JP2024121816A - Drugs containing anti-growth hormone antibodies - Google Patents

Abstract

[Problem] To provide a pharmaceutical composition that reduces blood insulin-like growth factor 1 (IGF-1) levels for treating or preventing diseases associated with hypersecretion or hyposecretion of growth hormone (GH). [Solution] To provide an antibody or antigen-binding fragment thereof that specifically binds to human GH. In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human GH, comprising a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises VH complementarity determining region (CDR) 1 (VHCDR1), VHCDR2, and VHCDR3, each having a specific amino acid sequence, and the VL comprises VL complementarity determining region (CDR) 1 (VLCDR1), VLCDR2, and VLCDR3, each having a specific amino acid sequence. [Selected Figure] Figure 1

Description

In one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to human growth hormone, nucleic acids encoding the same, a vector comprising said nucleic acid, a cell comprising said nucleic acid or vector, a pharmaceutical comprising said antibody or antigen-binding fragment thereof, or a method for reducing the level of insulin-like growth factor 1 (IGF-1) in blood, comprising the step of administering a therapeutically effective amount of said antibody or antigen-binding fragment thereof.

Growth hormone (GH) is a protein hormone synthesized and secreted from the pituitary gland. GH is known to have isoforms with molecular weights of 22 kDa and 20 kDa (GH-22K, GH-20K). The major molecular species in human serum is GH-22K, but the existence of several other isoforms, such as 20 kDa (GH-20K) is also known (Non-Patent Document 1). In pregnant women, GH isoforms with molecular weights of 22 kDa and 20 kDa (GH-V-22K, GH-V-20K) are secreted from the placenta (Non-Patent Document 2). GH exerts growth-related effects, such as bone elongation and muscle growth, as well as metabolic effects, such as metabolism promotion, blood glucose increase, and homeostasis maintenance, both through direct action on tissues and through stimulation of the release of insulin-like growth factor-1 (IGF-1) via its action on the growth hormone receptor (GHR) expressed in the liver and other tissues (Non-Patent Document 3).

GH hypersecretion or hyposecretion is related to many diseases. GH hypersecretion is caused by benign pituitary adenomas, and typical symptoms include acromegaly and gigantism. Acromegaly, also known as acromegaly, is a disease characterized by enlargement of the extremities of the body, such as the forehead, nose, chin, hands, feet, and tongue, coarse body hair, thickened skin, excessive sweating and unpleasant body odor due to enlargement of sebaceous and sweat glands, and a deepening of the voice due to proliferation of oral cartilage. If GH hypersecretion develops before puberty, it will lead to gigantism.

The prevalence of acromegaly is reported to be 4-24 people per 100,000 population, and the number of cases is not high, so it is considered a rare disease. However, if excessive GH secretion continues for a long period of time, in addition to the symptoms mentioned above, it can also cause metabolic disorders such as diabetes, high blood pressure, and hyperlipidemia, sleep apnea syndrome, and cardiac hypertrophy, and there is also a risk of developing angina, myocardial infarction, and cerebrovascular disorders. It is known that if excessive GH secretion is overlooked and not treated, the mortality rate will be high. It is also known that acromegaly increases the risk of developing colon cancer, thyroid cancer, and other conditions, so early detection and treatment are important.

Diseases caused by GH hypersecretion include acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, pulmonary inflammation, etc. Non-Patent Document 4 describes that GH expression in cancer cells is involved in the progression of endometrial cancer, breast cancer, etc., and Non-Patent Document 5 describes the tumor-suppressing effect of pegvisomant, a GH receptor antagonist, in tumor model mice. Non-Patent Document 6 describes the relationship between GH and diabetic nephropathy, Non-Patent Document 7 describes the relationship between GH and joint pain and arthritis, and Non-Patent Document 8 describes the relationship between IGF-1 and pulmonary inflammation.
Treatments for acromegaly and gigantism include surgery to remove the pituitary adenoma that secretes excessive GH, regression through radiation therapy, and drug therapy. Pituitary adenoma removal, like other surgical procedures, carries the risk of complications, including death, and requires advanced techniques. Radiation therapy also carries similar risks and can take several years to become effective. Currently, there are somatostatin analogues (octreotide acetate sustained-release formulations, lanreotide acetate sustained-release formulations, etc.), dopamine agonists (bromocriptine mesylate), and GH receptor antagonists (pegvisomant) on the market, but there is still no drug that is effective, safe, and convenient 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, anti-human GH mouse mAb (hGH-33) that binds to GH-20K but does not substantially bind to GH-22K, and anti-human GH mouse mAb (hGH-12) that binds to both GH-22K and GH-20K. In a GH-dependent proliferation test of Ba/F3 cell line in which human GHR/G-CSFR was forcibly expressed, it was shown that hGH-25 and hGH-26 inhibited only GH-22K-dependent cell proliferation, hGH-33 inhibited only GH-20K-dependent cell proliferation, and hGH-12 inhibited GH-22K- and GH-20K-dependent cell proliferation, but the strength of inhibition (IC ₅₀ : 50% inhibitory concentration) was not specified.

Non-patent literature 9 describes the preparation of eight types of anti-human GH mouse mAbs, and of these, clones EB1, EB2, and NA71, which cross-react with human placental lactotropic hormone (hCS) (human placental lactogen (hPL)), suppressed the proliferation of human GH and hCS-dependent Nb ₂ rat lymphoma cell line, but the strength of 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 promoted the chondrocyte proliferation in dwarf mice with human GH or hCS, and promoted the lactogenic activity in pigeons.

Non-patent literature 11 describes several anti-human GH mouse mAbs, and shows that clones EB1 and EB2 described in non-patent literature 9 and 10 inhibit the binding of human GH to human GHR and also inhibit the inhibitory activity of human GH against glucose uptake in the 3T3-F422A adipocyte cell line, but the strength of inhibition ( _IC50 ) is not specified.

Non-Patent Document 12 is related to Patent Document 1. Non-Patent Document 12 shows that in a GH-dependent proliferation test of Ba/F3 cell line in which human GHR/G-CSFR was forcibly expressed, hGH-25 and hGH-26 inhibited only GH-22K-dependent cell proliferation, hGH-33 inhibited only GH-20K-dependent cell proliferation, and hGH-12 inhibited GH-22K- and GH-20K-dependent cell proliferation, but does not specify the strength of inhibition (IC ₅₀ ).

Non-patent literature 13 describes three types of anti-human GH mouse mAbs, and shows that two of them (AC8, F11), which strongly inhibit the binding of human GH to the Nb ₂ rat lymphoma cell line, inhibited human GH-dependent Nb ₂ cell proliferation, but the strength of inhibition (IC ₅₀ ) is not specified.

Non-patent literature 14 describes 10 types of mouse mAbs specific to human GH, and describes that they suppressed T cell proliferation induced by mitogen stimulation (PHA), but does not demonstrate inhibitory activity against human GH.

Non-patent literature 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, but the strength of inhibition ( _IC50 ) is not specified.

Non-Patent Document 16 describes a novel method for measuring human GH activity in serum using Ba/F3 cell line (Ba/F3-hGHR) in which human GHR is forcibly expressed, and uses anti-human GH mouse mAb (clone 5801) as a positive control antibody to show that the proliferation of Ba/F3-hGHR is GH-dependent. The 5801 antibody at a molar amount 50-fold or 100-fold higher than that of human GH completely inhibited the GH-dependent proliferation of Ba/F3-hGHR, but the strength of inhibition ( _IC50 ) was not clearly stated.

Non-Patent Document 17 describes an anti-human GH mouse mAb (6J33) having neutralizing activity. Although it has been shown that the 6J33 antibody has neutralizing activity, the strength of inhibition (IC ₅₀ ) has not been clearly stated.

International Publication No. 1997/36929

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) Tumour-Derived Human Growth Hormone As a Therapeutic Target in Oncology (Jo K Perry et al., Trends Endocrinol Metab. 2017;28(8):587-596.) Antitumor activity of the growth hormone receptor antagonist pegvisomant against human meningiomas in nude mice (I E McCutcheon et al., J Neurosurg. 2001;94:487-492.) The glomerular podocyte as a target of growth hormone action: implications for the pathogenesis of diabetic nephropathy (P Anil Kumar et al., Curr Diabetes Rev. 2011;7(1):50-55.) Medical progress: Acromegaly (Shlomo Melmed, N Engl J Med 2006; 355:2558-2573) Adenovirus-mediated gene transfer of hIGF-IB in mouse lungs induced prolonged inflammation but no fibroproliferation (Caroline Leger et al., Am J Physiol Lung Cell Mol Physiol. 2010;298(4):L492-500.) Study of antigenic structure and inhibition of activity of human growth hormone and chorionic somatomammotropin by monoclonal antibodies. (Ivanyi J., Mol. Immunol. 19: 1611-1618, 1982. Potentiation of the somatogenic and lactogenic activity of human growth hormone with monoclonal antibodies. (Aston R. et al., J. Endocrinol. 110: 381-388, 1986) Actions of monoclonal antibodies on the activity of human growth hormone (GH) in an in vitro bioassay. (Burbridge D. et al., Mol. Cell. Endocrinol. 174: 11-19, 2001) Characterization of monoclonal antibodies specific for the human growth hormone 22K and 20K isoforms. (Mellado M. et al., J. Clin. Endocrinol. Metab. 81:1613-1618, 1996) Monoclonal antibodies to human growth hormone modulate its biological properties. (Roguin L.P. et al., Mol. Immunol. 32: 399-405, 1995) Monoclonal antibodies against recombinant human growth hormone as probes to study immune function. (Lattuada D. et al., Hybridoma 15: 211-217, 1996 Characterization of monoclonal antibodies to human growth hormone (hGH) (Sato Y., Tokyo Joshi Ika Daigaku Zasshi 54: 493-506, 1984) Novel Specific Bioassay for Serum Human Growth Hormone. (Ishikawa M. et al., J. Clin. Endocrinol. & Metab. 85: 4274-4279, 2000) GeneTex reagent: Catalog Number: GTX52773, https://www.genetex.com/Product/Detail/Growth-Hormone-antibody-6J33/GTX52773

In one embodiment, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human GH.

The present invention includes the following embodiments.
(1) 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 the amino acid sequence of SEQ ID NO:5 in which one or two amino acids are substituted, added, or deleted;
VHCDR2 comprises the amino acid sequence of SEQ ID NO:6 or the amino acid sequence of SEQ ID NO:6 in which one or two amino acids are substituted, added, or deleted;
VHCDR3 comprises the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence of SEQ ID NO: 7 in which one or two amino acids are substituted, added, or deleted;
VL comprises VL complementarity determining region (CDR) 1 (VLCDR1), VLCDR2, and VLCDR3;
VLCDR1 comprises the amino acid sequence of SEQ ID NO:8 or the amino acid sequence of SEQ ID NO:8 in which one or two amino acids are substituted, added, or deleted;
VLCDR2 comprises the amino acid sequence of SEQ ID NO:9 or the amino acid sequence of SEQ ID NO:9 in which one or two amino acids are substituted, added, or deleted;
The antibody or antigen-binding fragment thereof, wherein VLCDR3 comprises the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence of SEQ ID NO: 10 in which one or two amino acids have been substituted, added, or deleted.
(2) An antibody or an 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 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;
The antibody or antigen-binding fragment thereof, wherein the VL comprises the amino acid sequence of SEQ ID NO: 8 as VL complementarity determining region (CDR) 1 (VLCDR1), the amino acid sequence of SEQ ID NO: 9 as VLCDR2, and the amino acid sequence of SEQ ID NO: 10 as VLCDR3.
(3) The antibody or antigen-binding fragment thereof according to (1) or (2), which binds to human growth hormone with an equilibrium dissociation constant (K _D ) of 1.0×10 ⁻⁸ mol/L or less when measured by surface plasmon resonance.
(4) The antibody or antigen-binding fragment thereof according to any one of (1) to (3), which neutralizes the action of growth hormone.
(5) VH comprising 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 an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, and/or VL comprising 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: 4, 13, 14, or 20, or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20,
The antibody or antigen-binding fragment thereof according to any one of (1) to (4),
(6) The antibody or antigen-binding fragment thereof described in (5), comprising a VH having the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, and/or a VL having the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20.
(7) The antibody or antigen-binding fragment thereof according to any one of (1) to (6), wherein the antibody is a monoclonal antibody.
(8) The antibody or antigen-binding fragment thereof according to any one of (1) to (7), wherein the antibody is a chimeric antibody, a humanized antibody, a veneered antibody, or a fully human antibody.
(9) An antibody comprising a heavy chain constant region comprising an amino acid sequence in which one or several amino acids have been substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 17, and/or a light chain constant region comprising an amino acid sequence in which one or several amino acids have been substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 18, or 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),
(10) The antibody or antigen-binding fragment thereof 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.
(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 according to (12).
(14) A cell comprising the nucleic acid according to (12) or the vector according to claim (13).
(15) A medicine comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (11).
(16) The pharmaceutical according to (15) for reducing the level of insulin-like growth factor 1 (IGF-1) in blood.
(17) The pharmaceutical according to (15) or (16), for treating and/or preventing a disease selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, and pulmonary inflammation.
(18) A method for reducing the level of insulin-like growth factor 1 (IGF-1) in blood, comprising the step of administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof described in any one of (1) to (11).
(19) An antibody or an 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 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;
A pharmaceutical composition for injection comprising an antibody or an antigen-binding fragment thereof, wherein the VL comprises the amino acid sequence of SEQ ID NO: 8 as VL complementarity determining region (CDR) 1 (VLCDR1), the amino acid sequence of SEQ ID NO: 9 as VLCDR2, and the amino acid sequence of SEQ ID NO: 10 as VLCDR3.
(20) The pharmaceutical according to (19), wherein the antibody or antigen-binding fragment binds to human growth hormone with an equilibrium dissociation constant (K _D ) of 1.0×10 ⁻⁸ mol/L or less when measured by surface plasmon resonance.
(21) The pharmaceutical according to (19), wherein the antibody or antigen-binding fragment neutralizes the action of growth hormone.
(22) The pharmaceutical described in (19), wherein the antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, and/or a VL having the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20.
(23) The pharmaceutical according to any one of (19) to (22), wherein the antibody is a chimeric antibody, a humanized antibody, a veneered antibody, or a fully human antibody.
(24) The pharmaceutical according to any one of (19) to (22), 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.
(25) The pharmaceutical according to any one of (19) to (22), wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or scFv.
(26) The pharmaceutical agent according to any one of (19) to (22), for reducing the level of insulin-like growth factor 1 (IGF-1) in blood.
(27) The pharmaceutical agent according to any one of (19) to (22), for treating and/or preventing a disease selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, and pulmonary inflammation.
(28) The pharmaceutical according to any one of (19) to (22), wherein the dosage of the antibody or antigen-binding fragment thereof is 0.0001 to 100 mg/kg per day.

In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human GH.

Figure 1 shows serum IGF-1 levels in hypophysectomized/GH-replaced rats 2 days after administration of anti-GH mouse mAb (13H02). The normal control group (Normal) is hypophysectomized/GH-unreplaced rats, and the negative control group is hypophysectomized/GH-replaced rats administered control mouse IgG1. Figure 2 shows serum IGF-1 levels in hypophysectomized/GH-replaced rats 2 days after administration of anti-GH mouse-human chimeric mAb (Ch-13H02). The normal control group (Normal) consisted of hypophysectomized/GH-unreplaced rats, and the negative control group consisted of hypophysectomized/GH-replaced rats administered control human IgG1. Fig. 3 shows serum IGF-1 levels 2 days after administration of anti-GH humanized antibody Fc variant (Hu-13H02m) to hypophysectomized/GH-replaced rats. The normal control group (Normal) consisted of hypophysectomized/GH-non-replaced rats, and the negative control group consisted of hypophysectomized/GH-replaced rats administered control human IgG1.

1. Antibodies that Bind to Growth Hormone In one embodiment, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human growth hormone (GH) (hereinafter also referred to as "anti-GH antibody").

"Growth hormone" is a hormone secreted from cells in the anterior pituitary gland and is involved in the growth and metabolism of an individual. GH exerts growth-related effects such as bone elongation and muscle growth, as well as metabolic effects such as metabolic promotion, blood sugar increase, and homeostasis, both through direct action on tissues and through stimulating the release of insulin-like growth factor 1 (IGF-1) via its action on the GH receptor (GHR) expressed in the liver and other tissues. IGF-1 is a peptide hormone with a structure similar to insulin, and is secreted by various tissues including the liver. IGF-1 acts on many different organs, promoting cell proliferation and differentiation, protein synthesis, and inhibiting cell death, among other physiological functions.

Information on the amino acid sequence and mRNA sequence of human GH is available from publicly accessible databases such as GenBank. Similarly, sequence information for GH from other mammals, such as mice and monkeys, is also available from publicly accessible databases.

In this specification, the term "GH" refers to GH protein. In some cases, the gene encoding the GH protein may simply be referred to as GH. When "GH" refers to the gene encoding the GH protein, it will be clear from the context to those skilled in the art. In this specification, GH typically refers to human GH, but may also refer to GH of mammals other than humans (e.g., mice, rats, monkeys, etc.).

In this specification, an "antibody" is a glycoprotein that includes at least two heavy chains and two light chains interconnected by disulfide bonds, and in the case of IgM, also includes a J chain. The heavy chain includes a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region includes γ, μ, α, δ, and ε chains, and depending on the differences, there are isotypes such as IgG, IgM, IgA, IgD, and IgE. The heavy chain constant region includes three domains, namely CH1, CH2, and CH3, and in the case of IgE and IgM, it further includes CH4. The light chain includes a light chain variable region (VL) and a light chain constant region, and the light chain constant region includes one domain, namely CL. There are two types of light chain constant regions, called λ chains and κ chains. The VH and VL regions are further subdivided into four regions (FR1, FR2, FR3, FR4) that are relatively conserved in the variable region and called framework regions (FR), and three hypervariable regions (CDR1, CDR2, CDR3) that are called complementarity determining regions (CDR) and contribute to antigen binding. VH contains three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1 (CDRH1), FR2, CDR2 (CDRH2), FR3, CDR3 (CDRH3), FR4. VL contains three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1 (CDRL1), FR2, CDR2 (CDRL2), FR3, CDR3 (CDRL3), FR4. CDRs 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.).

The term "antibody" as used herein includes all classes and subclasses of intact immunoglobulins. The antibody described herein may be, for example, an IgG, more specifically, the constant region of which may be human IgG1, a variant in which the effector function of IgG1 has been eliminated, IgG2, IgG4, or IgG4 (S228P), and even more specifically, IgG1, or a variant in which the effector function of IgG1 has been eliminated. Generally, for antibodies that do not require effector function to exert their function, it is preferable to use an isotype with a weak effector function or a variant in which the effector function has been eliminated from the viewpoint of safety, but since the antibody described herein targets soluble GH, there is little need for such an isotype.

As used herein, "antigen-binding fragments" of antibodies include separated heavy chains, light chains, Fab, Fab', F(ab')2, Fv, scFv, etc.

In this specification, the term "antibody" or an antigen-binding fragment thereof includes derivatives thereof. Examples of derivatives of antibodies or antigen-binding fragments thereof include antibodies with artificial amino acid mutations in the constant region, antibodies with modified domain configurations in the constant region, antibodies with two or more Fc's per molecule, glycosylated antibodies, bispecific antibodies, antibody conjugates in which an antibody or an antigen-binding fragment thereof is linked to other components, antibody enzymes, tandem scFvs, bispecific tandem scFvs, trispecific tandem scFvs, and diabodies.

The antibodies described herein may be either polyclonal or monoclonal antibodies, with monoclonal antibodies being particularly preferred.

A "monoclonal antibody" (also written as "mAb") is an antibody obtained from a clone that is typically derived from a single antibody-producing cell. That is, mAbs are a homogeneous or substantially homogeneous population of individual antibodies whose amino acid sequences are identical except for possible naturally occurring mutations that may be present in small amounts. mAbs have high specificity by binding to a single antigenic determinant (epitope) on an antigen. In contrast, polyclonal antibodies are a mixture of multiple mAbs whose amino acid sequences differ at one or more positions.

As used herein, mAb includes fully human antibodies and non-human antibodies.

Non-human antibodies are antibodies other than fully human antibodies, and include, for example, antibodies from animals such as mice, rats, rabbits, and guinea pigs, chimeric antibodies, humanized antibodies, and veneered antibodies.

"Chimeric antibody" refers to an antibody whose variable region sequence is derived from one species and whose constant region sequence is derived from another species, for example, whose variable region sequence is derived from a non-human animal antibody and whose constant region sequence is derived from a human antibody.

"Humanized antibody" refers to an antibody in which only the CDR sequences and some of the framework amino acid residues in the variable regions are derived from a non-human animal antibody, and the other sequences, i.e., most of the framework sequences and all of the constant region sequences, are derived from a human antibody.

A "veneered antibody" is a type of humanized antibody that retains some, and usually all, of the CDRs of a nonhuman animal antibody and some of the nonhuman animal variable region framework residues, but replaces other variable region framework residues that may contribute to B or T cell epitopes, e.g., exposed residues (Padlan, Mol. Immunol. 28:489, 1991), with residues at the corresponding positions in a human antibody sequence.

A "fully human antibody" is an antibody that is entirely derived from a human antibody, including the CDR sequences.

As used herein, "specific binding" refers to an antibody binding to an antigen with an affinity that is significantly higher than the affinity for non-antigenic substances (non-specific interaction). Affinity can be measured by standard methods. Such methods are not particularly limited, but include, for example, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR). When measuring affinity, the antibody and/or antigen can be labeled with an enzyme, fluorescent substance, etc., and the labeled substance can be measured to determine affinity.

As parameters indicating affinity, K _D (equilibrium dissociation constant), k _on (binding rate constant), and k _off (dissociation rate constant) can usually be used.

As used herein, k _on refers to the rate constant for the binding of an antibody to an antigen, and k _off refers to the rate constant for the dissociation of an antibody from an antigen-antibody complex. Also, as used herein, K _D refers to the equilibrium dissociation constant of an antigen-antibody interaction, calculated as k _off /k _on .

In one embodiment, the antibodies described herein bind to human GH with a K _D of, e.g., 1×10 ^-8 mol/L or less, 1×10 ^-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, when measured according to the method described in Example 12 herein.

In one embodiment, the antibody described herein has high affinity for GH-22K among the human GH isoforms (GH-22K, GH-20K).

As used herein, "cross-reactivity" refers to reactivity with other proteins, such as GH derived from mammals other than humans, 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), when the reactivity with human GH (GH-22K) used as an antigen in the examples described herein is taken as 100%.

Cross-reactivity of anti-GH antibodies can be measured in vitro, for example, by competitive ELISA as described in Examples 3, 4, and 14 or SPR as described in Example 13.

In one embodiment, the antibody or antigen-binding fragment thereof described herein comprises a VH and a VL, wherein the VH comprises a VH complementarity determining region (CDR) 1 (VHCDR1), a VHCDR2, and a VHCDR3, and the VL comprises a VL complementarity determining region (CDR) 1 (VLCDR1), a VLCDR2, and a VLCDR3. In one embodiment, the VHCDR1 comprises or consists of the amino acid sequence of SEQ ID NO:5, or the amino acid sequence of SEQ ID NO:5 with one or two amino acid substitutions, additions, or deletions. In one embodiment, the VHCDR2 comprises or consists of the amino acid sequence of SEQ ID NO:6, or the amino acid sequence of SEQ ID NO:6 with one or two amino acid substitutions, additions, or deletions. In one embodiment, the VHCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:7, or the amino acid sequence of SEQ ID NO:7 with one or two amino acid substitutions, additions, or deletions. In one embodiment, VLCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 8, or the amino acid sequence of SEQ ID NO: 8 with one or two amino acid substitutions, additions or deletions. In one embodiment, VLCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 9, or the amino acid sequence of SEQ ID NO: 9 with one or two amino acid substitutions, additions or deletions. In one embodiment, VLCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 10, or the amino acid sequence of SEQ ID NO: 10 with one or two amino acid substitutions, additions or deletions. In one embodiment, VHCDR1 comprises or consists of an amino acid sequence of SEQ ID NO:5 with one amino acid substitution, addition, or deletion, VHCDR2 comprises or consists of an amino acid sequence of SEQ ID NO:6 with one amino acid substitution, addition, or deletion, VHCDR3 comprises or consists of an amino acid sequence of SEQ ID NO:7 with one amino acid substitution, addition, or deletion, VLCDR1 comprises or consists of an amino acid sequence of SEQ ID NO:8 with one amino acid substitution, addition, or deletion, VLCDR2 comprises or consists of an amino acid sequence of SEQ ID NO:9 with one amino acid substitution, addition, or deletion, and VLCDR3 comprises or consists of an amino acid sequence of SEQ ID NO:10 with one amino acid substitution, addition, or deletion.

In one embodiment, the antibody or antigen-binding fragment thereof described herein has a VH that comprises 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:7 as VHCDR3, and a VL that comprises 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 to CDR3 may each consist of the amino acid sequence of SEQ ID NO:5 to 7, and VLCDR1 to CDR3 may each consist of the amino acid sequence of SEQ ID NO:8 to 10.

The sequences of the framework regions of the antibodies or antigen-binding fragments thereof described herein are not limited as long as the antibodies or antigen-binding fragments thereof are capable of specifically binding to GH. In one embodiment, the antibodies or antigen-binding fragments thereof described herein have the following sequence:
VH comprising an amino acid sequence in which one or several amino acids have been 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 an amino acid sequence having 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 SEQ ID NO: 3, 11, 12 or 19, and the regions of CDR1 to CDR3 are each composed of SEQ ID NO: 5 to 7, respectively, and/or The amino acid sequence comprises an amino acid sequence in which one or several amino acids have been substituted, added or deleted in a region other than CDR1 to CDR3 of the amino acid sequence of SEQ ID NO: 4, 13, 14 or 20, or an amino acid sequence having 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 SEQ ID NO: 4, 13, 14 or 20, and the regions of CDR1 to CDR3 comprise VLs consisting of SEQ ID NOs: 8 to 10, respectively.

In one embodiment, the antibody or antigen-binding fragment thereof described herein comprises a VH comprising an amino acid sequence of SEQ ID NO: 3, 11, 12, or 19 in which one or several amino acids have been substituted, added, or deleted, or an amino acid sequence having 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 SEQ ID NO: 3, 11, 12, or 19; and/or a VL comprising an amino acid sequence of SEQ ID NO: 4, 13, 14, or 20 in which one or several amino acids have been substituted, added, or deleted, or an amino acid sequence having 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 SEQ ID NO: 4, 13, 14, or 20.

In this specification, "several" may mean, for example, 2 to 10, 2 to 8, or 2 to 5, such as 2 or 3.

As used herein, the term "identity" of an amino acid sequence refers to the percentage of identical amino acid residues. The identity of an amino acid sequence can be determined, for example, by BLAST (Basic Local Alignment Search Tool) analysis.

In one embodiment, the antibody or antigen-binding fragment thereof described herein comprises a VH having the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, and/or a VL having the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20.

In one embodiment, the antibody or antigen-binding fragment thereof described herein comprises:
A heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 17 in which one or several amino acids have been substituted, added, or deleted, or an amino acid sequence having 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 SEQ ID NO: 17, and/or a light chain constant region comprising an amino acid sequence of SEQ ID NO: 18 in which one or several amino acids have been substituted, added, or deleted, or a light chain constant region comprising an amino acid sequence having 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 SEQ ID NO: 18. Here, the amino acid sequence of SEQ ID NO: 17 may or may not have a C-terminal lysine residue.

In one embodiment, 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, in which a C-terminal lysine residue may or may not be present in the amino acid sequence of SEQ ID NO: 17.

The amino acid sequence of the constant region may include substitutions that increase serum half-life (see, e.g., Hinton P.R. et al., J. Biol. Chem. 279:6213-6216, 2004).

As methods for modifying one or more amino acids in the heavy chain constant region with the aim of extending the blood half-life of an antibody, see, for example, U.S. Patent Nos. 7,083,784, 7,217,797, and 8,088,376. Examples described in the above documents include, for example, a substitution of threonine at position 250 of the heavy chain with glutamine according to the EU numbering of Kabat, a substitution of methionine at position 428 of the heavy chain with leucine, and a substitution of asparagine at position 434 of the heavy chain with serine, and multiple combinations of the above amino acid substitutions are also possible.

In the present specification, an antibody or antigen-binding fragment thereof comprising a VH and/or VL containing an amino acid sequence in which one or several amino acids have been substituted, added, or deleted from the original amino acid sequence, or an amino acid sequence having 90% or more identity to the original amino acid sequence, may have biological activity equivalent to that of an antibody or antigen-binding fragment thereof having the original amino acid sequence. Here, "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.

In one embodiment, the antibodies described herein neutralize the action of GH. As used herein, "neutralization" refers to binding to a target protein and neutralizing or attenuating its action, and "neutralizing activity" refers to the degree of neutralizing or attenuating action. Since the antibodies described herein specifically bind to GH, unless otherwise specified or unless the context makes clear that it is against other proteins, neutralizing activity refers to the degree of neutralization or attenuation against GH. In one embodiment, the antibodies described herein specifically bind to GH and block the interaction with GHR, thereby reducing IGF-1 levels in plasma and/or tissues.

The neutralizing activity of the antibodies described herein can be measured, for example, in vitro (e.g., in a GH-GHR binding inhibition assay or a GH-dependent Nb ₂ cell proliferation inhibition assay as described in Example 2 herein). In one embodiment, the antibodies described herein have an IC50 of 10,000 pmol/L or less, 1,000 pmol/L or less, or 100 pmol/L or less, and/or 1 pmol/L or more, 0.1 pmol/L or more, or 0.01 pmol/L or more in the GH- _GHR binding inhibition assay described in Example 2. In one embodiment, the antibodies described herein have an _IC50 of 1,000 pmol/L or less, 100 pmol/L or less, or 10 pmol/L or less, and/or 1 pmol/L or more, 0.1 pmol/L or more, or 0.01 pmol/L or more in the GH-dependent Nb ₂ cell proliferation inhibition assay described in Example 2.

The antibodies described herein may be used to reduce serum IGF-1 concentrations and alleviate or treat symptoms of GH hypersecretion, or as prophylactic agents for people who may develop symptoms of GH excess in the future. The antibodies described herein may be used in patients who are refractory to conventional treatments for GH hypersecretion. In one embodiment, the antibodies described herein may be used in the treatment and/or prevention of disorders associated with GH hypersecretion, including, for example, acromegaly, gigantism, certain cancers, diabetic nephropathy, arthritis, and pulmonary inflammation.

In one embodiment, the antibodies described herein may have superior efficacy (e.g., equivalent to or better than pegvisomant), safety, and/or favorable blood kinetics.

In one embodiment, the antibodies described herein reduce serum IGF-1 levels in vivo. For example, the antibodies described herein dose-dependently reduce the increase in serum IGF-1 levels caused by continuous administration of exogenous human GH to rats that have been hypophysectomized to lack endogenous GH. In one embodiment, the antibodies described herein neutralize endogenous GH and continuously reduce serum IGF-1 levels when administered to a mammal, such as a monkey.

In one embodiment, the antibodies described herein have little or no toxicity to mammals.

In one embodiment, the antibodies described herein exhibit good physical properties (eg, at least one of water solubility, acid stability, thermal stability, and hydrophobic interactions).
2. Preparation of the Antibodies Described Herein The antibodies described herein can be selected and obtained from B cells obtained by immunizing an animal with an antigen by a wide variety of methods known in the art, such as the hybridoma method, various display methods, single-cell screening method, etc. Fully human monoclonal antibodies can be prepared by using genetically modified animals in which human antibody genes have been incorporated into antigen-immunized animals, or by using an antibody display library constructed from a pool of human antibody genes.

Hybridoma technology is a method for obtaining monoclonal antibodies by selecting and obtaining a target antibody-producing strain from among monoclonal hybridoma cell strains immortalized by fusing antibody-producing B cells obtained by immunizing animals such as mice or rats with an antigen with myeloma cells. The method is not limited, but can be carried out, for example, as follows.

Human GH or a fragment thereof can be used as an antigen for producing the antibodies described in this specification. These antigens are immunized multiple times with animals such as mice or rats together with various adjuvants, and animals with elevated serum antibody titers are given a final immunization with the antigen, after which the spleen and lymph nodes are removed and lymphocytes containing antibody-producing B cells are collected. The lymphocytes (B cells) are fused with a myeloma cell line using polyethylene glycol or an electric cell fusion device, and cultured in a selective medium (e.g., a selective medium containing HAT (hypoxanthine-aminopterin-thymidine)) in which only the fused cells of both can survive, to establish a hybridoma. The monoclonal hybridoma thus obtained is appropriately cultured, and the human GH binding activity and human GH neutralizing activity of the culture supernatant are measured, and a human GH-specific neutralizing antibody-producing hybridoma can be selected and obtained. Details of hybridoma production techniques are known, and reference can be made to, for example, Koehler, G. et al., Nature 256: 495-497, 1975, and Hnasko R.M. et al., Methods Mol. Biol. 1318: 15-28, 2015.

Examples of antibody production methods using display technology include phage display, yeast display, cDNA display, ribosome display, and animal cell display. Specifically, antibody fragments (such as scFv and Fab) encoded by genes isolated from B cell pools isolated from immunized or non-immunized animals, diseased patients, or healthy individuals, as described in the above hybridoma technology, are displayed on various display systems to construct a library. From this library, antibodies with affinity to the antigen and their genes can be concentrated, amplified, and isolated by a selection procedure called biopanning, to obtain the desired antibody. Details of the display technology are known, and are described, for example, in U.S. Pat. Nos. 5,223,409, 5,885,793, 6,582,915, 5,969,108, 6,172,197, 5,821,047, 6,706,484, and 6,753,136, as well as in Winter G.P. et al., Annu. Rev. Immunol. 12:433-455, 1994, Rothe C. et al., J. Mol. Biol. 376: 1182-1200, 2008, Scholler N., Methods Mol. Biol. 1827: 211-233, 2018, Yamaguchi J. et al., Nucleic Acids Res. 37: e108, 2009, Dreier B. et al., Methods Mol. Biol. 1827: 235-268, 2018; Zhou C. et al., Methods Mol. Biol. 907: 293-302, 2012.

Methods for producing antibodies using single-cell screening techniques for B cells include a method in which B cells isolated from the above-mentioned immunized animals or diseased patients are seeded, for example, in a very small well at one cell per well, and the antibody activity secreted by each cell is detected by a fluorescent reaction to select the B cells producing the desired antibody; or a method in which B cells are reacted with a fluorescently labeled antigen and the fluorescently stained B cells producing the desired antibody are selected at one cell per well by fluorescence-activated cell sorting (FACS). The antibody gene can be isolated from the B cells recovered in this way to obtain the desired antibody. Details of the single-cell screening technique for B cells are known, and reference can be made to, for example, Love J.C. et al., Nat. Biotechnol. 24:703-707, 2006, Tiller T. et al., J. Immunol. Methods 29:112-124, 2008, Jin A. et al., Nat. Med. 15:1088-1092, 2009, Starkie D.O. et al., PLoS ONE 11: 20152282, 2016, Winters A. et al., mAbs 11:1025-1035, 2019, and Josephides D. et al., SLAS Technol. 25:177-189, 2020.

Chimeric antibodies can be produced, for example, by genetically linking a non-human antibody variable region sequence with a human antibody constant region sequence, and producing the antibody using the method described below. Details of chimeric antibody production techniques are known, and reference can be made, for example, to U.S. Patent No. 5,482,856 and Morrison S.L. et al., Proc. Natl. Acad. Sci. USA 81: 6851-6855, 1984.

Humanized antibodies can be produced, for example, by grafting a part, and usually the whole, of the CDRs of a non-human antibody and a part of the non-human variable region framework residues into a human antibody variable region that is highly homologous to the non-human antibody variable region sequence. Details of humanized antibody production techniques are known, and reference can be made to, for example, U.S. Patent No. 5,530,101, U.S. Patent No. 5,585,089, U.S. Patent No. 5,225,539, U.S. Patent No. 6,407,213, U.S. Patent No. 5,859,205, U.S. Patent No. 6,881,557, Queen C.L. et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033, 1989, Tsurushita N. et al., Methods 36: 69-83, 2005, and Choi Y. et al., MAbs 7: 1045-1057, 2015.

Venerated antibodies can be made, for example, by retaining some, and usually all, of the CDRs of a non-human antibody and some of the non-human variable region framework residues, and replacing other variable region framework residues that may participate in B-cell or T-cell epitopes (e.g., exposed residues) (Padlan, Mol. Immunol. 28:489, 1991) with residues from the corresponding positions in a human antibody sequence. Details of venerated antibody production techniques are known, see, for example, U.S. Patent No. 5,585,089 and Riechmann L. et al., Nature 322: 323-327, 1988.

Fully human antibodies can be produced, for example, from B cells obtained by immunizing an animal transgenic for human antibody genes with an antigen, using the monoclonal antibody production techniques described above. They can also be produced from various antibody display libraries made from pools of B cells isolated from diseased patients or healthy individuals. Details of the technique for producing a fully human antibody are known, and are described, for example, in 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. et al., Cloning Stem Cells 4: 91-102, 2002; Osborn M.J. et al., J. Immunol. 190: 1481-1490, 2013; Lee E.C. et al., Nat. Biotechnol. 32: 356-363, 2014; Murphy A.J. et al., Proc. Natl. Acad. Sci. USA 111: 5153-5158, 2014, Winter G.P. et al., Annu. Rev. Immunol. 12:433-455, 1994, Rothe C. et al., J. Mol. Biol. 376: 1182-1200, 2008, U.S. Patent No. 5,223,409, U.S. Patent No. 5,885793, U.S. Patent No. 6,582,915, U.S. Patent No. 5,969,108, U.S. Patent No. 6,172,197, U.S. Patent No. 5,821,047, U.S. Patent No. 6,706,484, and U.S. Patent No. 6,753,136 can be referenced.

The heavy and light chain variable regions of a chimeric, humanized, veneered or fully human antibody can be linked to, for example, any human heavy and light chain constant regions, respectively. The choice of heavy chain constant region isotype depends on the target and whether complement-dependent cytotoxicity or antibody-dependent cytotoxicity is desired as the mechanism of action of the antibody. For example, human IgG1 and IgG3 generally have strong complement-dependent cytotoxicity and antibody-dependent cytotoxicity, while 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 natural allotype or any substitution of residues occupying polymorphic positions of natural allotypes. One or more amino acids at the amino or carboxy termini of the light and/or heavy chains of the antibody (e.g., the C-terminal lysine of the heavy chain) may be deleted or derivatized in a percentage or all of the molecules.

The antibody described herein can have a longer half-life in blood by substituting amino acid residues in the heavy chain constant region. Typical substitutions for extending the half-life in blood include, for example, substituting threonine at position 250 of the heavy chain with glutamine, substituting methionine at position 428 of the heavy chain with leucine, and substituting asparagine at position 434 of the heavy chain with serine, according to the EU numbering according to Kabat. Multiple amino acid substitutions can also be combined (see U.S. Patent Nos. 7,083,784, 7,217,797, and 8,088,376).
3. Producing the Antibodies Described Herein In one embodiment, the invention provides a method for producing the antibodies or antigen-binding fragments described herein.

In one embodiment, the invention provides isolated nucleic acids encoding the antibodies or antigen-binding fragments described herein. The nucleic acid molecules 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 isolated and sequenced, for example, from B cells, hybridomas, or isolated clones of various antibody displays (phage, yeast, animal cells, cDNA, etc.) that produce the antibodies described herein. For example, genes encoding the variable region amino acid sequences of the heavy and/or light chains of the antibodies can be isolated and sequenced using oligonucleotide probes that specifically bind to these constant region sequences.

In addition, antibodies with a known amino acid sequence (including chimeric, humanized, veneered antibodies, fully human antibodies, and antibodies with amino acid modifications in the variable or constant regions) can be produced by using a nucleic acid that encodes the amino acid sequence using known genetic recombination techniques or by chemical synthesis. Antibodies or antigen-binding fragments produced by genetic recombination techniques are also called recombinant antibodies or recombinant antigen-binding fragments.

In one embodiment, the present 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 typically comprises, for example, an expression control sequence capable of transforming or transfecting a eukaryotic host cell, operably linked to the nucleic acid sequence encoding the antibody or antibody-binding fragment (e.g., an origin of replication, a promoter, an enhancer (Queen C. et al., Immunol. Rev. 89:49-68, 1986), and necessary processing information sites (e.g., including ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription terminator sequences), and can be readily constructed by known recombinant gene techniques.

In one embodiment, the present invention provides a cell comprising a nucleic acid encoding an antibody or antigen-binding fragment described herein, or a vector comprising the nucleic acid. For example, a method for producing a hybridoma cell containing a nucleic acid encoding an antibody or antigen-binding fragment described herein is as described above.

For example, cells expressing a recombinant antibody or a recombinant antigen-binding fragment (hereinafter also referred to as "recombinant cells") can be obtained by introducing a vector containing a nucleic acid encoding the antibody or antigen-binding fragment into a host cell and expressing it transiently or stably. Examples of host cells include E. coli cells, yeast cells, insect cells, mammalian cells, etc., and among these, preferred hosts include mammalian cells, such as human HEK293 fetal kidney-derived cells, monkey COS cells, Chinese hamster ovary (CHO) cells, mouse myeloma cells (Sp2/0, NS0), etc. Details of the techniques for producing cells expressing a recombinant antibody or a recombinant antigen-binding fragment are publicly known.

The recombinant antibody or recombinant antigen-binding fragment can be produced by culturing the recombinant cells described above and purifying the cell extract or culture supernatant according to standard protein purification techniques, including various column chromatography and ultraconcentration. Details of the techniques for purifying recombinant antibodies or recombinant antigen-binding fragments are publicly known.

For the above-mentioned methods for producing antibodies with known amino acid sequences, methods for constructing vectors containing nucleic acids encoding antibodies or antigen-binding fragments, recombinant cell production techniques, and techniques for purifying recombinant antibodies or recombinant antigen-binding fragments, see, for example, Ossipow V. et al., Monoclonal Antibodies, Methods Mol. Biol. 1131 (2nd ed.), 2014.

Furthermore, recombinant antibodies or recombinant antigen-binding fragments can also be produced by expressing them in the milk of transgenic plants (e.g., tobacco, duckweed) or transgenic animals (e.g., cows, goats) that have incorporated vectors containing nucleic acids encoding these proteins into their chromosomes, and purifying them according to the same methods as described above. For methods of producing antibody transgenic plants and antibody transgenic animals, see, for example, Perdigones A.S. et al., Cell Engineering 7: 143-164, 2011, and Pollock D.P. et al., J. Immunol.Methods 231: 147-157, 1999.

In addition to the above-mentioned antibody production method, the antigen-binding fragment can also be produced by enzymatic digestion of the antibody or chemical synthesis (for example, in the case of a short polypeptide of about 50 amino acids or less). Methods for enzymatic digestion and chemical synthesis of antibodies are known. For enzymatic digestion, for example, details of a technique for preparing Fab by partial digestion of IgG with papain or the like are known, and reference can be made to, for example, Zao Y. et al., Protein Expression and Purification 67: 182-189, 2009. For chemical synthesis, for example, the fragment can be produced by an automated polypeptide synthesizer using a solid-phase method, and reference can be made to, for example, U.S. Patent No. 5,807,715; U.S. Patent No. 4,816,567; and U.S. Patent No. 6,331,415.
4. Medicaments and Methods In one embodiment, the invention relates to medicaments comprising the antibodies or antigen-binding fragments described herein.

In one embodiment, the antibody or antigen-binding fragment or medicament described herein can be used to reduce the level of IGF-1 in the blood. In one embodiment, the antibody or antigen-binding fragment or medicament described herein can be used to prevent and/or treat a condition associated with excess GH and/or excess IGF-1 in an individual.

Symptoms associated with excess GH and/or excess IGF-1 include acromegaly, gigantism, pituitary gigantism, impaired glucose tolerance, diabetic nephropathy, sleep apnea, loss of vitality, menstrual irregularities, cancer, increased incidence of tumors, malocclusion, excessive sweating, numbness in the hands and feet, arthritis, joint pain due to osteoarthritis, pulmonary inflammation, etc. Symptoms associated with excess GH and/or excess IGF-1 are preferably acromegaly, gigantism, impaired glucose tolerance, diabetic nephropathy, sleep apnea, more preferably acromegaly and gigantism.

In one embodiment, the antibody or antigen-binding fragment or medicament described herein reduces blood IGF-1 levels. The blood IGF-1 level may be, 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% or less of the level before administration.

In one embodiment, therapeutic administration of an antibody or antigen-binding fragment or medicament described herein results in the reduction or amelioration of one or more symptoms of acromegaly and/or reduced incidence. Symptoms of acromegaly include, but are not limited to, excess IGF-1, excessive sweating, unpleasant body odor, thickened skin, darkened skin, oily skin, skin tags, fatigue, muscle weakness, enlarged vocal cords and enlarged sinuses and/or deepening of the voice due to cartilage proliferation in the pharynx, severe snoring, sleep apnea, visual impairment, headaches, enlarged tongue, back pain, joint pain, limited range of motion in joints, menstrual cycle irregularities, decreased libido, erectile dysfunction, enlarged hands, enlarged feet, wide facial features, protruding lower dentition beyond upper dentition, and/or protruding lower jaw. These include: underbite (crossbite), enlarged liver, heart, kidneys, spleen, increased chest size (barrel chest), increased coarse body hair, improper processing of dietary sugars, diabetes, high blood pressure, increased calcium in the urine, kidney stones, gallstones, enlargement of the thyroid gland (goiter), heart disease, arthritis, precancerous growths (polyps) in the colon, carpal tunnel syndrome, hypopituitarism, uterine fibroids, peripheral neuropathy resulting in intraneural fibrous proliferation, necrosis and/or erosion of articular cartilage proliferation, hyperphosphatemia, and spinal cord compression.

Clinical criteria and prognostic indicators for acromegaly are well known in the art, and diagnosis or evaluation of acromegaly is established. Individuals suitable for treatment and/or prevention with the antibodies or antigen-binding fragments or medicaments described herein may be selected based on the above clinical criteria and evaluation. Assessment of acromegaly severity may be performed based on known tests, such as, for example, measurement of blood IGF-1 levels, measurement of GH before and after an oral glucose test, and magnetic resonance imaging (MRI) of the head to detect pituitary tumors. Relief, amelioration, regulation, reduction in occurrence, or delay in onset or progression of symptoms of acromegaly may be measured by testing blood IGF-1 levels.

In one embodiment, therapeutic administration of an antibody or antigen-binding fragment or medicament described herein results in the alleviation or amelioration of one or more symptoms of gigantism and/or a reduction in the occurrence of such symptoms. Examples of symptoms of gigantism include, but are not limited to, excess IGF-1, excess height growth, excess muscle growth, excess organ growth, delayed puberty, double vision, difficulty with peripheral vision, frontal bossing, prominent chin, headaches, increased sweating, menstrual irregularities, large hands, large feet, thick fingers, thick toes, breast milk release, facial thickening, weakness, adrenal insufficiency, diabetes insipidus, hypogonadism, and hypothyroidism.

Clinical criteria and prognostic indicators for gigantism are well known in the art, and diagnosis or evaluation of gigantism is established. Individuals suitable for treatment and/or prevention with the antibodies or antigen-binding fragments or medicaments described herein may be selected based on the above-mentioned clinical criteria and evaluation. Assessment of gigantism severity may be based on known tests, such as, for example, computed tomography (CT) or MRI of the head to detect pituitary tumors, measurement of GH before and after an oral glucose test, measurement of blood prolactin, measurement of blood IGF-1, measurement of blood cortisol, measurement of blood estradiol, measurement of blood testosterone, and measurement of thyroid hormones. Furthermore, reduction, amelioration, regulation, reduction in the occurrence, or delay in the onset or progression of the symptoms of gigantism may be measured by measuring blood IGF-1 levels.

As used herein, "treatment" includes all medical treatments related to GH excess, such as improvement of symptoms associated with excess GH, sustained improvement of symptoms, suppression of recurrence, reduction of other treatments, etc., and also includes improvement of the quality of life of individuals with symptoms due to GH excess. As used herein, "prevention" includes preventing the occurrence of symptoms associated with excess GH or reducing the risk of such symptoms.

In one embodiment, the pharmaceutical composition described herein further contains a pharma- ceutical acceptable carrier (such as an excipient, a filler, a binder, a lubricant, etc.) and/or a known additive (such as a buffer, an isotonicity agent, a chelating agent, a coloring agent, a preservative, a fragrance, a flavoring agent, a sweetener, etc.).
Examples of the carrier and/or known additives include water, dextrose, maltose, fructose, sucrose, sorbitol, mannitol, trehalose, lactose, starch, crystalline cellulose, methylcellulose, ethylcellulose, calcium silicate, calcium phosphate, light anhydrous silicic acid, calcium carbonate, polyvinylpyrrolidone, talc, hardened oil, stearic acid, magnesium stearate, agar, pectin, gelatin, alginate, gum arabic, glycerin, vegetable oils such as sesame oil, olive oil, and soybean oil, hydroxypropylcellulose, low viscosity hydrogels, and the like. Examples of the non-ionic surfactant include hydroxypropyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, polyalcohols such as polyethylene glycol and propylene glycol, polysorbates, polyoxyethylene hydrogenated castor oil, benzyl benzoate, benzyl alcohol, ethylenediamine, paraoxybenzoic acid esters, phenol, cresol, chlorobutanol, carmellose sodium, sodium alginate, aluminum monostearate, fat emulsions, lecithin, citrates, acetates, and phosphates.

The pharmaceutical compositions described herein may also contain one or more other drugs. In this specification, a pharmaceutical composition generally refers to a drug for treating or preventing a disease, or for testing or diagnosing a disease.

The pharmaceuticals described herein are usually administered systemically or locally, orally or parenterally. The pharmaceuticals described herein can be administered to a living body, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, suppository, or topical preparation. The dosage of the pharmaceuticals described herein varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, and other conditions, and varies depending on various conditions. For example, the pharmaceuticals or active ingredients can be administered to an individual at 0.0001 to 100 mg/kg per day, preferably 0.01 to 100 mg/kg per day.

The pharmaceuticals described herein can be in the form of pharma- ceutical acceptable preparations. The preparations can be made into injections (injectable compositions) such as sterile solutions, suspensions, and lyophilized preparations, or into tablets, granules, powders, capsules, emulsions, suspensions, syrups, and the like, according to commonly used procedures.

When the pharmaceutical of the present invention is made into an injection (injectable composition), it can be administered by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, etc., and intravenous injection includes one-shot intravenous injection and drip intravenous injection.

The pharma- ceutically acceptable formulation may be, for example, an injectable sterile solution or suspension in water or another pharma- ceutically acceptable liquid. For example, the antibody or antigen-binding fragment described herein may be formulated by appropriately combining it with a pharma- ceutically acceptable carrier or vehicle, specifically, sterile water, saline, vegetable oil, emulsifier, suspending agent, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative, binder, etc., and mixing in a unit dose form. In these formulations, the active ingredient may be mixed with other ingredients to obtain the desired amount.

Sterile compositions for injection can be formulated according to conventional pharmaceutical practice using vehicles such as distilled water for injection, aqueous solutions for injection, or oily solutions.

Examples of aqueous solutions for injection include isotonic solutions containing physiological saline, glucose, and other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride). In addition to the aqueous solutions for injection, the injectable preparations described herein may contain appropriate solubilizing agents, such as alcohol (e.g., ethanol), polyalcohol (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants (e.g., Polysorbate 80(TM), HCO-50, etc.).

Examples of oily liquids include sesame oil and soybean oil. In addition to the oily liquid, the injectable preparation described in this specification may contain benzyl benzoate and/or benzyl alcohol as a solubilizing agent.

The injectable preparations described herein may further include a buffer (e.g., phosphate buffer and sodium acetate buffer), a soothing agent (e.g., procaine hydrochloride), a stabilizer (e.g., benzyl alcohol and phenol), and an antioxidant. The injectable preparations described herein may be filled into suitable ampoules.

The pharmaceuticals described herein can be used alone or in combination with other conventional treatment methods. In this case, the administration timing and treatment timing of the pharmaceuticals described herein and other drugs are not limited, and they may be administered to the subject at the same time or at different times.

In one embodiment, the present invention relates to a method for reducing the level 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 a condition associated with excess GH and/or excess IGF-1 as described above. The method may further comprise identifying an individual at risk for a disease associated with excess GH and/or excess IGF-1, assessing the individual for risk factors, and/or assessing or diagnosing the individual for a condition.

The following provides a more detailed explanation of the embodiments of the present invention using examples, but these are not intended to limit the present invention and may be modified without departing from the scope of the present invention.

Example 1: GH binding activity measurement method
Recombinant human GH (Pharmaceutical and Medical Device Regulatory Science Foundation) diluted to 5 μg/mL in Dulbecco's phosphate buffer solution (PBS) was immobilized at 50 μL/well on a 96-well half-area immunoplate (Corning) and blocked with PBS containing 1% bovine serum albumin (BSA). Then, 50 μL/well of a positive control (anti-GH mouse mAb (MAB1067, R&D Systems)) or a sample containing anti-GH mouse mAb diluted in assay buffer (PBS containing 0.2% BSA and 0.05% Tween 20) was added and incubated at room temperature for 2 hours. After washing the plate with washing buffer (PBS containing 0.05% Tween 20), 50 μL/well of HRP (Horseradish peroxidase)-labeled anti-mouse IgG goat antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted to 0.1 μg/mL in assay buffer was added and left to stand at room temperature for 2 hours. After further washing the plate with washing buffer, 50 μL/well of 3,3',5,5'-tetramethylbenzidine (TMB) solution was added as a substrate for HRP and reacted at room temperature for 10 minutes. After adding 0.5 mol/L _{H2SO4 aqueous} solution to stop the reaction, the absorbance at 450 nm was measured using a plate reader.

Example 2: GH neutralizing activity measurement method
To evaluate the GH neutralizing activity of anti-GH antibodies, (a) a GH-GH receptor (GHR) binding inhibition assay and (b) a GH-dependent Nb ₂ cell proliferation inhibition assay were established.

(a) GH-GHR binding inhibition assay
The GH-GHR binding inhibitory activity was measured by calculating the inhibition rate of the binding signal of biotin-labeled GH to GHR extracellular domain protein-human Fc fusion (GHR-hFc) immobilized on an ELISA plate via anti-human Fcγ mouse antibody. Biotin-labeled GH was prepared by labeling recombinant GH using the EZ-Link Micro NHS-PEG4-Biotinylation kit (Thermo Fisher Scientific). The specific method is as follows. Anti-human Fcγ mouse antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted in 1 μg/mL PBS was immobilized at 50 μL/well on a 96-well half-area immunoplate, and blocked with 1% BSA-containing PBS. Then, GHR-hFc (R&D Systems) diluted to 0.1 μg/mL in assay buffer (PBS containing 0.2% BSA and 0.05% Tween 20) was added at 50 μL/well to capture the antibody on the plate. After washing the plate with washing buffer (PBS containing 0.05% Tween 20), 50μL/well of a mixture of equal volumes of biotin-labeled GH diluted to a final concentration of 5.5ng/mL with assay buffer and a sample (anti-GH mouse mAb-containing sample) diluted with assay buffer was added and left at room temperature for 2 hours. As a positive control, unlabeled GH or anti-GH mouse mAb (MAB1067) was used instead of the sample. After washing the plate four times with washing buffer, 50μL/well of HRP-labeled streptavidin (Jackson ImmunoResearch Laboratories, Inc.) diluted to 0.1μg/mL with assay buffer was added and left at room temperature for 2 hours. After washing the plate six times, 50μL/well of TMB solution was added as a substrate for HRP and allowed to react at room temperature for 10 minutes. After the reaction was stopped by adding 0.5 mol/ _{L H2SO4} aqueous solution, the absorbance at 450 nm was measured using a plate reader. The GH-GHR binding inhibition rate of each sample was converted into IC50, with the absorbance when only biotin-labeled GH was added being 0% inhibition, and the absorbance when biotin-labeled GH was not added being ₁₀₀ % inhibition. The _IC50 of the positive control antibody MAB1067 under the above conditions was 6,860 pmol/L.

(b) GH-dependent _Nb2 cell proliferation inhibition assay As a cell-based GH neutralizing activity measurement method, _Nb2 rat lymphoma cell line proliferation assay, which is known as a standard GH activity measurement method, was used (Omae Y. et al., Endocrinol Japon 36, 9-13, 1989). The 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 cultured in Fischer's Medium containing 1% HS in a _CO2 incubator (37℃, 5% _CO2 ) for about 24 hours and placed in a serum-starved state. The cells were seeded at 2 x ¹⁰⁴ cells/well on a 96-well culture plate, and human GH (Pharmaceutical and Medical Device Regulatory Science Foundation) diluted with 0.1% BSA-containing PBS to a final concentration of 9.1 pmol/L ( _EC80 : 80% effective concentration) and anti-GH mAb specimen (5 nmol/L) diluted ⁵⁰ to ⁷ times with the same buffer were added at 10 μL/well. As a positive control, anti-GH mouse mAb (MAB1067) was used instead of the specimen. After culturing these cells in a _CO2 incubator for about 72 hours, WST-8 (Cell counting kit-8, Dojindo Laboratories) was added at 10 μL/well and reacted at 37 °C for about 3 hours, and the absorbance at 450 nm was measured using a plate reader to serve as an index of the number of live cells. The _IC50 was calculated by taking the absorbance when neither GH nor anti-GH antibody was added as 100% inhibition, and the absorbance when GH was added but not anti-GH antibody was 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 with GH derived from experimental animals
The cross-reactivity of anti-GH mouse mAb to GH derived from monkeys, mice, and rats was measured by competitive ELISA. The specific method is described below.

(a) Preparation of recombinant cynomolgus monkey GH The DNA sequence of monkey GH (NM_001290284.1) cloned from cynomolgus monkey pituitary cDNA was inserted into pIEx-4 insect cell expression vector (Merck Millipore). The vector was transfected into HighFive cell line (Invitrogen), and after 72 hours of rotational culture at 28°C, the culture supernatant was collected by centrifugation. This culture supernatant was applied to CaptureSelect hGH Affinity Matrix (Thermo Fisher Scientific) equilibrated with 20 mmol/L Tris-HCl buffer (pH 8.0) containing 150 mmol/L NaCl, washed with the same buffer containing 500 mmol/L NaCl, and then monkey GH adsorbed to the carrier was eluted with 20 mmol/L citrate buffer (pH 3.0) containing 150 mmol/L NaCl. The eluate was dialyzed against 20 mmol/L Tris-HCl buffer (pH 8.0) containing 150 mmol/L NaCl and 10% glycerol to obtain the final preparation (monkey GH). The purity of the monkey GH was >85% as determined by SDS-PAGE and size exclusion chromatography using Superdex 75 Increase 10/300 GL (GE Healthcare), and it showed activity of _EC50 = 9.8 pmol/L in the _Nb2 cell line proliferation assay described in Example 2(b).

(b) Cross-reactivity test
Recombinant human GH (Pharmaceutical and Medical Device Regulatory Science Foundation) diluted to 5 μg/mL in PBS was immobilized at 50 μL/well on a 96-well half-area immunoplate (Corning) and blocked with 1% BSA-containing PBS. Then, human GH serially diluted in assay buffer (PBS containing 0.2% BSA and 0.05% Tween20), cynomolgus monkey GH, mouse GH (LifeSpan BioSciences), and rat GH (Protein Laboratories Rehovot) prepared above, and anti-GH mouse mAb diluted to a final concentration of 30 ng/mL in the same buffer were added at 50 μL/well and allowed to stand at room temperature for 2 hours. After washing the plate with washing buffer (PBS containing 0.05% Tween 20), 50 μL/well of HRP-labeled anti-mouse IgG goat antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted to 0.1 μg/mL in assay buffer was added and left to stand at room temperature for 2 hours. After further washing the plate with washing buffer, 50 μL/well of TMB solution was added as HRP substrate and the enzyme reaction was carried out at room temperature. After 10 minutes, 0.5 mol/L H ₂ SO ₄ was added to stop the reaction, and the absorbance at 450 nm was measured using a plate reader. IC ₂₀ (nmol/L) of anti-GH mouse mAb was calculated from the concentration-dependent curve of human GH and GH derived from experimental animals, and the cross-reactivity against each experimental animal-derived GH was calculated using the following formula 1.

Cross-reactivity to GH derived from experimental animals (%) = IC ₂₀ (human GH)/IC ₂₀ (GH derived from experimental animals) × 100 ... (Formula 1)

Example 4: Cross-reactivity test of anti-GH mouse mAb to GH-like proteins (proteins with structural similarity to GH)
The cross-reactivity of anti-GH mouse mAb to proteins that have structural similarity to GH (placental lactogen (PL) and prolactin (PRL)) was measured by competitive ELISA. The specific method is described below.

Recombinant human GH (Pharmaceutical and Medical Device Regulatory Science Foundation) diluted to 5 μg/mL in PBS was immobilized at 50 μL/well on a 96-well half-area immunoplate (Corning) and blocked with 1% BSA-containing PBS. Then, human GH, PL (Protein Laboratories Rehovot), and PRL (BBT Boster Immunoleader) serially diluted in assay buffer (PBS containing 0.2% BSA and 0.05% Tween20) were mixed with anti-GH mouse mAb diluted to a final concentration of 30 ng/mL in the same buffer, and 50 μL/well was added and left to stand at room temperature for 2 hours. After washing the plate with washing buffer (PBS containing 0.05% Tween20), 50 μL/well of HRP-labeled anti-mouse IgG goat antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted to 0.1 μg/mL in assay buffer was added and left to stand at room temperature for 2 hours. After washing the plate with washing buffer, 50 μL/well of TMB solution was added as HRP substrate and the enzyme reaction was allowed to proceed at room temperature. After 10 minutes, 0.5 mol/ _{L H2SO4} was added to stop the reaction, and the absorbance at 450 nm was measured using a plate reader. _IC20 (nmol/L) of anti-GH mouse mAb was calculated from the concentration-dependent curves of human GH and GH-like proteins, and cross-reactivity with GH-like proteins was calculated using the following formula 2.
Cross-reactivity to GH-like proteins (%) = _IC20 (human GH)/ _IC20 (GH-like protein) 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 subcutaneously immunized with 5 μg or 10 μg of recombinant human GH (FUJIFILM Wako Pure Chemical Corporation, Japan Pharmaceutical and Medical Device Regulatory Science Foundation) twice a week for 7 to 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 given a final immunization with 10 μg of recombinant human GH intravenously or intraperitoneally, and 3 to 4 days later, the mice were euthanized, and lymph nodes and spleens were removed. The isolated lymphocytes were electrofused with mouse myeloma cell line P3U1 (JCRB0708, National Institutes of Biomedical Innovation, Health and Nutrition) and cultured in a semi-solid selection medium (STEMCELL Technologies) containing hypoxanthine-aminopterin-thymidine (HAT) in a _CO2 incubator for 8 to 12 days. The grown monoclonal hybridomas were picked using a colony picker, transplanted into a 96-well culture plate containing hypoxanthine-thymidine (HT)-containing liquid medium (STEMCELL Technologies), and cultured in a _CO2 incubator for 3 to 4 days.

A 10-fold dilution of the hybridoma culture supernatant thus obtained was subjected to the ELISA described in Example 1 to measure GH binding activity. A 2-fold dilution of the culture supernatant was also subjected to the 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 70% or more inhibitory activity in the GH-GHR binding inhibition assay were expanded in 24-well plates, and those whose culture supernatants showed recapitulation of GH binding activity and GH-GHR binding inhibition activity and produced a single IgG subtype were selected as neutralizing mAb-producing hybridomas and frozen and stored.
The anti-GH mouse mAb-producing hybridomas selected as above were expanded in DMEM medium containing 10% Ultra Law IgG, and the mAb was purified from the culture supernatant using rProtein A Sepharose FF (GE Healthcare) to obtain anti-GH mouse mAb. The purity was confirmed by SDS-PAGE, and the antibody concentration was quantified based on the absorbance at 280 nm, assuming a molecular extinction coefficient (1 mg/mL) of 1.38.

The mouse mAb preparations thus prepared were examined in the GH-GHR binding inhibition assay described in Example 2, the GH-dependent Nb ₂ cell proliferation inhibition assay, the cross-reactivity to experimental animal-derived GH described in Example 3, and the cross-reactivity to GH-like proteins described in Example 4. As a result, an anti-GH mouse mAb, 13H02 (IgG2a/κ), which was superior overall in terms of GH binding properties and GH neutralizing activity, was selected and obtained.

(2) Profile of anti-GH mouse mAb, 13H02 For anti-GH mouse mAb (13H02), the GH-GHR binding inhibitory activity ( _IC50 ) and GH-dependent _Nb2 cell proliferation inhibitory activity ( _IC50 ) measured by the method of Example 2 are shown in Table 1, the cross-reactivity to GH derived from laboratory animals measured by the method of Example 3 is shown in Table 2, and the cross-reactivity to GH-like proteins measured by the method of Example 4 is shown in Table 3. When a commercially available anti-GH mouse mAb (MAB1067) was used as a positive control, the results are also shown.

13H02 potently inhibited GH activity with _IC50 = 65.3 pmol/L in the GH-GHR binding assay and _IC50 = 3.36 pmol/L in the GH-dependent Nb ₂ cell proliferation assay. 13H02 did not bind substantially to mouse GH or rat GH with cross-reactivity <1%, but it did bind significantly to monkey GH with cross-reactivity of 41%. 13H02 also did not bind substantially to GH-like proteins (PL, PRL) with cross-reactivity <1%.

Example 6: Efficacy of anti-GH mouse mAb (13H02) in hypophysectomized/GH-replaced 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) (hereafter referred to as "hypophysectomized rats") were hypophysectomized at 4 weeks of age and showed decreased serum IGF-I due to endogenous GH deficiency. A 2-week Alzet osmotic pump (Muromachi Kikai Co., Ltd.) containing recombinant human GH (Sandoz Co., Ltd., 30 μg/day) was implanted subcutaneously. One day after the start of GH release, these hypophysectomized/GH-supplemented rats were given a single subcutaneous dose of 0.01, 0.1, 1, 5, or 10 mg/kg of 13H02 (n=5 or 6). Hypophysectomized/GH-unsupplemented rats (hypophysectomized rats not administered recombinant human GH) served as normal controls, and hypophysectomized/GH-supplemented rats were given control mouse IgG1 (Leinco Technologies, Inc., M1411) as negative controls. Serum IGF-I levels two days after administration of 13H02 are shown in Figure 1 (mean and standard error). 13H02 significantly reduced serum IGF-I levels in the 1, 5, and 10 mg/kg administration groups (###: p<0.001 vs. normal control group/Aspin-Welch t-test, *: p<0.05 vs. negative control group/Steel multiple comparison test).

Example 7: Determination of amino acid sequence of variable region of 13H02
From the hybridoma producing the anti-GH mouse mAb (13H02) selected in Example 5, mRNA was prepared using Dynabeads mRNA DIRECT Kit (ThermoFischer Scientific) according to the manual attached to the kit. cDNA was synthesized using SMARTer RACE 5'/3' Kit (Takara Bio Inc.) with 5 μL of the mRNA solution as a template, and the variable region genes (VH, VL) of the antibody H chain and L chain were amplified by PCR using 10× Universal Primer A Mix included in the kit as a forward primer, 5'-RACE Reverse primer designed from the constant region sequence of a known mouse antibody gene as a reverse primer, and KOD-Fx Neo (TOYOBO) as a polymerase.

The amplified PCR products were incorporated into the cloning vector pCR4Blunt-TOPO (Thermo Fisher Scientific) and then introduced into Stellar competent cells (Takara Bio Inc.) to obtain transformants. Plasmids were prepared from clones in which VH and VL inserts were confirmed by colony PCR using SapphireAmp Fast PCR Master Mix (Takara Bio Inc.), and the amino acid sequences were deduced from the determined variable region DNA sequences. CDRs 1 to 3 in the amino acid sequences of the H and L chain variable regions were determined using Discovery Studio (Biovia) according to the method of Kabat et al. The DNA and amino acid sequences 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 and amino acid sequences of the L chain variable region (VL), respectively. The amino acid sequence of mature 13H02_VH from which the signal peptide starting from position 20 of SEQ ID NO:19 has been removed is shown in SEQ ID NO:3, and 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 shown in SEQ ID NO:4. Complementarity determining regions (CDRs) 1, 2, and 3 of 13H02 VH were SSEIS (SEQ ID NO:5), WIYPGTGSSKFNQKFTG (SEQ ID NO:6), and RGFFGGSFDY (SEQ ID NO:7), respectively. CDRs 1, 2, and 3 of 13H02 VL were TATSSVSSSYLH (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-chain and L-chain constant region genes of human IgG1/κ antibody were introduced into pcDNA3.4 (Thermo Fisher Scientific) to prepare mouse-human chimeric antibody H-chain and L-chain expression cassette vectors (hereinafter, the mouse-human chimeric antibody H-chain and mouse-human chimeric antibody L-chain expression cassette vectors containing the constant region genes of human IgG1/κ antibody are also referred to as "human IgG1/κ mouse-human chimeric antibody (H-chain, L-chain) cassette vectors"). The VH and VL genes of 13H02 isolated in Example 7 were inserted into the human IgG1/κ mouse-human chimeric antibody (H-chain, L-chain) cassette vector to construct recombinant mouse-human chimeric antibody (H-chain, L-chain) expression vectors. The recombinant mouse-human chimeric antibody (H chain, L chain) expression vector was co-expressed in the ExpiCHO-S cell line at a H chain:L chain = 1:1 (weight ratio) using the ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific), and the cells were shaken in a _CO2 incubator shaker (37°C, 8% _CO2 ) for 8 days. The culture supernatant was collected by centrifugation, and the antibody was purified using rProtein A Sepharose FF (GE Healthcare) to obtain the recombinant anti-GH mouse-human chimeric mAb (Ch-13H02). Protein purity was confirmed by SDS-PAGE, and the antibody concentration was quantified based on the absorbance at 280 nm, assuming 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 Nb ₂ cell proliferation inhibition assay described in Example 2(b). The results are shown in Table 4.

Ch-13H2 has an _IC50 of 6.27 pmol/L, which is almost equivalent to the GH-dependent _Nb2 cell proliferation inhibitory activity of the parent mouse antibody (13H02) derived from the hybridoma, and it was confirmed that the H-chain and L-chain genes contributing to the activity were isolated from the 13H02-producing hybridoma.

Example 10: Humanization of anti-GH mouse mAb (13H02)
The humanized design of VH and VL of 13H02 was performed according to the report by Queen C. et al. (Proc. Natl. Acad. Sci. USA 86, 10029-10033, 1989) and the method by Tsurushita N. et al. (Methods 36, 69-83, 2005). Briefly, the process is as follows. First, a three-dimensional molecular model of VH and VL of 13H02 was constructed using JN Biosciences' proprietary algorithm, and amino acid residues important for maintaining the CDR structure within the framework region were identified. In addition, the human VH and VL amino acid sequences with the highest homology to the VH and VL of this antibody were identified from public databases, respectively. Then, the three CDR sequences from VH and VL of 13H02, together with amino acid residues important for maintaining the CDR structure within the framework region, were grafted into the human antibody framework region sequence specified above to design a humanized version of 13H02 (Hu-13H02).
The amino acid sequence of the humanized designed Hu-13H02_VH is shown in SEQ ID NO: 11, and the amino acid sequence of mature Hu-13H02_VH from which the signal peptide starting at position 20 of SEQ ID NO: 11 has been removed is shown in SEQ ID NO: 12. The amino acid sequence of Hu-13H02_VL is shown in SEQ ID NO: 13, and the amino acid sequence of mature Hu-13H02_VL from which the signal peptide starting at position 23 of SEQ ID NO: 13 has been removed is shown in SEQ ID NO: 14.

Example 11: Preparation of anti-GH humanized antibody
DNA (sequence number 15) encoding the VH amino acid sequence (sequence number 11) and DNA (sequence number 16) encoding the VL amino acid sequence (sequence number 13) of the anti-GH humanized antibody (Hu-13H02) designed in Example 10 were inserted into the human IgG1/κ mouse-human chimeric antibody (H chain, L chain) cassette vector constructed in Example 8 to construct recombinant human antibody (H chain, L chain) expression vectors (hereinafter also referred to as "Hu-13H02 (H chain, L chain) expression vectors").
In addition, to improve hemodynamics, an Fc variant (Hu-13H02m) was prepared in which the H chain Met ⁴²⁸ of Hu-13H02 was replaced with a Leu residue. Specifically, a gene encoding a human IgG1 constant region in which Met ⁴²⁸ of the H chain was replaced with Leu ⁴²⁸ was introduced into pcDNA3.4 to produce an H chain expression cassette vector in which DNA encoding the VH amino acid sequence of the anti-GH humanized antibody (Hu-13H02) was inserted. The L chain expression vector for Hu-13H02m was used (hereinafter, also referred to as "Hu-13H02m (H chain, L chain) expression vector").

Hu-13H02 or Hu-13H02m (H chain, L chain) expression vectors were co-expressed in ExpiCHO-S cell line at a H chain:L chain = 1:1 (weight ratio) using ExpiFectamine CHO Transfection Kit (Thermo Fisher Scientific), and cultured according to the Standard protocol or Max titer protocol of the expression system. The culture supernatant was collected by centrifugation, and the antibody was purified using HiTrap rProtein A FF Column (GE Healthcare) to obtain Hu-13H02 or Hu-13H02m preparations. Purity was confirmed by SDS-PAGE, and antibody concentration was quantified based on absorbance at 280 nm with a molecular extinction coefficient (1 mg/ml) = 1.38. Endotoxin content of the preparations used for the rat efficacy test was measured using Endospecy ES-50M (Seikagaku Corporation), and confirmed to be <1EU/mg. The Hu-13H02 or Hu-13H02m specimens prepared in this manner were used in the subsequent tests. The amino acid sequence of the heavy chain constant region of Hu-13H02m is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain constant region of Hu-13H02m is shown in SEQ ID NO: 18.

Example 12: Inhibitory activity of anti-GH chimeric antibody and anti-GH humanized antibody on GH-dependent _Nb2 cell proliferation
The recombinant anti-GH humanized antibody (Hu-13H02), the Fc variant of the anti-GH humanized antibody (Hu-13H02m), and the chimeric antibody (Ch-13H02) prepared in Example 11 were subjected to the GH-dependent Nb ₂ cell proliferation inhibition assay described in Example 2(b), and the IC ₅₀ for human GH was determined from the concentration-dependent curve. In addition, the IC ₅₀ for monkey GH was determined using the monkey GH prepared in Example 3 instead of the human GH in Example 2(b). 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. The results are shown in Table 5.

Hu-13H02 and Hu-13H02m showed potent inhibitory activity against human and monkey GH on the proliferation of GH-dependent _Nb2 cells, similar to Ch-13H02.

Example 13: Binding properties of anti-GH humanized antibodies to human GH and monkey GH
The binding characteristics of the recombinant anti-GH humanized antibody (Hu-13H02) and its Fc variant (Hu-13H02m) prepared in Example 11, and the chimeric antibody (Ch-13H02) prepared in Example 8 to human GH (Japan Pharmaceutical and Medical Device Regulatory Science Foundation) and monkey GH (Example 3) were measured by SPR using BIACORE 8K+ (GE Healthcare). Anti-human IgG (Fc) antibody was immobilized on a CM5 sensor chip, and the three types of antibody preparations (1 μg/mL) (Hu-13H02, Hu-13H02m, Ch-13H02) were captured. Next, human GH at 0.13 to 8.0 nmol/L or monkey GH at 0.50 to 32 nmol/L was injected, and the binding and dissociation parameters were calculated. The results are shown in Table 6.

Hu-13H02 and Hu-13H02m retained human GH binding activity almost equivalent to that of Ch-13H02, and also showed strong binding activity to monkey GH.

Example 14: Cross-reactivity of anti-GH chimeric antibodies and anti-GH humanized antibodies with GH-like proteins
The cross-reactivity of the recombinant anti-GH humanized antibody (Hu-13H02) and its Fc variant (Hu-13H02m) prepared in Example 11, and the parent chimeric antibody (Ch-13H02) prepared in Example 8, against proteins showing structural similarity to GH (placental lactogen (PL), prolactin (PRL)), was measured by competitive ELISA in the same manner as in Example 4. For each anti-GH antibody, the _IC50 (nmol/L) was determined from the concentration-dependence curves of human GH and GH-like proteins, and the cross-reactivity against GH-like proteins was calculated according to the following formula 3.
Cross-reactivity to GH analogues (%) = IC ₅₀ (human GH)/IC ₅₀ (GH analogues) × 100 (Equation 3)
The results are shown in Table 7.

Like the parent mouse antibody 13H02, Ch-13H02, Hu-13H02, and Hu-13H02m showed cross-reactivity to PL and PRL of <1%, and did not substantially bind to these GH-like proteins.

Example 15: Efficacy of anti-GH mouse-human chimeric mAb (Ch-13H02) and anti-GH humanized antibody Fc variant (Hu-13H02m) in hypophysectomized/GH-replaced 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-replaced rats as described in Example 6.
Hypophysectomized SD rats (Japan SLC, 6 weeks old) that had been hypophysectomized at 4 weeks of age and exhibited decreased serum IGF-I due to endogenous GH deficiency were subcutaneously implanted with a 3-day Alzet osmotic pump (Muromachi Kikai Co., Ltd.) containing recombinant human GH (Sandoz Co., Ltd., 30 μg/day). One day after the start of GH release, these hypophysectomized/GH-supplemented rats were subcutaneously administered a single dose of anti-GH antibodies (Ch-13H02, Hu-13H02m) at 1, 3, and 10 mg/kg, respectively (n=6). Hypophysectomized/GH-unsupplemented rats (hypophysectomized rats not administered recombinant human GH) were used as normal controls, and hypophysectomized/GH-supplemented rats were administered control human IgG1 (Bio X Cell, Inc., BE0297) as negative controls. Serum IGF-I levels two days after administration of Ch-13H02 and Hu-13H02m are shown in Figure 2 and Figure 3, respectively (mean and standard error). Ch-13H02 significantly reduced serum IGF-I levels in the 3 and 10 mg/kg groups, and Hu-13H02m significantly reduced serum IGF-I levels in the 3 and 10 mg/kg groups (###: p<0.001 vs. normal control group/Aspin-Welch t-test, *: p<0.05 vs. negative control group/Steel multiple comparison test)."

The sequences described herein are shown below.

Claims (10)

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 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;
A pharmaceutical composition for injection comprising an antibody or an antigen-binding fragment thereof, wherein the VL comprises the amino acid sequence of SEQ ID NO: 8 as VL complementarity determining region (CDR) 1 (VLCDR1), the amino acid sequence of SEQ ID NO: 9 as VLCDR2, and the amino acid sequence of SEQ ID NO: 10 as VLCDR3. The pharmaceutical according to claim 1, wherein the antibody or antigen-binding fragment binds to human growth hormone with an equilibrium dissociation constant (K _D ) of 1.0×10 ⁻⁸ mol/L or less as measured by surface plasmon resonance. The pharmaceutical according to claim 1, wherein the antibody or antigen-binding fragment neutralizes growth hormone action. The pharmaceutical of claim 1, wherein the antibody or antigen-binding fragment comprises a VH having the amino acid sequence of SEQ ID NO: 3, 11, 12, or 19, and/or a VL having the amino acid sequence of SEQ ID NO: 4, 13, 14, or 20. The pharmaceutical according to any one of claims 1 to 4, wherein the antibody is a chimeric antibody, a humanized antibody, a veneered antibody, or a fully human antibody. The pharmaceutical according to any one of claims 1 to 4, 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. The pharmaceutical according to any one of claims 1 to 4, wherein the antigen-binding fragment is Fab, Fab', F(ab')2, Fv, or scFv. The pharmaceutical agent according to any one of claims 1 to 4 for reducing the level of insulin-like growth factor 1 (IGF-1) in the blood. The pharmaceutical agent according to any one of claims 1 to 4 for treating and/or preventing a disease selected from the group consisting of acromegaly, gigantism, cancer, diabetic nephropathy, arthritis, and pulmonary inflammation. The pharmaceutical according to any one of claims 1 to 4, wherein the dose of the antibody or antigen-binding fragment thereof is 0.0001 to 100 mg/kg per day.