Antibody to CXCL12 and composition for treating hair loss containing the same

Abstract

The present invention relates to a composition for treating hair loss, which comprises a neutralizing antibody against CXCL12. The neutralizing antibody against CXCL12 has the effect of suppressing CXCL12 and promoting hair growth, and is therefore useful for preventing or treating hair loss.

Description

[Technical Field]

[0001] The present invention relates to a novel antibody against CXCL12 and a composition for treating hair loss containing the same. Specifically, the present invention relates to a neutralizing antibody or humanized antibody against CXCL12, which has the effect of promoting hair growth by inhibiting CXCL12. [Background technology]

[0002] Mouse hair grows, maintains, and falls out through a three-phase cycle: anagen, catagen, and telogen. Generally, apoptosis of hair follicle cells occurs 10 to 14 days after the anagen phase, causing the hair follicle to shrink. The catagen phase is followed by the telogen phase, which lasts an average of three months and prepares for the next anagen phase, before the hair falls out. The reason for the difference in hair length between different regions is that the duration of the anagen phase, a unique characteristic of hair follicles, differs from region to region.

[0003] Human hair also has a fixed hair growth cycle, so a certain number of hairs are always maintained. However, as hair loss progresses, the hair papilla present in the hair root becomes smaller, and as the hair papilla becomes smaller, the thickness of the hair becomes thinner, and at the same time, the hair growth cycle becomes shorter. Therefore, as hair loss progresses, the hair becomes very thin and the hair growth cycle becomes even shorter, so it falls out after a short period of growth.

[0004] It is known that hair loss is caused not only by genetic factors and the effects of male hormones, but also by a combination of factors such as endocrine disorders, nutritional deficiencies, drug use, childbirth, fever, surgery, and other severe physical and mental stress. Recently, the number of women experiencing hair loss has been increasing, and the age of those experiencing hair loss is also getting younger, due not only to male pattern hair loss but also to increased stress caused by changes in diet and the social environment.

[0005] Currently, the most commonly used hair loss treatments in Korea include finasteride (trade name Propecia®), dutasteride (trade name Avodart®), and minoxidil (trade name Minoxil® or Rogaine®). Finasteride and dutasteride are 5α-reductase inhibitors that block the conversion of the male hormone testosterone to 5α-dihydrotestosterone (DHT). However, these products cause side effects such as decreased libido, erectile dysfunction, and impaired driving and performance. Meanwhile, minoxidil, while its mechanism of action is not yet fully understood, is a potassium channel opener that hyperpolarizes cell membranes. It is believed that vasodilation and potassium channel opening increase the supply of oxygen, blood, and nutrients to hair follicles, promoting their health. However, this product also causes side effects such as itching, erythema, skin irritation, and eye irritation at the application site, and unwanted hair growth on other body parts besides the head has also been observed. In recent years, hair transplantation has been attempted for patients with severe hair loss, but limitations due to high costs and side effects after the procedure have been pointed out.

[0006] Chemokines are leukocyte chemotactic factors that attract leukocytes to various tissues in the body, a process essential for all bodily responses to inflammation and infection. Chemokines and their receptors are not only central to immune regulation and the pathophysiology of inflammatory and infectious diseases, but in recent years, specific roles have been shown in specific diseases, including autoimmune diseases, depending on the type of chemokine and its receptor, and therapeutic approaches have been proposed that regulate the activity of specific chemokines or their receptors.

[0007] Therefore, the present inventors have endeavored to develop a novel hair loss treatment agent, and as a result have confirmed that treatment with an antibody against CXCL12, a type of chemokine, promotes the growth of hair follicles and hair, thereby completing the present invention. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Tran PB et al., Chemokine receptors in the brain: a developing story.J.Comp.Neurol.2003;457:1-6. [Non-patent document 2] Zou YR et al.,Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.Nature.1998;393:595-599. [Non-patent document 3] Michel et al.Study of gene expression alteration in male androgenetic alopecia: evidence of predominant molecular signaling pathways.British journal of dermatology.2017;177:1322-1336. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide an antibody against CXCL12.

[0010] An object of the present invention is to provide a composition for preventing or treating hair loss, which comprises an antibody against CXCL12. [Means for solving the problem]

[0011] The present invention provides an antibody that specifically binds to CXCL12, a pharmaceutical composition for preventing or treating hair loss, which comprises an antibody that specifically binds to CXCL12, as well as a polynucleotide encoding an antibody that specifically binds to CXCL12 and a vector comprising the same.

[0012] The antibody comprises a heavy chain variable region comprising: a CDRH1 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1; a CDRH2 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2; and a CDRH3 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3; a CDRL1 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 4; a CDRL2 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 5; The antibody may be a monoclonal antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6. ...

[0013] The antibody comprises a heavy chain variable region comprising: a CDRH1 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 1; a CDRH2 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 2; and a CDRH3 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 3; a CDRL1 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 4; a CDRL2 having a sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 5; The antibody may be a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6. Preferably, the antibody is a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3.

[0014] The antibody may be a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising a sequence that has at least 90%, 95%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 7 to 9, and a light chain variable region comprising a sequence that has at least 90%, 95%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 10 to 12; and is preferably a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising a sequence that has at least 90%, 95%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 7 to 9, and a light chain variable region comprising a sequence that has at least 90%, 95%, or 99% sequence identity with any of the amino acid sequences of SEQ ID NOs: 10 to 12.

[0015] The antibody may be a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; or a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0016] The antibody may be a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; or a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0017] The antibody may be a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; or a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12.

[0018] The hair loss may be alopecia areata, hereditary androgenetic alopecia, telogen effluvium, traumatic alopecia, trichotillomania, pressure-induced alopecia, anagen effluvium, pityriasis caudatus, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, congenital alopecia, or hair loss due to a side effect of a drug. The hair loss due to a side effect of a drug may also be hair loss due to an anticancer drug, an anticoagulant, an antidepressant, or a hormonal drug, and the anticancer drug may be an anticancer drug for treating a cancer selected from the group consisting of melanoma, epithelial cancer, breast cancer, cervical cancer, solid cancer, urothelial cell carcinoma, non-small cell lung cancer, pancreatic adenocarcinoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, and gastrointestinal cancer.

[0019] The present invention provides a composition for use in preventing or treating hair loss, comprising the antibody that specifically binds to CXCL12.

[0020] The present invention provides use of a composition comprising the antibody that specifically binds to CXCL12 for the prevention or treatment of hair loss.

[0021] The present invention provides a method for treating hair loss by administering to a subject suffering from hair loss an antibody that specifically binds to CXCL12. [Effects of the Invention]

[0022] The present invention relates to a novel antibody against CXCL12 and a composition for treating hair loss containing the same. The antibody against CXCL12 has the effect of suppressing CXCL12 and promoting hair growth, and is therefore useful for preventing or treating hair loss. [Brief explanation of the drawings]

[0023] [Figure 1] FIG. 1 shows the results of hair follicle growth in a human hair follicle culture model treated with a neutralizing antibody against CXCL12 of the present invention. [Figure 2] FIG. 1 shows the results of hair follicle growth in a human hair follicle culture model treated with a neutralizing antibody against CXCL12 of the present invention. [Figure 3]FIG. 1 shows the results of hair growth in a mouse anagen induction model as a result of treatment with a neutralizing antibody against CXCL12 of the present invention. [Figure 4] FIG. 1 shows the results of hair growth in a mouse anagen induction model as a result of treatment with a neutralizing antibody against CXCL12 of the present invention. [Figure 5] FIG. 1 shows the results of analyzing the antigen-binding affinity of the humanized antibody of the present invention against CXCL12. [Figure 6] FIG. 1 shows the results of hair follicle growth in a mouse whisker culture model treated with the humanized antibody against CXCL12 of the present invention. [Figure 7] FIG. 1 shows the results of hair follicle growth in a mouse whisker culture model treated with the humanized antibody against CXCL12 of the present invention. [Figure 8] FIG. 1 shows the results of hair growth in a mouse anagen induction model as a result of treatment with the humanized antibody against CXCL12 of the present invention. [Figure 9] FIG. 1 shows the results of hair growth in a mouse anagen induction model as a result of treatment with the humanized antibody against CXCL12 of the present invention. [Figure 10] FIG. 1 shows the results of hair growth in an animal model of alopecia areata treated with the humanized antibody against CXCL12 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0024] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will now be described in detail with reference to the accompanying drawings, in order to enable those skilled in the art to easily carry out the present invention. However, the present invention may be embodied in various forms and should not be construed as being limited to the embodiments and examples set forth herein.

[0025] Throughout this specification, when a part is said to "comprise" a certain element, this does not mean that it excludes other elements, but that it may further include other elements, unless otherwise specified.

[0026] The present invention relates to a neutralizing antibody or a humanized antibody against CXCL12, and a composition for treating hair loss comprising an antibody against CXCL12.

[0027] The neutralizing antibody against CXCL12 may be a monoclonal antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6; or a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising CDRH1 having the amino acid sequence of SEQ ID NO: 1, CDRH2 having the amino acid sequence of SEQ ID NO: 2, and CDRH3 having the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising CDRL1 having the amino acid sequence of SEQ ID NO: 4, CDRL2 having the amino acid sequence of SEQ ID NO: 5, and CDRL3 having the amino acid sequence of SEQ ID NO: 6; or a humanized antibody that specifically binds to CXCL12, comprising a heavy chain variable region comprising any of the amino acid sequences of SEQ ID NOs: 7 to 9, and a light chain variable region comprising any of the amino acid sequences of SEQ ID NOs: 10 to 12.

[0028] In the present invention, "CXCL12 (CXC motif chemokine 12)" refers to stromal cell-derived factor 1 (SDF1), a type of chemokine. It is known to interact with CXCR4 as a ligand for the CXC chemokine receptor CXCR4 and has also been reported as a ligand for CXCR7 (RDCI). CXCL12 is widely expressed in various tissue types, including the heart, liver, spleen, kidney, brain, skeletal muscle, endothelial cells, epithelial tissues, and stem cells.

[0029] In the present invention, "chemokine" refers to a basic, heparin-binding, low-molecular-weight protein that induces and activates leukocyte migration. Chemokine molecules contain four cysteine ​​residues, and they are classified into four subfamilies, CXC (CXCL), CC (CCL), CX3C (CX3CL), and C (XCL), depending on the arrangement of the cysteine ​​residues in the first two molecules. At least 40 species have been identified to date.

[0030] In the present invention, a "neutralizing antibody" means an antibody, whether particulate or molecular, that specifically binds to an antigen and eliminates or reduces the activity when the antigen has biological activity such as infectiousness, toxicity, or enzymatic activity in a living body.

[0031] In the present invention, the term "humanized antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by humans and / or an antibody produced by the human antibody production techniques disclosed in the present invention.

[0032] The term "antibody" refers to a natural immunoglobulin or a partially or wholly synthetically produced immunoglobulin. The term also includes any polypeptide or protein containing the antigen-binding domain of an antibody. Antibody fragments containing the antigen-binding domain include molecules such as Fab, scFv, Fv, dAb, Fd, and diabodies. The term "antibody" is used in a broad sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies. It also includes antibody fragments that exhibit the desired biological activity. Antibodies are composed of two heavy chains and two light chains and have variable regions whose amino acid sequences vary depending on the type of target antigen, and constant regions whose sequences do not change.

[0033] The light and heavy chain variable regions contain three highly variable regions called "complementarity-determining regions (CDRs)." CDRs bind to an epitope of an antigen, and each chain CDR typically has three regions (CDR1, CDR2, and CDR3).

[0034] As is well known to those skilled in the art, the term "specifically binding" in the present invention specifically means that an antigen and an antibody interact specifically to form an antigen-antibody complex and induce an immunological reaction.

[0035] In the present invention, "hair loss" refers to a state in which hair is not present in areas where hair should normally be present, regardless of the cause, and includes, but is not limited to, alopecia areata, hereditary androgenic alopecia, telogen effluvium, traumatic alopecia, trichotillomania, pressure-induced alopecia, anagen effluvium, pityriasis alopecia, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, congenital alopecia, or hair loss due to drug side effects.

[0036] In the present invention, "prevention" means any action of suppressing or delaying the onset of a disease by administering a composition, and "treatment" means any action of improving or favorably changing the symptoms of an individual who has developed a disease or an individual who is suspected of having a disease by administering a composition.

[0037] The pharmaceutical composition of the present invention may be formulated into any form suitable for preparation, including oral dosage forms such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, and aerosols, topical skin preparations such as ointments and creams, suppositories, injections, fillers, and sterile injection solutions, by a conventional method.

[0038] The present invention will be described in more detail below with reference to examples, but these examples are merely for the purpose of illustrating the present invention and are not intended to limit the present invention. [Example]

[0039] Generation of neutralizing antibodies against CXCL12 1-1. Preparation of immunized mice Fifty micrograms (50 μg) of recombinant human CXCL12 protein (cat#13511-HNCE) purchased from Sinobiologics (single dose per mouse) was mixed with the same volume of Freund's complete adjuvant (Sigma, USA) to prepare an emulsion. The emulsion was then injected intraperitoneally into six 7-week-old female BALB / C mice. Each mouse received 50 μg of antigen in a total volume of 500 μL. After one and two weeks, the mice were further injected intraperitoneally with emulsions prepared by mixing the antigen with incomplete Freund's adjuvant (Sigma, USA).

[0040] 1-2. Confirmation of antibody production Blood was collected from the eyeballs of mice immunized using the above method, placed in a 1.5 mL microcentrifuge tube, and then centrifuged at 13,000 rpm for 10 minutes. Serum was separated and stored at -20°C until experiments to confirm antibody production were performed. Antibody production was confirmed by enzyme immunoassay using the antigen protein, and then, 3 days before cell fusion, the mice were again intraperitoneally injected with an emulsion of the antigen mixed with incomplete Freund's adjuvant (Sigma, USA).

[0041] 1-3. Hybridoma production After confirming antibody production, the mice were sacrificed. Spleen cells were isolated and fused with myeloma cells P3X63Ag8.653 (ATCC CRL-1580) to generate hybridomas. Specifically, mouse P3X63Ag8.653 cells were cultured in culture plates using RPMI 1640 medium supplemented with 10% fetal bovine serum. To perform cell fusion, P3X63Ag8.653 cells were washed twice with serum-free RPMI 1640 medium (Hyclone, USA) and diluted to 1 × 10 7The cell concentration was adjusted to 100%. Mice were sacrificed by cervical dislocation, and the spleens were collected and then placed in a mesh container (Sigma, USA) for cell separation. A suspension of spleen cells was prepared, and the suspension was then washed by centrifugation. The spleen cell solution was exposed to Tris-NH4Cl (TRIS 20.6 g / L, NH4Cl 8.3 g / L) to lyse red blood cells. Completely separated antibody-producing cells were centrifuged at 400 × g for 5 minutes. They were then washed twice with serum-free medium and resuspended in 10 mL of medium. Lymphocytes were counted using a hemocytometer, and 1 × 10 lymphocytes were obtained. 8 The mixture was mixed with 1 x 10 P3X63Ag8.653 cells (10:1) in serum-free medium. After centrifugation at 400 x g for 5 minutes, 1 mL of the solution was added dropwise with 50% (M / V) polyethylene glycol 1500 (Sigma, USA) pre-warmed to 37°C and mixed for 1 minute. The resulting fusion mixture was diluted with serum-free RPMI 1640 and centrifuged at 400 x g for 3 minutes. The cells were suspended in 35 mL of RPMI 1640 selective medium supplemented with 20% fetal bovine serum and HAT (100 μM hypoxanthine, 0.4 μM aminopterin, 16 μM thymidine). 100 μL of the suspension was loaded onto a 96-well plate coated one day previously with feeder cells (macrophages isolated from the peritoneal cavity using RPMI 1640) and cultured at 37°C in 5% CO2. After 5 days, the HAT medium was replaced every 2–3 days and the cells were cultured for 14 days. After 14 days, the medium was replaced with RPMI 1640 supplemented with 20% fetal bovine serum and HT (HAT medium minus 0.4 μM aminopterin) for secondary culture.

[0042] 1-4. Selection and separation of antibody-producing fused cells The supernatant of the culture medium of the hybridoma cells prepared as described above was collected and tested for the production of antibodies specific to the antigen prepared as described above by enzyme immunoassay. The culture medium of the hybridoma cells showing an appropriate concentration of at least four times that of the negative control group was selected and transferred to a 24-well plate for culture. The cells were then diluted to a limiting dilution so that one cell was placed per well in a 96-well plate. The culture medium was then collected and coated with CXCL12 protein, used as the antigen, at 0.1 μg per well. Finally, hybridoma cells producing monoclonal antibodies were selected by enzyme immunoassay.

[0043] 1-5. Monoclonal antibody analysis The neutralizing antibody against CXCL12 obtained through the above process was named 2B12. Sequence analysis confirmed that the antibody contained a heavy chain variable region including CDRH1 having the sequence of SEQ ID NO: 1, CDRH2 having the sequence of SEQ ID NO: 2, and CDRH3 having the sequence of SEQ ID NO: 3, and a light chain variable region including CDRL1 having the sequence of SEQ ID NO: 4, CDRL2 having the sequence of SEQ ID NO: 5, and CDRL3 having the sequence of SEQ ID NO: 6, as shown in Table 1.

[0044] [Table 1] [Example]

[0045] Confirmation of the hair follicle growth-promoting effect of treatment with a neutralizing antibody against CXCL12 in a human hair follicle culture model The hair follicle growth-promoting effect of the neutralizing antibody against CXCL12 prepared in Example 1 was confirmed as follows.

[0046] Hair follicles from a man in his 30s were cultured in organ culture. The follicles were cultured in a 48-well plate with one follicle per well, and divided into six groups: a negative control group, a positive control group (commercial antibody 50 ng / mL, 500 ng / mL), and a 2B12 treatment group (0.05 ng / mL, 0.5 ng / mL, 5 ng / mL). The human hair follicles were then treated with each treatment and cultured in an incubator for four days.

[0047] As shown in Figures 1 and 2, the results of the human hair follicle culture experiment confirmed that treatment with the neutralizing antibody 2B12 against CXCL12 of the present invention promoted hair follicle growth, at a level equivalent to or superior to that of the positive control group.

[0048] Therefore, it is clear that the neutralizing antibody against CXCL12 of the present invention promotes the growth of hair follicles by suppressing CXCL12, and has an excellent hair growth effect. [Example]

[0049] Confirmation of the hair growth promoting effect of treatment with a neutralizing antibody against CXCL12 in a mouse anagen induction model The hair growth promoting effect of the neutralizing antibody against CXCL12 prepared in Example 1 was confirmed as follows.

[0050] Seventeen 7-week-old male C3H / HeN mice were mechanically depilated, and the remaining hair was completely removed with a hair remover. They were then allowed to acclimate for one day before use. Each group consisted of 5–6 mice. The mice were divided into four groups: a negative control group (IgG1, 5 μg / head), a positive control group (commercial antibody, 5 μg / head), and a 2B12 antibody treatment group (5 μg / head). Each group received four subcutaneous injections into the backs of the mice at 4-day intervals (total dose: 20 μg / head). The extent of hair growth on the backs of the mice was monitored for 17 days after each antibody injection. After 17 days, the backs of mice with hair growth were shaved with a razor, and the resulting hair was weighed.

[0051] As shown in Figures 3 and 4, the hair growth promoting effect in mice was evaluated, and it was confirmed that administration of the neutralizing antibody 2B12 against CXCL12 of the present invention promoted hair growth.

[0052] Therefore, it is clear that the neutralizing antibody against CXCL12 of the present invention promotes hair growth by suppressing CXCL12, and has an excellent hair growth effect. [Example]

[0053] Generation of a humanized antibody based on a neutralizing antibody against CXCL12 4-1. Production of humanized antibodies To prepare a humanized antibody by modifying the neutralizing antibody against CXCL12 in Example 1 to have a structure corresponding to that of a human, the CDR of a mouse antibody was grafted onto a human antibody using the CDR grafting method. The sequence of the prepared humanized antibody was analyzed, and the results are shown in Table 2.

[0054] [Table 2]

[0055] 4-2. Construction and production of humanized IgG To construct humanized IgG antibodies, DNA sequences encoding the heavy and light chains of chimeric and humanized antibodies were synthesized and inserted into the pcDNA3.4 vector to construct expression plasmids for full-length hIgG. The heavy and light chain plasmids were transfected according to GenScriptProbio's standard operating procedures (SOPs), and antibody expression was carried out in Expi293F cell culture. As shown in Table 3, a total of nine combinations of humanized antibodies against the heavy and light chain variable regions were expressed.

[0056] [Table 3]

[0057] 4-3. Confirmation of antigen-binding affinity of purified humanized IgG (ELISA) The antigen-binding affinities of the nine combinations of humanized antibodies were confirmed as follows.

[0058] All antibodies were initially diluted at 100 μg / mL and serially diluted by 1 / 3 to a total volume of 100 μL per well of a plate. The plates were incubated overnight at 4°C. The following day, the plates were incubated with 3% nonfat milk at 37°C for 1 hour. The plates were then washed with wash buffer and then incubated with 100 μL of 2 μg / mL antigen (SDF-1α) at ​​room temperature for 2 hours. After 2 hours, the wells were washed with wash buffer and then treated with secondary antibody (0.1 μg / mL His-[HRP]) for 45 minutes. The washed wells were then incubated with 100 μL of TMB-substrate for 10 minutes, followed by the addition of 50 μL of 1M HCl to terminate the reaction. Binding affinity was analyzed by measuring absorbance at 450 nm using a spectrophotometer.

[0059] As shown in Figure 5, the analysis of antigen-binding affinity confirmed that the nine humanized antibody combinations had excellent antigen-binding affinity, and in particular, four humanized antibodies had superior antigen-binding affinity to chimeric antibodies. [Example]

[0060] Confirmation of the hair follicle growth-promoting effect of humanized antibody against CXCL12 in a mouse whisker model The humanized antibody against CXCL12 prepared in Example 4 was examined for its effect of promoting hair follicle growth as follows.

[0061] Terminal hair from female C57BL / 6 mice was trimmed and cultured in organ culture. Hair follicles were cultured in a 48-well plate with one per well. The terminal hairs were divided into 21 groups: a negative control group, chimeric antibody treatment groups (50 ng / mL, 500 ng / mL), and IgG humanized antibody treatment groups (50 ng / mL, 500 ng / mL), and then cultured in an incubator at 37°C for 2 days.

[0062] As shown in FIGS. 6 and 7, the results of the mouse hair follicle culture experiment confirmed that treatment with the humanized antibody against CXCL12 of the present invention promoted the growth of all hair follicles.

[0063] Therefore, it was confirmed that the humanized antibody against CXCL12 of the present invention promotes the growth of hair follicles by suppressing CXCL12, and has an excellent hair growth effect. [Example]

[0064] Confirmation of the hair growth promoting effect of humanized antibody treatment against CXCL12 in a mouse anagen induction model The hair growth promoting effect of the humanized antibody against CXCL12 prepared in Example 4 was confirmed as follows.

[0065] The hair of 23 7-week-old male C3H / HeN mice was shortened using a hair removal machine, and the remaining hair was completely removed using a hair remover. The mice were then allowed to acclimate for one day. The experiment was conducted in groups of 5-6 mice each. The groups were as follows: Group 1: Control group (IgG1, 50 μg / head), 5 animals Group 2: Chimeric antibody (50 μg / head) administration group, 6 animals Group 3: (#1) VH1-VL1 antibody (50 μg / head) administration group, 6 animals Group 4: (#3) VH1-VL3 antibody (50 μg / head) administration group, 6 animals Group 5: (#4) VH2-VL1 antibody (50 μg / head) administration group, 6 animals

[0066] Each group of mice was subcutaneously injected once (50 μg / head) into the back of the mouse, and the degree of hair growth on the back of the mouse was monitored for 17 days after the injection of each antibody. After 17 days, the backs of mice with hair growth were shaved with a razor, and the weight of the resulting hair was measured.

[0067] As shown in Figures 8 and 9, the hair growth promoting effect in mice was evaluated, and it was confirmed that treatment with the humanized antibody against CXCL12 of the present invention promoted hair growth.

[0068] Therefore, it was confirmed that the humanized antibody against CXCL12 of the present invention promotes hair growth by inhibiting CXCL12, and has an excellent hair growth effect. [Example]

[0069] Confirmation of the hair growth promoting effect of humanized antibody against CXCL12 in an animal model of alopecia areata The hair growth promoting effect of the humanized antibody against CXCL12 prepared in Example 4 was confirmed as follows.

[0070] Eddy Hsi Chun Wang et al. conducted an experiment to establish an animal model of alopecia areata by transplanting skin-draining lymph node cells (LNCs) derived from animals with alopecia areata (alopecia areata) into a 10-week-old C3H / HeJ animal model. The animals were divided into groups of three. The groups were as follows: Group 1: Control group (IgG1, 100 μg / head), 3 animals Group 2: VH1-VL3 antibody (100 μg / head) administration group, 3 animals

[0071] On the first and tenth days of the experiment, each group of mice was given a subcutaneous injection into the abdominal skin twice (50 μg / head / time), and the hair loss sites were photographed over a period of six weeks to observe the state of hair growth on the mice's skin.

[0072] As shown in Figure 10, the hair growth promoting effect was evaluated in an animal model of alopecia areata, and it was confirmed that treatment with the humanized antibody against CXCL12 of the present invention promoted hair growth.

[0073] Therefore, it was confirmed that the humanized antibody against CXCL12 of the present invention promotes hair growth and has excellent hair growth effects by suppressing CXCL12, and in particular has therapeutic effects for alopecia areata.

Claims

1. a heavy chain variable region comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and a CDRH3 having the amino acid sequence of SEQ ID NO: 3; a light chain variable region comprising a CDRL1 having the amino acid sequence of SEQ ID NO:4, a CDRL2 having the amino acid sequence of SEQ ID NO:5, and a CDRL3 having the amino acid sequence of SEQ ID NO:6; A monoclonal antibody that specifically binds to CXCL12.

2. a heavy chain variable region comprising a CDRH1 having the amino acid sequence of SEQ ID NO: 1, a CDRH2 having the amino acid sequence of SEQ ID NO: 2, and a CDRH3 having the amino acid sequence of SEQ ID NO: 3; a light chain variable region comprising a CDRL1 having the amino acid sequence of SEQ ID NO:4, a CDRL2 having the amino acid sequence of SEQ ID NO:5, and a CDRL3 having the amino acid sequence of SEQ ID NO:6; A humanized antibody that specifically binds to CXCL12.

3. a heavy chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 7 to 9; and a light chain variable region comprising any one of the amino acid sequences of SEQ ID NOs: 10 to 12. A humanized antibody that specifically binds to CXCL12.

4. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

10. A humanized antibody that specifically binds to CXCL12.

5. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

11. A humanized antibody that specifically binds to CXCL12.

6. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

12. A humanized antibody that specifically binds to CXCL12.

7. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

10. A humanized antibody that specifically binds to CXCL12.

8. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

11. A humanized antibody that specifically binds to CXCL12.

9. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

12. A humanized antibody that specifically binds to CXCL12.

10. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

10. A humanized antibody that specifically binds to CXCL12.

11. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

11. A humanized antibody that specifically binds to CXCL12.

12. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising the amino acid sequence of SEQ ID NO:

12. A humanized antibody that specifically binds to CXCL12.

13. A pharmaceutical composition for preventing or treating hair loss, comprising the antibody according to any one of claims 1 to 12.

14. The hair loss is selected from the group consisting of alopecia areata, hereditary androgenetic alopecia, telogen effluvium, traumatic alopecia, trichotillomania, pressure-induced alopecia, anagen effluvium, pityriasis alopecia, syphilitic alopecia, seborrheic alopecia, symptomatic alopecia, cicatricial alopecia, congenital alopecia, and hair loss due to side effects of medication, The pharmaceutical composition for preventing or treating hair loss according to claim 13.

15. The hair loss caused by a drug side effect is hair loss caused by a drug selected from the group consisting of anticancer drugs, anticoagulants, antidepressants, and hormone drugs. The pharmaceutical composition for preventing or treating hair loss according to claim 14.

16. The anticancer agent is an anticancer agent for treating a cancer selected from the group consisting of melanoma, epithelial cancer, breast cancer, cervical cancer, solid cancer, urothelial cell carcinoma, non-small cell lung cancer, pancreatic adenocarcinoma, kidney cancer, ovarian cancer, pancreatic cancer, prostate cancer, esophageal cancer, and gastrointestinal cancer. The pharmaceutical composition for preventing or treating hair loss according to claim 15.

17. A polynucleotide encoding the antibody of any one of claims 1 to 12.

18. A vector comprising the polynucleotide of claim 17.

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