the anti-GH mouse mAb (13H02) selected in Example 5 was evaluated in hypophysectomized/GH supplemented rats simulating acromegaly.
- hypophysectomized SD rats Japan SLC, 6 weeks old exhibiting a decrease in serum IGF-I due to endogenous GH deficiency (hereinafter also referred to as "hypophysectomized rats")
- a 2-week type Alzet osmotic pump (Muromachi Kikai Co., Ltd.) containing recombinant human GH (Sando Co., Ltd., 30 ⁇ g/day) was subcutaneously implanted.
- the normal control group was hypophysectomized/GH non-supplemented rats (recombinant human GH non-administered hypophysectomized rats), and the negative control group was hypophysectomized/GH supplemented rats with control mouse IgG1 (Leinco Technologies). , Inc., M1411). Serum IGF-1 levels two days after administration of 13H02 are shown in FIG. 1 (mean and standard error). 13H02 significantly decreased serum IGF-I levels in the 1, 5 and 10 mg/kg administration groups (###: p ⁇ 0.001 vs normal control/Aspin-Welch t-test, *: p ⁇ 0.05 versus disease control group/Steel's multiple comparison test).
- Example 7 Determination of the variable region amino acid sequence of 13H02> Using the Dynabeads mRNA DIRECT Kit (ThermoFischer Scientific), mRNA was prepared from the anti-GH mouse mAb (13H02)-producing hybridoma selected in Example 5 according to the manual attached to the kit. Using 5 ⁇ L of the above mRNA solution as a template, cDNA was synthesized using the SMARTer RACE 5'/3' Kit (Takara Bio Inc.), 10 ⁇ Universal Primer A Mix attached to the kit was used as a forward primer, and a known mouse antibody gene was synthesized.
- VH, VL Antibody H chain and L chain variable region genes (VH, VL) were amplified by PCR using a 5'-RACE reverse primer designed from the constant region sequence as a reverse primer and KOD-Fx Neo (TOYOBO) as a polymerase.
- the DNA sequence and amino acid sequence of the H chain variable region (VH) of 13H02 are shown in SEQ ID NO: 1 and SEQ ID NO: 19, respectively, and the DNA sequence and amino acid sequence of the L chain variable region (VL) are shown in SEQ ID NO: 2 and SEQ ID NO: 20, respectively.
- the amino acid sequence of mature 13H02_VH from which the signal peptide starting at position 20 of SEQ ID NO: 19 has been removed is shown in SEQ ID NO: 3
- the amino acid sequence of mature 13H02_VL from which the signal peptide starting from position 23 of SEQ ID NO: 20 has been removed is arranged. Shown in number 4.
- Complementarity determining regions (CDRs) 1, 2, 3 of 13H02 VH were SSEIS (SEQ ID NO: 5), WIYPGTGSSKFNQKFTG (SEQ ID NO: 6), RGFFGGSFDY (SEQ ID NO: 7), respectively.
- CDRs 1, 2 and 3 of 13H02 VL were TATSVSSSYLH (SEQ ID NO: 8), STSNLAS (SEQ ID NO: 9) and HQYHHSPPT (SEQ ID NO: 10), respectively.
- Example 8 Preparation of anti-GH mouse-human chimeric mAb>
- the H and L chain constant region genes of human IgG1/ ⁇ antibody were introduced into pcDNA3.4 (Thermo Fisher Scientific) to create mouse-human chimeric antibody H and L chain expression cassette vectors (hereafter referred to as human IgG1/ ⁇ antibody
- a mouse-human chimeric antibody H chain and a mouse-human chimeric antibody L chain expression cassette vector containing the constant region gene are also referred to as "human IgG1/ ⁇ mouse-human chimeric antibody (H chain, L chain) cassette vectors").
- the culture supernatant was collected by centrifugation, and the antibody was purified using rProtein A Sepharose FF (GE Healthcare) to obtain a recombinant anti-GH mouse-human chimeric mAb (Ch-13H02).
- the protein purity was confirmed by SDS-PAGE, and the antibody concentration was quantified based on the absorbance at 280 nm with a molecular extinction coefficient (1 mg/mL) of 1.38.
- Example 9 GH-dependent Nb2 proliferation inhibitory activity of anti-GH mouse-human chimeric mAb (Ch-13H02)>
- the GH neutralizing activity of the recombinant anti-GH mouse-human chimeric mAb (Ch-13H02) prepared in Example 8 was measured in the GH-dependent Nb2 cell growth inhibition assay described in Example 2(b). Table 4 shows the results.
- Example 10 Humanization of anti-GH mouse mAb (13H02)> The humanized design of VH and VL of 13H02 was reported by Queen C. et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033, 1989) and Tsurushita N. et al. 2005). Briefly, it is as follows. First, we constructed a three-dimensional molecular model of VH and VL of 13H02 using JN Biosciences' unique algorithm, and identified amino acid residues important for maintaining the CDR structure within the framework region. In addition, human VH and VL amino acid sequences that are most homologous to the VH and VL of this antibody were identified from public databases, respectively.
- Example 11 Preparation of anti-GH humanized antibody> DNA (SEQ ID NO: 15) encoding the VH amino acid sequence (SEQ ID NO: 11) and DNA (SEQ ID NO: 13) encoding the VL amino acid sequence (SEQ ID NO: 13) of the anti-GH humanized antibody (Hu-13H02) designed in Example 10 16) was inserted into the human IgG1/ ⁇ mouse-human chimeric antibody (H chain, L chain) cassette vector constructed in Example 8 to construct a recombinant human antibody (H chain, L chain) expression vector (hereinafter , also referred to as "(H chain, L chain) expression vector for Hu-13H02").
- an Fc variant (Hu-13H02m) was prepared by substituting a Leu residue for the H chain Met 428 of Hu-13H02 for the purpose of improving blood kinetics.
- the H chain expression cassette vector in which the gene encoding the human IgG1 constant region in which the H chain Met 428 is replaced with Leu 428 was introduced into pcDNA3.4 was added to the VH of the anti-GH humanized antibody (Hu-13H02).
- a vector into which a DNA encoding the amino acid sequence was inserted was prepared (hereinafter also referred to as "Hu-13H02m (H chain, L chain) expression vector").
- the endotoxin content of the preparation used for the rat drug efficacy test was measured using Endospecie ES-50M (Seikagaku Corporation), and it was confirmed to be ⁇ 1 EU/mg.
- the Hu-13H02 or Hu-13H02m preparations prepared in this way were used in subsequent tests.
- the amino acid sequence of the heavy chain constant region of Hu-13H02m is shown in SEQ ID NO:17
- the amino acid sequence of the light chain constant region of Hu-13H02m is shown in SEQ ID NO:18.
- Example 12 GH-dependent Nb2 cell proliferation inhibitory activity of anti-GH chimeric antibody and anti-GH humanized antibody> Regarding the recombinant anti-GH humanized antibody (Hu-13H02) prepared in Example 11, the Fc variant of the anti-GH humanized antibody (Hu-13H02m), and the chimeric antibody (Ch-13H02) prepared in Example 8, The GH-dependent Nb2 cell growth inhibition assay described in Example 2(b) was performed and the IC50 against human GH was determined from the concentration-dependent curve. Also, the monkey GH prepared in Example 3 was used in place of the human GH in Example 2(b), and the IC 50 against monkey GH was determined. Human GH was added at a final concentration of 9.1 pmol/L, and monkey GH was added at a final concentration of 41 pmol/L. Table 5 shows the results.
- Hu-13H02 and Hu-13H02m showed strong GH-dependent Nb2 cell growth inhibitory activity against human and monkey GH, similar to Ch-13H02.
- Example 13 Binding characteristics of anti-GH humanized antibody to human GH and monkey GH> Recombinant anti-GH humanized antibody (Hu-13H02) prepared in Example 11 and its Fc variant (Hu-13H02m), chimeric antibody (Ch-13H02) prepared in Example 8 human GH (Regular Tri-Science Foundation), binding properties to monkey GH (Example 3) were measured by SPR using BIACORE 8K+ (GE Healthcare).
- An anti-human IgG (Fc) antibody was immobilized on a CM5 sensor chip, and antibody preparations (1 ⁇ g/mL) of these three types (Hu-13H02, Hu-13H02m, Ch-13H02) were captured. Next, 0.13 to 8.0 nmol/L of human GH or 0.50 to 32 nmol/L of monkey GH was flowed, and binding and dissociation parameters were calculated. Table 6 shows the results.
- Hu-13H02 and Hu-13H02m retained almost the same human GH-binding activity as Ch-13H02, and also showed strong binding activity to monkey GH.
- Example 14 Cross-reactivity of anti-GH chimeric antibody and anti-GH humanized antibody to GH-like proteins> Structural similarity of the recombinant anti-GH humanized antibody (Hu-13H02) and its Fc variant (Hu-13H02m) prepared in Example 11 and the parental chimeric antibody (Ch-13H02) prepared in Example 8 to GH Cross-reactivity to proteins (placental lactogen (PL), prolactin (PRL)) showing For each anti-GH antibody, IC 50 (nmol/L) was determined from the concentration-dependent curves of human GH and GH-like proteins, and cross-reactivity to GH-like proteins was calculated by Equation 3 below.
- Cross-reactivity to GH-like protein (%) IC 50 (human GH)/IC 50 (GH-like protein) ⁇ 100 (Formula 3) Table 7 shows the results.
- Ch-13H02, Hu-13H02, and Hu-13H02m did not substantially bind to these GH-like proteins, with ⁇ 1% cross-reactivity to PL and PRL, similar to parental mouse antibody 13H02.
- Example 15 Efficacy of anti-GH mouse-human chimeric mAb (Ch-13H02) and anti-GH humanized antibody Fc variant (Hu-13H02m) in hypophysectomized/GH supplemented rats> The efficacy of anti-GH mouse-human chimeric mAb (Ch-13H02) and anti-GH humanized antibody Fc variant (Hu-13H02m) was evaluated in hypophysectomized/GH supplemented rats as described in Example 6.
- hypophysectomized SD rats (Japan SLC, 6 weeks old), whose hypophysectomy was performed at 4 weeks of age and showed a decrease in serum IGF-I due to endogenous GH deficiency, were subcutaneously injected with recombinant human GH (Sand Co., Ltd.).
- a 3-day type Alzet osmotic pump (Muromachi Kikai Co., Ltd.) was implanted.
- the normal control group was hypophysectomized/GH non-supplemented rats (recombinant human GH non-administered hypophysectomized rats), and the negative control group was hypophysectomized/GH supplemented rats with control human IgG1 (Bio X Cell , Inc., BE0297).
- Serum IGF-1 levels 2 days after administration of Ch-13H02 are shown in FIG. 2, and serum IGF-1 levels 2 days after administration of Hu-13H02m are shown in FIG. 3 (average and standard error).
- Ch-13H02 significantly decreased serum IGF-I levels in the 3 and 10 mg/kg dose groups, and Hu-13H02m in the 3 and 10 mg/kg dose groups (###: p ⁇ 0.001 vs. normal controls).
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Abstract
Description
先端巨大症や巨人症の治療法としては、GHを過剰分泌する下垂体腺腫の摘出手術や放射線療法による退縮、そして薬物治療が施されている。下垂体腺腫摘出手術はその他の外科的手術と同様に死亡を含む合併症のリスクを伴い、高度な技術を要する。また放射線療法にも同様なリスクが伴い、効力を発揮するには数年を要することがある。現在、市場で使用できる治療薬としてはソマトスタチン類似体(オクトレオチド酢酸塩徐放性製剤、ランレオチド酢酸塩徐放性製剤など)、ドーパミン作動薬(メシル酸ブロモクリプチン)、GH受容体拮抗薬(ペグビソマント)があるが、有効性、安全性、利便性を同時に満たす薬剤はいまだ存在しない。
(1)ヒト成長ホルモンに特異的に結合する抗体又はその抗原結合断片であって、重鎖可変領域(VH)及び軽鎖可変領域(VL)を含み、
VHが、VH相補性決定領域(CDR)1(VHCDR1)、VHCDR2、及びVHCDR3を含み、
VHCDR1が、配列番号5のアミノ酸配列、又は配列番号5のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VHCDR2が、配列番号6のアミノ酸配列、又は配列番号6のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VHCDR3が、配列番号7のアミノ酸配列、又は配列番号7のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLがVL相補性決定領域(CDR)1(VLCDR1)、VLCDR2、及びVLCDR3を含み、
VLCDR1が、配列番号8のアミノ酸配列、又は配列番号8のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLCDR2が、配列番号9のアミノ酸配列、又は配列番号9のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLCDR3が、配列番号10のアミノ酸配列、又は配列番号10のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む、前記抗体又はその抗原結合断片。
(2)ヒト成長ホルモンに特異的に結合する抗体又はその抗原結合断片であって、重鎖可変領域(VH)及び軽鎖可変領域(VL)を含み、
VHがVH相補性決定領域(CDR)1(VHCDR1)として配列番号5のアミノ酸配列、VHCDR2として配列番号6のアミノ酸配列、及びVHCDR3として配列番号7のアミノ酸配列を含み、
VLがVL相補性決定領域(CDR)1(VLCDR1)として配列番号8のアミノ酸配列、VLCDR2として配列番号9のアミノ酸配列、VLCDR3として配列番号10のアミノ酸配列を含む、前記抗体又はその抗原結合断片。
(3)表面プラズモン共鳴法によって測定した場合に、1.0×10-8 mol/L以下の平衡解離定数(KD)でヒト成長ホルモンに結合する、(1)又は(2)に記載の抗体又はその抗原結合断片。
(4)成長ホルモン作用を中和する、(1)~(3)のいずれかに記載の抗体又はその抗原結合断片。
(5)配列番号3、11、12、又は19のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号3、11、12、又は19のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含むVH、及び/又は
配列番号4、13、14、又は20のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号4、13、14、又は20のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含むVL、
を含む、(1)~(4)のいずれかに記載の抗体又はその抗原結合断片。
(6)配列番号3、11、12、又は19のアミノ酸配列を有するVH、及び/又は配列番号4、13、14、又は20のアミノ酸配列を有するVLを含む、(5)に記載の抗体又はその抗原結合断片。
(7)抗体がモノクローナル抗体である、(1)~(6)のいずれかに記載の抗体又はその抗原結合断片。
(8)抗体がキメラ抗体、ヒト化抗体、ベニア化抗体又は完全ヒト抗体である、(1)~(7)のいずれかに記載の抗体又はその抗原結合断片。
(9)抗体が、配列番号17のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む重鎖定常領域、又は配列番号17のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含む重鎖定常領域、及び/又は
配列番号18のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む軽鎖定常領域、又は配列番号18のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含む軽鎖定常領域、
を含む、(1)~(8)のいずれかに記載の抗体又はその抗原結合断片。
(10)抗体が、配列番号17のアミノ酸配列を有する重鎖定常領域、及び/又は配列番号18のアミノ酸配列を有する軽鎖定常領域を含む、(1)~(9)のいずれかに記載の抗体又はその抗原結合断片。
(11)抗原結合断片が、Fab、Fab’、F(ab’)2、Fv、又はscFvである、(1)~(10)のいずれかに記載の抗体又はその抗原結合断片。
(12)(1)~(11)のいずれかに記載の抗体又はその抗原結合断片をコードする核酸。
(13)(12)に記載の核酸を含むベクター。
(14)(12)に記載の核酸若しくは請求項(13)に記載のベクターを含む細胞。
(15)(1)~(11)のいずれかに記載の抗体又はその抗原結合断片を含む医薬。
(16)血液中のインスリン様成長因子1(IGF-1)のレベルを低減させるための、(15)に記載の医薬。
(17)先端巨大症、巨人症、がん、糖尿病性腎症、関節炎、及び肺炎症からなる群から選択される疾患を治療及び/又は予防するための、(15)又は(16)に記載の医薬。
(18)血液中のインスリン様成長因子1(IGF-1)のレベルを低減させるための方法であって、治療有効量の(1)~(11)のいずれかに記載の抗体又はその抗原結合断片を投与するステップを含む方法。
一実施形態において、本発明は、ヒト成長ホルモン(growth hormone、GH)に特異的に結合する抗体又はその抗原結合断片(以下、「抗GH抗体」とも言う)を提供する。
配列番号3、11、12、又は19のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号3、11、12、又は19のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含み、CDR1~CDR3の領域は配列番号5~7からそれぞれなるVH、及び/又は
配列番号4、13、14、又は20のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号4、13、14、又は20のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含み、CDR1~CDR3の領域は配列番号8~10からそれぞれなるVLを含む。
配列番号3、11、12、又は19のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号3、11、12、又は19のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含むVH、及び/又は
配列番号4、13、14、又は20のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号4、13、14、又は20のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含むVLを含む。
配列番号17のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む重鎖定常領域、又は配列番号17のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含む重鎖定常領域、及び/又は
配列番号18のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む軽鎖定常領域、又は配列番号18のアミノ酸配列に対して90%以上、91%以上、92%以上、93%以上、94%以上、95%以上、96%以上、97%以上、98%以上、又は99%以上の同一性を有するアミノ酸配列を含む軽鎖定常領域を含む。ここで、配列番号17のアミノ酸配列においては、C末端のリジン残基が存在しても存在しなくてもよい。
2.本明細書に記載の抗体の作製
本明細書に記載の抗体は、例えば、動物への抗原免疫によって得られたB細胞からハイブリドーマ法、各種ディスプレー法、1細胞スクリーニング法等の当該技術分野で公知の多種多様な方法により選択取得することができる。また抗原免疫動物にヒト抗体遺伝子を組み込んだ遺伝子改変動物を用いたり、ヒト抗体遺伝子プールから構築された抗体ディスプレーライブラリーを用いたりすることにより、完全ヒトモノクローナル抗体を作製することができる。
一実施形態において、本発明は、本明細書に記載の抗体又は抗原結合断片を製造する方法を提供する。
4.医薬及び方法
一実施形態において、本発明は、本明細書に記載の抗体又は抗原結合断片を含む医薬に関する。
96ハーフウェルイムノプレート(Corning)にダルベッコリン酸緩衝液(PBS)で5μg/mLに希釈した組換えヒトGH(医薬品医療機器レギュラトリーサイエンス財団)を50μL/ウェルで固相化し、1 %ウシ血清アルブミン(BSA)含有PBSでブロッキングした。その後、アッセイバッファー(0.2% BSA、0.05% Tween 20含有PBS)で希釈した陽性コントロール(抗GHマウスmAb(MAB1067、R&D Systems))又は抗GHマウスmAb含有サンプルを50μL/ウェルずつ添加し、室温で2時間静置した。このプレートを洗浄バッファー(0.05% Tween 20含有PBS)で洗浄後、アッセイバッファーで0.1μg/mLに希釈したHRP(Horseradish peroxidase(ホースラディッシュ・ペルオキシダーゼ))標識抗マウスIgGヤギ抗体(Jackson ImmunoResearch Laboratories, Inc.)を50μL/wellずつ添加し、室温で2時間静置した。さらにプレートを洗浄バッファーで洗浄した後、HRPの基質として3,3’,5,5’-テトラメチルベンジジン(TMB)溶液を50μL/wellずつ添加し、室温で10分間反応させた。0.5 mol/L H2SO4水溶液を加えて反応を停止させた後、プレートリーダーを用いて450nmの吸光度を測定した。
抗GH抗体のGH中和活性評価を目的として、(a)GH-GH受容体(GHR)結合阻害アッセイ及び(b)GH依存的Nb2細胞増殖阻害アッセイを確立した。
GH-GHR結合阻害活性は、ELISAプレートに抗ヒトFcγマウス抗体を介して固定化したGHR細胞外領域タンパク-ヒトFc融合体(GHR-hFc)に対するビオチン標識GHの結合シグナルの阻害率を算出することにより測定した。ビオチン標識GHは、組換えGHをEZ-Link Micro NHS-PEG4-Biotinylation kit(Thermo Fisher Scientific)を用いて標識して調製した。具体的な方法は以下の通りである。96ハーフウェルイムノプレートに1μg/mL PBS に希釈した抗ヒトFcγマウス抗体(Jackson ImmunoResearch Laboratories, Inc.)を50μL/ウェルで固相化し、1 % BSA含有PBSでブロッキングした。その後、アッセイバッファー(0.2% BSA、0.05% Tween 20含有PBS)で0.1μg/mLに希釈したGHR-hFc(R&D Systems)を50μL/ウェルずつ加えてプレートに捕捉した。このプレートを洗浄バッファー(0.05% Tween 20含有PBS)で洗浄後、アッセイバッファーで終濃度5.5 ng/mLに希釈したビオチン標識GHと、アッセイバッファーで希釈した検体(抗GHマウスmAb含有サンプル)とを等容量混合したものを50μL/ウェルずつ添加し、室温2時間静置した。なお、陽性コントロールとして、検体の代わりに非標識GH、又は抗GHマウスmAb(MAB1067)を用いた。このプレートを洗浄バッファーで4回洗浄後、アッセイバッファーで0.1μg/mLに希釈したHRP標識ストレプトアビジン(Jackson ImmunoResearch Laboratories, Inc.)を50μL/ウェルずつ添加し、室温2時間静置した。さらにプレートを6回洗浄後、HRPの基質としてTMB溶液を50μL/ウェルずつ添加し、室温10分間反応させた。0.5 mol/L H2SO4水溶液を加えて反応を停止させた後、プレートリーダーを用いて450nmの吸光度を測定した。ビオチン標識GHのみ添加時の吸光度を0 %阻害、ビオチン標識GH非添加時の吸光度を100 %阻害として各サンプルのGH-GHR結合阻害率を換算し、IC50を算出した。上記条件における陽性コントロール抗体MAB1067のIC50は6,860 pmol/Lであった。
セルベースのGH中和活性測定法としては、標準的なGH活性測定法として知られているNb2ラットリンパ腫細胞株増殖アッセイを用いた(Omae Y. et al., Endocrinol Japon 36, 9-13, 1989)。具体的な方法は以下の通りである。10% 牛胎児血清及び10% ウマ血清(HS)含有Fischer’s Mediumに馴化させたNb2-11細胞株(EC90741101、ECACC)を、1% HS含有Fischer’s Medium中、CO2インキュベーター(37℃、5% CO2)で約24時間培養し、血清飢餓状態に置いた。この細胞を2×104 細胞/ウェルで96 ウェル培養プレートに播種し、0.1 % BSA含有PBSで終濃度9.1 pmol/L(EC80:80%効果濃度)となるよう希釈したヒトGH(医薬品医療機器レギュラトリーサイエンス財団)、及び抗GH mAb検体(5 nmol/L)を同bufferで50~7倍希釈したものをそれぞれ10μL/ウェルずつ添加した。なお、陽性コントロールとして、検体の代わりに抗GHマウスmAb(MAB1067)を用いた。これらの細胞をCO2インキュベーター中で約72時間培養後、WST-8 (Cell counting kit-8、株式会社同仁化学研究所)を10μL/ウェルずつ添加し37℃で約3時間反応させた後、プレートリーダーを用いて450nmの吸光度を測定し、生細胞数の指標とした。GH非添加かつ抗GH抗体非添加時の吸光度を100%阻害、GH添加かつ抗GH抗体非添加時の吸光度を0%阻害とし、IC50を算出した。上記条件における陽性コントロール抗体MAB1067のIC50は21,500 pmol/Lであった。
抗GHマウスmAbのサル、マウス、ラット由来GHに対する交差反応性を競合的ELISAにより測定した。具体的な方法を以下に記す。
カニクイザル脳下垂体cDNAよりクローニングしたサルGHのDNA配列(NM_001290284.1)をpIEx-4昆虫細胞発現ベクター(Merck Millipore)に挿入した。当該ベクターをHighFive細胞株(Invitrogen)に遺伝子導入し、28℃で72時間旋回培養後、遠心分離により培養上清を回収した。この培養上清を150 mmol/L NaCl含有20 mmol/L Tris-塩酸緩衝液(pH 8.0)で平衡化したCaptureSelect hGH Affinity Matrix(Thermo Fisher Scientific)に供し、500 mmol/L NaClを含む同緩衝液で洗浄した後、150 mmol/L NaCl含有20 mmol/L クエン酸緩衝液(pH 3.0)で担体に吸着したサルGHを溶出した。溶出液を、150 mmol/L NaCl、10% グリセロール含有20 mmol/L Tris-塩酸緩衝液(pH 8.0)に対し透析したものを最終標品(サルGH)とした。サルGHのSDS-PAGE及びSuperdex 75 Increase 10/300 GL(GE Healthcare)を用いたサイズ排除クロマトグラフィーによる純度は>85%であり、実施例2(b)に記載のNb2細胞株増殖アッセイにおいてEC50 = 9.8 pmol/Lの活性を示した。
96ハーフウェルイムノプレート(Corning)にPBSで5 μg/mLに希釈した組換えヒトGH(医薬品医療機器レギュラトリーサイエンス財団)を50 μL/ウェルで固相化し、1 % BSA含有PBSでブロッキングした。その後、アッセイバッファー(0.2% BSA, 0.05% Tween20含有PBS)で段階希釈したヒトGH、上記で調製したカニクイザルGH、マウスGH(LifeSpan BioSciences)、ラットGH(Protein Laboratories Rehovot)と同バッファーで終濃度30 ng/mLに希釈した抗GHマウスmAbを混合したものを50μL/ウェルずつ添加し、室温で2時間静置した。このプレートを洗浄バッファー(0.05% Tween20含有PBS)で洗浄後、アッセイバッファーで0.1μg/mLに希釈したHRP標識抗マウスIgGヤギ抗体(Jackson ImmunoResearch Laboratories, Inc)を50μL/ウェルずつ添加し、室温で2時間静置した。さらにプレートを洗浄バッファーで洗浄後、HRP基質としてTMB溶液を50μL/ウェルずつ添加し、室温で酵素反応させた。10分後、0.5 mol/L H2SO4を加えて反応を停止させ、プレートリーダーを用いて450nmの吸光度を測定した。抗GHマウスmAbについてヒトGH及び実験動物由来GHの濃度依存曲線よりIC20 (nmol/L)を求め、各実験動物由来GHに対する交差反応性を下記の式1により算出した。
・・・(式1)
抗GHマウスmAbの、GHと構造類似性を有するタンパク質(placental lactogen (PL)、prolactin (PRL))に対する交差反応性を競合的ELISAにより測定した。具体的な方法を以下に記す。
GH類似タンパクへの交差反応性(%) = IC20(ヒトGH)/IC20(GH類似タンパク)×100
・・・(式2)
(1)抗GHマウスmAbの作製
雌性マウス(7週齢)に、各種アジュバントと共に5μg又は10μgの組換えヒトGH(富士フイルム和光純薬株式会社、医薬品医療機器レギュラトリーサイエンス財団)を週2回、7~13回繰り返し皮下免疫した。これらのマウスから経時的に採血を行い、実施例1に記載のELISAを用いて血漿抗体価を測定した。血漿抗体価が十分に上昇したマウスの尾静脈内又は腹腔内に10μgの組換えヒトGHを最終免疫し、3~4日後に安楽死させて、リンパ節及び脾臓を摘出した。単離したリンパ球とマウスミエローマ細胞株P3U1(JCRB0708、国立研究開発法人 医薬基盤・健康・栄養研究所)とを電気融合し、ヒポキサンチン-アミノプテリン-チミジン(HAT)を含有するセミソリッド選択培地(STEMCELL Technologies)中、CO2インキュベーターで8~12日間培養した。増殖したモノクローナルなハイブリドーマをコロニーピッカーによりピッキングし、ヒポキサンチン-チミジン(HT)を含有する液体培地(STEMCELL Technologies)の入った96ウェル培養プレートに移植してCO2インキュベーターで3~4日間培養した。
前記で選択取得した抗GHマウスmAb産生ハイブリドーマを10 % Ultra Law IgG含有DMEM培地で拡大培養し、培養上清からrProtein A Sepharose FF(GE Healthcare)を用いてmAbを精製し、抗GHマウスmAbを得た。また、SDS-PAGEにより純度を確認し、280 nmの吸光度より分子吸光係数(1 mg/mL)=1.38として抗体濃度を定量した。
抗GHマウスmAb(13H02)について、実施例2の方法で測定したGH-GHR結合阻害活性(IC50)及びGH依存的Nb2細胞増殖阻害活性(IC50)を表1に、実施例3の方法で測定した実験動物由来GHに対する交差反応性を表2に、実施例4の方法で測定したGH類似タンパクに対する交差反応性を表3に示す。なお、陽性コントロールとして市販の抗GHマウスmAb(MAB1067)を用いた場合は、その結果も示す。
実施例5で選抜取得した抗GHマウスmAb(13H02)の薬効を、先端巨大症を模した下垂体摘除/GH補充ラットにおいて評価した。
実施例5で選抜した抗GHマウスmAb(13H02)の産生ハイブリドーマからDynabeads mRNA DIRECT Kit(ThermoFischer Scientific)を用い、キットに添付のマニュアルに従いmRNAを調製した。前記mRNA溶液5μLを鋳型として、SMARTer RACE 5'/3' Kit(タカラバイオ株式会社)を用いてcDNAを合成し、同キット付属の10× Universal Primer A Mixをフォワードプライマー、既知のマウス抗体遺伝子の定常領域配列から設計した5’-RACE Reverse primerをリバースプライマーとして、KOD-Fx Neo(TOYOBO)をポリメラーゼに用いたPCRにより抗体H鎖、L鎖の可変領域遺伝子(VH、VL)を増幅した。
ヒトIgG1/κ抗体のH鎖、L鎖定常領域遺伝子をpcDNA3.4(Thermo Fisher Scientific)に導入してマウス-ヒトキメラ抗体H鎖、L鎖発現カセットベクターを作製した(以下、ヒトIgG1/κ抗体の定常領域遺伝子を含むマウス-ヒトキメラ抗体H鎖、及びマウス-ヒトキメラ抗体L鎖発現カセットベクターを「ヒトIgG1/κマウス-ヒトキメラ抗体(H鎖、L鎖)カセットベクター」とも言う)。実施例7で単離した13H02のVH、VL遺伝子を前記ヒトIgG1/κマウス-ヒトキメラ抗体(H鎖、L鎖)カセットベクターに挿入し、組換えマウス-ヒトキメラ抗体(H鎖、L鎖)発現ベクターをそれぞれ構築した。組換えマウス-ヒトキメラ抗体(H鎖、L鎖)発現ベクターをExpiFectamine CHO Transfection Kit(Thermo Fisher Scientific)を用いてExpiCHO-S細胞株にH鎖:L鎖=1:1(重量比)で共発現させ、CO2インキュベーターシェーカー(37℃、8%CO2)中で8日間振盪培養した。培養上清を遠心分離により回収し、rProtein A Sepharose FF(GE Healthcare)を用いて抗体を精製し、組換え抗GHマウス-ヒトキメラmAb(Ch-13H02)を得た。なお、SDS-PAGEによりタンパク純度を確認し、280 nmの吸光度より分子吸光係数(1 mg/mL)=1.38として抗体濃度を定量した。
実施例8で調製した組換え抗GHマウス-ヒトキメラmAb(Ch-13H02)のGH中和活性を、実施例2(b)に記載のGH依存的Nb2細胞増殖阻害アッセイにおいて測定した。結果を表4に示す。
13H02のVH及びVLのヒト化デザインは、Queen C.らの報告(Proc. Natl. Acad. Sci. USA 86, 10029-10033, 1989)及びTsurushita N. らの方法(Methods 36, 69-83, 2005)に従って行った。簡潔に述べると以下の通りである。まず初めに、JN Biosciences社独自のアルゴリズムを用いて13H02のVH、VLの3次元分子モデルを構築し、フレームワーク領域内でCDR構造維持に重要なアミノ酸残基を特定した。また、本抗体のVH、VLと最も相同性の高いヒトVH、VLアミノ酸配列を公共データベースからそれぞれ特定した。そして、13H02のVH、VLそれぞれ3つのCDR配列をフレームワーク領域内でCDR構造維持に重要なアミノ酸残基と一緒に、上記で特定したヒト抗体フレームワーク領域配列に移植し13H02をヒト化デザインした(Hu-13H02)。
ヒト化デザインしたHu-13H02_VHのアミノ酸配列を配列番号11に、配列番号11のポジション20から始まるシグナルペプチドが除去された成熟型Hu-13H02_VHのアミノ酸配列を配列番号12に示す。またHu-13H02_VLのアミノ酸配列を配列番号13に、配列番号13のポジション23から始まるシグナルペプチドが除去された成熟型Hu-13H02_VLのアミノ酸配列を配列番号14に示す。
実施例10でデザインした抗GHヒト化抗体(Hu-13H02)のVHアミノ酸配列(配列番号11)をコードするDNA(配列番号15)及びVLアミノ酸配列(配列番号13)をコードするDNA(配列番号16)を、実施例8で構築したヒトIgG1/κマウス-ヒトキメラ抗体(H鎖、L鎖)カセットベクターに挿入し、組換えヒト抗体(H鎖、L鎖)発現ベクターをそれぞれ構築した(以下、「Hu-13H02用(H鎖、L鎖)発現ベクター」とも言う)。
また、血中動態改善を目的としてHu-13H02のH鎖Met428をLeu残基に置換したFc改変体(Hu-13H02m)を調製した。具体的には、H鎖のMet428をLeu428に置換したヒトIgG1定常領域をコードする遺伝子をpcDNA3.4に導入したH鎖発現カセットベクターに、抗GHヒト化抗体(Hu-13H02)のVHアミノ酸配列をコードするDNAを挿入したものを作製した(以下、「Hu-13H02m用(H鎖、L鎖)発現ベクター」とも言う)。
実施例11で調製した組換え抗GHヒト化抗体(Hu-13H02)、抗GHヒト化抗体のFc改変体(Hu-13H02m)、及び実施例8で調製したキメラ抗体(Ch-13H02)について、実施例2(b)に記載のGH依存的Nb2細胞増殖阻害アッセイを実施し、濃度依存性曲線からヒトGHに対するIC50を求めた。また、実施例2(b)におけるヒトGHの代わりに実施例3で作製したサルGHを用いて、サルGHに対するIC50を求めた。ヒトGHは終濃度として9.1 pmol/L 、サルGHは終濃度として41 pmol/Lを添加した。結果を表5に示す。
実施例11で調製した組換え抗GHヒト化抗体(Hu-13H02)及びそのFc改変体(Hu-13H02m)、実施例8で調製したキメラ抗体(Ch-13H02)のヒトGH(医薬品医療機器レギュラトリーサイエンス財団)、サルGH(実施例3)に対する結合特性をBIACORE 8K+(GE Healthcare)を用いたSPRにより測定した。CM5センサーチップに抗ヒトIgG(Fc)抗体を固定化し、これら3種類(Hu-13H02、Hu-13H02m、Ch-13H02)の抗体標品(1μg/mL)を捕捉した。次に0.13~8.0 nmol/LでヒトGHまたは0.50~32 nmol/LでサルGHを流し、結合、解離パラメータを算出した。結果を表6に示す。
実施例11で調製した組換え抗GHヒト化抗体(Hu-13H02)及びそのFc改変体(Hu-13H02m)と実施例8で調製した親キメラ抗体(Ch-13H02)の、GHと構造類似性を示すタンパク質(placental lactogen (PL)、prolactin (PRL))に対する交差反応性を実施例4に記載の方法と同様な方法で競合的ELISAにより測定した。各抗GH抗体についてヒトGH及びGH類似タンパクの濃度依存曲線よりIC50 (nmol/L)を求め、GH類似タンパクに対する交差反応性を下記の式3により算出した。
GH類似タンパクへの交差反応性(%) = IC50(ヒトGH)/IC50(GH類似タンパク)×100・・・(式3)
結果を表7に示す。
実施例6に記載の下垂体摘除/GH補充ラットにおいて抗GH マウス-ヒトキメラmAb(Ch-13H02)及び抗GHヒト化抗体Fc改変体(Hu-13H02m)の薬効を評価した。
4週齢で下垂体を摘除し、内在性のGH不全により血清IGF-I低下を呈する下垂体摘除SDラット(日本エスエルシー、6週齢)の皮下に、組換えヒトGH(サンド株式会社、30μg/day)を封入した3日間型アルゼット浸透圧ポンプ (室町機械株式会社)を埋め込んだ。この下垂体摘除/GH補充ラットに対し、GH徐放開始1日後に抗GH抗体(Ch-13H02、Hu-13H02m)を各々1、3、及び10 mg/kgで単回皮下投与した(n=6)。尚、正常対照群(Normal)は下垂体摘除/GH非補充ラット(組換えヒトGH非投与下垂体摘除ラット)とし、陰性対照群には下垂体摘除/GH補充ラットにcontrol humanIgG1(Bio X Cell, Inc.、BE0297)を投与した。Ch-13H02投与2日後の血清IGF-1値を図2、Hu-13H02m投与2日後の血清IGF-1値を図3に示す(平均値及び標準誤差)。Ch-13H02は3及び10 mg/kg投与群において、Hu-13H02mは3及び10 mg/kg投与群において、血清IGF-I値を有意に低下させた(###: p<0.001対 正常対照群/Aspin-Welchのt検定、*: p<0.05対 陰性対照群/Steelの多重比較検定)。」
Claims (18)
- ヒト成長ホルモンに特異的に結合する抗体又はその抗原結合断片であって、重鎖可変領域(VH)及び軽鎖可変領域(VL)を含み、
VHが、VH相補性決定領域(CDR)1(VHCDR1)、VHCDR2、及びVHCDR3を含み、
VHCDR1が、配列番号5のアミノ酸配列、又は配列番号5のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VHCDR2が、配列番号6のアミノ酸配列、又は配列番号6のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VHCDR3が、配列番号7のアミノ酸配列、又は配列番号7のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLがVL相補性決定領域(CDR)1(VLCDR1)、VLCDR2、及びVLCDR3を含み、
VLCDR1が、配列番号8のアミノ酸配列、又は配列番号8のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLCDR2が、配列番号9のアミノ酸配列、又は配列番号9のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含み、
VLCDR3が、配列番号10のアミノ酸配列、又は配列番号10のアミノ酸配列において1若しくは2個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む、前記抗体又はその抗原結合断片。 - ヒト成長ホルモンに特異的に結合する抗体又はその抗原結合断片であって、重鎖可変領域(VH)及び軽鎖可変領域(VL)を含み、
VHがVH相補性決定領域(CDR)1(VHCDR1)として配列番号5のアミノ酸配列、VHCDR2として配列番号6のアミノ酸配列、及びVHCDR3として配列番号7のアミノ酸配列を含み、
VLがVL相補性決定領域(CDR)1(VLCDR1)として配列番号8のアミノ酸配列、VLCDR2として配列番号9のアミノ酸配列、VLCDR3として配列番号10のアミノ酸配列を含む、前記抗体又はその抗原結合断片。 - 表面プラズモン共鳴法によって測定した場合に、1.0×10-8mol/L以下の平衡解離定数(KD)でヒト成長ホルモンに結合する、請求項1又は2に記載の抗体又はその抗原結合断片。
- 成長ホルモン作用を中和する、請求項1~3のいずれか一項に記載の抗体又はその抗原結合断片。
- 配列番号3、11、12、又は19のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号3、11、12、又は19のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含むVH、及び/又は
配列番号4、13、14、又は20のアミノ酸配列において、CDR1~CDR3以外の領域において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は、配列番号4、13、14、又は20のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含むVL、
を含む、請求項1~4のいずれか一項に記載の抗体又はその抗原結合断片。 - 配列番号3、11、12、又は19のアミノ酸配列を有するVH、及び/又は配列番号4、13、14、又は20のアミノ酸配列を有するVLを含む、請求項5に記載の抗体又はその抗原結合断片。
- 抗体がモノクローナル抗体である、請求項1~6のいずれか一項に記載の抗体又はその抗原結合断片。
- 抗体がキメラ抗体、ヒト化抗体、ベニア化抗体又は完全ヒト抗体である、請求項1~7のいずれか一項に記載の抗体又はその抗原結合断片。
- 抗体が、配列番号17のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む重鎖定常領域、又は配列番号17のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含む重鎖定常領域、及び/又は
配列番号18のアミノ酸配列において、1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列を含む軽鎖定常領域、又は配列番号18のアミノ酸配列に対して90%以上の同一性を有するアミノ酸配列を含む軽鎖定常領域、
を含む、請求項1~8のいずれか一項に記載の抗体又はその抗原結合断片。 - 抗体が、配列番号17のアミノ酸配列を有する重鎖定常領域、及び/又は配列番号18のアミノ酸配列を有する軽鎖定常領域を含む、請求項1~9のいずれか一項に記載の抗体又はその抗原結合断片。
- 抗原結合断片が、Fab、Fab’、F(ab’)2、Fv、又はscFvである、請求項1~10のいずれか一項に記載の抗体又はその抗原結合断片。
- 請求項1~11のいずれか一項に記載の抗体又はその抗原結合断片をコードする核酸。
- 請求項12に記載の核酸を含むベクター。
- 請求項12に記載の核酸若しくは請求項13に記載のベクターを含む細胞。
- 請求項1~11のいずれか一項に記載の抗体又はその抗原結合断片を含む医薬。
- 血液中のインスリン様成長因子1(IGF-1)のレベルを低減させるための、請求項15に記載の医薬。
- 先端巨大症、巨人症、がん、糖尿病性腎症、関節炎、及び肺炎症からなる群から選択される疾患を治療及び/又は予防するための、請求項15又は16に記載の医薬。
- 血液中のインスリン様成長因子1(IGF-1)のレベルを低減させるための方法であって、治療有効量の請求項1~11のいずれか一項に記載の抗体又はその抗原結合断片を投与するステップを含む方法。
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