Anti-IGF-I receptor humanized antibody

Abstract

The present invention provides a humanized antibody that increases muscle mass via an IGF-I receptor without lowering blood sugar levels. Such a humanized antibody is an anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof, which has each specific amino acid sequence of SEQ ID NO: 1 to 6 as each CDR sequence, and specifically binds to the extracellular domain of the IGF-I receptor.

Description

Technical Field

[0001] The present invention relates to an anti-IGF-I receptor humanized antibody, and more particularly, to an anti-IGF-I receptor humanized antibody that specifically binds to the IGF-I receptor of vertebrates. Background Art

[0002] 1. IGF-I

[0003] IGF-I is insulin-like growth factor, by the growth hormone (GH) secreted by the pituitary gland activating GH receptor, and mainly secreted by the liver, and with the IGF-I receptor effect, thereby embodying various physiological functions in various organs. Therefore, IGF-I is expected to treat various diseases. IGF-I has up to about 40% similarity compared with the amino acid sequence of proinsulin, so it is sometimes combined with insulin receptor and embodies insulin-like effect (non-patent literature 1). In addition, IGF-I receptor has up to about 60% similarity compared with the amino acid sequence of insulin receptor, so these two receptors sometimes form heterodimers (non-patent literature 1). It should be noted that insulin embodies potent hypoglycemic effect by acting with insulin receptor, so it is used for treatment as hypoglycemic drug.

[0004] 2. IGF-I receptor

[0005] IGF-I receptor is a transmembrane protein, which is composed of an α chain and a β chain, and contains six extracellular domains, namely L1, CR, L2, Fn1, Fn2 and Fn3, a transmembrane domain and an intracellular domain (Non-patent Document 2). The intracellular domain of the IGF-I receptor has a tyrosine kinase. The cysteine-rich domain (CR: cysteine-rich domain) as the extracellular domain is involved in the activation of intracellular tyrosine kinases accompanied by changes in the three-dimensional structure of the receptor when IGF-I binds to the IGF-I receptor. If the IGF-I receptor forms a homodimer complex (homotype) and IGF-I binds to it, the signal is transmitted by activating the receptor kinase. In addition, if a heterodimer complex (heterotype) is formed with the insulin receptor and insulin or IGF-I binds to it, the signal is transmitted by activating the receptor kinase (Non-patent Documents 3, 4).

[0006] 3. Physiological effects of IGF-I

[0007] IGF-I has been shown to have growth-promoting effects such as increasing height or weight, and insulin-like metabolic effects such as promoting sugar metabolism or hypoglycemic effects. It has been confirmed that Mecasermin, a human recombinant IGF-I, improves symptoms such as hyperglycemia, hyperinsulinemia, melanoma, and hirsutism in patients with insulin receptor abnormalities. In addition, it has been confirmed that it improves growth disorders in growth hormone-resistant dwarfism (Non-Patent Document 5).

[0008] 4. Growth-promoting effects of IGF-I

[0009] It is known that IGF-I increases the DNA synthesis ability of human chondrocytes. In addition, administration of IGF-I increases the body weight of rats from which pituitary glands have been removed and lengthens the femur (Non-Patent Document 5).

[0010] 5. IGF-I's muscle mass-increasing effect

[0011] The hyperactivity of cell proliferation activity mediated by IGF-I requires the continuous activation of IGF-I receptors (non-patent document 6). Muscle mass increases in animals in which IGF-I receptors are overexpressed (non-patent document 7). In addition, the continuous administration (administration) of IGF-I / IGFBP3 can increase the grip strength of patients with proximal femoral fractures and improve the ability to stand up from a seat without assistance (non-patent document 8). It is known that the IGF-I concentration in the muscles of elderly people and mice decreases compared to younger people (non-patent documents 9, 10), but in elderly mice in which IGF-I is specifically forced to be expressed in muscle tissue, muscle mass improves compared to wild-type mice (non-patent document 11).

[0012] 6. Existing products that increase muscle mass

[0013] Anamorelin, a ghrelin receptor agonist, increased lean body mass in a clinical trial for cachexia, a disuse muscular atrophy, but nausea and an increase in blood sugar levels were observed as side effects (Non-Patent Document 12).

[0014] Myostatin is a negative regulator of skeletal muscle formation that acts on activin receptor II (ActRII) to inhibit Akt / mTOR (Non-Patent Documents 13-15). LY2495655, an anti-myostatin antibody, increases muscle mass in patients who have undergone total hip replacement and the elderly (Non-Patent Documents 16, 17).

[0015] In addition, bimagrumab, an anti-ActRII antibody, increases muscle mass in patients with neuromuscular diseases (Non-Patent Document 18). However, there is currently no drug that can promote skeletal muscle formation and be used for treatment.

[0016] 7. Hypoglycemic effect of IGF-I

[0017] As an insulin-like action of IGF-I, it is known to have a hypoglycemic effect. IGF-I makes glucose uptake hyperactive in cells from rat muscle (Non-patent Document 5). In addition, administration of IGF-I can reduce the blood sugar level of rats (Non-patent Document 5).

[0018] It is reported that the hypoglycemic effect of IGF-I can cause hypoglycemia, which is a clinical side effect (Non-Patent Document 19). In addition, IGF-I causes hypoglycemia by administration to humans, so appropriate doses are administered sequentially from a low dose at the beginning of treatment, and various clinical findings including blood sugar levels after administration need to be observed (Non-Patent Document 5).

[0019] IGF-I manifests hypoglycemic effect through the hyperphosphorylation of Akt, which is the downstream signal of IGF-I receptor. The active mutant of Akt causes the glucose uptake of 3T3-L1 cells to be hyperactive (non-patent literature 20). On the other hand, the blood sugar level of mice with Akt2 deficiency rises (non-patent literature 21). In addition, in cells from rat muscle, Akt inhibitors inhibit glucose uptake based on insulin stimulation (non-patent literature 22). Moreover, it is known that IGF-I activates insulin receptors involved in hypoglycemic effect. It is believed from these results that: excessive activation of Akt and activation of insulin receptors are involved in the hypoglycemic effect of IGF-I.

[0020] 8. Short blood half-life of IGF-I

[0021] Since IGF-I has a short half-life in the blood, it needs to be administered frequently during treatment. In fact, the half-life of mecasermin, a human recombinant IGF-I, in the blood is about 11 to 16 hours, and it needs to be administered once or twice a day for the treatment of dwarfism (Non-Patent Document 5).

[0022] About 70 to 80% of IGF-I in the blood is bound to IGFBP3. Free IGF-I exhibits physiological activity. Binding to IGFBP3 maintains the half-life of IGF-I in the blood for about 10 to 16 hours (Non-Patent Document 1).

[0023] IPLEX, a combined preparation of IGF-I and IGFBP3, has a longer half-life in the blood of about 21 to 26 hours than IGF-I, and is a drug that can be administered once a day (Non-Patent Document 23). However, IPLEX is being withdrawn from the market.

[0024] Attempts have also been made to develop PEGylated IGF-I with improved kinetics of IGF-I, but no drug for treatment exists (Patent Document 1).

[0025] 9. Expected therapeutic effects through the action of IGF-I

[0026] It is known that IGF-I acts on a variety of organs and has a wide range of physiological functions (Non-Patent Document 19).

[0027] It is reported that IGF-I has mitochondrial protection and neuroprotective effects based on antioxidant effects via activation of IGF-I receptors in the central nervous system (Non-Patent Documents 24, 25). IGF-I promotes the formation of neurites after injury (Non-Patent Document 26).

[0028] IGF-I is a major factor for promoting growth (Non-Patent Documents 27 and 28). In fact, mecasermin, which is a human recombinant IGF-I, is clinically used as a therapeutic drug for dwarfism.

[0029] IGF-I is believed to be effective in treating liver cirrhosis. Liver cirrhosis is a disease that develops as a result of liver damage or chronic liver disease, and is accompanied by liver fibrosis. In liver cirrhosis model animals, administration of IGF-I inhibited liver fibrosis (Non-Patent Document 29).

[0030] IGF-I is also known to be involved in the development and function of the kidney. In renal mesangial cells, IGF-I has a protective effect against oxidative stress and apoptosis caused by glucotoxicity (Non-Patent Document 30). IGF-I is expected to be a therapeutic drug for kidney disease.

[0031] Pathological conditions that are expected to be improved by administering IGF-I include: dwarfism, Laron's disease, cirrhosis, liver fibrosis, aging, intrauterine growth retardation (IUGR), neurological diseases, stroke, spinal cord injury, cardiovascular protection, diabetes, insulin resistance, metabolic syndrome, kidney disease, osteoporosis, cystic fibrosis, wound healing, myotonic dystrophy, AIDS sarcopenia, HIV-associated fat redistribution syndrome, burns, Crohn's disease, Werner syndrome, X-linked combined immunodeficiency, hearing loss, anorexia nervosa and retinopathy of prematurity (Non-Patent Document 19).

[0032] IGF-I is expected to be used as a therapeutic drug for various diseases due to its rich physiological effects. However, its hypoglycemic effect as a side effect and the need for multiple administrations due to its short half-life have become issues in its clinical application.

[0033] 10. IGF-I receptor agonist antibody

[0034] Antibody preparations generally have a long half-life and are effective when administered once or twice a month. Although there are reports of IGF-I receptor agonist antibodies that activate the receptor in vitro, there are no reports of antibodies that exhibit agonist activity against the IGF-I receptor in vivo (Non-Patent Documents 31 to 35).

[0035] Antibody 3B7 and antibody 2D1 promote DNA synthesis in cells in vitro (Non-Patent Document 32).

[0036] Antibodies 11A1, 11A4, 11A11, and 24-57 enhance tyrosine phosphorylation of the IGF-I receptor in vitro (Non-Patent Document 33).

[0037] Antibodies 16-13, 17-69, 24-57, 24-60, 24-31, and 26-3 were shown to have effects of promoting cellular DNA synthesis and glucose uptake in vitro, and these antibodies are likely to have hypoglycemic effects (Non-Patent Documents 34 and 35).

[0038] However, to date, there are no reports of IGF-I receptor agonist antibodies showing cell proliferation activity in vitro using primary cultured cells, especially human myoblasts, and even fewer reports of IGF-I receptor agonist antibodies showing muscle mass increasing effects in vivo.

[0039] 11. IGF-I receptor antagonist antibody

[0040] Regarding antibodies that bind to the IGF-I receptor, attempts have been made to use them for the treatment of malignant tumors, etc., by utilizing their antagonist effect of inhibiting the binding of IGF-I to the IGF-I receptor. However, existing IGF-I receptor antagonist antibodies not only have many side effects such as hyperglycemia in single treatment (Non-Patent Document 36), but also increase the incidence of hyperglycemia when used in combination with other anticancer drugs (Non-Patent Document 37), and therefore their applicability in treatment is considered to be limited.

[0041] Prior art literature

[0042] Patent Literature

[0043] Patent Document 1: Japanese Patent Application No. 2011-518778;

[0044] Non-patent literature

[0045] Non-patent document 1: Ohlsson, C. et al., The role of liver-derived insulin-likegrowth factor-I. Endocr Rev, 2009. 30 (5): 494-535;

[0046] Non-patent literature 2: Kavran, JM et al., How IGF-I activates its receptor. Elife, 2014. 3;

[0047] Non-patent document 3: Bailyes, EM et al., Insulin receptor / IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting. Biochem J, 1997. 327 (Pt 1): 209-15;

[0048] Non-patent document 4: Pandini, G. et al., Insulin / insulin-like growth factor I hybrid receptors have different biological characteristics depending on theinsulin receptor isoform involved. J Biol Chem, 2002. 277 (42): p. 39684-95;

[0049] Non-patent document 5: Orphan Pacific, IF. 2015;

[0050] Non-patent document 6: Fukushima, T. et al., Phosphatidylinositol 3-kinase (PI3K) activity bound to insulin-like growth factor-I (IGF-I) receptor, which iscontinuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation. J Biol Chem, 2012. 287 (35): Page 29713-21;

[0051] Non-patent literature 7: Schiaffino, S. and C. Mammucari, Regulation of skeletal muscle growth by the IGF-I-Akt / PKB pathway: insights from genetic models. Skelet Muscle, 2011. 1 (1): Page 4;

[0052] Non-patent literature 8: Boonen, S et al., Musculoskeletal effects of the recombinanthuman IGF-I / IGF binding protein-3 complex in osteoporotic patients with proximal femoral fracture: a double-blind, placebo-controlled pilot study. JClin Endocrinol Metab, 2002. 87 (4): Page 1593-9;

[0053] Non-patent document 9: Barton-Davis, ER et al., Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function. Proc Natl Acad Sci USA, 1998. 95 (26): p. 15603-7;

[0054] Non-patent document 10: Lamberts, SW, AW van den Beld, and AJ van der Lely, The endocrinology of aging. Science, 1997. 278 (5337): 419-24;

[0055] Non-patent document 11: Musaro, A. et al., Localized IGF-I transgene expressions sustains hypertrophy and regeneration in senescent skeletal muscle. NatGenet, 2001. 27 (2): 195-200;

[0056] Non-patent literature 12: Temel, JS et al., Anamorelin in patients with non-small-cell lung cancer and cachexia (ROMANA 1 and ROMANA 2): results from two randomized, double-blind, phase 3 trials. Lancet Oncol, 2016. 17 (4): 519-31.

[0057] Non-patent literature 13: Glass, DJ, Signaling pathways perturbing muscle mass. Curr Opin Clin Nutr Metab Care, 2010. 13 (3): pp. 225-9;

[0058] Non-patent document 14: Lee, SJ and AC McPherron, Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci USA, 2001. 98 (16): pp. 9306-11;

[0059] Non-patent literature 15: Amirouche, A. et al., Down-regulation of Akt / mammalian target of rapamycin signaling pathway in response to myostatin overexpression in skeletal muscle. Endocrinology, 2009. 150 (1): 286-94;

[0060] Non-patent literature 16: Woodhouse, L. et al., A Phase 2 Randomized Study Investigating the Efficacy and Safety of Myostatin Antibody LY2495655 versus Placebo in Patients Undergoing Elective Total Hip Arthroplasty. J FrailtyAging, 2016. 5 (1): Pages 62-70;

[0061] Non-patent literature 17: Becker, C. et al., Myostatin antibody (LY2495655) in older weak fallers: a proof-of-concept, randomized, phase 2 trial. Lancet Diabetes Endocrinol, 2015. 3 (12): 948-57;

[0062] Non-patent literature 18: Amato, AA et al., Treatment of sporadic inclusion body myositis with bimagrumab. Neurology, 2014. 83 (24): pp. 2239-46;

[0063] Non-patent literature 19: Puche, JE and I. Castilla-Cortazar, Human conditions ofinsulin-like growth factor-I (IGF-I) deficiency. J Transl Med, 2012. 10: 224;

[0064] Non-patent document 20: Kohn, AD et al., Expression of a constitutively active Akt Ser / Thr kinase in 3T3-L1 adipocytes stimulates glucose uptake and glucosetransporter 4 translocation. J Biol Chem, 1996. 271 (49): p. 31372-8;

[0065] Non-patent document 21: Cho, H. et al., Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science, 2001. 292 (5522): 1728-31;

[0066] Non-patent document 22: Green, CJ et al., Use of Akt inhibitor and a drug-resistant mutant validates a critical role for protein kinase B / Akt in theinsulin-dependent regulation of glucose and system A amino acid uptake. J Biol Chem, 2008. 283 (41): p. 27653-67;

[0067] Non-patent literature 23: FDA application information, APPLICATION NUMBER 21-884;

[0068] Non-patent document 24: Garcia-Fernandez, M. et al., Low doses of insulin-like growth factor I improve insulin resistance, lipid metabolism, and oxidative damage in aging rats. Endocrinology, 2008. 149 (5): 2433-42;

[0069] Non-patent document 25: Puche, JE et al., Low doses of insulin-like growth factor-I induce mitochondrial protection in aging rats. Endocrinology, 2008.149 (5): 2620-7;

[0070] Non-patent literature 26: Joseph D'Ercole, A. and P. Ye, Expanding the mind: insulin-like growth factor I and brain development. Endocrinology, 2008. 149 (12): 5958-62;

[0071] Non-patent document 27: Abuzzahab, MJ et al., IGF-I receptor mutations resulting in intrauterine and postnatal growth retardation. N Engl J Med, 2003. 349(23): 2211-22;

[0072] Non-patent document 28: Woods, KA et al., Intrauterine growth retardation and postnatal growth failure associated with deletion of the insulin-like growth factor I gene. N Engl J Med, 1996. 335 (18): p. 1363-7;

[0073] Non-patent document 29: Perez, R. et al., Mitochondrial protection by low doses ofinsulin-like growth factor-I in experimental cirrhosis. World J Gastroenterol, 2008. 14 (17): pp. 2731-9;

[0074] Non-patent document 30: Kang, BP et al., IGF-I inhibits the mitochondrial apoptosis program in mesangial cells exposed to high glucose. Am J Physiol Renal Physiol, 2003. 285 (5): p. F1013-24;

[0075] Non-patent document 31: Bhaskar, V. et al., A fully human, allosteric monoclonal antibody that activates the insulin receptor and improves glycemic control. Diabetes, 2012. 61 (5): 1263-71;

[0076] Non-patent document 32: Xiong, L. et al., Growth-stimulatory monoclonal antibodies against human insulin-like growth factor I receptor. Proc Natl Acad Sci USA, 1992. 89 (12): 5356-60;

[0077] Non-patent document 33: Runnels, HA et al., Human monoclonal antibodies to theinsulin-like growth factor 1 receptor inhibit receptor activation and tumor growth in preclinical studies. Adv Ther, 2010. 27 (7): 458-75;

[0078] Non-patent document 34: Soos, MA et al., A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding, and biological activity. J Biol Chem, 1992. 267 (18): p. 12955-63;

[0079] Non-patent literature 35: Kato, H. et al., Role of tyrosine kinase activity in signal transduction by the insulin-like growth factor-I (IGF-I) receptor. Characterization of kinase-deficient IGF-I receptors and the action of anIGF-I-mimetic antibody (alpha IR-3). J Biol Chem, 1993. 268 (4): Page 2655-61;

[0080] Non-patent literature 36: Atzori, F. et al., A Phase I Pharmacokinetic and Pharmacodynamic Study of Dalotuzumab (MK-0646), an Anti-Insulin-like Growth Factor-1 Receptor Monoclonal Antibody, in Patients with Advanced Solid Tumors. Clin Cancer Res., 2011. 17 (19): 6304-12;

[0081] Non-patent literature 37: de Bono JS et al., Phase II randomized study of offigitumumab plus docetaxel and docetaxel alone with crossover formetastaticcastration-resistant prostate cancer. Clin Cancer Res., 2014. 20(7): 1925-34. Summary of the invention

[0082] Problems to be solved by the invention

[0083] One of the objects of the present invention is to provide an anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof, which specifically binds to the IGF-I receptor of a vertebrate. Another object of the present invention is to provide an antibody which increases muscle mass via the IGF-I receptor without lowering blood sugar levels.

[0084] Means for solving problems

[0085] That is, the present invention relates to the following contents.

[0086] [1] An anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof, comprising: the amino acid sequence of SEQ ID NO: 1 as the CDR-1 (CDR-H1) sequence of the heavy chain variable region, or an amino acid sequence in which one amino acid residue is substituted in the amino acid sequence of SEQ ID NO: 1; the amino acid sequence of SEQ ID NO: 2 as the CDR-2 (CDR-H2) sequence of the heavy chain variable region, or an amino acid sequence in which one or two amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 2; the amino acid sequence of SEQ ID NO: 3 as the CDR-3 (CDR-H3) sequence of the heavy chain variable region, or an amino acid sequence in which one or two amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 3; the amino acid sequence of SEQ ID NO: 4 as the CDR-1 (CDR-L1) sequence of the light chain variable region, or an amino acid sequence in which one or two amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 4 as the amino acid sequence in which one or two amino acid residues are substituted; the amino acid sequence of SEQ ID NO: 5 as the CDR-2 (CDR-L2) sequence of the light chain variable region, or an amino acid sequence in which one amino acid residue is substituted in the amino acid sequence of SEQ ID NO: 5; and the amino acid sequence of SEQ ID NO: 6 as the CDR-3 (CDR-L3) sequence of the light chain variable region, or an amino acid sequence in which one or two amino acid residues are substituted in the amino acid sequence of SEQ ID NO: 6, and specifically binds to the extracellular domain of SEQ ID NO: 14 (human IGF-I receptor).

[0087] [2] The anti-IGF-I receptor humanized antibody or its fragment or derivative thereof described in [1], wherein the anti-IGF-I receptor humanized antibody or its fragment or derivative thereof comprises: as the amino acid sequence of the heavy chain variable region, the amino acid sequence comprises the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 7 are substituted, deleted or added; and as the amino acid sequence of the light chain variable region, the amino acid sequence comprises the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence in which one or more amino acid residues in the amino acid sequence of SEQ ID NO: 8 are substituted, deleted or added.

[0088] [3] The anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof described in [1] or [2], wherein the anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof comprises: an amino acid sequence of SEQ ID NO: 7 as a heavy chain variable region; and an amino acid sequence selected from any one of SEQ ID NOs: 8, 9, 10, 11 and 12 as a light chain variable region.

[0089] [4] The anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof according to [1] to [3], further comprising a constant region of various types of human immunoglobulins as a constant region.

[0090] [5] The anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof according to [4], wherein the heavy chain constant region is a heavy chain constant region of the human IgG4 class.

[0091] [6] An anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof, according to any one of [1] to [5], which binds to an epitope or its vicinity comprising a peptide having an amino acid sequence (ProSerGlyPheIleArgAsnGlySerGlnSerMet) corresponding to amino acid numbers 308 to 319 of SEQ ID NO: 14 (human IGF-I receptor).

[0092] [7] The anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof according to any one of [1] to [6], which does not induce glucose uptake in cultured human or guinea pig myoblasts at a dose that induces proliferation of the cultured cells.

[0093] [8] The anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof according to any one of [1] to [7], which, when administered to a vertebrate at a dose that induces an increase in muscle mass and / or body length in the vertebrate, does not reduce the blood glucose level in the vertebrate.

[0094] [9] The anti-IGF-I receptor humanized antibody or its fragment, or its derivative, described in [8] does not reduce the blood glucose level of the vertebrate even at a blood exposure amount that is 10 times or more of the effective dose administered to the vertebrate to induce an increase in muscle mass and / or body length of the vertebrate.

[0095]

[10] A nucleic acid molecule consisting of a polynucleotide sequence encoding the anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof according to any one of [1] to [9].

[0096]

[11] A cloning vector or an expression vector comprising at least one nucleic acid molecule according to

[10] .

[0097]

[12] A recombinant cell (recombinant cell), which is obtained by introducing the vector described in

[11] into a host cell.

[0098]

[13] A method for producing an anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof according to any one of [1] to [9], the method comprising the steps of culturing the recombinant cell according to

[12] and purifying the anti-IGF-I receptor humanized antibody or a fragment thereof, or a derivative thereof produced by the recombinant cell.

[0099]

[14] A pharmaceutical composition comprising as an active ingredient the anti-IGF-I receptor humanized antibody or fragment thereof, or derivative thereof according to any one of [1] to [9], the nucleic acid molecule according to

[10] , the vector according to

[11] , or the recombinant cell according to

[12] .

[0100]

[15] The pharmaceutical composition described in

[14] is used to treat muscular dystrophy or dwarfism.

[0101]

[16] The pharmaceutical composition of

[15] , wherein the muscle wasting disease is disuse muscle atrophy, sarcopenia or cachexia.

[0102]

[17] The pharmaceutical composition of

[15] , wherein the dwarfism is Laron dwarfism or growth hormone-resistant dwarfism.

[0103] Effects of the Invention

[0104] The antibody or fragment thereof or derivative thereof of the present invention has the effect of specifically binding to the IGF-I receptor of vertebrates. BRIEF DESCRIPTION OF THE DRAWINGS

[0105] [ Figure 1 ] Figure 1 It is a diagram showing the results of comparison of the amino acid sequences (amino acid sequences are shown in single-letter symbols) of the CR domain of the IGF-I receptor in mouse, rat, human, guinea pig and rabbit.

[0106] [ Figure 2 ] Figure 2 The graph shows the weight of the extensor digitorum longus muscle of guinea pigs 2 weeks after continuous administration of IGF-I by an osmotic pump or a single intravenous administration of the anti-IGF-I receptor humanized antibody R11-16B.

[0107] [ Figure 3 ] Figure 3This is a graph showing the changes over time in the blood dynamics of guinea pigs under fasting conditions after a single subcutaneous administration of IGF-I.

[0108] [ Figure 4 ] Figure 4 This is a graph showing the changes in blood dynamics over time when the anti-IGF-I receptor humanized antibody R11-16B was administered intravenously once to guinea pigs under fasting conditions. DETAILED DESCRIPTION

[0109] The present invention is described below based on specific embodiments, but the present invention is not limited to these embodiments. It should be noted that all documents cited in this specification, including patent gazettes, patent application publication gazettes and non-patent gazettes, are incorporated into this specification by reference in their entirety for all purposes.

[0110] [IGF]

[0111] In the present disclosure, "IGF" refers to insulin-like growth factor, and there are IGF-I and IGF-II. IGF-I and IGF-II are ligands in vivo with agonist activity, which bind to the IGF-I receptor (Insulin-like Growth Factor-I Receptor) described later and transmit cell division or metabolism signals to the cell. It is known that IGF-I and IGF-II also weakly cross-bind to the insulin receptor (INSR), which has structural similarities with the IGF-I receptor. In this specification, although the more well-known IGF-I, such as physiological functions, is mainly described, in the case of studying the effects or diseases mediated by the binding of the IGF-I receptor to the ligand, the effects of both IGF-I and IGF-II are sometimes also recorded.

[0112] IGF-I, also known as somatomedin C, is a hormone of a single polypeptide consisting of 70 amino acids. The sequence of human IGF-I can be obtained with reference to UniProtKB-Accession No. P50919 of EMBL-EBI. SEQ ID NO: 13 of the sequence table shows the amino acid sequence of mature IGF-I. The sequence consisting of 70 amino acids is preserved in multiple species. In the present invention, when it is only recorded as "IGF-I", unless otherwise specified, it means an IGF-I protein with hormonal activity.

[0113] IGF-I is produced in various cells in the organism headed by hepatocytes, and is also present in blood or other body fluids. Therefore, natural IGF-I can be purified from the body fluid of animals, or from cultures of primary cultured cells or strains of cells (cell lines) isolated from animals. In addition, IGF-I is produced in cells by growth hormone induction, so it can also be obtained by purifying IGF-I from the body fluid of animals given growth hormone, or from cultures of primary cultured cells or strains of cells isolated from animals in the presence of growth hormone. In addition, as other methods, recombinant cells or transgenic animals or transgenic plants into which IGF-I genes are introduced (introduced) can also be used to manufacture IGF-I, and the above-mentioned recombinant cells are obtained by inserting a nucleic acid molecule encoding the amino acid sequence of IGF-I into an expression vector, and then introducing it into a host of prokaryotes such as Escherichia coli or eukaryotic cells such as yeast, insect cells or cultured cells from mammals. In addition, human IGF-I can also be used as a research reagent (Enzo Life Sciences, catalog: ADI-908-059-0100; Abnova, catalog: page 3452, etc.), a drug (Somazon (注册商标) , mecasermin, INCRELEX (注册商标) The in vivo and in vitro activities of the IGF-I used can be evaluated by comparing the activity in the IGF-I standard substance with the NIBSC code: 91 / 554 of the National Institute for Biological Standards and Control (NIBSC) of the World Health Organization as 1 international unit / microgram. The IGF-I in the present invention is described as an IGF-I having the same specific activity as the IGF-I with the NIBSC code: 91 / 554.

[0114] [IGF-I receptor]

[0115] In the present disclosure, "IGF-I receptor" refers to insulin-like growth factor-I receptor (Insulin-like Growth Factor-I Receptor). In this specification, unless otherwise specified, "IGF-I receptor" means IGF-I receptor protein. IGF-I receptor is a protein with a structure formed by the association of two subunits consisting of an α chain and a β chain. In the amino acid sequence of the human IGF-I receptor shown in SEQ ID NO: 14, the part consisting of the amino acid sequence from the 31st to the 735th position in its amino acid sequence is equivalent to the α chain, and the β chain is equivalent to the sequence after the 740th position. The α chain of the IGF-I receptor has a binding part for IGF-I, and the β chain is a transmembrane structure that plays a role in transmitting signals into the cell. The α chain of the IGF-I receptor is divided into the domains of L1, CR, L2, FnIII-1 and FnIII2a / ID / FnIII2b. In the amino acid sequence of human IGF-I receptor shown in SEQ ID NO: 14, the portion from position 31 to position 179 corresponds to the L1 domain, the portion from position 180 to position 328 corresponds to the CR domain, the portion from position 329 to position 491 corresponds to the L2 domain, the portion from position 492 to position 607 corresponds to the FnIII-1 domain, and the portion from position 608 to position 735 corresponds to the FnIII-2a / ID / FnIII-2b domain. Among them, the CR domain (cysteine-rich domain) is involved in the activation of the intracellular tyrosine kinase of the β chain accompanied by the change of the three-dimensional structure of the receptor when IGF-I binds to the IGF-I receptor. The amino acid sequence of human IGF-I receptor can be referred to UniProtKB-Accession No. P08069 of EMBL-EBI, etc., and is also shown in SEQ ID NO: 14 in the sequence table.

[0116] Known IGF-I receptor is expressed in the wide range of tissues or cells in vivo, and accepts stimulation such as induction of cell proliferation based on IGF-I or activation of intracellular signal. Particularly, myoblast can be used for evaluating the effect of IGF-I mediated by IGF-I receptor with cell proliferation activity as index. It can be known from this that myoblast is effective in analyzing the effect of antibodies combined with IGF-I receptor. In addition, in the recombinant cell obtained by inserting the nucleic acid molecule of the amino acid sequence of IGF-I receptor of coding people or other vertebrates into the expression vector, then importing into the host of eukaryotic cells such as insect cells or cultured cells from mammals, by making the IGF-I receptor encoded by the nucleic acid imported into its cell membrane express, the cell of IGF-I receptor of expression people or other vertebrates can be artificially manufactured. This IGF-I receptor expression cell can be used for analyzing the binding of antibodies or studying the signal transmission to the cell, etc.

[0117] [Anti-IGF-I receptor humanized antibody]

[0118] According to one aspect of the present invention, a novel anti-IGF-I receptor humanized antibody (hereinafter, for convenience, referred to as "the antibody of the present invention") is provided.

[0119] In the present disclosure, "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains bound to each other by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region, and the heavy chain constant region comprises three domains, namely CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region comprises one domain, namely CL. In the constant region of the light chain, there are two types called λ chain and κ chain. In the constant region of the heavy chain, there are γ chain, μ chain, α chain, δ chain and ε chain, and according to the difference of its heavy chain, there are antibody isotypes such as IgG, IgM, IgA, IgD and IgE. VH and VL are further subdivided into: 4 more conserved regions (FR-1, FR-2, FR-3, FR-4) called framework regions (FR) and 3 regions of hypervariability (CDR-1, CDR-2, CDR-3) called complementarity determining regions (CDR). VH contains 3 CDRs and 4 FRs arranged in the order of FR-1, CDR-1 (CDR-H1), FR-2, CDR-2 (CDR-H2), FR-3, CDR-3 (CDR-H3), FR-4 from the amino terminus to the carboxyl terminus. VL contains 3 CDRs and 4 FRs arranged in the order of FR-1, CDR-1 (CDR-L1), FR-2, CDR-2 (CDR-L2), FR-3, CDR-3 (CDR-L3), FR-4 from the amino terminus to the carboxyl terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0120] The antibody of the present invention may be a fragment and / or derivative of an antibody. As a fragment of an antibody, there may be listed: F(ab')2, Fab, Fv, etc. As a derivative of an antibody, there may be listed: an antibody obtained by artificially introducing amino acid mutations into the constant region, an antibody obtained by modifying the structure of the domain of the constant region, an antibody in the form of two or more Fc per molecule, an antibody consisting only of a heavy chain or only of a light chain, a sugar chain-modified antibody, a bispecific antibody, an antibody conjugate obtained by binding to an antibody or an antibody fragment compound or a protein other than an antibody, an abzyme, a nanobody, a tandem scFv, a bispecific tandem scFv, a diabody, a VHH, etc. It should be noted that in the present invention, when referred to as "antibody", as long as there is no additional explanation, it also includes a fragment and / or derivative of the antibody.

[0121] In the present disclosure, “antigen-antibody reaction” refers to the reaction of an antibody with an equilibrium dissociation constant (KD) of 1×10 -7 The antibody of the present invention preferably binds to the IGF-I receptor with an affinity of less than 1×10 -5 M or less, of which 1×10 -6 M or less, and further 1×10 -7 The KD below M binds to IGF-I receptor.

[0122] In the present disclosure, "antibody specificity" for an antigen means that a high antigen-antibody reaction occurs between the antibody and its antigen. In particular, in the present disclosure, "IGF-I receptor-specific antibody" means that the antigen-antibody reactivity to INSR having a high similarity to the primary structure (amino acid sequence) and higher-order structure of the IGF-I receptor is less than 1.5 times the reactivity to Mock cells at a concentration at which an antigen-antibody reaction is clearly shown to cells expressing the IGF-I receptor.

[0123] Regarding the determination of antigen-antibody reaction, if a person skilled in the art can appropriately select a binding assay in a solid phase or liquid phase system to conduct the determination. Such methods include: enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), surface plasmon resonance (SPR), fluorescence resonance energy transfer (FRET), luminescence resonance energy transfer (LRET), etc., but are not limited to these. In addition, when determining such antigen-antibody binding, the antibody and / or antigen can be labeled with enzymes, fluorescent substances, luminescent substances, radioactive isotopes, etc., and the antigen-antibody reaction can be detected by a determination method suitable for the physical and / or chemical properties of the labeled substance.

[0124] The antibody of the present invention is considered to activate a homotypic receptor formed by a dimer of the IGF-I receptor or a heterotypic receptor formed by a dimer of the IGF-I receptor and INSR by binding to the CR domain of the IGF-I receptor.

[0125] The antibody of the present invention preferably has a specific amino acid sequence as each CDR sequence. Specifically as follows. It should be noted that, in the present invention, the "identity" of an amino acid sequence means the ratio of identical amino acid residues, and the "similarity" means the ratio of identical or similar amino acid residues. Similarity and identity can be determined, for example, by the BLAST method (default conditions of PBLAST of NCBI).

[0126] Here, "similar amino acid residues" means amino acid residues having side chains with the same chemical properties (for example, charge or hydrophobicity). Examples of similar amino acid residues include the following combinations.

[0127] 1) Amino acid residues with aliphatic side chains: glycine, alanine, valine, leucine and isoleucine residues;

[0128] 2) amino acid residues with aliphatic hydroxyl side chains: serine and threonine residues;

[0129] 3) amino acid residues with amide-containing side chains: asparagine and glutamine residues;

[0130] 4) Amino acid residues with aromatic side chains: phenylalanine, tyrosine and tryptophan residues;

[0131] 5) Amino acid residues with basic side chains: lysine, arginine and histidine residues;

[0132] 6) Amino acid residues with acidic side chains: aspartic acid and glutamic acid residues;

[0133] 7) Amino acid residues with sulfur-containing side chains: cysteine ​​and methionine residues.

[0134] In the present invention, the CDR-1 (CDR-H1) sequence of the heavy chain variable region is preferably SEQ ID NO: 1 (SerTyrTrpMetHis) or an amino acid sequence in which the amino acid residue at any one of SEQ ID NO: 1 is substituted. In addition, the CDR-H1 sequence of the heavy chain variable region preferably has 80% or more similarity (preferably identity) with SEQ ID NO: 1.

[0135] As the CDR-2 (CDR-H2) sequence of the heavy chain variable region, SEQ ID NO: 2 (GluThrAsnProSerAsnSerValThrAsnTyrAsnGluLysPheLysSer) or an amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 2 are substituted is preferred. In addition, the CDR-H2 sequence of the heavy chain variable region preferably has 88% or more similarity (preferably identity) with SEQ ID NO: 2, of which 94% is preferred.

[0136] As the CDR-3 (CDR-H3) sequence of the heavy chain variable region, SEQ ID NO: 3 (GlyArgGlyArgGlyPheAlaTyr) or an amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 3 are substituted is preferred. In addition, the CDR-H3 sequence of the heavy chain variable region preferably has 75% or more similarity (preferably identity) with SEQ ID NO: 3, wherein 87% or more.

[0137] As the CDR-1 (CDR-L1) sequence of the light chain variable region, SEQ ID NO: 4 (ArgAlaSerGlnAsnIleAsnPheTrpLeuSer) or an amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 4 are substituted is preferred. In addition, the CDR-L1 sequence of the light chain variable region preferably has 81% or more similarity to SEQ ID NO: 4, wherein 90% or more similarity (preferably identity) is preferred.

[0138] As the CDR-2 (CDR-L2) sequence of the light chain variable region, SEQ ID NO: 5 (LysAlaSerAsnLeuHisThr) or an amino acid sequence in which the amino acid residue at any one of SEQ ID NO: 5 is substituted is preferred. In addition, the CDR-L2 sequence of the light chain variable region preferably has 85% or more similarity (preferably identity) with SEQ ID NO: 5.

[0139] As the CDR-3 (CDR-L3) sequence of the light chain variable region, SEQ ID NO: 6 (LeuGlnGlyGlnSerTyrProTyrThr) or an amino acid sequence in which any one or two amino acid residues of SEQ ID NO: 6 are substituted is preferred. In addition, the CDR-L3 sequence of the light chain variable region preferably has a similarity (preferably identity) of 77% or more, particularly 88% or more, to SEQ ID NO: 6.

[0140] In particular, the antibody of the present invention preferably has a combination of the following CDR sequences: an amino acid sequence of SEQ ID NO: 1 as a CDR-H1 sequence, an amino acid sequence of SEQ ID NO: 2 as a CDR-H2 sequence, an amino acid sequence of SEQ ID NO: 3 as a CDR-H3 sequence, an amino acid sequence of SEQ ID NO: 4 as a CDR-L1 sequence, an amino acid sequence of SEQ ID NO: 5 as a CDR-L2 sequence, and an amino acid sequence of SEQ ID NO: 6 as a CDR-L3 sequence.

[0141] It should be noted that, as a method for identifying each sequence of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 or CDR-L3 in an antibody, for example, the Kabat method (Kabat et al., The Journal of Immunology, 1991, Vol. 147, No. 5, pp. 1709-1719) or the Chothia method (Al-Lazikani et al., Journal of Molecular Biology, 1997, Vol. 273, No. 4, pp. 927-948) can be cited. These methods are common technical knowledge for those skilled in the art, and an overview can also be obtained from the Internet homepage of Dr. Andrew CR Martin's Group (http: / / www.bioinf.org.uk / abs / ).

[0142] The immunoglobulin framework sequence as the antibody of the present invention is preferably a framework sequence of each type of human immunoglobulin.

[0143] The antibody of the present invention preferably has a specific amino acid sequence as a heavy chain variable region and a light chain variable region. The details are as follows. It should be noted that, in the present disclosure, unless otherwise specified, "1 or more" refers to any one of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0144] As the heavy chain variable region of the antibody of the present invention, SEQ ID NO: 7, an amino acid sequence in which any one or more amino acid residues of SEQ ID NO: 7 are substituted, or an amino acid sequence having 90% or more similarity (preferably identity) with SEQ ID NO: 7 is preferred. In addition, as the light chain variable region of the antibody of the present invention, SEQ ID NO: 8, an amino acid sequence in which any one or more amino acid residues of SEQ ID NO: 8 are substituted, or an amino acid sequence having 90% or more similarity (preferably identity) with SEQ ID NO: 8 is preferred.

[0145] In particular, the antibody of the present invention preferably has SEQ ID NO: 7 as a heavy chain variable region, and has a sequence comprising SEQ ID NO: 8, or an amino acid sequence in which position 36 (Kabat numbering: L36) of SEQ ID NO: 8 is substituted by Tyr to Ala, Ser, Phe or Cys (SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively) as a light chain variable region.

[0146] The amino acid sequences of each framework region and / or each constant region of the heavy chain and light chain of the antibody of the present invention can be selected from various types of human IgG, IgA, IgM, IgE and IgD and their mutants. According to one example, the heavy chain constant region of the antibody of the present invention is the heavy chain constant region of human IgG4.

[0147] [Epitope of anti-IGF-I receptor humanized antibody]

[0148] The antibody of the present invention uses the CR domain of the IGF-I receptor as an epitope. Preferably, the antibody of the present invention binds to an epitope or its vicinity comprising a peptide having an amino acid sequence (ProSerGlyPheIleArgAsnGlySerGlnSerMet) corresponding to amino acid numbers 308 to 319 of SEQ ID NO: 14 (human IGF-I receptor). It is believed that the antibody of the present invention activates the following receptors by binding to the CR domain of the IGF-I receptor: a homotypic receptor obtained by forming a dimer of the IGF-I receptor, or a heterotypic receptor obtained by forming a dimer of the IGF-I receptor and INSR. However, the agonist antibody of the present invention described later (the antibody of the present invention as an agonist antibody) does not bind to INSR, which has a high similarity in primary structure (amino acid sequence) and higher-order structure with the IGF-I receptor.

[0149] [IGF-I receptor agonist antibodies and antagonist antibodies]

[0150] The antibodies of the present invention include both agonist antibodies and antagonist antibodies (hereinafter, for convenience, the antibodies of the present invention as agonist antibodies are referred to as "agonist antibodies of the present invention", and for convenience, the antibodies of the present invention as antagonist antibodies are referred to as "antagonist antibodies of the present invention"). The agonist antibodies of the present invention, when acting alone, have the effect of enhancing the proliferation activity of myoblasts based on IGF-I. On the other hand, the antagonist antibodies of the present invention, when acting simultaneously with IGF-I, have the effect of inhibiting the proliferation activity of myoblasts based on IGF-I.

[0151] The agonist antibody of the present invention is preferably a human IgG class or a mutant thereof, preferably a human IgG4 subclass or a mutant thereof, or a human IgG1 subclass or a mutant thereof. In one example, the stabilized IgG4 constant region comprises a proline at position 241 of the hinge region according to the Kabat system. According to the EU numbering method (Kabat et al., Sequences of Proteins of Immunological Interest, DIANE Publishing, 1992, Edelman et al., Proc. Natl. Acad. Sci USA, 63, 78-85, 1969), this site corresponds to position 228 of the hinge region. In human IgG4, this residue is usually serine, and stabilization can be induced by replacing serine with proline. In one example, the N297A mutation can be inserted into the constant region of IgG1 to suppress the ability to bind to Fc receptors and / or fix complement as much as possible.

[0152] The agonist antibody of the present invention strongly binds to a specific domain of the IGF-I receptor and has an effect of enhancing the proliferation activity of myoblasts in vitro.

[0153] In addition, the agonist antibody of the present invention has the following characteristics: it has no effect on enhancing glucose uptake by differentiated myocytes in vitro. Specifically, the agonist antibody of the present invention has no effect on enhancing glucose uptake by cultured differentiated myocytes in vitro even at an action concentration that enhances the proliferation activity of myoblasts (e.g., myoblasts from humans or guinea pigs), more preferably at a concentration 10 times higher than the action concentration, and even more preferably at a concentration 100 times higher than the action concentration.

[0154] In addition, the agonist antibody of the present invention also has the following characteristics: it does not have a hypoglycemic effect at a dose that shows a muscle mass increasing effect. IGF-I has a significant hypoglycemic effect when administered at a dose that shows a muscle mass increasing effect. However, the agonist antibody of the present invention does not have an effect of lowering the blood sugar level of the vertebrate at a dose that induces an increase in the muscle mass and / or body length of the vertebrate. Preferably, the agonist antibody of the present invention does not have an effect of lowering the blood sugar level of the vertebrate even when administered in a manner that achieves a blood exposure amount that is 10 times or more of the effective dose that induces an increase in the muscle mass and / or body length of the vertebrate.

[0155] Furthermore, the agonist antibody of the present invention has an in vivo activity equivalent to the muscle mass increasing effect of continuous administration of IGF-I when administered to guinea pigs once. In addition, the agonist antibody of the present invention has a long half-life in the blood and exhibits a muscle mass increasing effect by a single administration to animals.

[0156] From the above, it can be seen that the agonist antibody of the present invention has the potential to become a therapeutic or preventive drug for various diseases related to IGF-I receptors, such as disuse muscular atrophy and dwarfism, which are expected to be caused by IGF-I, and can overcome the hypoglycemic effect that is a problem of IGF-I and prolong its half-life in the blood.

[0157] On the other hand, the antagonist antibody of the present invention has the effect of inhibiting the binding of IGF-I to IGF-I receptor.

[0158] One embodiment of the antagonist antibody of the present invention is an antibody that activates the IGF-I receptor but inhibits the action of IGF-I on the IGF-I receptor. In this case, the antibody has the effect of eliminating the synergistic agonist activity of IGF-I, such as the proliferation-inducing activity of myoblasts based on IGF-I.

[0159] Another embodiment of the antagonist antibody of the present invention is an antibody that binds to the IGF-I receptor but does not activate the IGF-I receptor. Examples of antagonist antibodies that do not activate the IGF-I receptor by cross-linking include monovalent antibodies such as Fab and scFv, antibodies that have bivalent binding sites such as bispecific antibodies but only one side of the binding site binds to the specific domain of the IGF-I receptor, or antibodies obtained by changing the distance between the bivalent binding sites using a linker or the like. However, the antagonist antibodies of this embodiment are not limited to these.

[0160] Among the antagonist antibodies of the present invention, regarding the embodiment of antibodies that bind to the IGF-I receptor but do not have agonist activity, it can be confirmed that they have binding properties to the IGF-I receptor by a method of measuring the antigen-antibody reaction between the antibody and the IGF-I receptor, and it can be confirmed that they do not have cell proliferation inducing activity by a cell proliferation test in cells such as myoblasts.

[0161] In addition, the antagonist antibody of the present invention has no effect on glucose uptake by differentiated muscle cells in vitro or blood glucose levels in vivo. Therefore, the antagonist antibody of the present invention may be used as a therapeutic or preventive drug for malignant tumors such as breast cancer, colorectal cancer, sarcoma, lung cancer, prostate cancer, thyroid cancer, myeloma, etc. in the form of an anti-IGF-I receptor humanized antibody that does not show side effects such as hyperglycemia.

[0162] [Cross-reactivity]

[0163] The antibody of the present invention preferably has cross-reactivity to IGF-I receptors of other vertebrates. Cross-reactivity means that the antibody shows binding to antigens of animal species other than the animal species (e.g., human) of the IGF-I receptor with which the antibody undergoes antigen-antibody reaction. It is preferred that the antibody has cross-reactivity with IGF-I receptors of humans or non-human animals other than the animal species of the IGF-I receptor with which the antibody undergoes antigen-antibody reaction, and the non-human animals include guinea pigs, monkeys, rabbits, cattle, pigs, horses, sheep (sheep), dogs or chickens. In Example 4 described below, it is shown that R11-16B, an anti-IGF-I receptor humanized antibody, binds to the sequence of ProSerGlyPheIleArgAsnGlySerGlnSerMet in the CR domain of the human IGF-I receptor. The sequence of ProSerGlyPheIleArgAsnGlySerGlnSerMet is conserved in the same part of the IGF-I receptor of monkeys (cynomolgus monkeys), rabbits, guinea pigs, cattle, sheep, horses and dogs, and has cross-binding properties with the IGF-I receptors of these species. In addition, in mice and rats, the amino acid sequence of the same part is ProSerGlyPheIleArgAsnSerThrGlnSerMet, and by obtaining an anti-IGF-I receptor antibody that binds to this part, an antibody that binds to the IGF-I receptor of mice or rats, etc. and has the same properties or functions as R11-16B can be obtained.

[0164] Furthermore, by using an animal species that does not cross-react with the antibody of the present invention and modifying its cells or animals by genetic engineering, cells or animals expressing an IGF-I receptor that cross-reacts with the antibody of the present invention can also be prepared.

[0165] [Activity of inducing proliferation of cells derived from vertebrates, activity of inducing increase in muscle mass and / or stature]

[0166] The humanized anti-IGF-I receptor antibody in one embodiment of the present invention has proliferation-inducing activity in cells derived from vertebrates. Although the existence of IGF-I receptor agonist antibodies is known, there are no reports of antibodies showing cell proliferation-inducing activity in primary cultured cells, especially in myoblasts. In addition, there are no reports of antibodies showing cell proliferation-inducing activity in vitro at EC values ​​lower than IGF-I. 50 The antibody having cell proliferation inducing activity at a certain dose is reported.

[0167] It should be noted that, in the present disclosure, "cells from vertebrates" are preferably cells from mammals, birds, reptiles, amphibians or fish, more preferably cells from mammals or birds, and even more preferably cells from humans, monkeys, rabbits, guinea pigs, cattle, pigs, sheep, horses or dogs. Among the cells from these species, if the cells express the IGF-I receptor and the antibodies of the present invention cross-react with the IGF-I receptor, the cell proliferation can be induced by the antibodies of the present invention. In addition, the "cells from vertebrates" in the present disclosure also include cells or animals modified in a manner to express the IGF-I receptor of a species that has cross-reactivity with the antibodies of the present invention, or cells from the modified animals.

[0168] As cells used for studying the proliferation-inducing activity of vertebrate-derived cells in vitro, primary cultured cells, established cell lines, or transformed cells (transformed cells) of these cells can be used.

[0169] In the present disclosure, "primary culture cells" refer to cells separated from the organs or tissues of organisms, and the cells can usually be subcultured at a certain number of passages. Primary culture cells from vertebrates can be obtained from the organs or tissues of vertebrates by methods such as enzyme treatment, dispersion based on physical methods, or explant methods. In addition, organs or tissues obtained from vertebrates, or fragments thereof, can also be used. As organs or tissues for modulating these primary cells, preferably, they can be listed: endocrine tissues such as thyroid, parathyroid or adrenal glands; immune tissues such as appendix, tonsils, lymph nodes or spleen; respiratory organs such as trachea or lungs; digestive organs such as stomach, duodenum, small intestine or large intestine; urinary organs such as kidneys or bladder; male reproductive organs such as vas deferens, testicles or prostate; female reproductive organs such as breasts or fallopian tubes; muscle tissues such as myocardium or skeletal muscle, etc., more preferably liver, kidneys or digestive organs or muscle tissue, etc., and further preferably muscle tissue. As the primary culture cells used to study the proliferation inducing activity of the antibody of the present invention, cells expressing IGF-I receptors and inducing proliferation by IGF-I binding to the IGF-I receptors are used. As a representative example, skeletal muscle myoblasts, which are primary culture cells isolated from muscle tissue, are used. As primary culture cells derived from humans or animals, cells that are separately sold or commercially available can also be obtained and used. Human primary culture cells can be obtained, for example, from ATCC (注册商标) 、ECACC、Lonza、Gibco (注册商标) , CellApplications, ScienCell research laboratories, PromoCell and other institutions or companies.

[0170] In the present disclosure, "cell lines" refer to cells from organisms that are immortalized and retain certain properties, and can be cultured cells that can be semi-permanently proliferated. Cell lines include cells from non-tumor origin and cells from tumor origin. As vertebrate cell lines used to study the proliferation inducing activity of the antibody of the present invention, cells expressing IGF-I receptors and induced to proliferate by IGF-I binding to their IGF-I receptors are used. As cell lines expressing IGF-I receptors and inducing cell proliferation by IGF-I, for example, human neuroblastoma SH-SY5Y, human epidermal keratinocyte line HaCaT, human alveolar basal epithelial adenocarcinoma cell line A549, human colon adenocarcinoma cell line Caco-2, cell lines from human liver cancer HepG2, human cervical cancer cell line Hela, human cervical cancer cell line SiHa, human breast cancer cell line MCF7, human pluripotent embryonal carcinoma NTERA-2 or human osteosarcoma cell line U-2-OS, etc., but are not limited to these.

[0171] In the present disclosure, as transformant cells that can be used to study the proliferation inducing activity of the antibody of the present invention, there can be cited: the transformant cells of the above-mentioned primary culture cells or cell lines. As an example of such transformant cells, iPS cells made from primary culture cells, or cells or tissues induced by differentiation of iPS cells, etc. can be cited. In addition, as an example of other transformant cells, cells obtained by introducing genes into primary culture cells or cell lines and expressing them transiently or continuously are also included in the transformant cells. As genes introduced into the cells and expressed, genes of IGF-I receptors of humans or other species can be included.

[0172] As methods for studying the cell proliferation activity induced by the antibody of the present invention in cells derived from vertebrates, there are examples of cell number measurement, measurement of DNA synthesis amount, measurement of changes in metabolic enzyme activity, etc. Cell number measurement methods include methods using a hemocytometer or a method using a cell counting device such as a Coulter counter, methods for measuring DNA synthesis amount include measurement methods based on the uptake of [3H]-thymidine or 5-bromo-2'-deoxyuridine (BrdU), and methods for observing changes in metabolic enzyme activity include MTT method, XTT method, or WST method, etc., and other appropriate methods can also be used by those skilled in the art.

[0173] The cell proliferation induction activity can be determined by the fact that when the antibody of the present invention is reacted with the cultured cells used in the test, the cell proliferation is increased compared to when the antibody is not reacted. In this case, as a control for the induction activity, if the measurement is performed under the same conditions in which the original IGF-I receptor ligand, that is, IGF-I, is reacted, it is convenient to evaluate the activity. When the concentrations of the antibody of the present invention and IGF-I are changed and the cultured cells used in the test are reacted, the concentration at which 50% of the maximum proliferation induction activity is shown is expressed as EC 50 When evaluating proliferation induction activity using human skeletal muscle myoblasts, it is preferred that the cell proliferation induction activity of the antibody of the present invention is equivalent to that of IGF-I. 50 value, more preferably the EC value of the antibody of the present invention 50 The EC value of the antibody of the present invention is preferably less than 1 / 10 of that of IGF-I, more preferably less than 1 / 20. 50 The value is less than 1 / 50 of that of IGF-I. In addition, when human skeletal muscle myoblasts are used to evaluate the proliferation induction activity, the EC value of the antibody of the present invention is 50 The value is preferably 0.5 nmol / L or less, more preferably 0.3 nmol / L or less, and even more preferably 0.1 nmol / L or less.

[0174] As a method for studying the proliferation-inducing activity of cells from vertebrates in vivo, the antibody of the present invention can be administered to the vertebrate, and the weight, size, cell number, etc. of the entire individual or the organ or tissue in the individual to which the antibody is administered can be measured, or, using an animal transplanted with the vertebrate cells, the weight, size, cell number, etc. of a transplant containing the transplanted vertebrate cells can be measured in the individual to which the transplant is performed, thereby conducting the study. In the measurement of the entire individual, the body weight, height, limb circumference, etc. are measured, or the body composition is measured by impedance method, using creatinine, height (height) coefficient, etc. Regarding the measurement of the organ or tissue or transplant in the individual, for animals other than humans, the target organ, tissue or transplant is directly recovered, and the weight, size or number of cells contained is calculated, etc. In addition, as a method for non-invasively measuring the organ, tissue or transplant of the individual, image analysis based on X-ray photography images, CT, MRI, and angiography using tracers based on isotopes or fluorescent substances are used. When the tissue as the object is skeletal muscle, changes in muscle strength, etc. become indicators. In addition, if it is a person skilled in the art, the effect of the antibody of the present invention on the proliferation induction activity of cells from vertebrates in vivo can be studied by adopting other appropriate methods. In order to study the proliferation induction activity of the antibody of the present invention on cells from vertebrates in vivo, it can be measured according to the method shown above, etc. between individuals given the antibody of the present invention and individuals given antibodies other than the antibody of the present invention or other control substances, and the obtained measurement results can be compared to evaluate.

[0175] The antibody of the present invention has the following characteristics: compared with natural IGF-I, the duration of its proliferation induction effect is longer than the time of contact with cells. In the in vitro cell proliferation induction activity, if the natural IGF-I is washed with a medium without IGF-I after contact with cells, the cell proliferation induction activity disappears. In contrast, the mouse antibody IGF11-16 antibody (Japanese patent application: Special Application 2017-106529) which is the design basis of the antibody of the present invention, even if it is washed with a medium without IGF11-16 antibody after contact with cells, the cell proliferation induction activity will continue. In addition, in Example 8 described later, the blood dynamics of IGF-I and the antibody of the present invention, namely the R11-16B antibody, were compared. Natural IGF-I disappeared from the blood by about 50% or more by 24 hours after administration to animals. In contrast, the R11-16B antibody was present in the blood by more than 60% even 48 hours after administration to animals, thereby showing that the R11-16B antibody is present in the blood for a long time. These results show that the antibody of the present invention exhibits a long-term cell proliferation inducing effect both in vitro and in vivo.

[0176] In addition, the effects of the antibody of the present invention in vivo include: the effect of increasing muscle mass and / or height. In addition to acting on the proliferation or differentiation of the above-mentioned myoblasts in skeletal muscle, IGF-I also has the effect of thickening muscle fibers. As such a comprehensive effect, it is believed that it has the effect of increasing muscle mass. The antibody of the present invention, like IGF-I, has the effect of increasing the muscle mass of the animal by administering it to an animal. As an effect of an IGF-I receptor agonist antibody, it is the first time that the antibody of the present invention has been shown to have a muscle-increasing effect in vivo.

[0177] As a method for measuring the increase effect of the muscle mass of the antibody of the present invention, in the measurement of the whole individual, body weight, height, limb circumference, etc. are measured, or body composition is measured according to the impedance method, using creatinine, height (height) coefficient, etc., and image analysis based on CT and MRI, angiography based on isotopes or fluorescent substances, etc. are also used. In addition, changes in muscle strength, etc. also become indicators. In addition, in animals other than humans, it can also be a method of directly collecting muscles and measuring their weight or size, etc. The increase effect of muscle mass can be evaluated by comparing the increase in muscle mass in individuals given the antibody of the present invention and individuals not given the antibody, or by comparing muscle mass before and after giving the antibody of the present invention in an individual. About the increase effect of muscle mass, if the difference in muscle mass increase is found by giving the antibody of the present invention, the effect can be known, but by confirming the difference in muscle mass between individuals given the antibody of the present invention and individuals not given the antibody, or before and after giving the antibody of the present invention, preferably 103% or more, more preferably 104% or more, the effect produced by giving the antibody of the present invention can be judged. IGF-I is also involved in bone growth and has the effect of increasing stature (height in the case of humans). Therefore, the antibody of the present invention also has the effect of increasing stature by administering it to animals. The stature-increasing effect of the antibody of the present invention can be measured by measuring the body weight, stature, limb circumference, etc. of an individual.

[0178] [Effect on glucose uptake in cells derived from vertebrates and / or blood sugar levels in animals]

[0179] According to one embodiment, the antibody of the present invention has the following characteristics: it has no effect on intracellular glucose uptake in differentiated muscle cells from vertebrates and / or blood sugar levels in vertebrates. It is known that IGF-I causes an increase in intracellular glucose uptake or a decrease in blood sugar levels as part of its agonist action on the IGF-I receptor. However, the agonist antibody of the present invention, which functions as an IGF-I receptor agonist antibody, has an unexpected effect, namely, it has the following characteristics: even in vitro ECs with proliferation-inducing activity in cells from vertebrates, it has no effect on the proliferation-inducing activity in vitro ECs. 50The antagonist antibody of the present invention, which functions as an IGF-I receptor antagonist antibody, does not induce glucose uptake in differentiated muscle cells even at a dose of 100 times or more of the effective dose for inducing muscle mass increase, and does not change blood glucose levels even at a blood exposure of 10 times or more of the effective dose for inducing muscle mass increase when administered parenterally to animals. In addition, the antagonist antibody of the present invention, which functions as an IGF-I receptor antagonist antibody, also has no effect on glucose uptake in differentiated muscle cells from vertebrate cells and / or blood glucose levels in vertebrates, and this feature is a beneficial effect in terms of avoiding hyperglycemia, which is a problem that is not fully met when using existing IGF-I receptor antagonist antibodies in human treatment. It should be noted that the cells from vertebrates in the present disclosure are as described above.

[0180] As cells used to study the characteristic of the antibody of the present invention that it has no effect on the glucose uptake into cells from vertebrates in vitro, primary cultured cells, cell lines, or transformed cells of these cells can be used. It should be noted that the primary cultured cells, cell lines, and transformed cells in the present disclosure are all as described above.

[0181] As a method for studying the effect of the antibody of the present invention on glucose uptake in cells from vertebrates, there are the following methods: determination of intracellular glucose concentration, determination of the intracellular uptake of glucose-like tracer substances, determination of changes in glucose transporters, etc. The determination method of glucose concentration includes absorbance determination methods such as enzymatic methods, and as a method for determining the intracellular uptake of glucose-like tracer substances, there is determination of the uptake of [3H]-2'-deoxyglucose, and as a method for observing changes in glucose transporters, there are cell immunostaining methods, protein blotting methods, etc. If it is a person skilled in the art, it can also be implemented by other appropriate methods, etc. The effect on intracellular glucose uptake can be determined by the same degree of glucose uptake into the cell when the antibody of the present invention is reacted with the cultured cells used for the test and when the antibody is not reacted. In this case, as a control for the induction activity, if the determination of the reaction of the ligand of the original IGF-I receptor, i.e., IGF-I, is carried out under the same conditions, it is convenient to evaluate its activity.

[0182] After changing the concentrations of the antibody of the present invention and IGF-I, the concentrations of the antibody of the present invention and IGF-I are changed to react with the cultured cells tested, and the glucose uptake into the cells of the untreated group (untreated group) is displayed as 100%. When human differentiated muscle cells are used to evaluate the glucose uptake, the glucose uptake of the antibody of the present invention is preferably less than the glucose uptake of IGF-I at the same concentration, more preferably less than 110% of the untreated group, and further preferably less than 100% of the glucose uptake of the antibody of the present invention, which is the same as the untreated group. In addition, when human differentiated muscle cells are used to evaluate the glucose uptake, the glucose uptake when 100 nmol / L of the antibody of the present invention is added is preferably less than 110%, more preferably less than 105%, and further preferably 95 to 100%.

[0183] As a method for studying glucose uptake in vivo by cells from vertebrates, the antibody of the present invention can be administered to the vertebrate, and then the changes in the glucose content in the organs or tissues of the administered individual can be measured. In the measurement of the entire individual, the determination of blood sugar value or hemoglobin A1C with glycated protein as an indicator is adopted. In the determination of the glucose uptake in the organs or tissues of the individual, the target organs or tissues are directly recovered from animals other than humans, and the glucose content or tracer is calculated. In addition, as a method for non-invasively measuring glucose uptake in the organs or tissues of the individual, X-ray angiography images or image analysis based on CT and MRI, angiography using tracers based on isotopes or fluorescent substances, etc. are adopted. In the case where the tissue as the object is skeletal muscle, glucose clamps, etc. also become indicators. In addition, if it is a person skilled in the art, the effect of the antibody of the present invention on the glucose uptake in vivo by cells from vertebrates can be studied by adopting other appropriate methods.

[0184] In addition, the antibody of the present invention is characterized in that, when administered to a vertebrate, the blood sugar level of the vertebrate is not changed even at the same dose, preferably 10 times or more, relative to the effective dose for inducing an increase in muscle mass of the vertebrate. Animals used to study changes in blood sugar levels of the antibodies of the present invention in vertebrates are preferably mammals, birds, reptiles, amphibians or fish, more preferably mammals or birds, and even more preferably humans, monkeys, rabbits, guinea pigs, cattle, pigs, sheep, horses or dogs. In addition, animals used to study changes in blood sugar levels of the antibodies of the present invention in vertebrates also include animals modified in such a way as to express IGF-I receptors of species that have cross-reactivity with the antibodies of the present invention. In the determination of blood sugar levels, as a blood-observing method, a colorimetric method or an electrode method is used, and enzymes used for detection include glucose oxidase method (GOD method), glucose dehydrogenase method (GDH method), etc. As a non-blood-observing method, there are optical measurement methods, etc., and other appropriate methods can also be selected by those skilled in the art. In the case of human blood sugar levels, the normal range of fasting blood sugar levels is 100 mg / dL to 109 mg / dL. Adverse events caused by the administration of drugs to blood sugar levels (Common Terminology Standards for Adverse Events v4.0) are defined as follows: hypoglycemia is defined as a blood sugar level lower than the range of 77 mg / dL to 55 mg / dL, and hyperglycemia is defined as a blood sugar level higher than the range of 109 mg / dL to 160 mg / dL. The fact that there is no effect on blood sugar levels by administering drugs means that the blood sugar level after the administration of the drug is greater than 55 mg / dL and less than 160 mg / dL, more preferably greater than 77 mg / dL and less than 109 mg / dL. However, the normal value of blood sugar levels or their fluctuation ranges vary depending on the animal to which they are administered. In addition, even for humans, the blood sugar level at the time of administration is not necessarily limited to the range of normal values, so the disclosure does not change the blood sugar level of vertebrates, which means that the blood sugar level of vertebrates administered with the antibodies of the present invention is preferably within 30%, more preferably within 20%, and further preferably within 10%.

[0185] [Method for producing humanized anti-IGF-I receptor antibodies]

[0186] The antibody of the present invention can be obtained by humanizing the mouse monoclonal antibody IGF11-16 (mouse IGF11-16 antibody, Japanese patent application: Special Application 2017-106529) against IGF-I receptor. An example of a method for obtaining such a humanized antibody is described in Example 1 described below. As the humanized antibody obtained therefrom, there can be listed: a humanized antibody (R11-16B, R11-16C, R11-16D, R11-16E or R11-16F) having a VH amino acid sequence of SEQ ID NO: 7 and a VL amino acid sequence of SEQ ID NO: 8, 9, 10, 11 or 12. However, the antibody of the present invention is not limited to these.

[0187] Regarding the obtained anti-IGF-I receptor humanized antibody, a nucleic acid molecule having a nucleotide sequence encoding the amino acid of the protein of the antibody can also be obtained, and the antibody can also be prepared by genetic engineering using such a nucleic acid molecule. The H chain, L chain, or their variable regions in the gene information of the antibody can be modified with reference to information such as CDR sequences to improve the binding or specificity of the antibody.

[0188] As a method for producing the antibody of the present invention, mammalian cells, insect cells, Escherichia coli, etc. into which genes encoding the amino acids of the protein of the desired antibody can be respectively cultured, and the antibody can be obtained by purifying the antibody from the obtained culture supernatant by conventional methods. As a specific method, it is not limited to this, and the following method can be exemplified.

[0189] First, a nucleic acid molecule encoding an H chain signal peptide and a nucleic acid molecule encoding an H chain constant region are combined with a nucleic acid molecule encoding an H chain variable region. A nucleic acid molecule encoding an L chain signal peptide and a nucleic acid molecule encoding an L chain constant region are combined with a nucleic acid molecule encoding an L chain variable region.

[0190] These H chain genes and L chain genes are inserted into a vector suitable for expression in the selected host cell, such as a cloning vector or an expression vector. Here, the H chain gene and the L chain gene may be inserted into one vector or into different vectors if they are two genes to be expressed.

[0191] Next, the vector into which the H chain gene and the L chain gene are inserted is introduced into a host cell. Examples of host cells include eukaryotic cells such as mammalian cells, insect cells, yeast cells, or plant cells, or bacterial cells. As a method for introducing genes into host cells, it can be appropriately selected from the following methods: chemical methods such as calcium phosphate or lipid transfection, physical methods such as electroporation or particle gun methods, or methods based on infection by viruses or bacteriophages, etc. The host cells into which the H chain gene and the L chain gene are introduced can be used for culture without selection, or can be selectively concentrated by using properties such as drug resistance or nutritional requirements, or further used to culture cloned recombinant cells established from a single host cell into which the gene is introduced.

[0192] The host cell into which the H chain gene and the L chain gene are introduced is cultured under appropriate culture medium and culture conditions. Here, the products of the H chain gene and the L chain gene expressed in the host cell are usually secreted into the culture medium in the form of antibody protein, and the antibody protein produced can be obtained by recovering the culture medium. However, by utilizing the combination of gene and host, the host cell can be destroyed if necessary to recover the antibody protein accumulated in the cell, and in the case of prokaryotic cells, the antibody protein can also be recovered from the periplasmic component. As a method for purifying antibodies from samples such as culture medium containing the recovered antibody protein, salt precipitation method, concentration based on dialysis or ultrafiltration method or solvent exchange, affinity chromatography using a carrier fixed with protein A, protein G or antigen, etc., etc. are usually adopted, and in addition, ion exchange chromatography, hydrophobic chromatography, mixed mode chromatography or size exclusion chromatography can also be appropriately adopted for purification. Various methods used for these steps are known to those skilled in the art.

[0193] Here, for the genes encoding the heavy chain and / or light chain of immunoglobulin, genetic modification is performed to introduce the desired traits, or the structural information of the variable region or CDR region of the heavy chain and / or light chain of immunoglobulin is used to produce antibody chimeric proteins, low molecular weight antibodies, scaffold antibodies, etc., which can be implemented by those skilled in the art using known techniques. In addition, in order to improve the performance of the antibody or avoid side effects, modifications are introduced into the structure of the antibody constant region, or modifications are performed in the sugar chain portion, which can also be appropriately performed using techniques well known to those skilled in the art.

[0194] [Medicament containing humanized anti-IGF-I receptor antibody]

[0195] The antibody of the present invention can be used as a therapeutic or preventive drug for a state associated with IGF-I or a disease caused by the action on the IGF-I receptor. Specifically, as a state associated with IGF-I or a disease to be treated or prevented using an IGF-I receptor agonist antibody, there can be listed: muscle wasting diseases (e.g., disuse muscle atrophy, sarcopenia, cachexia, etc.), dwarfism (e.g., Laron type dwarfism, growth hormone resistant dwarfism, etc.), cirrhosis, liver fibrosis, diabetic nephropathy, chronic renal insufficiency, aging, intrauterine fetal growth retardation (IUGR), cardiovascular protection, diabetes, insulin resistance, metabolic syndrome, osteoporosis, cystic fibrosis, myotonic dystrophy, AIDS muscle atrophy, HIV associated fat redistribution syndrome, Crohn's disease, Werner syndrome, X-linked combined immunodeficiency, hearing loss, anorexia nervosa and retinopathy of prematurity, Turner syndrome, Prader-Willi syndrome, Silver-Russell syndrome, idiopathic dwarfism, obesity, multiple sclerosis, ulcerative colitis, low muscle mass, myocardial ischemia, low bone density; diseases that can be treated or prevented using IGF-I receptor antagonist antibodies include neuroblastoma (neuroblastoma), rhabdomyosarcoma, osteosarcoma, childhood cancer, acromegaly, ovarian cancer, pancreatic cancer, benign prostatic hypertrophy, breast cancer, prostate cancer, bone cancer, lung cancer, colorectal cancer, neck cancer, synovial sarcoma, bladder cancer, gastric cancer, Wilms' tumor, metastatic carcinoid and angiogenesis Diarrhea associated with VIP-secreting tumors, VIPoma, Verner-Morrison syndrome, Beckwith-Wiedemann syndrome, renal cancer, renal cell carcinoma, transitional cell carcinoma, Ewing's sarcoma, leukemia, acute lymphoblastic leukemia, brain tumors, glioblastoma, non-glioblastic brain tumors, meningioma, pituitary adenoma, vestibular schwannoma, primitive neuroectodermal tumors, medulloblastoma (medulloblastoma), astrocytoma, oligodendroglioma, ependymoma, choroid plexus papilloma, gigantism, psoriasis, atherosclerosis, vascular smooth muscle restenosis, inappropriate microvascular proliferation, diabetic retinopathy, Griff's disease, systemic lupus erythematosus, Hashimoto's thyroiditis, myasthenia gravis, autoimmune thyroiditis, and Behcet's disease. In particular, the antibody of the present invention is preferably used as a therapeutic or preventive drug for amyotrophic diseases (e.g., disuse amyotrophy, sarcopenia, cachexia, etc.) and / or dwarfism (e.g., Laron type dwarfism, growth hormone resistant dwarfism, etc.). In addition, the antibody of the present invention does not cause changes in blood sugar levels due to its administration, and is excellent in this respect.

[0196] The drug containing the antibody of the present invention can be prepared as a pharmaceutical composition, which contains a pharmaceutically acceptable carrier and / or other additives in addition to the antibody of the present invention. The preparation using a pharmaceutically acceptable carrier and / or other additives can be implemented according to the method described in University of the Sciences in Philadelphia, "Remington: The Science and Practice of Pharmacy, 20th Edition", Lippincott Williams & Wilkins, 2000.

[0197] As a form of such a therapeutic or preventive medicine, it is supplied in the form of a liquid preparation or a freeze-dried preparation prepared by dissolving, suspending or emulsifying in a sterile aqueous solution or oily solution. As such a solvent or dissolving liquid, distilled water for injection, physiological saline, etc. can be cited as an aqueous solution. In addition, when an osmotic pressure regulator (e.g., D-glucose, D-sorbitol, D-mannitol, sodium chloride, etc.) is added, sometimes an appropriate cosolvent, such as alcohol (e.g., ethanol), polyol (e.g., propylene glycol, polyethylene glycol), nonionic surfactant (e.g., polysorbate 80, polyoxyethylene hydrogenated castor oil 50), etc., is also used. In addition, as a solvent or dissolving liquid, an oily solution is sometimes used, and as an example of the oily solution, sesame oil, soybean oil, etc. can be cited, and sometimes benzyl benzoate, benzyl alcohol, etc. are also used as cosolvents. In such preparations, additives such as buffers (e.g., phosphate buffers, acetate buffers), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives (e.g., ascorbic acid, isoascorbic acid and salts thereof), colorants (e.g., copper chlorophyll, β-carotene, Red No. 2, Blue No. 1, etc.), preservatives (e.g., parabens, phenol, benzethonium chloride, benzalkonium chloride, etc.), thickeners (e.g., hydroxypropyl cellulose, carboxymethyl cellulose and salts thereof), stabilizers (e.g., human serum albumin, mannitol, sorbitol, etc.), and flavor enhancers (e.g., menthol, citrus flavor, etc.) are sometimes used as appropriate.

[0198] As other forms, therapeutic drugs or preventive drugs for mucosal application can also be listed. In this preparation, the main purpose is to impart adsorption and retention to the mucosa, and sometimes adhesives, adhesion enhancers, thickeners, thickening agents, etc. (for example, mucin, agar, gelatin, pectin, carrageenan, sodium alginate, locust bean gum, xanthan gum, tragacanth gum, gum arabic, chitosan, pullulan, waxy starch, sucralfate, cellulose, and its derivatives (for example, hydroxypropyl methylcellulose, polyglycerol fatty acid esters, acrylic acid (meth) alkyl acrylate copolymers, or their salts, polyglycerol fatty acid esters, etc.) are also contained as additives. However, the form of the therapeutic drug or preventive drug supplied to the organism and the solvent or additive are not limited to these, and those skilled in the art can select appropriately.

[0199] In addition to the antibody of the present invention, the drug containing the antibody of the present invention may also contain other existing drugs (active ingredients). In addition, the drug containing the antibody of the present invention can be combined with other existing drugs to form a kit. As active ingredients combined with the IGF-I receptor agonist antibody, there can be listed: growth hormone or its analogs, insulin or its analogs, IGF-II or its analogs, anti-myostatin antibodies, myostatin antagonists, anti-activin type IIB receptor antibodies, activin type IIB receptor antagonists, soluble activin type IIB receptors or their analogs, growth hormone releasing peptides or their analogs, follistatin or its analogs, β2 agonists, and selective androgen receptor modulators. In addition, examples of active ingredients to be combined with IGF-I receptor antagonist antibodies include corticosteroids, antiemetics, ondansetron hydrochloride, granisetron hydrochloride, metoclopramide, domperidone, haloperidol, cyclizine, lorazepam, prochlorperazine, dexamethasone, levomepromazine, tropisetron, cancer vaccines, GM-CSF inhibitors, GM-CSF DNA vaccines, cell-based vaccines, dendritic cell vaccines, recombinant virus vaccines, heat shock protein (HSP) vaccines, allogeneic tumor vaccines, autologous tumor vaccines, analgesics, ibuprofen, naproxen, choline magnesium salicylate, oxycodone hydrochloride, antiangiogenic drugs, antithrombotic drugs, anti-PD-1 antibodies, nivolumab, pembrolizumab, anti-PD-L1 antibodies, atezolizumab, anti-CTLA4 antibodies, ipilimumab, Ipilimumab, anti-CD20 antibody, Rituximab, anti-HER2 antibody, Trastuzumab, anti-CCR4 antibody, Mogamulizumab, anti-VEGF antibody, Bevacizumab, anti-VEGF receptor antibody, soluble VEGF receptor fragment, anti-TWEAK antibody, anti-TWEAK receptor antibody, soluble TWEAK receptor fragment, AMG 706, AMG 386, antiproliferative drugs, farnesyl protein transferase inhibitors, αvβ3 inhibitors, αvβ5 inhibitors, p53 inhibitors, Kit receptor inhibitors, ret receptor inhibitors, PDGFR inhibitors, growth hormone secretion inhibitors, angiogenin inhibitors, tumor infiltrating macrophage inhibitors, c-fms inhibitors, anti-c-fms antibodies, CSF-1 inhibitors, anti-CSF-1 antibodies, soluble c-fms fragments, Pegvisomant, Gemcitabine, Panitumumab, Irinotecan and SN-38.The dosage of drugs other than the antibody of the present invention to be added can be the dosage used for general treatment, and can be increased or decreased according to the situation.

[0200] The therapeutic or preventive medicine of the present invention can be administered parenterally for the purpose of improving symptoms. In the case of parenteral administration, for example, a nasal preparation can be prepared, and a liquid preparation, a suspension, a solid preparation, etc. can be selected. In addition, as other forms of parenteral administration, an injection can be prepared, and as an injection, a subcutaneous injection, an intravenous injection, a drip injection, an intramuscular injection, an intraventricular injection, or an intraperitoneal injection, etc. can be selected. In addition, as other preparations for parenteral administration, suppositories, sublingual preparations, transdermal preparations, and transmucosal administration preparations other than nasal preparations, etc. can also be listed. Moreover, it is also possible to locally administer the intravascular administration in a scheme containing or coating in a stent or an intravascular embolic agent.

[0201] The dosage of the therapeutic or preventive drug of the present invention varies depending on the patient's age, sex, body weight, symptoms, therapeutic effect, administration method, treatment time, or the type of active ingredient contained in the pharmaceutical composition. Generally, the main agent can be administered to each adult in a range of 0.1 mg to 1 g, preferably in a range of 0.5 mg to 300 mg, once every 1 to 4 weeks, or once every 1 to 2 months. However, since the dosage and the number of administrations vary according to various conditions, there are cases where an amount and number of administrations less than the above dosage and number of administrations are sufficient, and there are also cases where the dosage and number of administrations exceeding the above range are required.

[0202] [Cell culture method using anti-IGF-I receptor humanized antibody]

[0203] IGF-I or its derivatives are mostly used in cell culture techniques for maintaining, proliferating and / or differentiating cells from vertebrates in vitro, and are commercially available as reagents for cell culture. Due to stability issues, the effect of IGF-I may weaken over time in long-term culture, and in order to carry out stable cell culture, appropriate concentration adjustment and other countermeasures are required. In addition, since IGF-I induces glucose uptake into cells, it is possible to induce changes in cell metabolism or characteristics as the concentration of glucose in the cell rises, or the concentration of glucose in the culture medium decreases, causing the culture environment to change. The antibody of the present invention has the following characteristics: compared with IGF-I, it has high stability, a long time to induce cell proliferation after contact with cells, and shows cell proliferation induction activity at a concentration lower than IGF-I, and does not induce glucose uptake into cells. In addition to adding an appropriate amount to the culture medium in cell culture, the antibody of the present invention can also be adsorbed or fixed on the solid phase of the container for culture, thereby reducing the amount used or effectively inducing cell proliferation in cells attached to the solid phase. It should be noted that the cells from vertebrates in the present disclosure are as described above. Furthermore, the cultured objects using the antibody of the present invention also include organs or tissues from vertebrates or genetically modified animals thereof. The antibody of the present invention can be used in the culture for substance production using cells or in the culture step of cell medicine / regenerative medicine using cells themselves. Example

[0204] The present invention will be described in more detail below with reference to the following examples, but the present invention is not limited to the following examples and can be implemented in any manner without departing from the spirit of the present invention.

[0205] [Example 1] Preparation of humanized antibody gene of mouse IGF11-16 antibody:

[0206] A template human antibody as a transplantation destination for the amino acids in the complementarity determining region (CDR) of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-IGF-I receptor mouse monoclonal antibody IGF11-16 (mouse IGF11-16 antibody, Japanese patent application: Special Application 2017-106529) produced by the hybridoma method of Kohler et al. (Nature, 256:495-497, 1975) is selected from the germline of human antibodies having an amino acid sequence with high similarity to the amino acid sequence of the framework region (FR) of VH and VL of the mouse IGF11-16 antibody.

[0207] The humanized antibody is prepared by transplanting the amino acid sequences required for VH and VL of the mouse IGF11-16 antibody into the FRs of the above-mentioned template human antibody VH and VL. Specifically, with respect to VH, the CDR amino acid sequence and multiple FR amino acids of the above-mentioned template human antibody VH are replaced with the corresponding amino acid sequences in VH of the mouse IGF11-16 antibody, and the amino acid sequence of VH obtained by humanizing the mouse IGF11-16 antibody, namely R11-16VH (SEQ ID NO: 7), is designed, and the nucleotide sequence of the DNA encoding these amino acids is then designed.

[0208] Regarding VL, the amino acid sequence of the CDR amino acid sequence and FR amino acids of the above-mentioned template human antibody VL were replaced with the amino acid sequence in the VL of the mouse IGF11-16 antibody at multiple locations, and the amino acid sequence of the VL obtained by humanizing the mouse IGF11-16 antibody, namely R11-16VL, was designed.

[0209] The amino acid at position 36 of R11-16VL is cysteine ​​in mouse IGF11-16 antibody, but in ordinary human antibodies, cysteine ​​is less present at this position, and a disulfide bond that would not otherwise be produced is formed, which is considered to be the cause of aggregation. Therefore, the amino acid sequences of five types of R11-16VL, namely R11-16VL-C36, R11-16VL-C36Y, R11-16VL-C36A, R11-16VL-C36S, and R11-16VL-C36F, whose amino acids at position 36 are composed of cysteine, tyrosine, alanine, serine, and phenylalanine (SEQ ID NO: 12, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11), were designed, and the nucleotide sequences of DNA encoding these amino acids were designed. The structures of each humanized antibody and its amino acid are shown in Table 1 below.

[0210] [Table 1]

[0211]

[0212] [Example 2] Preparation of humanized antibodies:

[0213] DNA encoding the heavy chain variable region R11-16VH of the designed humanized antibody and DNA encoding the mutant obtained by stabilizing the human IgG4 subclass, namely the human IgG4S228P mutant, were synthesized separately, inserted into the pCAGGS1 expression vector and ligated to form a plasmid for expressing the humanized antibody heavy chain.

[0214] Regarding the designed humanized antibody light chain variable region R11-16VL, DNA encoding the humanized antibody light chain region linked to the κ chain constant region was synthesized and inserted into the pCAGGS1 expression vector to serve as a plasmid for expressing the humanized antibody light chain.

[0215] Mix these humanized antibody heavy chain expression plasmids and humanized antibody light chain expression plasmids and use Expi293 TM The expression system (Thermo Fisher Scientific) was introduced into the cells to express various antibodies obtained by humanizing the mouse IGF11-16 antibody. At this time, the humanized antibody obtained by combining the heavy chain expression plasmid with R11-16VH inserted and the light chain expression plasmid with R11-16VL-C36Y inserted was named R11-16B antibody, the humanized antibody obtained by combining the heavy chain expression plasmid with R11-16VH inserted and the light chain expression plasmid with R11-16VL-C36A inserted was named R11-16C antibody, and the humanized antibody obtained by combining the heavy chain expression plasmid with R11-16VH inserted and the light chain expression plasmid with R11-16VL-C36A inserted was named R11-16C antibody. The humanized antibody obtained by combining the expression plasmid of the heavy chain inserted with R11-16VH and the light chain inserted with R11-16VL-C36F was named R11-16D antibody, the humanized antibody obtained by combining the expression plasmid of the heavy chain inserted with R11-16VH and the light chain inserted with R11-16VL-C36F was named R11-16E antibody, and the humanized antibody obtained by combining the expression plasmid of the heavy chain inserted with R11-16VH and the light chain inserted with R11-16VL-C36 was named R11-16F antibody. The humanized antibody was obtained by affinity purification of the culture supernatant of cells into which the humanized antibody heavy chain and humanized antibody light chain expression plasmids were introduced using a protein A column.

[0216] [Example 3] Binding activity to IGF-I receptor (ELISA):

[0217] In order to study the binding activity of IGF-I receptor agonist antibodies to IGF-I receptors of humans (SEQ ID NO: 14, NP_000866), guinea pigs (SEQ ID NO: 16, XP_003475316), cynomolgus monkeys (SEQ ID NO: 18, XP_005560575) and rats (SEQ ID NO: 20, NP_434694), Cell ELISA was performed using cells expressing various IGF-I receptors.

[0218] The pEF1 expression vector (Thermofisher) containing human (SEQ ID NO: 15), guinea pig (SEQ ID NO: 17), cynomolgus monkey (SEQ ID NO: 19), and rat (SEQ ID NO: 21) IGF-I receptor genes was introduced into HEK293T cells by lipid transfection. HEK293T cells cultured overnight after lipid transfection were incubated at 4×10 4 Cells / well were added to a 96-well plate (coated with poly-D-lysine), and cultured overnight, and the obtained cells were used for ELISA.

[0219] In ELISA, 100 μL of each humanized antibody solution prepared with 1% BSA / 1% FBS / PBS to 2nM was added to each well and reacted at 37°C for about 1 hour. Washed 3 times with washing solution. 100 μL of anti-human IgG antibody HRP conjugate solution prepared with 1% BSA / 1% FBS / PBS to each well was added, reacted at 37°C for about 1 hour, and washed 3 times with washing solution. 100 μL of substrate (TMB) was added to each well to initiate the reaction. After about 30 minutes, 100 μL of 1M sulfuric acid was added to each well, the absorbance at 450nm and 650nm was measured, and the absorbance 450-650nm was calculated. The binding activity was calculated as 1 relative to the absorbance 450-650nm of HEK293T cells (control cells) into which the IGF-I receptor gene was not introduced. The results are shown in Table 2 below.

[0220] [Table 2]

[0221]

[0222] In cells expressing human, cynomolgus monkey and guinea pig IGF-I receptors, each humanized antibody increased the binding activity by more than 3 times compared to control cells (HEK293T). On the other hand, the binding activity to cells expressing rat IGF-I receptors was equivalent to that of control cells. This shows that each humanized antibody binds to human, cynomolgus monkey and guinea pig IGF-I receptors, but does not bind to rat IGF-I receptors.

[0223] [Example 4] Determination of the epitope of humanized antibody R11-16B:

[0224] R11-16B binds to the IGF-I receptor of humans, cynomolgus monkeys, and guinea pigs, but not to the IGF-I receptor of rats. In addition, the mouse antibody (Japanese patent application: Special Application 2017-106529) that serves as the design basis for R11-16B, namely the mouse IGF11-16 antibody, binds to the CR domain of the human IGF-I receptor, but not to the IGF-I receptor of rats. It is inferred that the epitope of R11-16B is an amino acid sequence in the CR domain of the IGF-I receptor that is common to humans, cynomolgus monkeys, and guinea pigs, but different from that of rats.

[0225] In order to determine which amino acid site in the CR domain of the human IGF-I receptor R11-16B binds to, the binding of the CR domain to various amino acid substitutions was measured by ELISA. Cell ELISA was performed using cells expressing IGF-I receptors in which the amino acid sequence in the CR domain that is presumed to bind to R11-16B was mutated.

[0226] The following two kinds of amino acid substitutions were used for the CR domain. In addition, as a positive control, an amino acid substitution was used in which a DNA (SEQ ID NO: 22) encoding an amino acid sequence in which a FLAG tag (tag) (AspTyrLysAspAspAspAspLys) was bound to the C-terminus of the wild-type human IGF-I receptor (SEQ ID NO: 14, NP_000866). Cells treated with only the pEF1 expression vector were used as mocks.

[0227] (CR domain substitution 1)

[0228] The aspartic acid, alanine and glutamic acid at positions 245, 247 and 294 of the amino acid sequence of human IGF-I receptor (SEQ ID NO: 14, NP_000866) were substituted with asparagine, threonine and aspartic acid, respectively. A DNA (SEQ ID NO: 23) encoding an amino acid sequence with a FLAG tag bound to the C-terminus of the amino acid sequence of the substituted 1 receptor was inserted into the pEF1 expression vector.

[0229] (CR domain substitution 2)

[0230] The glycine and serine at positions 315 and 316 of the amino acid sequence of human IGF-I receptor (SEQ ID NO: 14, NP_000866) were substituted with serine and threonine, respectively. DNA (SEQ ID NO: 24) encoding the amino acid sequence of the substituted receptor with a FLAG tag attached to the C-terminus was inserted into the pEF1 expression vector.

[0231] 293T cells were cultured at 6 × 10 6 293T cells were seeded in a 10 cm poly-D-lysine coated dish. The next day, each plasmid DNA was introduced into the cells using lipid transfection. The next day, 293T cells were detached using 0.05% trypsin / EDTA and suspended in culture medium. 293T cells were cultured at 2×10 4 10 cells / well were added to a 96-well plate (coated with poly-D-lysine) and cultured overnight at 37°C and 5% CO2. The culture medium was removed from the 96-well plate and the plate was incubated with 10% buffered formalin (Mildform (注册商标) 10NM, Wako) for fixation, and then replaced with blocking buffer (3% BSA / PBS / 0.02% sodium azide), and the obtained cells were used for ELISA.

[0232] In ELISA, add 50 μL of R11-16B prepared at 5 nM with blocking buffer to each well and react at room temperature for about 1 hour. Wash twice with washing solution. Add 50 μL of anti-human IgG antibody ALP conjugate solution (2087-04, Southern Biotech) diluted to 2500 times with blocking buffer to each well and react at room temperature for about 1 hour. Wash 3 times with washing solution. Add 100 μL of substrate (pNPP) to each well to initiate the reaction. Measure the absorbance at 405 nm and 550 nm after about 1 hour. The difference obtained by subtracting the value of the untreated group from the R11-16B treated group was used as the binding activity for evaluation. The results are shown in Table 3 below.

[0233] [Table 3]

[0234]

[0235] Compared with Mock, the binding activity of R11-16B to human IGF-I receptor was enhanced (increased) by more than 2 times. In addition, the binding activity to Substitute 1 was equivalent to its binding activity to human IGF-I receptor. On the other hand, the binding activity to Substitute 2 was equivalent to that of Mock, and enhancement of binding activity was not confirmed. This shows that the amino acids at positions 315 and 316 of the IGF-I receptor are more important in the binding activity of R11-16B to the human IGF-I receptor.

[0236] Based on the above results, it is speculated that the binding site of R11-16B to human IGF-I receptor is near Gly (glycine) and Ser (serine) at positions 315 and 316. Based on the fact that the number of amino acids recognized by antibodies is usually 8 residues (average value of 6 to 10 residues), and the cross-reactivity of R11-16B (it has no binding to rat IGF-I receptor, but has binding to human and guinea pig IGF-I receptor), it is believed that the binding site sequence of human IGF-I receptor to R11-16B is ProSerGlyPheIleArgAsnGly * Ser * GlnSerMet(Gly * Ser * represents the amino acid sequence at positions 315 and 316).

[0237] [Example 5] Cell proliferation activity in human myoblasts:

[0238] In order to examine the proliferation activity of IGF-I receptor agonist antibodies on human myoblasts, the drug was added to human myoblasts and the intracellular ATP level was measured 4 days later.

[0239] Normal human skeletal muscle myoblast cells (HSMM, Lonza) were cultured at 0.1 mL / well (2 × 10 3 Cells were seeded in 96-well plates (type I collagen coated) at 37°C and 5% CO2. Various agents were added at 25 μL / well on the second day of cell seeding and cultured at 37°C and 5% CO2 for 4 days. (注册商标) The luminescent cell viability assay (Promega) was used to measure the amount of ATP in the cells as an indicator of cell proliferation. The supernatant was removed from the 96-well plate after 4 days of culture to make the culture medium reach 50 μL / well, and the plate was left to stand at room temperature for more than 30 minutes. CellTiter-Glo was added at 50 μL / well. (注册商标) The reagent was added and allowed to react for more than 10 minutes, and then the luminescent signal was measured using a luminometer (Berthold). The activity of the group to which only the solvent was added was calculated as 100%. The results are shown in Tables 4 to 6 below.

[0240] [Table 4]

[0241]

[0242] R11-16B at 0.0000005, 0.000005, 0.00005, 0.0005, 0.005, 0.05, 0.5, 5 and 50 nM concentrations enhanced the proliferation activity of human myoblasts in a concentration-dependent manner. EC values ​​of myoblast proliferation activity of R11-16B, mouse IGF11-16 antibody and IGF-I 50 The concentrations of R11-16B were 0.002, 0.002 and 0.95 nM respectively. R11-16B showed an activity equivalent to that of mouse IGF11-16 antibody and more than 100 times stronger than that of IGF-I.

[0243] [Table 5]

[0244]

[0245] 0.00005, 0.0005, 0.005, 0.05, 0.5, 5 and 50 nM of R11-16B, R11-16C, R11-16D, R11-16E and R11-16F increased the proliferation activity of human myoblasts in a concentration-dependent manner. The EC values ​​of the proliferation activity of human myoblasts of R11-16B, R11-16C, R11-16D, R11-16E and R11-16F were 50 All humanized antibodies showed an activity more than 100 times stronger than that of IGF-I.

[0246] [Table 6]

[0247]

[0248] IGF-I enhanced cell proliferation activity compared to a control antibody (FLAG M2, Sigma-Aldrich).

[0249] The 16-13 antibody and the 26-3 antibody described in Non-Patent Document 35 (agonist antibodies showing an effect of enhancing DNA synthesis and glucose uptake in cells in vitro) did not show significant cell proliferation activity compared to the solvent control (containing sodium azide), and were weaker than the activity of R11-16B.

[0250] [Example 6] In vivo efficacy (muscle mass increasing effect in guinea pigs):

[0251] In order to confirm the in vivo efficacy of the IGF-I receptor agonist antibody, the effect was compared with the effect of continuous administration of IGF-I.

[0252] R11-16B was administered to guinea pigs once, and the muscle mass was measured 2 weeks later. The muscle mass increasing effect refers to an increase in the muscle weight of guinea pigs by 5% or more compared to the control group. R11-16B (0.1 and 0.3 mg / kg) was administered intravenously to normal guinea pigs once. As a positive control, human recombinant IGF-I (Mecasermin) was implanted subcutaneously using an osmotic pump (Alzet) and continuously administered to reach 0.3 and 1 mg / kg / day. Two weeks after the administration of the drug, the guinea pigs were bled to death under anesthesia, and the weight of the extensor digitorum longus muscle was measured. The results are shown in Figure 2 .

[0253] Compared with the control group (vehicle) treated with only a solvent, the groups (R11-16B) to which 0.1 and 0.3 mg / kg of R11-16B were intravenously administered increased muscle mass in a dose-dependent and significant manner.

[0254] The amount of muscle gain in the group given a single dose of 0.3 mg / kg of R11-16B was equivalent to that of the group given human recombinant IGF-I continuously at 1 mg / kg / day (IGF-I). This shows that R11-16B has the same efficacy as continuous administration of IGF-I through a single dose. The dosage of IGF-I (Mecasermin) in clinical practice is once to twice a day. On the other hand, in vivo R11-16B showed the same effectiveness as continuous administration of IGF-I by administering it once every 2 weeks, thus showing that its sustainability is superior to IGF-I.

[0255] [Example 7] In vivo hypoglycemic effect (hypoglycemic effect in guinea pigs):

[0256] In order to confirm whether the IGF-I receptor agonist antibody has a hypoglycemic effect in vivo, R11-16B was administered once to guinea pigs, and blood glucose levels were measured over time to compare with the hypoglycemic effect of a single administration of IGF-I. The hypoglycemic effect refers to an effect that reduces blood glucose levels to 50 mg / dL or below, or produces hypoglycemic symptoms.

[0257] The hypoglycemic effect of IGF-I was studied. Guinea pigs were fasted for 12 hours, and human recombinant IGF-I (Mecasermin) was administered subcutaneously at 0.3, 1, 3, and 10 mg / kg. The guinea pigs were fasted until 24 hours after administration. Blood was collected from awake guinea pigs before administration (0 hours), 1, 2, 4, 8, and 24 hours after administration, and blood glucose levels were measured using a Glutest sensor (Sanwa Chemical Research Institute). The results are shown in Table 7 below.

[0258] [Table 7]

[0259]

[0260] IGF-I significantly lowers blood sugar levels starting at 0.3 mg / kg, hypoglycemia symptoms are observed at 1 mg / kg or more, and death is observed at 3 mg / kg or more.

[0261] The effects of R11-16B, R11-16C, R11-16D, R11-16E and R11-16F on blood glucose levels were studied. Guinea pigs were fasted for 12 hours, and each humanized antibody was administered intravenously at a single dose of 10 mg / kg. Guinea pigs were fasted until 24 hours after administration. Blood was collected from awake guinea pigs before administration (0 hours), 1, 2, 4, 8 and 24 hours after administration, and blood glucose levels were measured using a Glutest sensor (Sanwa Chemical Research Institute). The results are shown in Tables 8 to 10 below.

[0262] [Table 8]

[0263]

[0264] [Table 9]

[0265]

[0266] [Table 10]

[0267]

[0268] No significant difference was observed in blood glucose levels between each humanized antibody and the solvent control group to which only the solvent was administered, and the blood glucose levels after administration were all above 50 mg / dL. This indicates that each humanized antibody does not have a significant hypoglycemic effect like IGF-I and has no effect on blood glucose levels, thus showing the potential as a drug to overcome hypoglycemia, a side effect of IGF-I.

[0269] [Example 8] Hemodynamics of IGF-I and R11-16B (Hemodynamics in Guinea Pigs):

[0270] ・Hemodynamics of IGF-I:

[0271] Guinea pigs were fasted for 12 hours and human recombinant IGF-I was subcutaneously administered at 0.3, 1, 3, and 10 mg / kg. Guinea pigs were fasted until 24 hours after administration. Blood was collected from awake guinea pigs before administration (0 hours), 1, 2, 4, 8, 10, and 24 hours after administration, and the concentration of human IGF-I in plasma was measured by ELISA (DG100, R&D). The results are shown in Figure 3 .

[0272] The IGF-I concentration in plasma increased depending on the dose, and the IGF-I concentration in plasma 24 hours after administration decreased to less than about 50% of the peak value. The IGF-I concentration in the 0.3 mg / kg administration group 24 hours after administration was below the lower limit of determination. In addition, the 10 mg / kg administration group died of hypoglycemia 4 hours after administration, so plasma could not be collected.

[0273] ・Hemodynamics of humanized antibodies:

[0274] The guinea pigs were fasted for 12 hours, and humanized antibody R11-16B was administered intravenously at a single dose of 1.5 mg / kg and 10 mg / kg. The guinea pigs were fasted for 24 hours after administration, and then fed again 24 hours later. Blood was collected from awake guinea pigs before administration (0 hours), 2, 4, 8, 24, 48, and 72 hours after administration, and the concentration of humanized antibody in plasma was determined by ELISA. The results are shown in Figure 4 .

[0275] The concentration of humanized antibody in plasma increased depending on the administered dose, and even after 48 hours of administration, the concentration of humanized antibody in plasma was maintained at about 50% or more compared to that after 24 hours of administration, indicating that the persistence of hemodynamics of humanized antibody was superior to that of IGF-I.

[0276] Industrial Applicability

[0277] The present invention can provide an antibody that specifically binds to the IGF-I receptor of vertebrates and increases muscle mass via the IGF-I receptor without lowering blood sugar levels, so it can be used for the treatment, prevention or diagnosis of diseases associated with anti-IGF-I receptor humanized antibodies.

Claims

1. An anti-IGF-I receptor humanized antibody or a derivative thereof, comprising: an amino acid sequence of SEQ ID NO: 7 as a heavy chain variable region; and an amino acid sequence selected from any one of SEQ ID NO: 8, 9, 10, 11 and 12 as a light chain variable region; and the anti-IGF-I receptor humanized antibody or a derivative thereof specifically binds to a binding site comprising amino acid residues corresponding to the 315th and 316th amino acids in the amino acid sequence of SEQ ID NO: 14 as a human IGF-I receptor.

2. The anti-IGF-I receptor humanized antibody or the derivative thereof according to claim 1, further comprising a constant region of each type of human immunoglobulin as a constant region.

3. The anti-IGF-I receptor humanized antibody or its derivative according to claim 2, wherein: The heavy chain constant region is that of human IgG4 class.

4. The anti-IGF-I receptor humanized antibody or a derivative thereof according to claim 1, which binds to an epitope or its vicinity comprising a peptide having an amino acid sequence corresponding to amino acid numbers 308 to 319 of SEQ ID NO: 14 for human IGF-I receptor, i.e., ProSerGlyPheIleArgAsnGlySerGlnSerMet.

5. The anti-IGF-I receptor humanized antibody or its derivative according to claim 1, wherein: It did not induce glucose uptake in myoblasts from humans or guinea pigs at doses that induced proliferation of the cells.

6. The anti-IGF-I receptor humanized antibody or a derivative thereof according to any one of claims 1 to 5, wherein At a dose administered to a vertebrate to induce an increase in muscle mass and / or body length in the vertebrate, it does not reduce blood glucose levels in the vertebrate.

7. The anti-IGF-I receptor humanized antibody or its derivative according to claim 6, wherein: Even at a blood exposure amount that is 10 times or more of the effective dose administered to a vertebrate to induce an increase in muscle mass and / or body length of the vertebrate, it does not lower the blood sugar level of the vertebrate.

8. A nucleic acid molecule consisting of a polynucleotide sequence encoding the anti-IGF-I receptor humanized antibody or a derivative thereof according to any one of claims 1 to 7.

9. A cloning vector or an expression vector comprising at least one nucleic acid molecule according to claim 8.

10. A recombinant cell obtained by introducing the vector according to claim 9 into a host cell.

11. A method for producing an anti-IGF-I receptor humanized antibody or a derivative thereof according to any one of claims 1 to 7, the method comprising the steps of culturing the recombinant cell according to claim 10, and purifying the anti-IGF-I receptor humanized antibody or a derivative thereof produced by the recombinant cell.

12. A pharmaceutical composition comprising the anti-IGF-I receptor humanized antibody or a derivative thereof according to any one of claims 1 to 7, the nucleic acid molecule according to claim 8, the vector according to claim 9 or the recombinant cell according to claim 10 as an active ingredient.

13. The pharmaceutical composition of claim 12, which is used for treating muscular dystrophy or dwarfism.

14. The pharmaceutical composition according to claim 13, wherein Muscle wasting diseases include disuse muscle atrophy, sarcopenia or cachexia.

15. The pharmaceutical composition according to claim 13, wherein The dwarfism is either Laron-type dwarfism or growth hormone-resistant dwarfism.

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