Treatment of hair loss

Abstract

The present invention provides compounds, compositions, and methods for treating alopecia areata (AA) in patients who require it.

Description

[Technical Field]

[0001] (Cross-reference of related applications)

[0002] This application claims priority and benefit to U.S. Provisional Patent Application No. 63 / 516,534, filed on 31 July 2023, the entirety of which is incorporated herein by reference. [Background technology]

[0003] Interleukin-7 (IL-7) is a member of the common gamma chain (γc) family of cytokines, which also includes IL-2, IL-4, IL-9, IL-15, and IL-21. Like its other members, IL-7 signals via a triple complex formed by its intrinsic α-receptor, IL-7Rα (CD127), and common gamma receptor (γc, CD132). IL-7 regulates T cell homeostasis by enhancing the survival and proliferation of naive and memory T cells, but is considered essential for B cell development in humans. IL-7Rα is expressed on T cells, including most mature T cells, as well as on dendritic cells (DCs) and activated monocytes.

[0004] The IL-7 / IL-7R interaction stimulates Janus kinase (JAK) and signaling and transcriptional activator (STAT) proteins, promoting target gene transcription through subsequent activation of phosphoinositol 3 kinase (PI3K) / Akt or the Src pathway. IL-7 Rα is also utilized by thymic stromal lymphopoietin (TSLP) as part of a complex containing a second receptor chain, TSLPR.

[0005] The TSLPR complex, containing CD127 and TSLPR(CRLF2), is expressed on several types of immune cells, including B cells, T cells, monocytes, and dendritic cells (DCs). TSLP has been shown to be a potent activator of antigen-presenting cells, such as DCs, for inducing TH2-mediated immune responses. For example, TSLP-stimulated DCs increase OX40L expression and production of TH2 chemokines (such as CCL17 and CCL21), leading to priming of TH2 cell development.

[0006] IL-7 plays a crucial role in the development of a normal immune system because it is essential for thymic development, peripheral maintenance, and lymphocyte survival. Thymic T cell precursors require IL-7 for proliferation, differentiation, and survival. In the periphery, IL-7 regulates T cell hemostasis by enhancing the survival and proliferation of naive and memory T cells.

[0007] IL-7 is a tissue-derived cytokine, primarily produced by stromal and epithelial cells in various tissues. For example, in the small and large intestines, IL-7 is produced by goblet epithelial cells and has been described as essential for the persistence of chronic colitis in animal models. IL-7 has also been shown to interfere with the immunosuppressive capacity of regulatory T cells (Tregs). Therefore, drugs that can modulate IL-7 activity in vivo, thereby reducing the survival / function of pathogenic T cells and / or increasing the induction of regulatory T cells, are highly desirable for the treatment of inflammatory diseases such as inflammatory bowel disease.

[0008] Alopecia areata, also known as patchy baldness, is a condition characterized by hair loss in part or all areas of the body. Often, this results in several bald patches on the scalp, each approximately coin-sized. Generally, alopecia areata is characterized by bald patches on the scalp, beard, and sometimes above the eyebrows, and eyelashes may also fall out. However, alopecia areata differs from the more common pattern baldness seen in men.

[0009] Psychological stress and illness can be contributing factors to alopecia areata in at-risk individuals, but in most cases, there is no obvious trigger. People with alopecia areata are generally otherwise healthy. In a small number of cases, all hair on the scalp is lost (alopecia totalis) or all body hair is lost (alopecia universalis). Hair loss can be permanent or temporary.

[0010] Alopecia areata is thought to be an autoimmune disease resulting from a breach of the immune privilege of hair follicles. Risk factors include a family history of the condition. In identical twins, if one is affected, the other is approximately 50% likely to be affected as well. The underlying mechanism may involve the body being unable to recognize its own cells, followed by immune-mediated destruction of the hair follicles.

[0011] Dai et al. (Sci. Adv. 2021;7: eabd1866) described that the interleukin-7 (IL-7) signaling pathway plays a crucial role in regulating T cell function and survival, and that exogenous IL-7 accelerated the onset of AA by enhancing the proliferation of hair loss-related T cells, while IL-7 blockade halted the progression of AA and reversed early AA in C3H / HeJ mice.

[0012] Currently, there is no known cure for this condition. Some treatments, particularly triamcinolone injections and 5% minoxidil topical cream, are effective in accelerating hair regrowth. Sunscreen, head coverings to protect from cold and sunlight, and glasses are also recommended if eyelashes are missing. In over 50% of cases of the suddenly onset “patchy” disease, hair regrows within one year. In patients with only one or two patches, this one-year recovery occurs in up to 80% of cases. However, most patients experience two or more episodes in their lifetime. In many patients, hair loss and regrowth occur simultaneously over several years. In those who have lost all body hair, the recovery rate is less than 10%.

[0013] Baricitinib, a Janus kinase (JAK) inhibitor, is the only FDA-approved treatment for alopecia. While JAK inhibitors have shown hair regrowth in patients with severe alopecia (≥50% hair loss), the increased risk of serious side effects may deter this option for some patient populations. Other standard treatments for alopecia include topical corticosteroids, immunotherapy, and phototherapy. The lack of efficacy and side effects of these latter treatments limit their usefulness across the overall patient population. The development of safe and effective selective treatments for severe alopecia could provide an alternative treatment option for patients who cannot tolerate baricitinib or for whom baricitinib is contraindicated.

[0014] Approximately 0.15% of people experience alopecia areata at one point in their lives, and 2% experience it at some point in time. The incidence of alopecia areata appears to increase almost linearly with age, but the median age of onset seems to be between 25 and 36 years old. Women are more affected than men.

[0015] Therefore, there is a need to develop effective treatments to alleviate hair loss in alopecia areata and other hair loss disorders. [Overview of the project]

[0016] This disclosure provides a method for reducing alopecia disorders (including alopecia areata (AA) and autoimmune-driven AA) or alopecia in a mammalian subject (e.g., human) that requires it, the method comprising administering an effective amount of a composition comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence comprising the sequence of SEQ ID NO: 1 (DHAMH), a VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and a VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), a VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and a VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT), thereby treating an alopecia disorder (e.g., AA) and / or reducing alopecia in a mammalian subject.

[0017] Furthermore, this specification provides compositions for use in the treatment of alopecia disorders (e.g., alopecia areata (AA), including autoimmune-driven AA) or for use in reducing alopecia disorders (e.g., reducing alopecia associated with AA) in mammalian subjects where such use is required, comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence comprising the sequence of SEQ ID NO: 1 (DHAMH), a VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and a VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), a VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and a VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT).

[0018] Furthermore, a method for inducing hair growth in mammalian subjects requiring it is provided herein, the method comprising administering an effective amount of a composition containing an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) containing a VH CDR1 sequence of SEQ ID NO: 1 (such as any one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) containing a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6, thereby inducing hair growth in mammalian subjects.

[0019] Furthermore, compositions for use in inducing hair growth in mammals requiring such induction are provided herein, the compositions comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence containing SEQ ID NO: 1 (such as any one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6.

[0020] Also provided herein is a composition for use in a therapeutic method for inducing hair growth in mammals that require it, the composition comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence of SEQ ID NO: 1 (such as one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6.

[0021] Furthermore, this disclosure provides a pharmaceutical composition comprising an antibody having two heavy chains and two light chains, each of which the heavy chains has the amino acid sequence of SEQ ID NO: 23, and each of the light chains has the amino acid sequence of SEQ ID NO: 24, and the antibody is formulated as a pharmaceutically acceptable solution having 50 to 200 mg / mL (e.g., about 100 mg / mL) of antibody in about 50 to 200 mg / mL (e.g., about 15 to 25 mM (e.g., about 20 mM)) histidine, 200 to 300 mM (e.g., about 260 mM) sucrose, and about 0.03 to 0.07% (w / v) (e.g., about 0.05% (w / v) at pH 6.0) polysorbate 80 at pH 6.0.

[0022] Furthermore, this disclosure provides a container (e.g., a polycarbonate bottle) containing an extractable volume of about 2 mL of the pharmaceutical composition described herein.

[0023] Any embodiment described herein, including those described only in the examples, may be combined with any one or more other embodiments unless such combination is expressly excluded or inappropriate. Therefore, as used herein, the term “embodiment” should not be considered to exclude features enumerated in other embodiments. [Brief explanation of the drawing]

[0024] [Figure 1] Figure 1 is a schematic diagram of the Phase II clinical trial described in Example 1. Abbreviations used include: EOS - End of Study, Q2W - Once every two weeks, SALT - Alopecia Severity Tool, SC - Subcutaneous. The primary endpoint is the percentage change from baseline in the SALT score. The final dose of compound A or placebo is administered at week 24. [Figure 2]Figure 2 shows the SALT assessment tool by Olson et al. (J Am Acad Dermatol. 2004;51(3):440-447. doi: 10.1016 / j.jaad.2003.09.032). The total SALT score is calculated based on the formula [(LRS×0.18)+(RRS×0.18)+(TRS×0.40)+(BRS×0.24)], where LRS is the left quadrant score, TRS is the upper quadrant score, RRS is the right quadrant score, and BRS is the posterior quadrant score. [Figure 3] Figure 3 shows SKINDEX-16 for AA. [Figure 4] Figures 4A-4C show the effects of prophylactic therapy on weight loss (Figure 4A), plasma cytokine levels (ELISA) (Figure 4B), and the percentage of human T cells in the blood (CD45+ CD3+) (Figure 4C), as measured by flow cytometry, in a Hu-NSG mouse model of GvHD using compound A (Comp. A) and the control drug IL-7Rα antibody (A3312F). Measurements were determined 21 days after PBMC migration. Abbreviations: CCL1: antimicrobial protein with bactericidal activity, CD: cluster differentiation, GvHD: graft-versus-host disease, Hu-NSG: humanized nod / SCID / IL2rγnull, IFN: interferon, IL-7Rα: interleukin-7 receptor alpha, IP: intraperitoneal, PBMC: peripheral blood mononuclear cells, TNF: tumor necrosis factor. [Figure 5A]Figures 5A-5B show targeted binding and inhibition of pSTAT5 levels on human T cells in the blood (Figure 5A) and spleen (Figure 5B) of Hu-NSG mice treated with the control drug IL-7Rα antibody (A3312F) or vehicle (PBS). The antibody was administered in vivo at concentrations of 0.2, 1, and 5 mg / kg. IL-7-induced pSTAT5 was evaluated by ex vivo assay, including untargeted IL-7-negative control samples (CTL Unstim). Abbreviations: CTL: control, Hu-NSG: humanized nod / SCID / IL2rγnull, IL-7Rα: interleukin-7 receptor alpha, MFI: mean fluorescence intensity, PBS: phosphate-buffered saline, pSTAT5: phosphorylation signaling and transcriptional activator 5. [Figure 5B] Figures 5A-5B show targeted binding and inhibition of pSTAT5 levels on human T cells in the blood (Figure 5A) and spleen (Figure 5B) of Hu-NSG mice treated with the control drug IL-7Rα antibody (A3312F) or vehicle (PBS). The antibody was administered in vivo at concentrations of 0.2, 1, and 5 mg / kg. IL-7-induced pSTAT5 was evaluated by ex vivo assay, including untargeted IL-7-negative control samples (CTL Unstim). Abbreviations: CTL: control, Hu-NSG: humanized nod / SCID / IL2rγnull, IL-7Rα: interleukin-7 receptor alpha, MFI: mean fluorescence intensity, PBS: phosphate-buffered saline, pSTAT5: phosphorylation signaling and transcriptional activator 5. [Figure 6A] Figures 6A and 6B show data from human Phase I clinical trials after administration of single-dose escalation doses (SAD; 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg) and multiple-dose doses (MAD; 1 mg / kg) (see Example 8). Figure 6A shows the percentage of IL-7 receptor occupancy (RO) in CD3+ T cells, and Figure 6B shows the percentage of inhibition of phosphorylated STAT5 (pSTAT5)-mediated IL-7 signaling in T cells. [Figure 6B]Figures 6A and 6B show data from human Phase I clinical trials after administration of single-dose escalation doses (SAD; 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg) and multiple-dose doses (MAD; 1 mg / kg) (see Example 8). Figure 6A shows the percentage of IL-7 receptor occupancy (RO) in CD3+ T cells, and Figure 6B shows the percentage of inhibition of phosphorylated STAT5 (pSTAT5)-mediated IL-7 signaling in T cells. [Figure 7A] Figure 7A shows data (mean ± SD) regarding serum concentration profiles in patients with atopic dermatitis (AD) after subcutaneous administration of compound A at a dose of 2 or 3 mg / kg every two weeks. SD: standard deviation. 5 μg / mL represents the concentration with the predicted maximum receptor occupancy in the skin. [Figure 7B] Figure 7B shows preliminary data (mean ± SD) on IL-7Rα receptor occupancy (RO) on circulating CD3+ T cells in patients with atopic dermatitis (AD) after subcutaneous administration of compound A at a dose of 2 or 3 mg / kg every two weeks. RO: receptor occupancy, SD: standard deviation. The dashed vertical line represents the administration day. [Modes for carrying out the invention]

[0025] The present invention provides a method for treating alopecia disorders (such as alopecia areata (AA)) in mammals (e.g., humans) that require it, or a method for reducing hair loss in said alopecia disorders (such as reducing hair loss associated with AA, including autoimmune-driven AA), the method comprising administering an effective amount of a composition comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence of SEQ ID NO: 1 (DHAMH) (such as one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and a VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), a VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and a VL CDR1 sequence of SEQ ID NO: 6 (QQFNSYPLWIT) The CDR3 sequence comprises a light chain variable region (LCVR) and, as a result, treatment of alopecia disorders (e.g., AA) and / or reduction of alopecia in mammalian subjects (e.g., humans).

[0026] Furthermore, this specification provides compositions for use in mammals (e.g., humans) that require such use for alopecia disorders (including alopecia areata (AA), autoimmune-driven AA), or for reducing hair loss in alopecia disorders (such as reducing hair loss associated with AA), the compositions comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence containing the sequence of SEQ ID NO: 1 (DHAMH), a VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and a VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), a VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and a VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT).

[0027] In some embodiments, the alopecia disorder includes androgenic alopecia, telogenous alopecia, alopecia areata, tinea capitis, hypotrichosis, or hereditary simple trichorrhizosis.

[0028] Furthermore, the present disclosure provides a method for inducing hair growth in a mammal (e.g., human) subject that requires it, the method comprising administering an effective amount of a composition comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence of SEQ ID NO: 1 (such as one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6, thereby inducing hair growth in a mammal (e.g., human) subject.

[0029] Also provided herein are compositions used to induce hair growth in mammals (e.g., humans) that require it, the compositions comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence of SEQ ID NO: 1 (such as one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6.

[0030] In some embodiments, the method is a cosmetic or non-therapeutic method.

[0031] This specification also provides compositions used in therapeutic methods for inducing hair growth in mammals (e.g., humans) that require it, the compositions comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising a VH CDR1 sequence of SEQ ID NO: 1 (such as one of SEQ ID NOs from 7 to 22), a VH CDR2 sequence of SEQ ID NO: 2, and a VH CDR3 sequence of SEQ ID NO: 3, and (b) a light chain variable region (LCVR) comprising a VL CDR1 sequence of SEQ ID NO: 4, a VL CDR2 sequence of SEQ ID NO: 5, and a VL CDR3 sequence of SEQ ID NO: 6. In some embodiments, the subject (e.g., human) has or is suffering from a hair loss disorder.

[0032] In some embodiments, the subject suffers from a hair loss disorder, which is selected from androgenetic alopecia, telogen effluvium, alopecia areata, tinea capitis, hypotrichosis, cutaneous lupus such as discoid lupus erythematosus, bald folliculitis, frontal fibrous alopecia, and hereditary simple trichorrhizosis.

[0033] In some embodiments, hair loss disorders are associated with autoimmune conditions / diseases such as alopecia areata, alopecia totalis, systemic lupus erythematosus (SLE), and lupus discoid; hypothyroidism such as Hashimoto's thyroiditis; hyperthyroidism such as Graves' disease; dermatomyositis; scleroderma; and certain autoimmune disorders of the skin (such as pemphigus vulgaris).

[0034] In some embodiments, the hair loss disorder is androgenic alopecia.

[0035] In some embodiments, the hair loss disorder is alopecia areata.

[0036] In some embodiments, alopecia areata is alopecia totalis or alopecia universalis.

[0037] In some embodiments, the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24.

[0038] In some embodiments, the antibody is administered subcutaneously (sc) to the subject.

[0039] In some embodiments, the antibody is administered once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every eight weeks (Q8W), once every twelve weeks (Q12W), or any intermittent PRN (as needed).

[0040] In some embodiments, each dose starting with the second dose is separated from the immediately preceding dose by the same number of days, preferably by approximately the same time on the day of administration.

[0041] In some embodiments, the antibody is administered once every two weeks, preferably starting with the second dose, and each dose is separated from the immediately preceding dose by 14 days, preferably at approximately the same time on the day of administration.

[0042] In some embodiments, the antibody is administered in 10 to 15 consecutive doses (e.g., 11, 12, or 13 doses). In some embodiments, the antibody is administered in 10 consecutive doses. In some embodiments, the antibody is administered in 11 consecutive doses. In some embodiments, the antibody is administered in 12 consecutive doses. In some embodiments, the antibody is administered in 13 consecutive doses. In some embodiments, the antibody is administered in 14 consecutive doses. In some embodiments, the antibody is administered in 15 consecutive doses.

[0043] In some embodiments, antibodies are administered for up to 10 weeks, 13 weeks, 26 weeks, 52 weeks, 2 years, 3 years, 5 years, 10 years, or for life / permanently. In some embodiments, antibodies are administered as needed when hair loss occurs.

[0044] In a particular embodiment, the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24 and is administered subcutaneously (sc) to mammalian subjects (e.g., humans) at a dose of approximately 200 mg per dose in 10 to 15 consecutive doses (e.g., 11, 12, or 13 doses) every two weeks (Q2W).

[0045] In some embodiments, the subjects have moderate to severe AA, or severe to very severe AA (e.g., with overall scalp hair loss of ≥50% as defined by the SALT score).

[0046] As used herein, “moderate AA” has a SALT score of 21–49. “Severe AA” has a SALT score of 50–94. “Very severe AA” has a SALT score of 95–100. “Moderate to severe AA” has a SALT score of 21–94. “Severe to very severe AA” has a SALT range of 50–100.

[0047] In some embodiments, hair loss in a subject is assessed by the mean percentage relative change in the Alopecia Severity Tool (SALT) score at 12 weeks (week 12), 18 weeks (week 18), 24 weeks (week 24), 26 weeks (week 26), 48 weeks (week 48), and / or 52 weeks (week 52) compared to the baseline SALT score obtained immediately before the first dose in week 0. As used herein, this refers to the Saseline SALT score assessment obtained within 1 day, 12 hours, 6 hours, or 1 hour of the first dose in week 0.

[0048] In some embodiments, a relative reduction of ≥50% of the SALT score is achieved in subjects at 12 weeks (week 12), 18 weeks (week 18), and / or 24 weeks (week 24) of the first dose at week 0.

[0049] In some embodiments, the target absolute SALT score is ≤5, 10, 20, 30, or 50 after 12 weeks (week 12), 18 weeks (week 18), and / or 24 weeks (week 24) of the first dose in week 0.

[0050] In some embodiments, subjects have a relative reduction of ≥50% in their SALT score from baseline to week 36 and / or week 24.

[0051] In some embodiments, the target absolute SALT score is ≤5, ≤10, ≤20, ≤30, or ≤50 at week 36.

[0052] In some embodiments, hair loss includes eyelash loss and / or eyebrow hair loss.

[0053] In some embodiments, the reduction in eyebrow alopecia is based on a score of 0 or 1 (total coverage or minimal gap) on the ClinRO Scale for eyebrow alopecia, indicated by an improvement of ≥2 points from baseline at 24 weeks and / or 36 weeks.

[0054] In some embodiments, the reduction in eyelash loss is based on a ClinRO measure score of 0 or 1 (total coverage or minimal gap) for eyelash depilation, indicated by an improvement of ≥2 points from baseline at 24 weeks and / or 36 weeks.

[0055] In some embodiments, patients showed improved quality of life (QOL) after treatment, as measured by improvement in SKINDEX-16 AA compared to baseline at weeks 12, 18, 24, and / or 36.

[0056] In some embodiments, the antibody is formulated at pH 6.0 as a 100 mg / mL solution in 20 mM histidine, 260 mM sucrose, and 0.05% (w / v) polysorbate 80, optionally together with 0.05 mM pentetic acid.

[0057] In some embodiments, antibodies are administered to subjects based on a body weight-based dosing regimen.

[0058] In some embodiments, the antibody is administered to the subject in doses of approximately 0.1 mg / kg to 10 mg / kg, approximately 0.2 mg / kg to 8 mg / kg, approximately 0.5 mg / kg to 5 mg / kg, approximately 1 mg / kg to 4 mg / kg, or approximately 2 mg / kg to 3 mg / kg.

[0059] In some embodiments, the antibody is administered to the subject at doses of approximately 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0060] In some embodiments, antibodies are administered to subjects based on body surface area (BSA)-based dosing regimens. For example, doses for adult male patients of any weight (kg) and height (cm) can be calculated / converted based on the BSA-based dose used for a standard male with a weight of 91 kg and a height of 175 cm, resulting in doses of approximately 10 mg to 800 mg, approximately 20 mg to 500 mg, approximately 50 mg to 300 mg, and approximately 100 mg to 250 mg per dose.

[0061] In some embodiments, the dose for an adult male patient of any weight (kg) and height (cm) can be calculated / converted based on the BSA-based dose used for a standard male with a weight of 91 kg and a height of 175 cm, resulting in doses of approximately 25 mg per dose, approximately 50 mg per dose, approximately 100 mg per dose, approximately 150 mg per dose, approximately 200 mg per dose, approximately 250 mg per dose, approximately 300 mg per dose, or approximately 350 mg per dose.

[0062] In some embodiments, doses for adult female patients of any weight (kg) and height (cm) can be calculated / converted based on the BSA-based dose used for a woman with a standard weight of 77.5 kg and height of 160 cm, resulting in doses of approximately 10 mg to 800 mg per dose, approximately 20 mg to 500 mg per dose, approximately 50 mg to 300 mg per dose, or approximately 100 mg to 250 mg per dose.

[0063] In some embodiments, the dose for an adult female patient of any weight (kg) and height (cm) can be calculated / converted based on the BSA-based dose used for a woman with a standard weight of 77.5 kg and height of 160 cm to reach a dose of approximately 25 mg per dose, approximately 50 mg per dose, approximately 100 mg per dose, approximately 150 mg per dose, approximately 200 mg per dose, approximately 250 mg per dose, approximately 300 mg per dose, or approximately 350 mg per dose.

[0064] In some embodiments, the BSA dose is based on the Dubois formula for BSA dose, where Dose = BSA-based dose × 0.007184 × Height (cm) 0.725 ×Weight (kg) 0.425 That is the case.

[0065] In some embodiments, the BSA dose is based on the Monteler formula for BSA dose, where dose = BSA-based dose × square root [(height (cm) × weight (kg)) / 3600].

[0066] In some embodiments, antibodies are administered to subjects based on a fixed / flat dosing regimen.

[0067] In some embodiments, the antibody is administered to the subject in doses of approximately 10 mg to 800 mg, 20 mg to 500 mg, 50 mg to 300 mg, or 100 mg to 250 mg.

[0068] In some embodiments, the antibody is administered to the subject in doses of approximately 25 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg per dose.

[0069] In some embodiments, antibodies are administered to subjects based on a weight-group dosing regimen, in which patients within a specific weight range (weight zone) are administered a fixed dose for that particular weight zone. In some embodiments, patients weighing approximately 10–25 kg are grouped into the same weight zone and administered the same fixed dose. In some embodiments, patients weighing approximately 25–50 kg are grouped into the same weight zone and administered the same fixed dose. In some embodiments, patients weighing approximately 50–75 kg are grouped into the same weight zone and administered the same fixed dose. In some embodiments, patients weighing approximately 75–100 kg are grouped into the same weight zone and administered the same fixed dose. In some embodiments, the fixed dose for each weight zone is based on the average weight of the weight zone (for example, all patients in the 50–75 kg weight zone are administered a fixed dose based on the dose for the intermediate weight, i.e., 62.5 kg for this weight zone). In some embodiments, the fixed doses for the 50-75 kg weight range are approximately 150 mg, 200 mg, 250 mg, 300 mg, or 350 mg. In some embodiments, the fixed dose for a weight range is proportional to the fixed dose for the 50-75 kg weight range (for example, the fixed dose for the 10-25 kg weight range is based on a median weight of 17.5 kg, which is 17.5 / 62.5 = 28% of the fixed dose for the 50-75 kg weight range).

[0070] In some embodiments, the antibody is administered to the subject based on a dosing regimen including a loading dose or an accelerated initial dosing schedule, followed by a maintenance dose, the maintenance dose being administered according to one of the aforementioned weight-based, body surface area (BSA)-based, or fixed-dose dosing regimens. In some embodiments, the loading dose includes two doses of the maintenance dose (for example, if the maintenance dose is administered as a fixed dose of 200 mg once every two weeks (Q2W), a loading dose of a fixed dose of 400 mg may be administered once every two weeks (Q2W)). In some embodiments, an initial accelerated dosing schedule is used (for example, if the maintenance dose of 200 mg is administered once every two weeks (Q2W), the initial accelerated dosing schedule includes administering 200 mg once a week (Q1W) over two weeks).

[0071] In some embodiments, the maintenance dose includes an unlimited number of doses, which may be useful for managing chronic conditions such as alopecia areata. In some embodiments, the maintenance dose is administered intermittently (as needed).

[0072] In some embodiments, the subjects have renal impairment (e.g., mild renal impairment (e.g., glomerular filtration rate <60 mL / min and / or presence of albuminuria >30 mg / day), moderate renal impairment (e.g., glomerular filtration rate <45 mL / min), or severe renal impairment (e.g., glomerular filtration rate <30 mL / min). In other embodiments, the subjects are normal, for example, normal in terms of renal function.

[0073] In some embodiments, the subjects are adults (e.g., 18 years of age or older).

[0074] In some embodiments, the subjects are not adults, or are pediatric or adolescent patients (for example, patients under 18, under 16, under 14, under 12, under 10, under 5, under 3, under 2, under 1, under 6 months, or under 3 months).

[0075] In some embodiments, the subjects are Caucasian. In some embodiments, the subjects are Asian. In some embodiments, the subjects are African. In some embodiments, the subjects are Native Americans. In some embodiments, the subjects are of mixed race or ethnic groups. In some embodiments, the subjects are biological males. In some embodiments, the subjects are biological females.

[0076] In some embodiments, the subject is further treated or treated with a second therapeutic agent effective in treating alopecia areata. In some embodiments, the second therapeutic agent includes glucocorticoids, minoxidil, anthraline, bimatoprost, methotrexate, DMARDs, dupilumab, and / or JAK inhibitors (e.g., tofacitinib, ruxolitinib, upadacitinib, abrocitinib, and baricitinib).

[0077] In some embodiments, the antibody is administered to the subject at doses of approximately 25, 50, 100, 150, 200, or 250 mg, and optionally, the antibody is administered to the subject at doses of 10, 11, 12, 13, 14, or 15 doses, once every two weeks.

[0078] In some embodiments, this method reduces the measurement of AA-related biomarkers after treatment (compared to before treatment).

[0079] In some embodiments, the AA-related biomarker is a gene expression signature, which is an Alopecia Areata Disease Activity Index (ALADIN).

[0080] In some embodiments, the AA-related biomarker is an alopecia areata gene signature (AAGS) comprising one or more genes as described below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13]

[0081] In some embodiments, the method further includes detecting AA-related biomarkers in an individual either before treatment, after treatment, or both.

[0082] In some embodiments, AA-related biomarkers indicate the severity of alopecia areata.

[0083] In some embodiments, AA-related biomarkers indicate a tendency of an individual to respond to treatment with the target anti-IL-7Rα antibody, its antigen-binding fragment, or a pharmaceutically acceptable salt thereof.

[0084] In some embodiments, the method further includes selecting individuals based on the level of an AA-related biomarker before administering a target anti-IL-7Rα antibody, its antigen-binding fragment, or a pharmaceutically acceptable salt thereof to the individuals.

[0085] In some embodiments, administration of a target anti-IL-7Rα antibody, its antigen-binding fragment, or a pharmaceutically acceptable salt thereof results in a change in the level of AA-related biomarkers in an individual.

[0086] In some embodiments, the subject requiring it satisfies one or more of the selection criteria specified in Example 1. In some embodiments, the subject requiring it satisfies all of the selection criteria specified in Example 1.

[0087] In some embodiments, the subjects requiring it do not meet any of the exclusion criteria specified in Example 1 (i.e., are not excluded).

[0088] In some embodiments, subjects have had alopecia areata for more than 6 months but less than 10 years prior to antibody administration.

[0089] In a related embodiment, a method is also provided for treating alopecia areata (AA) in mammalian subjects requiring it, the method comprising administering an effective amount of a composition comprising an antibody against IL-7Rα, the antibody comprising (a) a heavy chain variable region (HCVR) comprising the sequence of SEQ ID NO: 1 (DHAMH, one of SEQ ID NOs from 7 to 22, etc.), the VH CDR1 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and the VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) comprising the VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), the VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and the VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT), the subject having ≥50% total hair loss of the scalp as defined by a baseline SALT score, the baseline SALT score being determined 24 hours prior to antibody administration, and the antibody being IgG1 without effector function The antibody, containing Fc, is administered subcutaneously every two weeks at a dose of 200 mg.

[0090] In some embodiments, VH CDR1 includes sequence number 7 (GFTFDDHAMH).

[0091] In some embodiments, subjects have had alopecia areata for more than 6 months but less than 10 years prior to antibody administration.

[0092] In some embodiments, the composition comprises 20 mM histidine, 260 mM sucrose, and 100 mg / ml of antibody in 0.05% (w / v) polysorbate 80, optionally together with 0.05 mM pentetic acid, at pH 6.0.

[0093] In some embodiments, the subject has an absolute lymphocyte count (ALC) of ≥ 800 per microliter.

[0094] In some embodiments, antibody administration is stopped when the target ALC is <500 per microliter.

[0095] In some embodiments, the antibody-containing composition is administered to a subject to maintain a serum antibody concentration of ≥5 μg / mL throughout the entire administration.

[0096] The serum concentration of an antibody can be measured using any method known in the art. For example, the level of anti-IL-7Rα antibody in serum can be determined using a targeted capture approach. Specifically, serum flows through a surface (e.g., a column) pre-coated with a capture agent specific to anti-IL-7Rα antibody (e.g., the extracellular domain of human IL-7R), and the antibody in the serum is captured on the surface. After washing the unbound material, a labeled secondary antibody that specifically binds to anti-IL-7Rα antibody but does not compete for binding with the capture agent (e.g., a labeled secondary antibody that binds to the Fc region of the anti-IL-7Rα antibody) is added to detect the presence of anti-IL-7Rα antibody captured by the pre-coated substrate. The signal intensity from the labeled secondary antibody is proportional to the concentration of anti-IL-7Rα antibody in the serum, and the serum concentration of the antibody is determined by comparing its measured signal with the signal produced by a standard with a known concentration of the antibody.

[0097] In non-limiting cases, serum antibody concentrations can be measured using the Gyrolab® system, which is a flow through an immunoassay platform. In this purpose-appropriate assay, antibodies present in serum are captured on a Gyrolab® CD column pre-coated with the biotinylated recombinant human IL-7R extracellular domain (target). The bound anti-IL-7Rα antibody is detected with AlexaFluor®-647 labeled Cyno anti-human IgG Fc mAb (Pur clone 10C7). Immunofluorescence measured using a Gyrolab® laser-guided fluorescence (LIF) detector is proportional to the concentration of anti-IL-7Rα antibody in the sample, standard, and control.

[0098] In another non-limiting example, serum antibody concentrations can be measured using the Gyrolab® system, which is a flow through an immunoassay platform. In this validated assay, antibodies present in serum are captured on a Gyrolab® CD column pre-coated with a biotinylated anti-idiotype monoclonal antibody (mAb) that specifically binds to anti-IL-7Rα antibody. The bound anti-IL-7Rα antibody is detected with an AlexaFluor®-647-labeled mAb, which is a non-competitive clone but also binds to anti-IL-7Rα antibody. Immunofluorescence measured using a Gyrolab® LIF detector is proportional to the concentrations of anti-IL-7Rα antibody in the sample, standard, and control.

[0099] In some embodiments, the antibody is administered to the subject at a dose of approximately 200 mg.

[0100] In some embodiments, the antibody is administered once every two weeks (Q2W).

[0101] In some embodiments, the antibody is administered for at least about 13 doses.

[0102] In some embodiments, the antibody comprises IgG1 Fc without effector function.

[0103] In a particular embodiment, the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24 and is administered subcutaneously (sc) to mammalian subjects (e.g., humans) in a continuous dose of approximately 200 mg per dose, once every two weeks (Q2W) for 10 to 15 doses (e.g., 11, 12, or 13 doses).

[0104] Furthermore, a pharmaceutical composition is also provided which comprises an antibody having two heavy chains and two light chains, wherein each of the heavy chains has the amino acid sequence of SEQ ID NO: 23, and each of the light chains has the amino acid sequence of SEQ ID NO: 24, and the antibody is formulated as a pharmaceutically acceptable solution having 50 to 200 mg / mL (e.g., about 100 mg / mL) of antibody in approximately 50 to 200 mg / mL (e.g., about 15 to 25 mM (e.g., about 20 mM)) histidine, 200 to 300 mM (e.g., about 260 mM) sucrose, and approximately 0.03 to 0.07% (w / v) (e.g., about 0.05% (w / v) at pH 6.0) polysorbate 80 at pH 6.0.

[0105] In some embodiments, the solution further comprises about 0.01–0.10 mM (e.g., about 0.05 mM) of pentetic acid.

[0106] In some embodiments, the pharmaceutical composition essentially comprises, or comprises, 20 mM histidine, 260 mM sucrose, and an antibody formulated as 100 mg / mL of antibody in 0.05% (w / v) polysorbate 80 at pH 6.0.

[0107] In some embodiments, the pharmaceutical composition essentially comprises, or comprises, 20 mM histidine, 260 mM sucrose, 0.05 mM pentetic acid, and an antibody formulated as 100 mg / mL of antibody in 0.05% (w / v) polysorbate 80 at pH 6.0.

[0108] A container (e.g., a polycarbonate bottle) containing the pharmaceutical composition described herein in an extractable volume of approximately 2 mL is also provided.

[0109] In some embodiments, the container contains or comprises about 2.26 mL of the pharmaceutical composition described herein.

[0110] The definitions and methods provided define this disclosure and guide those skilled in the art in implementing it. Unless otherwise stated, terms should be understood by those skilled in the art in accordance with their prior use.

[0111] Unless otherwise indicated, all numbers expressing quantities of components, reaction conditions, etc., used herein and in the claims should be understood in all cases to be modified by the term "approximately".

[0112] Where used herein, the term “approximately” is to be interpreted as including all values ​​that are less than the full increment of the least significant digit claimed, and greater than the full decrement. In other words, where used herein, the term “approximately” is intended to qualify the numbers it corrects, indicating such values ​​as variables within the margin of error. Where no specific margin of error, such as the standard deviation to the mean, is enumerated in a chart or table of data, the term “approximately” should be understood to mean the range that encompasses the enumerated values, and the range that is rounded to, or included by rounding, in that chart, taking into account any significant figure.

[0113] As used herein, “alopecia areata” or “AA” is a chronic T-cell-mediated autoimmune skin disease that leads to hair loss. Hair loss can be more diffuse across the entire scalp, in which case the condition is called diffuse alopecia areata. Solitary alopecia areata describes hair loss in only one area. This can occur anywhere on the head. Multiple alopecia areata refers to hair loss in multiple areas. Ophiasis refers to wavy hair loss around the scalp. The disease may be limited to the beard only, in which case it is called beard alopecia areata. If all hair on the scalp is lost, the disease is called alopecia totalis. If all body hair is lost, the diagnosis is alopecia universalis. Severe alopecia areata refers to alopecia totalis and alopecia universalis involving 50% or more of the entire scalp. Moderate alopecia areata refers to alopecia areata involving at least 30% and up to 50% of the entire scalp.

[0114] As used herein, “SALT score” refers to an alopecia severity tool, a tool for determining the degree of hair loss based on the proportion of scalp surface area involved in the upper, posterior, and lateral parts of the scalp. The SALT II score is an updated tool that includes smaller increments of scalp coverage, facilitating the assessment of hair loss where small patches of hair loss are predominant. The SALT score is determined by adding up the proportions of hair loss in different areas of the scalp, using either the original SALT I or SALT II.

[0115] As used herein, with respect to alopecia areata, “clinical response” refers to achieving more than 50% hair regrowth from baseline, based on the SALT score at the end of treatment.

[0116] As used herein, “Alopecia Areata Disease Activity Index” or “ALADIN” is a three-dimensional quantitative composite gene expression score intended for use as a biomarker to track disease severity and response to treatment.

[0117] As used herein, “AASIS” or “Alopecia Areata Symptom Impact Scale (AASIS)” may refer to a 13-item disease-specific scale that asks patients with AA about their AA-related symptoms and how these symptoms interfere with their daily functioning. Patients may be asked to rate how severe each of the following seven AA symptoms was in the past week, using an 11-point scale ranging from 0 “Not present” to 10 “Imaginably bad”: 1) hair loss on the scalp, 2) hair loss on the body or eyelashes, 3) tingling / numbness of the scalp, 4) itching or pain of the skin, 5) skin irritation, 6) feeling anxious or worried, or 7) feeling sad. Patients may also be asked to rate how severely AA interfered with each of the following six areas of daily functioning in the past week, using an 11-point scale ranging from 0 = “Not interfering” to 10 “Completely interfering”: work, enjoyment of life, social interaction, daily living activities, sexual relationships, and quality of life. The overall scoring system ranges from 0 to 130, with higher scores indicating a greater impact of AA symptoms.

[0118] As used herein, the Alopecia Areata-Related Quality of Life Index (AA-QLI) refers to a disease-specific questionnaire developed to assess the impact of AA on quality of life. Results are presented on a scale ranging from 0 to 84, where a score of 0 represents the best quality of life and a score of 84 represents the worst quality of life outcome. The AA-QLI consists of questions covering three areas of daily living: subjective symptoms, relationships, and objective signs.

[0119] As used herein, the term “AA-related biomarker” means any biological response, cell type, parameter, protein, polypeptide, enzyme, enzyme activity, metabolite, nucleic acid, carbohydrate, or other biomolecule present or detectable in AA patients at levels or amounts different from (e.g., above or below) those of markers present or detectable in non-AA patients. The term “AA-related biomarker” also includes genes or gene probes known in the art that are differentially expressed in subjects having AA compared to subjects without A. Alternatively, “AA-related biomarker” also includes genes downregulated by AA.

[0120] In some embodiments, the biomarker is evaluated using histology. In some embodiments, the biomarker is evaluated using RNA-seq. In some embodiments, the biomarker is evaluated using proteomic analysis. In some embodiments, the biomarker is evaluated using enzyme-linked immunosorbent assay. In some embodiments, the biomarker is evaluated using mass spectrometry. In some embodiments, the biomarker is evaluated using blood samples. In some embodiments, the biomarker is evaluated using serum samples. In some embodiments, the biomarker is evaluated using plasma samples. In some embodiments, the biomarker is evaluated using tissue samples. In some embodiments, the biomarker is evaluated using punch biopsy.

[0121] In some embodiments, the biomarker is selected from Th2 / IL-13, Th22 / IL-22, Thr / IFN-γ, and Thl7 / IL-17A. In some embodiments, the biomarker is selected from IFN-g, IL-2, IL-12, IL-13, IL-10, and IL-17. In some embodiments, the biomarker is selected from at least one of IL-2, IL-10, IL-12, IL-13, IL-17, IL-17A, IL-22, and IFN-γ. In some embodiments, the biomarker is IL-2. In some embodiments, the biomarker is IL-10. In some embodiments, the biomarker is IL-12. In some embodiments, the biomarker is IL-13. In some embodiments, the biomarker is IL-17. In some embodiments, the biomarker is IL-17A. In some embodiments, the biomarker is IL-22. In some embodiments, the biomarker is IFN-γ.

[0122] In some embodiments, the biomarker is a gene expression signature. In some embodiments, the gene expression signature includes gene expression information from one or more of the following gene groups: hair keratin (KRT) related genes, cytotoxic T lymphocyte infiltration (CTL) related genes, and interferon (IFN) related genes. In some embodiments, the KRT related genes include DSG4, HOXC31, KRT31, KRT32, KRT33B, KRT82, PKP1, and / or PKP2. In some embodiments, the CTL related genes include CD8A, GZMB, ICOS, and / or PRFl. In some embodiments, the IFN related genes include CXCL9, CXCL10, CXCL11, STAT1, and / or MX1. In some embodiments, AA-related biomarkers are selected from IL-15, CCL2, CCL3, CXCL10, IL-13, CCL13, CCL17, CCL22, CCL26, CCL4, and CCL11, and the levels of the biomarkers are increased in serum from individuals with AA compared to serum from healthy patients. In some embodiments, AA-related biomarkers are selected from IL-15 and eotaxin / CCL11, and the levels of the biomarkers are associated with SALT scores.

[0123] In some embodiments, AA-related biomarkers are selected from scalp TH2-related markers (CCL13 and IL-13) and serum T cell / NK cell activation markers (IL-15).

[0124] Furthermore, biomarkers for monitoring the reversal of disease are also provided by administering the target antibody or a pharmaceutically acceptable formulation thereof. Methods for detecting and / or quantifying such AA-related biomarkers are known in the art, kits for measuring such AA-related biomarkers are available from various commercial sources, and various commercial diagnostic laboratories provide services that measure such biomarkers.

[0125] As used herein, “TSLP” refers to a growth factor closely similar to IL-7 that plays a role in the maturation and activation of bone marrow cells (e.g., monocytes and dendritic cells). TSLP is produced by various cell types, including fibroblasts, epithelial cells, and stromal cells. Elevated levels of TSLP are associated with diseases such as asthma, atopic dermatitis, and inflammatory arthritis, and are also known to be associated with abnormal regulation of IL-7.

[0126] As used herein, the term “antibody” refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). In certain antibodies, such as naturally occurring IgG antibodies, the heavy chain constant region consists of a hinge and three domains, CH1, CH2, and CH3. In certain antibodies, such as naturally occurring IgG antibodies, each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain (abbreviated herein as CL). The VH and VL regions may be further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and to host tissues or factors including the first component (C1q) of the classical complement system. The heavy chain may or may not have a C-terminal lysine. Unless otherwise specified herein, amino acids in the variable region are numbered using the Rabat numbering system, and amino acids in the constant region are numbered using the EU system. "Antibodies" include, as an example, both natural and non-natural antibodies, monoclonal and polyclonal antibodies, chimeric and humanized antibodies, human and non-human antibodies, and fully synthetic antibodies.

[0127] As used herein, “IgG antibodies” such as, for example, human IgG1, IgG2, IgG3, and IgG4 antibodies, in some embodiments, have the structure of a naturally derived IgG antibody, i.e., have the same number of heavy and light chains and disulfide bonds as a naturally derived IgG antibody of the same subclass. For example, an anti-IL-7R IgG1, IgG2, IgG3, or IgG4 antibody consists of two heavy chains (HC) and two light chains (LC), and the two heavy and light chains are linked by the number and position of disulfide bridges that occur in naturally derived IgG1, IgG2, IgG3, and IgG4 antibodies, respectively (so long as the antibody is not mutated to modify the disulfide bridges).

[0128] Antibodies typically bind specifically to their cognate antigen with high affinity, reflected by a dissociation constant (K -5 ~10 -11 ) of 10 D M or less. Any K -4 greater than about 10 D M is generally considered to exhibit non-specific binding.

[0129] In some embodiments, anti-IL-7Rα antibodies that are used in the methods described herein or are useful bind specifically to IL-7Rα, such as with a K -5 of 10 -11 M or less (e.g., 10 -5 M or less, 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 M or less). D

[0130] In some embodiments, anti-IL-7Rα antibodies that are used in the methods described herein or are useful bind specifically to IL-7Rα with a K -7 of 10 -8 M or less, 10 -9 M or less, 5×10 -8 M or less, or 10 -10 M to 10 D ​IL-7Rα (human IL- 7 It specifically binds to antigens such as Rα, but does not bind to unrelated antigens with high affinity.

[0131] An antigen is "substantially identical" to a given antigen if it exhibits a high degree of sequence identity with respect to that given antigen, for example, if it exhibits at least 80%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity with respect to the sequence of the given antigen. As an example, an antibody that specifically binds to human IL-7Ra may, in some embodiments, cross-reactive with IL-7Ra antigens from a specific primate species (e.g., cynomolgus monkey IL-7Ra), but may not cross-reactive with IL-7Ra antigens from other species, or with antigens other than IL-7Ra.

[0132] As used herein, “isotype” refers to an antibody class encoded by a heavy chain constant region gene (e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE antibodies). IgG isotypes are divided into subclasses in specific species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. In some embodiments, the anti-IL-7R antibodies described herein are of the IgG1 isotype. Immunoglobulins, such as IgG1, exist in several allotypes, which differ from each other by up to a few amino acids.

[0133] As used herein, the term “allotype” refers to a native variant within a particular group of isotypes, which differ by a few amino acids. The anti-IL-7R antibodies described herein may be any allotype. As used herein, antibodies referred to as the “IgG1f,” “IgG1.If,” or “IgG1.3f” isotypes are allotype “f” IgG1, IgG1.1 without effector function, and IgG1.3 antibody without effector function, i.e., 214R, 356E, and 358M according to the EU index (e.g., Kabat, as shown in SEQ ID NO: 23).

[0134] The term “antigen-binding moiety” of an antibody, as used herein, refers to one or more fragments of an antibody that possess the ability to specifically bind to an antigen (e.g., human IL-7Ra). It has been shown that the antigen-binding function of an antibody may be carried out by fragments of a full-length antibody. Examples of binding fragments encompassed by the term “antigen-binding moiety” of an antibody, for example, as described herein for anti-IL-7R antibodies, include: (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of VL, VH, LC domains and CH1 domain; (ii) an F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment containing two Fab fragments linked by disulfide crosslinking at the hinge region; and (iii) an F consisting of the VH domain and CH1 domain. d Examples include (iv) an Fv fragment consisting of the VL and VH domains of a single arm of the antibody, (v) a dAb fragment consisting of the VH domain, (vi) an isolated complementarity-determining region (CDR), and (vii) a combination of two or more isolated CDRs that can be optionally bound by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be bound by a synthetic linker that allows the VL and VH regions to be paired and produced as a single protein chain forming a monovalent molecule (known as single-stranded Fv (scFv)) using a recombination method. Such a single-stranded antibody is also intended to be encompassed within the term "antigen-binding portion" of the antibody.

[0135] These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for usefulness in the same manner as intact antibodies. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0136] As used herein, the term “monoclonal antibody” refers to an antibody from a substantially homogeneous population of antibodies, i.e., the individual antibodies contained within the population are substantially similar, except for any variants that may arise during the production of the monoclonal antibody, and bind to the same epitope (e.g., the antibody exhibits a single binding specificity and affinity), with such variants generally present in small amounts. The modifier “monoclonal” indicates the characteristic of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method.

[0137] The term "human monoclonal antibody" refers to an antibody from a substantially homogeneous population of antibodies that exhibit a single binding specificity and have variable and optional constant regions derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by hybridomas, such as B cells obtained from transgenic non-human animals having a genome containing human heavy chain transgenes and light chain transgenes fused to immortalized cells, for example, transgenic mice.

[0138] The term “recombinant human antibody,” as used herein, encompasses all human antibodies that are prepared, expressed, produced or isolated by recombinant means, such as (a) antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomes to human immunoglobulin genes or hybridomas prepared therefrom; (b) antibodies isolated from host cells transformed to express such antibodies, such as from transfectomas; (c) antibodies isolated from recombinant combinatorial human antibody libraries; and (d) antibodies prepared, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, such recombinant human antibodies include variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent reconfigurations and mutations that occur, for example, during antibody maturation. As is known in the art, the variable region contains an antigen-binding domain, which is encoded by various genes that are reconfigured to form antibodies specific to foreign antigens. In addition to rearrangement, the variable region can be further modified by multiple single-amino acid changes (referred to as somatic mutations or hypermutations) to increase the affinity of antibodies to foreign antigens. The constant region changes in further response to the antigen (i.e., isotype switch). Therefore, rearranged and somatically mutant nucleic acid molecules encoding light-chain and heavy-chain immunoglobulin polypeptides in response to an antigen cannot have sequence identity with the original nucleic acid molecule, but instead are substantially identical or similar (i.e., have at least 80% identity).

[0139] A “human” antibody (HuMAb) refers to an antibody having a variable region in which both the framework region and the CDR region are derived from a human germline immunoglobulin sequence. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. Anti-IL-7R antibodies described herein may have a configuration that includes amino acid residues not encoded by a human germline immunoglobulin sequence (e.g., mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo). However, as used herein, the term “human antibody” is not intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as mouse, is transplanted onto a human framework sequence. The terms “human” antibody and “fully human” antibody are used synonymously.

[0140] A "humanized" antibody is one in which some, almost all, or all of the amino acids outside the CDR domain of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one embodiment of the humanized form of an antibody, some, almost all, or all of the amino acids outside the CDR domain are replaced with amino acids derived from human immunoglobulins, but some, almost all, or all of the amino acids within one or more CDR regions remain unchanged. Small additions, deletions, insertions, substitutions, or modifications of amino acids are acceptable as long as they do not interfere with the antibody's ability to bind to a particular antigen. Humanized antibodies retain similar antigen specificity to the original antibody.

[0141] A "chimeric antibody" refers to an antibody in which the variable region originates from one species and the constant region originates from another species; for example, an antibody in which the variable region originates from a mouse antibody and the constant region originates from a human antibody.

[0142] The phrases "antibody that recognizes an antigen" and "antibody that is specific to an antigen" are used interchangeably with the term "antibody that specifically binds to an antigen."

[0143] As used herein, “isolated antibody” is intended to refer to an antibody that substantially contains no other proteins or cellular material.

[0144] As used herein, “antibody that inhibits the binding of IL-7 to IL-7Rα” is intended to refer to an antibody that inhibits the binding of IL-7Rα to its ligand, for example, in a binding assay using T cells from whole blood expressing interleukin-7 (IL-7), for example, IL-7Rα, with an EC50 of approximately 1 pg / mL or less, for example, approximately 0.9 pg / mL or less, approximately 0.85 pg / mL or less, approximately 0.8 pg / mL or less, approximately 0.75 pg / mL or less, approximately 0.7 pg / mL or less. Methods recognized in the art, such as those for approximately 0.65 pg / mL or less, approximately 0.6 pg / mL or less, approximately 0.55 pg / mL or less, approximately 0.5 pg / mL or less, approximately 0.45 pg / mL or less, approximately 0.4 pg / mL or less, approximately 0.35 pg / mL or less, approximately 0.3 pg / mL or less, approximately 0.25 pg / mL or less, approximately 0.2 pg / mL or less, approximately 0.15 pg / mL or less, approximately 0.1 pg / mL or less, or approximately 0.05 pg / mL or less, include, for example, the FACS-based binding assay described herein.

[0145] "Effector function" refers to the interaction of the antibody Fc region with Fc receptors or ligands, or the biochemical events resulting from these interactions. Examples of effector functions include FcyR-mediated effector functions such as C1q binding, complement-dependent cell-mediated cytotoxicity (CDC), Fc receptor binding, ADCC, and antibody-dependent cell-mediated phagocytosis (ADCP), as well as the downregulation of cell surface receptors (e.g., B cell receptors, BCRs). Such effector functions generally require an Fc region combined with a binding domain (e.g., an antibody variable domain).

[0146] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of immunoglobulins. FcRs that bind to IgG antibodies include the FcγR family of receptors, which include allele variants and, alternatively, splicing forms of these receptors. The FcγR family consists of three activating receptors (FcγRI, FcγRIII, and FcγRIV in mice, and FcRIA, FcRIIA, and FcyRIIIA in humans) and one inhibitory receptor (FcγRIIB). Various properties of human FcyRs are known in the art. Most native effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIBs, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans), but do not express inhibitory FcγRIIB in mice or humans. Human IgG1 binds to most human Fc receptors and is considered equivalent to mouse IgG2a in terms of the types of activated Fc receptors it binds to.

[0147] The "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. Therefore, the Fc region includes the constant region of the antibody excluding the first constant-region immunoglobulin domain (e.g., CHI or CF). In IgG, IgA, and IgD antibody isotypes, the Fc region contains two identical protein fragments derived from the second constant domain (CH2) and third constant domain (CH3) of the two heavy chains of the antibody, while the IgM and IgE Fc regions contain three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. For IgG, the Fc region includes the immunoglobulin domains CH2 and CH3, as well as the hinge between the CH1 and CH2 domains. The definition of the boundary of the Fc region of an immunoglobulin heavy chain may vary as defined herein, but the human IgG heavy chain Fc region is defined as extending from amino acid residue D221 of IgG1, V222 of IgG2, IgG2 and F221 of IgG3, and P224 of IgG4 to the carboxy terminus of the heavy chain, with numbering following the EU index as in Kabat. The CH2 domain of the human IgG Fc region extends from amino acid 237 to amino acid 340, and the CH3 domain is located on the C-terminal side of the CH2 domain within the Fc region, i.e., extending from amino acid 341 of IgG to amino acids 447 or 446 (if a C-terminal lysine residue is absent) or 445 (if C-terminal glycine and lysine residues are absent). As used herein, the Fc region may be a native sequence Fc containing any homogeneous variant, or a variant Fc (e.g., a non-native type Fc). Fc can also refer, either alone or in context, to an Fc-containing protein polypeptide, such as a binding protein containing an Fc region, also known as an Fc fusion protein (e.g., an antibody or immunoadhesion).

[0148] A "natural sequence Fc region" or "natural sequence Fc" contains an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. Natural sequence human Fc regions include the natural sequence human IgG1 Fc region, the natural sequence human IgG2 Fc region, the natural sequence human IgG3 Fc region, and the natural sequence human IgG4 Fc region, as well as their natural variants. Natural sequence Fc includes various allotypes of Fes.

[0149] The term “epitope” or “antigenic determinant” refers to a site on an antigen (e.g., IF-7Rα) to which an immunoglobulin or antibody specifically binds, and is defined, for example, by the specific method used to identify it. Epitopes can be formed from both continuous amino acids (usually linear epitopes) or discontinuous amino acids juxtaposed by the tertiary folding of a protein (usually conformational epitopes). Epitopes formed from continuous amino acids are typically retained upon exposure to denaturing solvents, but not always, while epitopes formed by tertiary folding are typically lost upon processing with denaturing solvents. Epitopes typically contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial configuration. Methods for determining which epitopes are bound by a given antibody (i.e., epitope mapping) are known in the art, and include, for example, immunoblotting and immunoprecipitation assays in which duplicate peptides or serial peptides (e.g., from IF-7Rα) are tested for reactivity with a given antibody (e.g., anti-IF-7R antibody). Methods for determining the spatial structure of epitopes include, for example, X-ray crystallography, antigen mutation analysis, two-dimensional nuclear magnetic resonance, and HDX-MS.

[0150] When used herein, "k assoc " or "k a The term "k" is intended to refer to the association rate of a particular antibody-antigen interaction, whereas "k" is used herein to mean "k dis" or "k d The term "K" is intended to refer to the dissociation rate of a particular antibody-antigen interaction. When used herein, "K" is used to mean "K D The term "kD / ka" is intended to refer to the dissociation constant, which is derived from the kd to ka ratio (i.e., kd / ka) and expressed as molar concentration (M). D The value can be determined using a method well established in the art. The K of the antibody D Available methods for determining this include surface plasmon resonance, biosensor systems such as the BIACORE® system or flow cytometry, and scachard analysis.

[0151] As used herein, the term “high affinity” for an IgG antibody means 10% affinity for the target antigen. -8 M or less, 10 -9 M or less, or 10 -10 K below M D This refers to antibodies that possess high affinity. However, "high affinity" binding can vary depending on other antibody isotypes. For example, "high affinity" binding to IgM isotypes is 10 -10 M or less, or 10 -8 K below M D This refers to antibodies that possess [a certain characteristic].

[0152] In the context of in vitro or in vivo assays using antibodies or their antigen-binding fragments, the term "EC50" refers to the concentration of the antibody or its antigen-binding portion that elicits a response that is 50% of the maximum response, i.e., an intermediate response between the maximum response and the baseline.

[0153] As used herein, the term “autoimmune disease” refers to a disease or disorder in which the immune system produces an immune response (e.g., a B-cell or T-cell response) to an antigen (i.e., an autoantigen) that is part of a normal host, resulting in tissue damage. Autoantigens may originate from host cells or from symbiotic organisms, such as microorganisms that normally colonize mucosal surfaces (known as symbionts).

[0154] As used herein, the terms “inflammation” or “inflammatory process” refer to a complex set of events including dilation of arterioles, capillaries, and venules with increased permeability and blood flow, exudation of fluids containing plasma proteins, and migration of leukocytes to the inflammatory focus. Inflammation can be measured by many methods well known in the art, such as the number of leukocytes, the number of polymorphonuclear neutrophils (PMNs), measures of the degree of PMN activation such as lumen-enhancing chemiluminescence, or the amount of pro-inflammatory cytokines present (e.g., IL-6 or TNF-α).

[0155] As used herein, the term “regulatory T cells” (Treg) refers to a population of T cells that have the ability to reduce or suppress the induction and proliferation of effector T cells, thereby modulating the immune response. In some embodiments, Tregs can suppress the immune response by secreting anti-inflammatory cytokines such as IL-10, TGF-β, and IL-35, which can interfere with the activation and differentiation of naive T cells into effector T cells. In some embodiments, Tregs can also produce cell-lysing molecules such as granzyme B, which can induce apoptosis of effector T cells. In some embodiments, regulatory T cells are innate regulatory T cells (nTregs) (i.e., arising in the thymus). In some embodiments, regulatory T cells are induced regulatory T cells (i.e., naive T cells that differentiate into Tregs in peripheral tissues when exposed to specific stimuli. Methods for identifying Tregs are known in the art. For example, Tregs express specific phenotypic markers (e.g., CD25, Foxp3, or CD39) that can be measured using flow cytometry. In some embodiments, Treg cells are CD45RA-CD39+ T cells.

[0156] As used herein, “administration” means the physical introduction of a composition containing a therapeutic agent into a subject using any of the various methods and delivery systems known to those skilled in the art. Different routes of administration of anti-IL-7R antibodies described herein include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral routes, for example, by injection or infusion. As used herein, the term “parenteral administration” means a mode of administration other than enteral and topical administration, and is usually by injection, and is not limited to intravenous, intraperitoneal, intramuscular, intraarterial, subarachnoid, intralymphatic, intravesical, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described herein may be administered via topical, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or non-enteral extra-enteral routes. Administration may also be carried out, for example, once, multiple times, and / or over one or more extended periods.

[0157] As used herein, the terms “inhibit” or “block” (for example, referring to the inhibition / blockage of the binding of IL-7 to IL-7Ra on cells) are used interchangeably and encompass both partial and complete inhibition / blockage. In some embodiments, an anti-IL-7R antibody inhibits the binding of IL-7 to IL-7Ra by at least about 50%, for example, about 60%, 70%, 80%, 90%, 95%, 99%, or 100%, as determined, for example, as further described herein.

[0158] As used herein, the terms “treat,” “treating,” and “treatment” refer to any type of intervention or process performed on a subject or involving the administration of an active agent for the purpose of reversing, mitigating, improving, inhibiting, delaying, or preventing the progression, onset, severity, or recurrence of disease-related symptoms, complications, conditions, or biochemical signs, or enhancing overall survival. These terms do not include prophylactic interventions.

[0159] The term "preventive intervention" refers to treating individuals who do not yet have a disease, for preventative purposes.

[0160] The term "effective dose" or "effective dosage" is defined as the amount sufficient to achieve, or at least partially achieve, the desired effect.

[0161] The “therapeutic effective dose” or “therapeutic effective dose” of a drug or therapeutic agent (e.g., the anti-IL-7Ra antibody of interest) is any amount of the drug that promotes disease regression, as demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or the prevention of impairment or impairment due to disease, when used alone or in combination with another therapeutic agent. The therapeutic effective dose or dosage of a drug also includes the “preventive effective dose” or “preventive effective dose,” which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering a disease relapse, inhibits the onset or relapse of the disease. The ability of a therapeutic agent to promote disease regression or inhibit the onset or relapse of the disease can be evaluated using a variety of methods known to those skilled in the art, for example, by assassinating the activity of the drug in human subjects in clinical trials (including the methods described in the examples), animal model systems to predict efficacy in humans, or in vitro assays.

[0162] The term “patient” includes human and other mammalian subjects receiving either prophylactic or therapeutic treatment. In some embodiments, the patient is human.

[0163] As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions described herein can be used to treat a subject having cancer. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cattle, chickens, amphibians, reptiles, etc.

[0164] The terms “body weight-based” dose or dose as used herein mean that the dose administered to a patient is calculated based on the patient’s body weight. For example, if a patient weighing 60 kg requires 3 mg / kg of anti-IL-7R antibody, an appropriate amount of anti-IL-7Ra antibody (i.e., 180 mg) can be calculated and used for administration.

[0165] A method for treating alopecia areata is provided, comprising administering to subjects in need of such treatment an isolated antibody or its antigen-binding portion that specifically binds to the alpha chain of the human IL-7 receptor ("anti-IL-7Rα antibody," or abbreviated as "anti-IL-7R antibody"), comprising heavy chain CDR1, CDR2, CDR3 and light chain CDR1, CDR2, and CDR3, respectively, wherein the antibody is (a) EC of approximately 5 nM or less (for example, less than approximately 3 nM) 50 In whole blood containing T cells (CD4 + CD45RA + CD4 + CD45RA - CD8 + CD45RA + , and / or CD8 + CD45RA - ) can be joined, (b) Unable to bind to non-T cells in whole blood, (c) Do not stimulate IL-7 receptor signaling upon binding to the IL-7 receptor, e.g., minimal pSTAT5 activation, or (d) Any combination of those.

[0166] In some embodiments, the heavy chain CDR3 of the target anti-IL-7Rα antibody (e.g., those disclosed herein) contains the amino acid sequence described in SEQ ID NO: 3 (DEYSRGYYVLDV).

[0167] In some embodiments, the anti-IL-7Rα antibody is (a) Capable of selectively binding to the alpha chain of the human and cynomolgus monkey IL-7 receptor (IL-7R), (b) Soluble and capable of binding to the alpha chain of membrane-bound IL-7R, (c) When administered to a target that requires it, it can block the proliferation and / or survival of pathogenic T cells. (d) When administered to subjects in need, it can restore T regulatory cell (Treg) function and / or promote Treg survival. (e) It can maintain drug-free remission for a longer period than remission with CTLA4-Ig (ORENCIA®), (f) In the intestinal tissue of the target that requires it, for example, it can block inflammation and mucosal damage induced by pathogenic T cells. (g) In subjects where it is necessary, it is possible to reduce the frequency of T effector cells in the mesenteric lymph nodes (MLN) and / or lamina propria (LP), (h) T cells (e.g., CD4 + CD45RA + ) can reduce or inhibit IL-7-mediated pSTAT activation. (i) Can block the proliferation of IL-17 and / or IFN-gamma producing cells, (j) It can treat subjects with inflammatory diseases (e.g., inflammatory bowel disease), and (k) Having one or more properties selected from the group consisting of any combination thereof.

[0168] In some embodiments, the target anti-IL-7Rα antibody includes a heavy chain CDR1, which includes one of the amino acid sequences presented in SEQ ID NO: 1 (DHAMH), for example, the amino acid sequences described in SEQ ID NOs. 7 to 22. In some embodiments, the anti-IL-7Rα antibody includes a heavy chain CDR2, which includes the amino acid sequence presented in SEQ ID NO: 2 (GISWNSRGIGYADSVKG). In some embodiments, the anti-IL-7Rα antibody includes a heavy chain CDR3, which includes the amino acid sequence presented in SEQ ID NO: 3 (DEYSRGYYVLDV). In some embodiments, the anti-IL-7Rα antibody includes a light chain CDR1, which includes the amino acid sequence presented in SEQ ID NO: 4 (RASQGISSALA). In some embodiments, the anti-IL-7Rα antibody includes a light chain CDR2, which includes the amino acid sequence presented in SEQ ID NO: 5 (DASSLES). In some embodiments, the anti-IL-7Rα antibody comprises a light chain CDR3, the light chain CDR3 comprising the amino acid sequence presented in SEQ ID NO: 6 (QQFNSYPLWIT).

[0169] In some embodiments, the target anti-IL-7Rα antibody comprises a heavy chain CDR1 containing the amino acid sequence described in SEQ ID NO: 1 (e.g., any one of SEQ ID NOs from 7 to 22, such as SEQ ID NO: 7 (GFTFDDHAMH)), a heavy chain CDR2 containing the amino acid sequence described in SEQ ID NO: 2 (GISWNSRGIGYADSVKG), a heavy chain CDR3 with the amino acid sequence described in SEQ ID NO: 3 (DEYSRGYYVLDV), a light chain CDR1 with the amino acid sequence described in SEQ ID NO: 4 (RASQGISSALA), a light chain CDR2 with the amino acid sequence described in SEQ ID NO: 5 (DASSLES), and a light chain CDR3 with the amino acid sequence described in SEQ ID NO: 6 (QQFNSYPLWIT).

[0170] In some embodiments, the target anti-IL-7Rα antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence described in SEQ ID NO: 25. In some embodiments, VL comprises an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% identical to the amino acid sequence described in SEQ ID NO: 26.

[0171] In some embodiments, the target anti-IL-7Rα antibody disclosed herein is 24 SQLEVNGSQHSLTCAF 39 (Sequence ID 27) 73 FIETKKFLLIGKSNIC 88 (Sequence No. 28) 89 VKVGEKSLTCKKIDLTT 105 (Sequence ID 29) 136 QKKYVKVLMHDVAY 149 (Sequence ID 30) 181 YEIKVRSIPDHYFKGF 196 It specifically binds to the alpha chain of the human IL-7 receptor in epitopes selected from the group consisting of (SEQ ID NO: 31) and combinations thereof.

[0172] In some embodiments, the anti-IL-7Rα antibody is an epitope comprising one or more amino acid residues selected from the group consisting of H33, E75, F79, 182, K84, M144, R186, H191, Y192, and combinations thereof, which specifically binds to the alpha chain of the human IL-7 receptor.

[0173] In some embodiments, the anti-IL-7R antibody is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and their variants.

[0174] In some embodiments, the IL-7R antibody is an IgG1 antibody. In some embodiments, the anti-IL-7R antibody comprises an IgG1 Fc without effector function.

[0175] In some embodiments, the target anti-IL-7Rα antibody or its antigen-binding moiety includes a heavy chain variable region (VH) and a light chain variable region (VL), where VH includes the amino acid sequence described in SEQ ID NO: 25 and VL includes the amino acid sequence described in SEQ ID NO: 26.

[0176] In some embodiments, the target anti-IL-7Rα antibody or its antigen-binding moiety comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence described in SEQ ID NO: 23, and the light chain comprising the amino acid sequence of SEQ ID NO: 24.

[0177] In some embodiments, the anti-IL-7Rα antibody is measured by surface plasmon resonance and has a K content of 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3nM, 2nM, or less than 1nM (e.g., 1.3nM). D It binds to the alpha chain of the human IL-7 receptor. In some embodiments, the anti-IL-7Rα antibody, when measured by surface plasmon resonance, has a K content of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or less than 1 nM (e.g., 1.7 nM). D It binds to the alpha chain of the cynomolgus monkey IL-7 receptor. In some embodiments, binding to the alpha chain of the human IL-7 receptor or the alpha chain of the cynomolgus monkey IL-7 receptor is pH-dependent. In some embodiments, the anti-IL-7Rα antibody is approximately 1.3 nM K at pH 7.4. D And, at pH 6, approximately 5.3 nM of K D Then, it binds to the alpha chain of the human IL-7 receptor. In some embodiments, the anti-IL-7Rα antibody is approximately 1.7 nM K at pH 7.4. D And, at pH 6, approximately 7.0 nM of K D Then, it binds to the alpha chain of the IL-7 receptor in cynomolgus monkeys.

[0178] In some embodiments, an isolated antibody or its antigen-binding moiety that specifically binds to the alpha chain of a human IL-7 receptor (anti-IL-7Rα antibody) comprising heavy chain (HC)CDR1, CDR2, and CDR3 and light chain (LC)CDR1, CDR2, and CDR3 includes: (i) heavy chain CDR1 comprising the amino acid sequence shown in any one of SEQ ID NOs. 7 to 22; (ii) heavy chain CDR2 comprising the amino acid sequence described in SEQ ID NO. 2 (GISWNSRGIGYADSVKG); (iii) heavy chain CDR3 comprising the amino acid sequence described in SEQ ID NO. 3 (DEYSRGYYVLDV); (iv) light chain CDR1 comprising the amino acid sequence described in SEQ ID NO. 4 (RASQGISSALA); (v) light chain CDR2 comprising the amino acid sequence described in SEQ ID NO. 5 (DASSLES); and (vi) light chain CDR3 comprising the amino acid sequence described in SEQ ID NO. 6 (QQFNSYPLWIT). In certain embodiments, the heavy chain CDR1 comprises the amino acid sequence described in SEQ ID NO: 8 (GYTFDDHAMH), SEQ ID NO: 19 (GFDFDDHAMH), or SEQ ID NO: 20 (GFEFDDHAMH).

[0179] The relevant sequences of exemplary antibodies are provided below. [Table 2-1] [Table 2-2]

[0180] In some embodiments, the anti-IL-7Rα antibody, when measured by surface plasmon resonance, has a K content of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or less than 1 nM (e.g., 1.3 nM). D It binds to the alpha chain of human IL-7 7Rα. In some embodiments, the anti-IL-7Rα antibody has a K content of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or less than 1 nM (e.g., 1.7 nM) when measured by surface plasmon resonance. DIt binds to the alpha chain of the cynomolgus monkey IL-7 receptor. In some embodiments, binding to the alpha chain of the human IL-7 receptor or the alpha chain of the cynomolgus monkey IL-7 receptor is pH-dependent. In some embodiments, the anti-IL-7Rα antibody is approximately 1.3 nM K at pH 7.4. D And, at pH 6, approximately 5.3 nM of K D Then, it binds to the alpha chain of the human IL-7 receptor. In some embodiments, the anti-IL-7R antibody is at approximately 1.7 nM K at pH 7.4. D And, at pH 6, approximately 7.0 nM of K D Then, it binds to the alpha chain of the IL-7 receptor in cynomolgus monkeys.

[0181] In some embodiments, the anti-IL-7Rα antibody is formulated for administration to a subject in a flat dose or a body weight-based dose. In some embodiments, the anti-IL-7Rα antibody is formulated for administration to a subject in a body weight-based dose (e.g., mg / kg). In some embodiments, the anti-IL-7Rα antibody is formulated for intravenous, subcutaneous, intramuscular, intradermal, or intraperitoneal administration. In some embodiments, the anti-IL-7Rα antibody is formulated for intravenous (iv) or subcutaneous (sc) administration.

[0182] Compositions comprising the anti-IL-7Rα antibody or its antigen-binding moiety as described herein, having a desired purity in a physiologically acceptable carrier, excipient, or stabilizer, are provided herein. The acceptable carrier, excipient, or stabilizer is nontoxic to the recipient at the dose and concentration employed and includes buffers such as phosphoric acid, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, and Examples include proteins such as immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides such as glucose, mannose, or dextrin, disaccharides, and other carbohydrates, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0183] In certain embodiments, the pharmaceutical composition comprises an antibody or its antigen-binding moiety, a bispecific molecule, or an immune complex as described herein, and optionally one or more additional prophylactic or therapeutic agents in a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition comprises an effective amount of the antibody or its antigen-binding moiety as described herein, and optionally one or more additional prophylactic therapeutic agents in a pharmaceutically acceptable carrier. In some embodiments, the antibody is the sole active ingredient in the pharmaceutical composition. The pharmaceutical compositions described herein may be useful for regulating (e.g., reducing or inhibiting) IL-7 activity in T cells (e.g., pathogenic T cells), and for treating diseases or disorders such as inflammatory diseases, e.g., inflammatory bowel disease.

[0184] As used herein, pharmaceutically acceptable carriers include any and all physiologically compatible solvents, dispersion media, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc. In some embodiments, carriers are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epithelial administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, immune complex, or bispecific molecule, may be coated with a material to protect the compound from the action of acids and other natural states that can inactivate the compound.

[0185] Furthermore, pharmaceutical formulations are provided that improve the stability of anti-IL-7Rα antibodies and thus enable their long-term storage. In some embodiments, the pharmaceutical formulations disclosed herein comprise (a) an anti-IL-7R antibody, (b) a buffer, (c) a stabilizer, (d) a salt, (e) a volume extender, and / or (f) a surfactant. In some embodiments, the pharmaceutical formulations are stable for at least one month, at least two months, at least three months, at least six months, at least one year, at least two years, at least three years, at least five years, or longer. In some embodiments, the formulations are stable when stored at 4°C, 25°C, or 40°C.

[0186] A buffer can be a weak acid or base used to maintain the acidity (pH) of a solution near a selected value after the addition of another acid or base. A suitable buffer can maximize the stability of a pharmaceutical formulation by maintaining pH control of the formulation. A suitable buffer can also ensure physiological compatibility or optimize solubility. Rheology, viscosity, and other properties may also depend on the pH of the formulation. Common buffers include, but are not limited to, histidine, citrate, succinate, acetate, and phosphate. In some embodiments, the buffer includes histidine having an isotonic property (e.g., L-histidine) and the possibility of pH adjustment using acids or bases known in the art. In some embodiments, the buffer is L-histidine. In some embodiments, the pH of the formulation is maintained at about 2 to about 10, or about 4 to about 8.

[0187] Stabilizers are added to pharmaceuticals to stabilize the product. These agents can stabilize proteins in many different ways. Common stabilizers include, but are not limited to, amino acids such as glycine, alanine, lysine, arginine, or threonine; carbohydrates such as glucose, sucrose, trehalose, rhaumos, or maltose; polyols such as glycerol, mannitol, sorbitol, cyclodextrin, or dextran of any type and molecular weight; or PEG. In some embodiments, the stabilizer is selected to maximize the stability of the FIX polypeptide in the lyophilized preparation. In some embodiments, the stabilizer is sucrose and / or arginine.

[0188] Volume extenders can be added to pharmaceuticals to increase their volume and mass, thereby facilitating their accurate measurement and handling. Common volume extenders include, but are not limited to, lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, or magnesium stearate.

[0189] A surfactant is an amphiphilic substance having a hydrophilic group and a hydrophobic group. Surfactants can be anionic, cationic, zwitterionic, or nonionic. Examples of nonionic surfactants include, but are not limited to, alkyl ethoxyates, nonylphenol ethoxyates, amine ethoxyates, polyethylene oxide, polypropylene oxide, fatty alcohols such as cetyl alcohol or oleyl alcohol, cocamide MEA, cocamide DEA, polysorbates, or dodecyldimethylamine oxide. In some embodiments, the surfactant is polysorbate 20 or polysorbate 80.

[0190] In some embodiments, the pharmaceutical formulation comprises (a) about 0.25 mg / mL to 250 mg / mL (e.g., 10 to 200 mg / mL) of anti-IL-7Rα antibody, (b) about 20 mM histidine, (c) about 260 mM sucrose, (d) about 0.5 mM DTPA, and (e) about 0.05% Tween-80.

[0191] In some embodiments, the pharmaceutical formulation comprises 20 mM histidine, 260 mM sucrose, and approximately 100 mg / mL of anti-IL-7Rα antibody in 0.05% (w / v) polysorbate 80, together with an optional 0.05 mM pentetate, at pH 6.0.

[0192] The formulation may further comprise one or more of a buffer system, preservatives, isotonic agents, chelating agents, stabilizers, and / or surfactants, as well as various combinations thereof. The use of preservatives, isotonic agents, chelating agents, stabilizers, and surfactants in pharmaceutical compositions is known to those skilled in the art.

[0193] In some embodiments, the pharmaceutical formulation is an aqueous formulation. Such formulations are typically solutions or suspensions, but may also include colloids, dispersions, emulsions, and multiphase materials. The term “aqueous formulation” is defined as a formulation containing at least 50% w / w water. Similarly, the term “aqueous solution” is defined as a solution containing at least 50% w / w water, and the term “aqueous suspension” is defined as a suspension containing at least 50% w / w water.

[0194] In some embodiments, the pharmaceutical formulation is a lyophilized formulation to which a physician or patient adds a solvent and / or diluent before use.

[0195] The pharmaceutical compositions described herein may also be administered in combination therapy, i.e., in combination with other agents. For example, combination therapy may include the IL-7R antibody described herein in combination with at least one other therapeutic agent. Examples of therapeutic agents that may be used in combination therapy include other compounds, agents, and / or agents used to treat diseases or disorders (e.g., inflammatory disorders). Examples of such compounds, agents, and / or agents include anti-inflammatory agents or antibodies that block or reduce the production of inflammatory cytokines. In some embodiments, the therapeutic agent may comprise anti-IP-10 antibodies, anti-TNF-α antibodies (e.g., adalimumab (HUMIRA®), golimumab (SIMPONI®), infliximab (REMICADE®), certolizumab pegol (CIMZIA®)), interferon beta-1a (e.g., AVONEX®, REBIF®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), glatiramer acetate (e.g., COPAXONE®, GLATOPA®), mitoxantrone (e.g., NOVANTRONE®), nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, corticosteroids, and combinations thereof. In some embodiments, the therapeutic agent may comprise compounds, drugs, and / or agents capable of inducing the production of regulatory T cells (e.g., induced regulatory T cells). Non-exclusive examples of such therapeutic agents include TGF-β, IL-10, IL-2, and combinations thereof.

[0196] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. A “pharmaceutically acceptable salt” means a salt that retains the desired biological activity of the parent compound and does not produce undesirable toxic effects. Examples of such salts include acid addition salts and base addition salts. Acid addition salts are derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorus, as well as non-toxic organic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanos, hydroxyalkanoics, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts are derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, as well as non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine.

[0197] The pharmaceutical compositions described herein may also contain pharmaceutically acceptable antioxidants. Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants such as cysteine ​​hydrochloride such as ascorbic acid, sodium bisulfate, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and similar substances; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0198] Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, and polyethylene glycol), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0199] These compositions may also contain adjuvants such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization procedures described above, as well as by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenolsorbic acid, and similar substances. It may also be desirable to include isotonic agents such as sugars and sodium chloride in the composition. Furthermore, long-term absorption of injectable pharmaceutical forms can be achieved by including absorption-delaying agents such as aluminum monostearate and gelatin.

[0200] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The use of such cultures and agents for pharmaceutically active substances is known in the art. Unless any conventional culture or agent is incompatible with the active compound, its use in the pharmaceutical compositions described herein is intended. The pharmaceutical compositions may or may not contain preservatives. Auxiliary active compounds may be incorporated into the compositions.

[0201] Therapeutic compositions typically must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. Carriers can be solvents or dispersion media containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Often, compositions may contain isotonic agents, such as sugars, mannitol, sorbitol, or polyalcohols such as sodium chloride. Sustained absorption of injectable compositions can be achieved by including absorption-delaying agents in the composition, for example, monostearate and gelatin.

[0202] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent having one or a combination of the components listed above, as needed, followed by sterile microfractionation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other necessary components from those listed herein. In the case of sterile powders for the preparation of sterile injectable solutions, some preparation methods are vacuum drying and freeze-drying (lyophilization), which yield the active ingredient powder and any additional desired components from the previously sterile filtered solution.

[0203] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form varies depending on the target being treated and the specific mode of administration. Generally, the amount of active ingredient that can be combined with a carrier material to produce a single dosage form is the amount of the composition that produces the therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of the active ingredient, about 0.1 percent to about 70 percent, or about 1 percent to about 30 percent of the active ingredient when combined with a pharmaceutically acceptable carrier.

[0204] The administration regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and uniformity of dosage, it is particularly advantageous to formulate parenteral compositions into unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the unit dosage forms described herein are determined by and directly depend on (a) the inherent characteristics and specific therapeutic effects of the active compound to be achieved, and (b) the inherent limitations of the technique for formulating such active compound for the treatment of susceptibility in an individual.

[0205] In some embodiments, the anti-IL-7Rα antibody is administered in a flat dose (flat dose regimen).

[0206] In some embodiments, for example, with respect to the administration of the anti-IL-7Rα antibody described herein, the dosage range for each dose is approximately 10 mg to 800 mg per dose, approximately 20 mg to 500 mg per dose, approximately 50 mg to 300 mg per dose, or approximately 100 mg to 250 mg per dose.

[0207] In some embodiments, the anti-IL-7Rα antibody is administered to the subject in doses of approximately 25, 50, 100, 150, 200, 250, 300, or 350 mg.

[0208] In some embodiments, the anti-IL-7Rα antibody is administered in a dose based on body weight.

[0209] In some embodiments, for example, the dose range for the administration of the anti-IL-7Rα antibody described herein is approximately 0.1 mg / kg to 10 mg / kg per dose, approximately 0.2 mg / kg to 8 mg / kg per dose, approximately 0.5 mg / kg to 5 mg / kg per dose, approximately 1 mg / kg to 4 mg / kg per dose, or approximately 2 mg / kg to 3 mg / kg per dose.

[0210] In some embodiments, the anti-IL-7Rα antibody is administered to the subject at doses of approximately 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, or 10 mg / kg.

[0211] In some embodiments, the anti-IL-7Rα antibody is administered in a fixed dose along with another antibody.

[0212] Antibodies are usually administered multiple times. The interval between single doses can be, for example, weekly, bi-weekly (i.e., every two weeks), monthly, every three months, or annually. The interval can also be irregular, as indicated by measuring the blood levels of antibodies against the target antigen in the patient.

[0213] Exemplary treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months, or once every three to six months.

[0214] In some embodiments, an anti-IL-7Rα antibody, such as the antibody described herein, is administered once a week.

[0215] In some embodiments, an anti-IL-7Rα antibody, such as the antibody described herein, is administered once every two weeks.

[0216] In some embodiments, the treatment comprises 10 to 15 consecutive doses (e.g., 11, 12, or 13 doses) administered once every two weeks. For example, the treatment may comprise 10, 11, 12, 13, 14, or 15 consecutive doses, each dose administered every two weeks.

[0217] In some embodiments, antibodies are administered for up to 10 weeks, 13 weeks, 26 weeks, 52 weeks, 2 years, 3 years, 5 years, 10 years, or for life / permanently. In some embodiments, antibodies are administered as needed when hair loss occurs.

[0218] In some methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the indicated range.

[0219] In some methods, the dose is adjusted to achieve plasma antibody concentrations of approximately 0.1–10 μg / mL, 0.2–9 μg / mL, 0.5–8 μg / mL, 1–7 μg / mL, 2–6 μg / mL, and 4–5 μg / mL.

[0220] In some methods, the dose is adjusted to achieve plasma antibody concentrations of approximately 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, or 8 μg / mL.

[0221] Antibodies can be administered as sustained-release formulations, in which case less frequent administration is required. Dosage and frequency vary depending on the antibody's half-life in the patient. Generally, human antibodies have the longest half-lives, followed by humanized antibodies, chimeric antibodies, and non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. For prophylactic use, relatively low doses are administered over a long period at relatively infrequent intervals. Some patients continue treatment for life. For therapeutic use, relatively high doses at relatively short intervals may be required until disease progression is reduced or terminated, and until the patient shows partial or complete improvement in disease symptoms. The patient may then receive a maintenance regimen.

[0222] The actual dose levels of the active ingredients in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. The selected dose level will depend on various pharmacokinetic factors, including the activity of the particular composition described herein being employed, or esters, their salts or amides, route of administration, time of administration, excretion rate of the particular compound used, duration of treatment, other agents, compounds and / or materials used in combination with the particular composition used, age, sex, weight, condition, general health and prior medical history of the patient being treated, and similar factors well known in the medical technology.

[0223] The compositions described herein can be administered via one or more routes of administration using one or more of the various methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired result. The routes of administration of the anti-IL-7R antibodies described herein may comprise intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, or other parenteral routes of administration, for example, by injection or infusion. As used herein, the term "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraspinal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intraspinal, epidural, and intrasternal injection and infusion.

[0224] Alternatively, the antibodies described herein can be administered via non-parenteral routes such as local, epidermal, or mucosal routes of administration, for example, intranasal, oral, intravaginal, intrarectal, sublingual, or topical.

[0225] The active compounds can be prepared with carriers that protect the compound from rapid release, such as controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Many methods for the preparation of such formulations are generally known to those skilled in the art.

[0226] The therapeutic composition can be administered with a medical device known in the art. For example, in certain embodiments, the therapeutic composition described herein can be administered with a needleless subcutaneous injection device. Examples of well-known implants and modules for use with the anti-IL-7R antibodies described herein include implantable microinjection pumps for dispensing drugs at a controlled rate, therapeutic devices for administering drugs through the skin, drug infusion pumps for delivering drugs at an accurate infusion rate, variable flow implantable infusion devices for continuous drug delivery, osmotic drug delivery systems with multi-chamber compartments, and osmotic drug delivery systems. Many other such implants, delivery systems, and modules are known to those of skill in the art.

[0227] Additional treatable disease indications The examples relate to the treatment of alopecia areata (AA), but the invention described herein is not so limited to hair loss disorders treatable by the compounds and compositions described herein. Non-limiting examples of other hair loss disorders treatable according to the methods described herein: androgenetic alopecia, alopecia areata, telogen effluvium, alopecia areata, tinea capitis, alopecia totalis, hypotrichosis, hereditary simplex hypotrichosis, and alopecia universalis.

[0228] Combination therapy In some embodiments, a second / additional therapeutic agent effective for the treatment of hair loss disorders is administered to the subject.

[0229] In some embodiments, the second / additional therapeutic agent comprises a corticosteroid. The corticosteroid may be formulated, for example, as a topical formulation or a topical injection formulation (e.g., injection into the bald area / diseased skin area).

[0230] In some embodiments, the second / additional therapeutic agent comprises minoxidil (commercially available under the brand name ROGAINE®).

[0231] In some embodiments, the second / additional therapeutic agent comprises anthralin.

[0232] In some embodiments, the second / additional therapeutic agent includes methotrexate.

[0233] In some embodiments, hair loss includes eyelash shedding, and the second / additional therapeutic agent includes bimatoprost (or a similar agent).

[0234] In some embodiments, the second / additional therapeutic agent includes an inhibitor of a protein tyrosine kinase (PTK) involved in cytokine signaling. In some embodiments, the inhibitor is a Jak / Stat inhibitor. In further embodiments, the inhibitor is INCB 018424. In some embodiments, the Jak3 inhibitor is an antibody that specifically binds to the Jak3 protein or a fragment thereof, an antisense RNA or antisense DNA that reduces the expression of the gene encoding the Jak3 protein, an antisense RNA or antisense DNA that reduces the expression of the Jak3 protein, an siRNA that specifically targets the Jak3 gene, a small molecule, or a combination thereof. In some embodiments, the inhibitor is a Jak3 inhibitor. In further embodiments, the inhibitor is tofacitinib (CP690550). In further embodiments, the small molecule is Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 Inhibitor IV, NSC114792, or R348. In some embodiments, the Stat inhibitor is a Stat 1 inhibitor, such as an antibody or antibody fragment against Stat 1. In some embodiments, the Stat inhibitor is a Stat 2 inhibitor, such as an antibody or antibody fragment against Stat 2.

[0235] Non-exclusive examples of Jak1 / Jak2 inhibitors include baricitinib, tofacitinib, ruxolitinib, baricitinib, upadacitinib, abrocitinib, pacritinib, fedatinib, AG490;CYT387;SB1518;LY3009104(INCB28050), TG101348, and BMS-911543.

[0236] JAK1 / 2 inhibitors currently in clinical development that could potentially serve as a second therapeutic agent include a) topical and oral INCB018424 and oral incyte, a) 5 nM activity (Incyte), b) CEP-701 (Cephalon), and c) TG101348.

[0237] In some embodiments, the second / additional therapeutic agent includes a JAK3 inhibitor. Non-limiting examples of JAK3 inhibitors include: Janex 1 (WHI-P131), PF-956980, WHI-P154, VX-509, JAK3 inhibitor IV (ZM-39923), NSC114792, tofacitinib (CP690550), and R348.

[0238] Non-restrictive JAK3 inhibitors include a) Janex 1, oral and topical; b) PF-956980, intravenous infusion; c) WHI-P154; d) ZM-39923; e) NSC114792; f) tofacitinib (CP690550), oral.

[0239] Other non-limiting examples of JAK inhibitors useful as a second therapeutic agent include, for example, type I and type II Jak inhibitors.

[0240] Non-exclusive examples of Stat inhibitors that may be used as a secondary therapeutic agent include: WP-1034, fludarabine, epigallocatechin-3-gallate (EGCG), and hyperforin.

[0241] Additional second therapeutic agents that may be used in the manner described herein include any and all RTK inhibitors, such as JAK / STAT inhibitors. [Examples]

[0242] Example 1 Phase IIa randomized, double-blind, placebo-controlled trial of compound A for the treatment of severe alopecia. Compound A (an antibody possessing the heavy-chain amino acid sequence of SEQ ID NO: 23 and the light-chain amino acid sequence of SEQ ID NO: 24) is a fully human high-affinity anti-interleukin-7 receptor α (IL-7Rα) antagonist monoclonal antibody (mAb) developed from human immunoglobulin transgenic mice. It exhibits high affinity as an anti-interleukin-7 receptor α (IL-7Rα) and reduced affinity for FcγR (type I, type II, or type III), the receptor mediating effector function, and complement component 1 (C1q), a mediator of complement cell lysis. Compound A is demonstrated herein as an effective treatment for alopecia areata.

[0243] Alopecia areata is an autoimmune condition that affects hair follicles, leading to hair loss. This condition can develop at any age and in any sex, and its incidence is estimated to be 2% of the world's population. While some patients recover spontaneously, approximately 25% progress to alopecia totalis (complete hair loss on the scalp) or alopecia universalis (complete hair loss on the entire body). Although the pathophysiology is not fully described, the onset of the condition is mediated by inflammatory mechanisms.

[0244] The onset of alopecia is thought to originate from genetic factors (i.e., hereditary predisposition) and environmental factors (e.g., inconsistencies between identical twins, toxic agents mediating AA cases). The cascade of inflammatory events involves the recruitment of autoreactive CD8+ T cells around the hair follicles of AA skin lesions. These CD8+ T cells are locally activated by protein MICA binding on surface NKG2D and presented by ectopic MHC I molecules in immune-privileged hair follicles. Cytotoxic T cell-associated and IFNg-associated gene expression signatures were identified in lesions compared to normal individual skin, which reflect local T cell activation, subsequently inducing the recruitment of other immune cell types (CD4+ Th1 cells, macrophages, NK cells, and eosinophils) and leading to a breakdown of hair follicle immune privilege.

[0245] Non-clinical pharmacology studies confirm IL-7Rα receptor occupancy (RO) and IL-7Rα-mediated pharmacodynamic (PD) responses and inhibition of T cell-mediated biology. Both the interleukin (IL)-7 receptor and the thymic stromal lymphopoietin (TSLP) receptor are heterodimers that share a common IL-7Rα subunit.

[0246] In a Phase I trial in healthy participants, single doses of up to 4 mg / kg of Compound A and repeated dosing of 1 mg / kg once every two weeks were safe and well tolerated. The results of that Phase I trial showed that doses of Compound A that achieved a receptor occupancy of ≥95% were associated with >90% inhibition of IL-7 signaling via phosphorylation of signal transducer and activator of transcription-5 (STAT5). Repeated dosing of 1 mg / kg of Compound A showed sustained inhibition of IL-7Rα after the second dose over a two-week dosing regimen. Furthermore, lymphocyte subset and T cell immune function assessments showed modest and dose-dependent effects on these biomarkers. Based on available non-clinical data and Phase I safety data, no expected adverse events were identified. Thus, the Phase I trial showed that Compound A has acceptable tolerability and safety, confirms the expected pharmacology, enables the generation of a predictive pharmacokinetic (PK)-PD model, and supports the initiation of this Phase IIa trial.

[0247] Mechanistically, Compound A is a high-affinity effector-minimized anti-IL-7Rα antagonist antibody that blocks IL-7 cytokine binding to human anti-IL-7Rα (CD127) and inhibits the IL-7 receptor (IL-7R) (CD127 / CD132)-mediated intracellular signaling pathway.

[0248] This embodiment demonstrates that the anti-IL7Ra antibody compound A is effective in reducing hair loss in patients with severe alopecia (AA) based on the dosing regimen described herein, at least by week 22 and / or week 24 after the initiation of treatment. The primary objective of the study is to demonstrate the effect of compound A versus placebo on the reduction of hair loss at week 24 after the initiation of treatment in participants with AA. This primary objective is assessed based on the primary endpoint of the mean relative percentage change in Alopecia Severity Tool (SALT) score at week 24 compared to baseline.

[0249] The primary efficacy endpoint, the proportion of participants with a relative change in SALT score compared to baseline at week 24, is an established measure of alopecia severity and is clinically relevant to measuring the efficacy of compound A in this patient population.

[0250] A secondary objective of the study is to demonstrate the effect of compound A versus placebo on reducing hair loss over 24 weeks of treatment in participants with AA. This secondary objective will be assessed by the number of secondary endpoints analyzed, including the following: • Mean relative percentage change in SALT score compared to baseline at weeks 12 and 18. • Mean relative percentage change in SALT score at 24 weeks compared to the lowest therapeutic point. • Percentage of participants who achieved a relative reduction of ≥30% and ≥50% in SALT score compared to baseline at weeks 12, 18, and 24. • Percentage of participants with absolute SALT scores ≤ 5, 10, 20, 30, and 50 at weeks 12, 18, and 24.

[0251] Patients receiving treatment (e.g., baseline compound A group) showed statistically significant improvement in the mean relative percentage change in SALT scores at weeks 12, 18, 22, and / or 24 compared to the matched control group (e.g., patients receiving placebo).

[0252] Furthermore, more than 50% of patients receiving treatment showed statistically significant improvement in the mean relative percentage change in SALT scores at weeks 12, 18, 22, or 24 compared to the matched control group (e.g., patients receiving placebo).

[0253] Furthermore, the majority of participants have an absolute SALT score of ≤5 at weeks 12, 18, 22, and / or 24, and at weeks 10, 20, 20, 30, or 50.

[0254] Treatment also has a post-treatment effect on reducing alopecia in participants with AA, which is (1) a significant proportion of treated patients who maintained a relative percentage change from baseline in SALT score at 36 weeks compared to baseline and at 24 weeks, (2) achieved a relative reduction of ≥50% from baseline in SALT score at 36 weeks compared to baseline and at 24 weeks, and / or (3) the proportion of participants who achieved an absolute SALT score ≤5, 10, 20, 30, and 50 at 36 weeks.

[0255] The pharmacokinetics (PK) and immunogenicity of compound A in treated patients are also desirable, based on assays including serum compound A concentration and anti-drug antibody (ADA) rate according to an assessment schedule (SoA).

[0256] Finally, the safety and tolerability of compound A are evaluated, taking into account clinical laboratory assessments, physical examinations, vital signs, and 12-lead electrocardiograms (ECGs), including the tolerability of the injection site, and the acceptable number or percentage of adverse events (AEs), serious adverse events (SAEs), and adverse events of particular interest (AESIs).

[0257] The tests described herein also include some exploratory purposes.

[0258] One of the exploratory objectives is to evaluate the effect of compound A versus placebo on reducing eyebrow and eyelash loss in participants with AA. These exploratory objectives will be assessed by evaluating one or more of the following exploratory endpoints: • Change from baseline in physician-reported outcomes (ClinRO) for eyebrow alopecia at weeks 12, 18, 24, and 36; • Changes from baseline in ClinRO for eyelash loss at weeks 12, 18, 24, and 36; • The proportion of participants who achieved ClinRO for eyebrow alopecia score of 0 or 1 (complete coverage or minimal gap) with an improvement of ≥2 points from baseline at 24 and 36 weeks (one of participants with a baseline score of ≥2 [significant gap to significant hairlessness]); • The proportion of participants who achieved ClinRO for a trichiasis score of 0 or 1 (complete coverage or minimal gap) with an improvement of ≥2 points from baseline at 24 and 36 weeks (baseline score ≥2 [significant gap to significant hair loss]).

[0259] Another exploratory objective is to assess the impact on patients' quality of life (QOL), which can be evaluated by measuring the improvement in SKINDEX-16 AA compared to baseline at weeks 12, 18, 24, and 36.

[0260] Further exploratory objectives include investigating the pharmacokinetics (PK), immunogenicity, receptor occupancy (RO), and pharmacodynamics (PD) of compound A in subjects with AA. These investigations will be facilitated by evaluating the following: • Serum PK profiles of SC-administered compound A, evaluated using nonlinear mixed-effects modeling and population PK analysis. • Incidence and titer of serum anti-compound A antibodies, and related effects on PK, PD, efficacy, and safety ·Circulating CD3 + Changes in whole blood IL-7 receptor (IL-7R)-α RO on T cells • Changes in T cell subsets • Collect serum, plasma, peripheral blood mononuclear cells (PBMCs), and whole blood RNA and DNA (DNA is optional) and store them for potential future evaluation of biomarkers related to the treatment response to compound A and / or disease biomarkers of compound AA. Any relationships between the PK, RO, PD, and AE profiles of compound A, exploratory biomarkers, clinical laboratory data, and clinical activity may be investigated.

[0261] Clinical trial design: The clinical trial was designed as a Phase IIa, multicenter, placebo-controlled, proof-of-concept trial to evaluate the preliminary efficacy, safety, tolerability, pharmacokinetic (PK), and PD of compound A in participants with severe to very severe alopecia areata (AA), with ≥50% total scalp hair loss as defined by the SALT score on day 1. Approximately 40 participants with alopecia areata (AA) will be enrolled. Participants will be considered to have completed the trial if they complete all trial periods, including their last visit.

[0262] Participants were aged 18–75 years (inclusive) and had a current episode of severe alopecia >6 months–<10 years, along with investigator assessment of the possibility of hair regrowth, no evidence of regrowth at baseline, no known history of significant regrowth in the past 3 months, and a current episode of 10% or less regrowth in the past 6 months. Approximately 40 participants were enrolled and randomly assigned (3:1) to receive subcutaneous administration of 200 mg of compound A or the corresponding placebo for 24 weeks (i.e., SC Q2W for 24 weeks, a total of 13 doses). Randomization was stratified based on the SALT score on day 1 (≥50–<95, ≥95–100). Enrollment of participants in the ≥95–100 SALT score stratification was limited to approximately n=14. See Figure 1.

[0263] The trial includes a screening period of up to 30 days, with registration on day 1 (baseline). Participants will be treated with compound A or placebo every two weeks (Q2W) from day 1 (baseline) until week 24. Based on previous studies, trough exposure at week 24 remains within the predicted therapeutic range. The primary endpoint will be assessed at the week 24 visit. All participants will have a follow-up period of 12 weeks after the last dose (up to week 36) to assess long-term safety and observe the duration of efficacy after the treatment period.

[0264] Efficacy is assessed by SALT score evaluation, ClinRO Scale® for eyebrow alopecia, ClinRO Scale® for eyelash depilation, PRO measurement for eyebrow alopecia, PRO measurement for eyelash depilation, and SKINDEX16-AA.

[0265] Blood samples will be collected from all participants to characterize the PK, PD, and immunogenicity of compound A. Safety will be assessed by collecting AEs, SAEs, and AESIs, performing local injection site tolerability assessments, recording vital signs, performing physical examinations and ECGs, and evaluating laboratory results.

[0266] Therefore, the total duration of the trial was approximately 40 weeks, with a screening period of up to 30 days and a treatment period of 24 weeks, during which participants received either compound A or a corresponding placebo subcutaneously every two weeks, followed by a follow-up period of approximately 12 weeks. However, the treatment period itself was only about 24 weeks.

[0267] Dosage and frequency (dosage regimen) Participants will be administered either compound A or the corresponding placebo.

[0268] Compound A is formulated as a 100 mg / mL subcutaneous injection drug solution and supplied in sterile-filled 2R borosilicate glass vials with a Flurotec serum stopper and mat cap flip-off seal. Each vial contains 2 mL of Compound A, which is an extractable volume. That is, each dose of Compound A contains 200 mg of Compound A antibody, which is administered as a flat dose once every two weeks for a total of 13 doses, according to the activity schedule (SoA) detailed below (see also Figure 1). This dosing regimen (i.e., 200 mg of Compound A administered subcutaneously, Q2W) is expected to result in a steady-state serum trough concentration >5 μg / mL and a maximum RO on circulating T cells in >90% of subjects throughout the dosing period. Furthermore, post-200 mg exposure is expected to fall within the range of exposures previously tested at 3 mg / kg and is expected to be safe and well-tolerated.

[0269] Preliminary PK and RO data support this assessment. Steady-state (29.5 μg / mL) and AUC in patients with AA after a dose of 200 mg SC Q2W. ss Predicted maximum concentration (C) at (8531 μg*h / mL) max The exposure levels were >100 times and >50 times lower, respectively, than the NOAEL exposure to 150 mg / kg / week of SC compound A in cynomolgus monkeys in a 6-month GLP study (AUC of 3260 μg / mL and 466,000 μg*h / mL in non-sexually mature animals, and AUC of 3780 μg / mL and 540,000 μg*h / mL in sexually mature animals).

[0270] The placebo is formulated identically to compound A in a volume corresponding to the active dose. The formulation is stored in a safe, environmentally controlled, and monitored (manual or automated) area according to labeled storage conditions with restricted access to authorized personnel until use.

[0271] The subject's experienced TEAEs, standard safety clinical laboratory data, and systematic evaluation of the ADA are regularly monitored to ensure the subject's safety and identify the occurrence of events that meet the criteria for discontinuing IP (see below).

[0272] Stopping criteria [Table 3]

[0273] The sponsor will conduct blinded, periodic safety reviews as the trial progresses, as outlined in the safety monitoring plan.

[0274] Patient selection criteria Participants who meet all of the following criteria are eligible to take the test at the time of screening and / or on their first day visit, as described in the criteria. 1. Adult men and women aged 18-75 at the time of informed consent. 2. A clinical diagnosis of severe to very severe AA, defined as the presence of ≥50% overall scalp hair loss at screening and baseline (Day 1), as measured by the SALT score. 3. The current episode of hair loss has lasted for >6 months and <10 years, without any spontaneous improvement over the past 6 months, and the investigator assessed that the hair loss was stable for at least 3 months and regrowth was possible at the time of the screening visit. 4. In the judgment of the principal investigator, there is no evidence of active regrowth at baseline over the past three months, and there is no history of significant regrowth. 5. Throughout the trial period, I am willing to maintain the same hairstyle, including length and color (e.g., hair products, process, and timing of salon appointments), as directed by the principal investigator.

[0275] Patient exclusion criteria: Participants will be ineligible to participate in the study if, as necessary, they meet any of the following criteria at the time of screening and / or on their first day visit. 1. Weight at screening is <48kg or >120kg. 2. Evidence of active forms of other inflammatory skin diseases or other skin conditions (e.g., psoriasis, seborrheic dermatitis, lupus) at the time of screening and up to day 1 may, in the opinion of the principal investigator, interfere with the assessment of AA and the assessment of disease activity scales. 3. A history of or diagnosis of another type of alopecia (excluding androgenic alopecia) at the time of screening, based on the evaluation by the principal investigator. 4. A history of male or female pattern alopecia with Hamilton stage > III or Ludwig stage > II. 5. History (lifetime) or current history of hair transplantation. 6. History (lifetime) or presenting of micropigmentation on the scalp (Note: microblading of the eyebrows is acceptable). 7. Systemic, topical, or device-based therapies for AA, or immunotherapy required for any other condition unless otherwise noted: a. Use of steroids (systemic and intra-lesional), anthraline, sulfate, diphenylcyclopropenone, dinitrochlorobenzene, tacrolimus, minoxidil, or any other medications that, in the opinion of the principal investigator, may affect hair regrowth within four weeks of the Day 1 visit. Note: Intranasal and inhaled corticosteroids are permitted, as are eye drops and ear drops containing corticosteroids. b. Use of platelet-rich plasma injection 12 weeks prior to day 1. c. Use of topical medications that may affect AA, including but not limited to topical corticosteroids, minoxidil, calcineurin inhibitors, antibacterial agents, and medical devices, within two weeks prior to the first day of visit. Note: Topical corticosteroids are acceptable on the scalp, eyebrows, and outer eyelids. d. Prior treatment within 12 months of day 1 with any biological B-cell depletion therapy (e.g., rituximab, ocrelizumab, or ofatumumab) or other B-cell targeted therapy (e.g., belimumab). e. A history of plasmacytoid dendritic cell inhibitor therapy (e.g., anti-ILT7 [immunoglobulin-like transcript 7], anti-BDCA2 [blood dendritic cell antigen 2]) throughout one's life. f. If the last dose has been taken, use of any conventional disease-modifying antirheumatic drug, immunosuppressant (e.g., cyclosporine, methotrexate, or azathioprine), or JAK inhibitor (e.g., baricitinib): (i) within 8 weeks prior to day 1, or (ii) within 5 drug-specific half-lives (if more than 8 weeks). g. The patient has received a commercially available biological agent or investigational biological agent within 12 weeks of day 1 or within 5 half-lives (whichever is longer). h. Currently receiving non-biological IP or device therapy, or having received it within 4 weeks of day 1 or within 5 published half-lives, whichever is longer. i. You have received ultraviolet (UV)-B phototherapy (including on a yellowish-brown bed), psoralen-UV-A treatment, or excimer laser treatment within four weeks of day 1. j. Allergen immunotherapy was initiated within four weeks prior to randomization. Patients receiving a stable allergy immunotherapy regimen may be eligible if that regimen remains unchanged throughout the study period. 8. You have had, have recently had, or are currently at risk of a clinically serious viral, bacterial, fungal, parasitic, or mycobacterial infection, or any of the following serious infections: a. History of organ transplantation or bone marrow transplantation. b. A history of primary immunodeficiency disorder. c. There is a history of an infected joint prosthesis or other implanted device remaining in place. d. A history of opportunistic infections such as pneumonia. e. A history of disseminated Staphylococcus aureus infection or herpetic eczema. 9. Recent (within two months of informed consent unless otherwise stated) or currently clinically severe viral, bacterial, fungal, or parasitic infection, or mycobacterial infection, including but not limited to: a. Any history of symptomatic herpes zoster infection or disseminated / complication herpes zoster within 12 weeks prior to screening (e.g., polydermal involvement, ocular herpes zoster, central nervous system involvement, postherpetic neuralgia). b. Active herpes simplex virus type 1 or 2 at the time of screening or from the screening period up to day 1. This includes individuals with clinically active disease who are asymptomatic on suppressive therapy. c. A positive serological test for HIV-1 or HIV-2 at the time of screening. d. Evidence of active hepatitis A virus (HAV). Participants who are positive for total HAV antibodies should reflexively undergo HAV immunoglobulin (Ig) M testing. Participants who are positive for HAV IgM will be excluded. d. Evidence of active hepatitis A virus (HAV). Participants who are positive for total HAV antibodies should reflexively undergo HAV immunoglobulin (Ig) M testing. Participants who are positive for HAV IgM will be excluded. If HAV IgM is unavailable at the time of randomization, randomization may proceed if there is a reliable history of HAV vaccination or if there is no clinical or laboratory evidence of HAV infection, including normal liver function tests. e. Evidence of active hepatitis C virus (HCV) infection. Participants who are HCV antibody positive will require reflective HCV RNA testing. Participants with positive HCV RNA will be excluded. Participants who are HCV antibody positive but HCV RNA negative and have been previously treated for HCV may be enrolled with continuous monitoring to ensure they remain negative. f. Evidence of active hepatitis B virus (HBV) infection. Participants who are positive for hepatitis B surface antigen (HBsAg) will be excluded. Participants who are positive for hepatitis B core antibody and negative for HBsAg will require HBV DNA reflex testing. Participants with positive HBV DNA will be excluded. g. Evidence of active cytomegalovirus (CMV) or Epstein-Barr virus infection by polymerase chain reaction. PCR is available only if clinical CMV / EBV infection is suspected by the principal investigator, and must be negative before enrollment. h. You are receiving any therapy for a chronic infection, such as CMV, herpes zoster, or atypical mycobacteria. 10. Participants must have a negative chest X-ray and have not been treated with isoniazid or an appropriate treatment regimen for 9 months in accordance with World Health Organization or national guidelines, unless they have a positive QuantiFERON-TB Gold test at screening or in a history of tuberculosis (TB). The principal investigator is responsible for reviewing medical records and documenting the appropriateness of previous anti-TB treatments. The QuantiFERON-TB Gold trial may be repeated once for participants with inconclusive test results, and participants with two inconclusive test results may be enrolled after consultation with the medical monitor and sponsor. 11. You have been exposed to a live vaccine within 12 weeks prior to the randomization program, or you are scheduled to receive a live vaccine during the trial. 12. I received a COVID-19 vaccine (e.g., RNA-based vaccine, protein-based vaccine, and viral vector-based non-replicating vaccine) within four weeks of day 12.1. 13. Screening of clinical laboratory results at a central laboratory that meets one of the following criteria. These may be repeated once before declaring a participant unsuccessful in screening. Furthermore, participants with clinically significant laboratory values ​​based on the investigator's judgment, or with clinical laboratory abnormalities of grade 1 or higher according to the modified National Cancer Institute Common Terminology Criteria for Adverse Events v5.0, will not be enrolled. a. Alanine aminotransferase (ALT) or aspartate aminotransferase (AST) > 1.5 × upper limit of normal (ULN). b. Bilirubin ≥ 1.5 × ULN (If bilirubin is fractionated and direct bilirubin < 35%), then bilirubin ≥ 1.5 × ULN is acceptable. c. Hemoglobin < 10 g / dL. d. White blood cell count <3.0×10 9 / L. e. Platelet count < 150 × 10 9 / L. f. Absolute lymphocyte count <LLN。 14. Evidence of an uncontrolled metabolic disorder, including type 2 diabetes mellitus, as demonstrated by hemoglobin A1c > 8 or associated non-alcoholic steatohepatitis (NASH) / non-alcoholic fatty liver disease (NAFLD), independently of treatment, based on prior histological or non-invasive assessment including transient elastography, magnetic resonance imaging proton density lipid fraction, Fibrosis-4 index, AST / ALT ratio, or AST / platelet ratio. 15. The heart rate-adjusted QT interval (Fridericia method) at screening is >470 milliseconds (ms) for women and >450 milliseconds for men. 16. In the opinion of the principal investigator, other screening clinical test values ​​outside the central laboratory's reference range for populations or research sites that pose an unacceptable risk to individual participation in the trial. 17. Any other cardiovascular, respiratory, hepatic, gastrointestinal, endocrine, hematological, immunological, neurological, or psychiatric disorders, or any other serious and / or uncontrolled medical history or presence that, in the opinion of the principal investigator, constitutes a risk to IP administration or may interfere with the interpretation of data. 18. Current or historical kidney disease, or individual estimated glomerular filtration rate, calculated using the CKD-EPI (Chronic Kidney Disease Epidemiological Collaboration) equation for <60 mL / min at screening. 19. A serious comorbidity that may require the use of systemic corticosteroids, prevent participation in the study, or require active and frequent monitoring. 20. A history of any malignant tumor, such as non-melanoma skin cancer or excised cervical carcinoma in situ, as determined by the principal investigator. 21. Other active autoimmune diseases not listed above that make it difficult to adequately assess AA disease activity or pose a risk to individual participation in the study.

[0276] Efficacy analysis SALT evaluation To participate in this study, participants must have a SALT score of ≥50 at the time of screening and at baseline (Day 1).

[0277] The SALT score is a well-validated and widely used tool for determining the degree of hair loss in AA, based on the percentage of scalp surface area involved in the upper, posterior, and lateral parts of the scalp. The principal investigator determines the percentage of hair loss in a given quadrant and records the findings in the Canfield software program. The software program calculates the total percentage of hair loss by multiplying this value by the total scalp area and summing the resulting numbers for each quadrant, with a maximum possible score of 100. See Olson et al. (J Am Acad Dermatol. 2004;51(3):440-447. doi: 10.1016 / j.jaad.2003.09.032, incorporated herein by reference). SALT assessments are scheduled for week 0 (first injection on day 1), weeks 6, 12, 18, and 24 (end of treatment (EOT), week 13, and final injection), week 26 (first follow-up visit after treatment), week 30, and week 36 (end of study or EOS). The SALT assessment tool is provided in Figure 2 according to the following criteria. [Table 4]

[0278] Eyelash / Eyebrow Evaluation (ClinRO) The ClinRO scales for eyebrow alopecia and eyelash depilation are assessed by the principal investigator and include a single-item, 4-point, Likert response scale (0-3) to assess the incremental severity of each ClinRO scale. Responses range from 0 = normal appearance / no alopecia to 3 = severe appearance / severe alopecia. See Wyrwich et al. (Am J Clin Dermatol. 2020;21(5):725-732.doi:10.1007 / s40257-020-00545-9, incorporated herein by reference). ClinRO assessments are scheduled for week 0 (first injection on day 1), weeks 6, 12, 18, and 24 (end of treatment (EOT), week 13, and final injection), week 26 (first follow-up visit after treatment), week 30, and week 36 (end of study or EOS). The following is a summary of ClinRO. [Table 5]

[0279] Eyelash / Eyebrow Evaluation (PRO) The PRO scales for eyebrows and eyelashes were developed to provide effective and clinically meaningful scales that reflect the perspective of patients with AA. These scales have a severity scale ranging from 0 to 3, where 0 corresponds to complete eyebrows / eyelashes and 3 corresponds to no or very little eyebrow / eyelash hair. See Wyrwich et al. (Am J Clin Dermatol. 2020;21(5):725-732.doi:10.1007 / s40257-020-00545-9, incorporated herein by reference). The PRO scales for eyebrows and eyelashes are scheduled to be administered at week 0 (first injection on day 1), week 6, week 12, week 18, week 24 (end of treatment (EOT), week 13 and final injection), week 26 (first follow-up visit after treatment), week 30, and week 36 (end of study or EOS). The evaluation questionnaires for the PRO scales for eyebrows and eyelashes are shown in the table above.

[0280] SkinDex 16-AA (Psychosocial Rating Scale) The Skindex-16 is a PRO tool designed to assess how much a skin condition burdens a patient's quality of life across three areas: symptoms, emotions, and function. The Skindex-16 is inherently general and can be used in patients regardless of their specific skin condition. The Skindex-16 for AA is adapted for use in adults with AA. Using a Likert scale from 0 (never) to 6 (always), patients assess how much their condition has bothered them in the past week. The Skindex-16 for AA provides a single score for each area (symptoms, emotions, and function), as well as an overall total score. The Skindex-16 for AA is scheduled at week 0 (first injection on day 1), week 6, week 12, week 18, week 24 (end of treatment (EOT), week 13 and last injection), week 26 (first follow-up visit after treatment), week 30, and week 36 (end of study or EOS). The Skindex-16 for AA is shown in Figure 3.

[0281] Period and frequency of collecting AE and SAE information An AE is defined as any adverse medical event in a clinical trial participant that is time-related to the use of the IP, regardless of whether it is considered to be related to the IP. Therefore, an AE may be any undesirable unintended sign (including abnormal clinical laboratory findings), symptom, or disease (new or worsening) that is time-related to the use of the IP.

[0282] Events that satisfy the definition of AE include the following: • Conditions newly detected or diagnosed after signing the ICF. This includes conditions that may have existed before the start of the examination but were not detected. • Conditions that worsened after signing the ICF, but were known to have existed before the start of the examination. • Signs, symptoms, or clinical sequelae suggesting a drug interaction • Signs, symptoms, or clinical sequelae suggesting an overdose of either the IP or a concomitant medication (overdose of the IP itself should not be reported as an AE). • Abnormal clinical laboratory results (blood tests, clinical chemistry tests, or urine tests) or other safety assessments (ECG, radiographs, vital signs measurements, etc.) that are considered clinically important in the medical and scientific judgment of the principal investigator (i.e., not related to the progression of the underlying disease or more severe than expected for the participant's condition). Lack of efficacy or failure of the expected pharmacological effect itself is not reported as an AE or SAE. Such cases are included in the efficacy assessment. However, signs, symptoms, and / or clinical sequelae resulting from a lack of efficacy are reported as an AE or SAE if they meet the definition of an AE or SAE.

[0283] Events that do not fit the definition of AE include the following: Unless the principal investigator determines that the abnormality of the clinical laboratory findings related to the underlying disease or other abnormalities in safety assessments is more severe than expected for the participant's condition, these abnormalities will not be considered. • The disease / disorder being studied, or the progression, signs, or symptoms of the disease / disorder being studied, unless it is more severe than expected for the participant's condition. • Medical or surgical procedures (endoscopy, cecal removal, etc.): Conditions leading to the procedure are adverse events (AEs). • Circumstances in which no undesirable medical events occurred (hospitalization for social and / or convenience reasons) • Daily fluctuations in pre-existing diseases or conditions present or detected at the start of the study that are not expected to worsen.

[0284] Although it may have started before the introduction of IP, medical occurrences that occur after informed consent has been obtained are recorded as medical history / current medical condition, not as adverse events (AEs).

[0285] A SAE is defined as any adverse medical event that meets one or more of the listed criteria at any dose. • To lead to death • Threatening life

[0286] In the definition of severity, the term "life-threatening" refers to an event in which there was a risk of death for the participant at the time of the event. This does not refer to an event that, if more severe, could have hypothetically caused death. • Requires hospitalization or extension of hospital stay Generally, hospitalization means that a participant is admitted to a hospital or emergency department for observation and / or treatment that would not be appropriate in a clinic or outpatient setting (usually involving at least an overnight stay). Complications that occur during hospitalization are AEs. This event is serious if the complication prolongs hospitalization or meets other serious criteria. If you are unsure whether hospitalization occurred or was necessary, consult your sponsor's medical monitor. Hospitalization for selective treatment of a pre-existing condition that did not worsen from baseline is not considered an adverse event (AE). • Causes persistent or significant damage / disability The term "disability" refers to a substantial disruption of the ability to perform normal daily living functions. □This definition is not intended to include experiences of relatively minor medical significance, such as uncomplicated headaches, nausea, vomiting, diarrhea, flu, and accidental trauma (e.g., a sprained ankle), that interfere with or may interfere with daily functioning but do not constitute substantial disruption. • It is a congenital abnormality / birth abnormality. Other situations: Medical or scientific judgment is required of the principal investigator when determining whether SAE reporting is appropriate in other circumstances, such as significant medical events that, while not immediately life-threatening, do not result in death or hospitalization, could endanger a participant or require medical or surgical intervention to prevent one of the other outcomes listed above. These events should generally be considered serious. Examples of such events include invasive or malignant cancer, emergency treatment or intensive care at home for allergic bronchospasm, blood disorders, seizures that do not result in hospitalization, or the occurrence of intervention dependence or abuse. If you are unsure whether an event qualifies as a significant medical event, consult your sponsor's medical monitor.

[0287] Adverse events of particular note AESI (serious or non-serious) is defined as an AE or SAE of scientific and medical concern specific to the sponsor's product or program, and may require ongoing monitoring and prompt communication from the principal investigator to the sponsor.

[0288] The following events are considered AESIs in this study and will be reported using the same process as AEs. • Injection site reaction (also recorded in the injection site reaction CRF). • Infections including viral reactivation infections. Lymphopenia CTCAE grade ≥3 (<500 μL).

[0289] biomarkers Blood samples will be collected to evaluate the PD properties of compound A, including, but not limited to, changes in immune cell subsets and the therapeutic effect of compound A on AA disease biomarkers (including serum CCL17 (TARC)). Immune cell subsets may include, but are not limited to, total T cells, helper T cells, cytotoxic T cells, total natural killer cells, and total B cells. Additional T cell subsets may be evaluated, including, but are not limited to, regulatory, naive, memory, and regulatory T cell subsets. Blood samples collected in this trial may be used to measure other biomarkers that may be identified in the future and to support the development and validation of biomarker assays relevant to this trial.

[0290] The following exploratory biomarker collection will be conducted: • Serum and plasma samples will be collected and stored for potential testing of exploratory markers related to the treatment response to compound A. Exploratory markers may include, but are not limited to, soluble IL-7Rα, chemokines, cytokines, and / or other markers of inflammation, compound A activity, or AA disease biomarkers. Samples will be analyzed at the discretion of the sponsor. To assess the potential inhibition of additional immune cell subsets associated with AA disease and / or immune function related to the ADX914 treatment response, such as IL-7 and TSLP-mediated effects, peripheral blood mononuclear cells will be isolated from whole blood samples and frozen for later testing. Samples will be tested at the sponsor's discretion. • Whole blood cell samples (i.e., PAXgene® tubes) are collected and stored for potential ribonucleic acid (RNA) expression profiling of the treatment response to compound A. The purpose of RNA collection is to potentially evaluate the effect of compound A on transcripts associated with the IL-7Rα pathway (e.g., immune cell signature, IL7 / TSLP pathway signature, etc.). RNA samples may be used for genome-wide or candidate gene expression studies. Samples are tested at the discretion of the sponsor.

[0291] Genetics Deoxyribonucleic acid (DNA) samples may be collected in whole blood cell form for exploratory studies related to AA disease activity and / or response to ADX 914, and may be analyzed at the sponsor's discretion.

[0292] Genetic polymorphisms in genes encoding drug targets or downstream pathways may affect the efficacy and safety of investigational drugs. In cases of unexplained abnormal reactions or observations of adverse events, DNA samples may be used to determine whether there is a pharmacogenetic link between the drug response and the observed reaction. In the future, as our understanding of the roles of the IL-7 and TSLP pathways in AA increases, additional genetic analysis may be required to refine our knowledge of the molecular basis of the disease and drug response and to advance the development of novel therapeutic agents. In particular, but not limited to, sequencing of the T cell receptor beta chain can identify the effect of compound A on T cell compartment clonality.

[0293] Genetic polymorphism testing is a one-time blood draw, as T-cell clonality testing requires pre- and post-treatment blood draws, which are listed in the activity schedule and governed by the primary ICF of the test. Furthermore, if permitted by local, regional, and national regulatory authorities and ethics committees, subjects are offered the option to retain residual DNA samples for future exploratory unspecified genetic research, which may be used to understand the biology of other diseases and traits of interest to the sponsor, as well as to develop diagnostic and analytical tests. Subjects who choose to retain samples for future unspecified use will need to sign a separate ICF.

[0294] Example 2: Stability of Compound A formulation Compound A (an antibody having the heavy-chain amino acid sequence of SEQ ID NO: 23 and the light-chain amino acid sequence of SEQ ID NO: 24) was formulated as a 100 mg / mL pharmaceutical product in 20 mM histidine, 260 mM sucrose, and 0.05% (w / v) polysorbate 80 at pH 6.0. Based on ongoing long-term stability studies, this formulation has a shelf life of approximately 24 months when stored in the absence of direct light at -20°C (±5°C).

[0295] Formulation characterization tests were performed on compound A bulk active pharmaceutical ingredient (BDS) excipients, as well as formulations in 2R glass vials, including those exhibiting photostability, thermal resistance, agitation, and freeze-thaw cycles. The tests were conducted in 2R glass vials to generate formulation data using a pharmaceutical container closure system.

[0296] The photostability of compound A was evaluated at pH 6.0 and room temperature for up to 5 days in 10 and 60 mg / mL 2R glass vials in BDS formulations of 20 mM histidine, 260 mM sucrose, 0.05% (w / v) polysorbate 80, and 0.05 mM pentetate. Compared to samples kept at room temperature away from direct light, the appearance (A280, SE-HPLC, cIEF, pH, and number of particles invisible to the naked eye) and protein concentration were similar, indicating that compound A BDS at these concentrations and formulations is stable under these photostress conditions.

[0297] The buffer histidine and excipient sucrose were shown to have good steric stability in the pH range of 5.5–6.5 compared to other buffers such as citrate, phosphate, and excipient arginine and sodium chloride. Steric stability was evaluated using differential scanning fluorescence to assess unfolding temperature and dynamic light scattering, and to determine the hydrodynamic radius.

[0298] The addition of polysorbate 80 as a stabilizing surfactant in compound A BDS was evaluated by stirring and freeze-thaw tests at a protein concentration of 60 mg / mL. This study showed that stirring stress for up to one week had no observable effect on aggregation or a significant increase in microscopic particles in both formulations with and without polysorbate 80. Up to five freeze-thaw cycles at -80°C of compound A BDS at 60 mg / mL were also evaluated in formulations with and without polysorbate 80. In the freeze-thaw test, an increase in microscopic particles, as measured by microflow imaging (MFI), was observed in the formulation without polysorbate 80. This indicates that polysorbate 80 is an important excipient for the freeze-stabilization of compound A.

[0299] Development stability tests of compound A in BDS formulations were conducted over 12 weeks at pH 5.5, 6.0, 6.5, 10, and 60 mg / mL at 2–8°C, 25°C, and 40°C. Trend analysis was performed on pH, SE-HPLC, cIEF, CE-SDS, and particle analysis based on the number of MFI particles in the samples. The data indicate that the samples showed similar trends across all stability conditions.

[0300] The formulation of compound A at 100 mg / mL rBDS was determined through various screening studies. This formulation development was intended to explore the design space for a compound A 70 mg / mL BDS formulation without introducing significant changes to pH and buffering systems. Therefore, histidine buffer at pH 6.0 was tested with several combinations of excipients targeting a protein concentration of 100 mg / mL. Combinations of formulations tested included sucrose, trehalose, mannitol, sorbitol, sodium chloride, and arginine. Pentetic acid, a chelating agent that was part of the BDS formulation, was also evaluated and compared with ethylenediaminetetraacetic acid (EDTA). Polysorbate 80 was included in each formulation combination. Based on the results of the initial studies, it was determined that histidine buffer containing sucrose / pentetic acid in polysorbate 80 has the ability to stabilize compound A at 100 mg / mL.

[0301] The combination of formulations and the role of pentetic acid were further evaluated. Accelerated stability tests were conducted comparing 100 mg / mL formulations of compound A with and without pentetic acid at 40°C for 14 days and at 50°C for 7 days. The data from these studies are shown in Tables 2-1, 2-2, 2-3, and 2-4 below. [Table 6-1] [Table 6-2] [Table 7-1] [Table 7-2] [Table 8] [Table 9-1] [Table 9-2]

[0302] Due to instrument availability, the CEX method was used in the accelerated stability test instead of the cIEF method used in the long-term stability test. The number of assays was also reduced, focusing on the assay expected to show the most stability. For the accelerated stability test, protein concentration by A280 and efficacy by ELISA binding were not monitored, although these tests are performed for all time points and conditions during the long-term stability test. In addition, particle size by dynamic light scattering (DLS) and polydispersity index (PDI) were monitored for the accelerated stability test because growth in particle size and / or PDI can indicate protein aggregation, especially under accelerated stability conditions. Particle size by DLS and PDI is not part of the shipment and stability test panel for the long-term stability test.

[0303] The presence of pentetic acid had no positive or negative effect on stability in accelerated stability tests at 50°C for 7 days and at 40°C for 14 days. Furthermore, increasing the concentration of compound A from 70 mg / mL to 100 mg / mL did not affect accelerated stability over the same period at 50°C for 7 days and at 40°C for 14 days.

[0304] A freeze-thaw stability test was also conducted comparing compound A 25 mg / mL formulations containing and without pentetic acid over up to five freeze-thaw cycles. Appearance and purity tests were evaluated using SEC. The data from these studies are presented below.

[0305] Tables 2-5 and 2-6. [Table 10] [Table 11]

[0306] No instability was observed across all freeze-thaw cycles for both the pentetate-containing and pentetate-free formulations. Both 125 mg / mL formulations were considered stable for up to five freeze-thaw cycles.

[0307] Based on the results of the formulation process development, pentetic acid was determined to be optional in the formulation. The formulation of compound A 100 mg / mL rBDS was determined to consist of 20 mM histidine, 260 mM sucrose, and 0.05% polysorbate 80 at pH 6.0. The concentrated 100 mg / mL rBDS without chelating agents is expected to be stable at the intended storage temperature of -20 ± 5°C for the intended shelf life.

[0308] Furthermore, clinical suitability testing was conducted during development to verify the stability of the compound A drug over a range of potential doses over 4 hours at ambient temperature without light exposure under clinical conditions (polypropylene syringe and stainless steel needle). The drug was demonstrated to be compatible and stable with the administration material over 4 hours by binding efficacy against antibody purity, protein concentration, and enzyme-linked immunosorbent assay (ELISA) analysis using size exclusion high-performance liquid chromatography.

[0309] Example 3: Bonding properties of compound A SPR analysis of the binding of compound A precursor to FcγR Fcγ receptors are found on hematopoietic cells and mediate antibody-antigen-induced effector functions. These include antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell phagocytosis. Binding of the specific invariant region of IgG1 to FcγR expressed on immune cells mediates the effector function of antigen-antibody complexes.

[0310] The precursor of compound A (C1GM / 2hIgG1) was tested for its binding to human and monkey Fcγ receptors (types I, II, and III) by surface plasma resonance (SPR). Screened FcγRs included FcγRI (hCD64 and cynoCD64), FcγRII (hCD32a-H131 and hCD32a-R131, hCD32b, cynoCD32a, and cynoCD32b), and FcγRIII (hCD16a-V158, hCD16b-NA2, and cynoCD16) subtypes. The C1GM / 2hIgG1 antibody showed expected binding profiles for human and cynomolgus monkey FcγRI, FcγRII, and FcγRIII (data not shown).

[0311] Absence of binding of compound A to cynomolgus monkey and human Fcγ receptors Three amino acid substitutions were manipulated in the Fc chain of the precursor CICM / 2hG1 to attenuate effector function and lead to compound A. The mutations include a leucine-to-alanine substitution at position 234 (L234A), a leucine-to-glutamic acid substitution at position 235 (L235E), and a glycine-to-alanine substitution at position 237 (G237A). Compound A was expressed in HEK293 and CHO cells.

[0312] Compound A (1 μM) was purified from cell supernatant and its binding to human and cynomolgus monkey FcγR was evaluated by SPR. The FcγRs screened included the subtypes FcγRI (hCD64 and cynoCD64), FcγRII (hCD32a~H131, hCD32a-R131, hCD32b, cynoCD32a, and cynoCD32b), and FcγRIII (hCD16a~V158, hCD16b-NA2, and cynoCD16).

[0313] The binding of compound A to all FcγRs was below the undetectable limit when tested at high concentrations (1 μM) (data not shown). In contrast, the IgG1-positive control antibody showed the expected high degree of binding to human FcγRIs, with moderate binding to both FcγRII and FcγRIII (data not shown).

[0314] In summary, these data confirm that the combined substitution of compounds L234A, L235E, and G237A effectively minimizes potential effector functions.

[0315] Lack of binding to C1q by compound A C1q binding to the Fc domain of IgG1 antibody can mediate complement-mediated cytolysis. The ability of compound A to bind to C1q was evaluated using an ELISA assay. Briefly, microtiter plates were coated with human IL-7Rα, and compound A from two different lots was added to the coated plates at various concentrations, followed by the addition of a saturated concentration of C1q purified from human serum. Binding C1q was detected with sheep anti-human C1q antibody. Human IgG1 was included as a positive control. While the positive control IgG1 showed the expected affinity for C1q, neither lot of compound A showed measurable binding to C1q at concentrations up to 1000 ng / mL (6.7 nM).

[0316] This data indicates that compound A can avoid C1q-mediated complement-mediated cytolysis when administered to humans.

[0317] Binding properties of compound A to human and cynomolgus monkey FcRn at pH 6.0 and 7.4 Neonatal Fc receptors (FcRn), expressed on endothelial cells and bone marrow-derived cells, extend the half-life of IgG by facilitating its transport from epithelial cell endosomes (pH 6.0), which would otherwise be degraded and returned to the bloodstream as functional immunoglobulins (pH 7.4). Endosomal recycling is facilitated by a pH shift in the IgG-FcRn interaction (tight binding at pH 6.0 and low binding / high dissociation at pH 7.4).

[0318] The binding of compound A to a panel of neonatal Fc receptors (hFcRn and cFcRn, respectively) in humans and cynomolgus monkeys was evaluated using a single-cycle Biacore analysis. The Kd values ​​of compound A binding to human and cynomolgus monkey FcRn were 686 and 866 nM, respectively, at pH 6.0. Compound A did not bind to FcRn from either humans or monkeys at pH 7.4. This pH-dependent shift in antigen affinity facilitates the release of complexed compound A from lysosomes after lysosomal degradation. Unrelated human IgG1 and IgG4 wild-type antibodies were used as controls.

[0319] These findings suggest that endosomal recycling, which enhances the biostability / half-life of compound A in vivo, is expected.

[0320] SPR analysis of binding to human and cynomolgus monkey IL-7Rα at pH 6.0 and 7.4 The binding of compound A to purified recombinant human and cynomolgus monkey IL-7Rα via SPR was evaluated. The Kd of compound A to human IL-7Rα was 1.3 nM, and the Kd of compound A to cynomolgus monkey IL-7Rα was 1.7 nM at pH 7.4. The binding affinity of compound A to both human and cynomolgus monkey IL-7Rα was slightly less than 4 times at pH 6.0, with Kd values ​​of 5.3 nM for human IL-7Rα (hIL-7Rα) and 7.0 nM for cynomolgus monkey IL-7Rα (cIL-7Rα) (see Table 3 below). [Table 12]

[0321] These data indicate that compound A bound to human and cynomolgus monkey IL-7Rα with comparable high affinity at pH 6.0 and 7.4. For both species, the binding affinity was approximately four times higher at pH 7.4 compared to pH 6.0. This pH-dependent shift in antigen affinity may further facilitate the release of complexed compound A from lysosomes to avoid lysosomal degradation of compound A.

[0322] Example 4: In vitro evaluation of the efficacy of compound A Receptor occupancy and inhibition of IL-7-stimulated phosphorylation of STAT5 (pSTAT5) in CD4 T cells from healthy volunteers The effect of compound A on IL-7-induced pSTAT5 expression was investigated in healthy human volunteers using CD4 + Evaluation was performed in T cells (n=3). Whole blood of anticoagulants from healthy volunteers was incubated with increasing concentrations of compound A in the presence of recombinant human IL-7 (10 ng / mL). CD4 + The proportion of pSTAT5 in T cells, CD3+, CD4 + CD45RA + , and were measured by flow cytometry using a fluorescent probe for pSTAT5.

[0323] Compound A was obtained from naive CD4 from all three healthy volunteers (n=3). + It showed potent inhibition of pSTAT5 against T cells, and average IC 50 The values ​​ranged from 0.16 to 0.27 nM (23.7 to 40.4 ng / mL, respectively).

[0324] Inhibition of pSTAT5 in naive CD4+ and CD8 T cells from healthy volunteers. Compound A was evaluated for its target binding and inhibition of IL-7-induced IL-7R signaling on human T cells from normal, healthy volunteers. Freshly collected blood was incubated with IL-7 (100 ng / mL) ex vivo for 15 minutes, and pSTAT5 levels were measured by flow cytometry.

[0325] As shown in Table 4, compound A had an IC50 value in the range of 0.38 to 0.54 nM (corresponding to approximately STAT 57 to 81 ng / mL), and was found to be naive (CD45RA) from normal healthy volunteers. + ) and memory (CD45RA - It strongly inhibited IL-7-induced phosphorylation of STAT5 in T cells (both CD4 and CD8 phenotypes). [Table 13]

[0326] Compound A is an IL-7R antagonist antibody that does not possess partial agonist activity. To determine whether compound A possesses partial agonist ability, whole blood or purified peripheral blood monocytes (PBMCs) were collected from healthy volunteers (n=3). Compound A was incubated in each matrix at eight concentrations ranging from 1 to 100 nM. Signal transduction was evaluated by AlphaLISA® SureFire® Ultra® pSTAT5, and pSTAT5 was detected. A non-targeted IgG isotype was used as the negative control, and IL-7 (2 nM) was the positive control. IL-7 increased pSTAT5 levels by an average of 4.7-fold (±1.6) and 21.1-fold (±2.8) in whole blood and PBMCs, respectively. In both whole blood and PBMCs, no significant induction of pSTAT5 was observed at any concentration of compound A (1, 2, 3, 6, 13, 25, 50, or 100 nM), demonstrating that compound A is a complete antagonist at IL-7R and does not possess partial agonist activity. These data are shown in Table 5. [Table 14]

[0327] Compound A inhibits TSLP-induced TARC production by PBMCs. Thymus activation-regulating chemokines (TARC, also known as CCL17) are important chemokines released from monocytes and dendritic cells in response to thymic interstitial lymphapoietin (TSLP) stimulation. TARC is involved in dendritic cell maturation and IL-23 production, as well as CD4 + It drives T cell proliferation. TSLP is highly expressed in atopic dermatitis (AD) skin lesions. Signaling via IL-7Rα / TLSPR and IL-7Rα / CD132 has been demonstrated to produce additive inflammatory effects.

[0328] Compound A binds to the IL-7Rα subunit common to both the IL-7 receptor (IL-7Ra / CD132) and the TSLP receptor complex (IL-7Rα / TSLPR). Compound A inhibits TSLP-induced production of TARC from monocyte-rich PBMCs (in three independent assays using 1-2 donors) and has a mean IC50. 50 The concentration was 2.9 nM (428 ng / mL).

[0329] Therefore, in addition to blocking IL-7-induced signaling, compound A also inhibits TSLP-induced signaling at low nM concentrations.

[0330] Example 5: Evaluation of the efficacy of compound A in a humanized mouse model Efficacy of compound A in a humanized mouse graft-versus-host disease model Graft-versus-host disease (GvHD) is a systemic disease, and the skin is often one of the target organs.

[0331] Compound A is used in the humanized immune system of GvHD (NOD-SCID-IL2γr null[Hu-NSG] mice were evaluated in a prophylactic treatment in a humanized non-obese diabetic severe combined immunodeficient mouse model. NSG mice are immunodeficient due to the lack of mature mouse lymphocytes and natural killer (NK) cells that allow for efficient engraftment of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were intravenously (IV) injected into NSG mice, which engrafted and resulted in the development of a robust xenogeneic GvHD response, recapitulating many aspects of human disease.

[0332] The reference IL-7Rα antagonist antibody (A3312F, whose epitope residues appear to include all of the epitope residues of Compound A) was evaluated in two treatment regimens (prophylactic and therapeutic modes), and data from the prophylactic regimen are presented herein, where A3312F serves as the positive control for Compound A in this model of GvHD.

[0333] In the prophylactic treatment study, Compound A was administered subcutaneously (5 mg / kg) twice weekly for 3 weeks starting on day -1 (the day prior to adoptive PBMC transplantation) to NSG mice (n = 7 female), and showed robust inhibition of GvHD as indicated by decreased weight loss, inflammatory cytokine expression, and immune cell proliferation (Figures 4A - 4C). A3312F, tested in the same trial, showed efficacy equivalent to Compound A.

[0334] In another experiment, target engagement was evaluated by assessing the levels of pSTAT5 in cells from whole blood and spleen. Compound A treatment (0.2, 1, or 5 mg / kg 2× / week SC) was initiated 5 days after human PBMC transplantation into NSG mice. Whole blood and spleen were collected at 72 hours and 120 hours, respectively, after the fourth administration of Compound A. Cells were collected, stimulated with human IL-7 for 15 minutes, and pSTAT5 levels were determined by flow cytometry in human CD4 + and CD8 +Analysis was performed using T cells. Compound A showed complete target binding and inhibition of IL-7-induced pSTAT5 at treatments of 1 mg / kg and 5 mg / kg (Figures 5A-5B). This confirms that inhibition of IL-7Rα signaling by compound A is protective and can achieve complete target binding in a humanized mouse model of GvHD.

[0335] Several related experiments used a mouse surrogate antibody (SB14) to further evaluate the role of IL-7Rα inhibition in treating other disease indications in a mouse model of such disease indications (compound A was not used in these experiments in part because compound A does not cross-react with mouse IL-7Rα (data not shown)). Taken together, these additional in vivo studies demonstrate that short-term inhibition of IL-7Rα signaling provides a sustained and effective response, suggesting the potential of a low-dosage paradigm that maintains clinical efficacy. Furthermore, the importance of IL-7R signaling in driving T cell-mediated disease processes was demonstrated.

[0336] Interestingly, in at least one such disease model, CTLA4-Ig, a different inhibitor of T cell activation, did not demonstrate efficacy in that disease model using the same therapeutic strategy (data not shown), thus demonstrating a clear differentiation between anti-IL-7Rα mAb and CTLA4-Ig treatment and highlighting the potential for a durable, drug-free response with IL-7Rα mAb treatment.

[0337] Example 6: Pharmacokinetics / pharmacodynamics (PK / PD) in cynomolgus monkeys Single-dose intravenous pharmacokinetics and pharmacodynamics This study was conducted to provide data supporting the use of compound A in humans by comparing the PK and PD profiles of low-dose compound A administered to cynomolgus monkeys with those of a reference-positive control IL-7Rα antagonist antibody (A3312F). Compounds A and A3312F were administered by IV injection to groups of one or two monkeys per sex at doses of 0 (vehicle), 0.1 (low dose), 0.5 (medium dose), or 3.0 (high dose) mg / kg. All doses were administered at 1 mL / kg in a vehicle / carrier consisting of 20 mM histidine, 260 mM sucrose, 0.05 mM diethylenetriaminepentaacetic acid (DTPA), and 0.05% Tween 80, pH 6.0.

[0338] Criteria for PD evaluation included total IL-7R levels and soluble IL-7R levels in blood and plasma, inhibition of IL-7-induced pSTAT5, receptor occupancy (RO), and peripheral lymphocyte phenotypic analysis. Pharmacokinetic endpoints included AUC and C12. max , and T max This included, and the immunogenicity evaluation item included anti-drug antibodies (ADA).

[0339] On day 1, 100% RO was achieved 4 hours after administration and maintained for all doses of both compound A and A3312F from days 2 to 4. The vehicle control group did not consistently show RO throughout the study period. For the remainder of the study, RO decreased, with low-dose A3312F falling below 95% RO on day 4, while the other drug treatment groups maintained 100% RO. On day 7, RO in both low-dose groups fell to less than 50%. On day 10, RO for low-dose A3312F returned to vehicle control levels, with medium doses below 50% and high doses below 90%. Low-dose compound A also returned to vehicle control levels, with medium doses averaging less than 50% (with variability) and high doses remaining at 100% RO. On day 14, all A3312F dose groups returned to vehicle control levels (no RO), while both low and medium doses of compound A returned to vehicle levels, although the high dose of compound A maintained an RO of >50%. All doses of both compound A and A3312F were equivalent to vehicle control levels by day 17.

[0340] IL-7-induced pSTAT5 in cynomolgus monkeys was strongly inhibited by both compound A and A3312F. A low dose of 0.1 mg / kg showed significant inhibition of pSTAT5 (≥80%) at early time points (days 1-3), with compound A being slightly more effective than A3312F. An intermediate dose of compound A (0.5 mg / kg) was more effective than A3312F in maintaining complete inhibition of pSTAT5 until day 5, showing approximately four times higher inhibition at day 5. At a high dose of 3 mg / kg, compound A treatment maintained complete inhibition of pSTAT5 activity until day 8, while A3312F treatment showed less inhibition of pSTAT5 over the same period. Furthermore, compound A continued to substantially inhibit IL-7-induced pSTAT5 on day 11, while A3312F showed complete loss of this inhibitory activity.

[0341] Both mean total (blood) IL-7R concentrations and soluble (plasma) IL-7R concentrations, encompassing both free and bound IL-7R, increased after administration of compound A, with peak levels generally reaching 8–11 days. Mean soluble (plasma) IL-7R concentrations increased more significantly compared to total (blood) IL-7R levels. Cell surface IL-7R levels, measured using flow cytometry, did not change significantly after administration of compound A or A3312F, suggesting that reduced removal of the soluble IL-7R-compound A complex is responsible for the increase in total IL-7R levels in the blood.

[0342] On day 8, both compound A and A3312F-treated animals showed a decreasing trend in peripheral lymphocytes compared to baseline, which was most pronounced at the highest dose (3 mg / kg). However, no clear differences in peripheral lymphocyte subsets were observed for either compound A or A3312F during the study. Nevertheless, while there was variability in lymphocyte populations among animals in the same group, it is suggested that this variability may be attributable to stress (i.e., handling of monkeys, etc.) and the small sample size (n=3) of the study per dose group.

[0343] Exposure to compound A was evaluated after a single IV dose of ≤3 mg / kg and compared to A3312F. The mean exposure (AUC) of 3 mg / kg in the first week before anti-drug antibody (ADA) formation was expressed as the mean AUC from 0 to infinity. inf ) ≤ 287 μg*day / mL and AUC inf The ADA formation was higher for compound A (≤135 μg / day / mL) compared to A3312F (see Table 6). ADA formation for both compound A and A3312F was accompanied by a simultaneous enhanced decrease in serum concentration, RO, and pSTAT5 inhibition. [Table 15]

[0344] ADA was detected in all monkeys administered with compound A. ADA was detected by day 11 in 3 out of 3 monkeys administered with 0.1, 0.5, and 3 mg / kg of A3312F, respectively, and in 2 out of 3 monkeys. The presence of ADA resulted in generally lower serum concentrations of compound A and A3312F, as well as lower systemic exposure to individual compounds A and A3312F, in all monkeys at day 11 (240 hours post-administration).

[0345] In summary, compounds A and A3312F were well-tolerated in monkeys after a single IV dose of up to 3 mg / kg. No drug-related clinical findings or effects on body weight were observed during the study. A good correlation was observed between inhibited pSTAT5 and serum concentrations of anti-human IL-7R antibody in the CD4+CD45RA+ T cell population. A return of over 95% resulted in 90% inhibition of pSTAT5. Inhibition of pSTAT5 levels, a proximal biomarker of IL-7R signaling, by compound A was similar to that by A3312F, but more prolonged, suggesting that compound A shows improved PK compared to A3312F, but with similar in vivo potency. No significant changes in lymphocyte phenotype were observed for either compound A or A3312F after a single dose. Significant exposure and assessment of toxicity endpoints were achieved despite the influence of ADA on compound A concentrations.

[0346] Single-dose subcutaneous pharmacokinetics and pharmacodynamics Systemic toxicity profiles, tolerability, RO, and PD changes as measures of target binding were assessed for the determined immune response to subcutaneous (SC) administration of compound A at doses of 0 (vehicle), 3, and 30 mg / kg to groups of two male and two female cynomolgus monkeys. Criteria for PD assessment included IL-7-induced pSTAT5, IL-7 RO, peripheral blood lymphocyte phenotyping (T cells, B cells, and NK cells [T / B / NK]), T cell-dependent antibody response (TDAR) to keyhole limpet hemosinian (KLH) immunization, and assessment of total IL-7R levels and soluble IL-7R levels.

[0347] In the 3 mg / kg SC administration group, RO was 100% until day 8, 98% on day 11, 16% on day 15, 12% on day 22, and below the limit of quantitation (BLQ) on day 36. In the 30 mg / kg SC administration group, RO was 100% until day 11, 70% on day 15, and at the BLQ on day 22.

[0348] Inhibition of IL-7-induced pSTAT5 in CD3+ T cells was observed at 4 hours after dosing (mean inhibition rate 92% - 99%) in all dose groups, indicating significant PD activity. In the 3 mg / kg SC group, the mean % of pSTAT5 levels was 100% until day 8, 79% on day 11, and returned to baseline levels on day 22. In the 30 mg / kg SC administration group, the % of pSTAT5 inhibition was 100% until day 11, 30% on day 15, and returned to baseline on day 22. Inhibition of pSTAT5 generally correlated with the extent of IL-7 RO and compound A systemic exposure reduced by the presence of ADA in all monkeys by day 11.

[0349] Average exposure (AUC 0~336h and C max ) generally increased approximately dose-proportionally with SC injection of 3 - 30 mg / kg. No substantial gender differences were observed. Systemic exposure after a single SC dose of 3 mg / kg was equivalent to that after a single IV dose of 3 mg / kg, and the bioavailability was 78% calculated from the data of the first week.

[0350] Overall, compound A had a single SC dose ≤ 30 mg / kg (AUC 0-336hThe compound A was well-tolerated in monkeys after doses of ≤61.314 μg*h / mL. Compound A-related PD activity was observed at all doses, as indicated by inhibition of IL-7-induced pSTAT5, and generally correlated with IL-7 RO and compound A exposure, which decreased by day 11 in all monkeys due to the presence of ADA. The decrease in lymphocyte subpopulation observed early on day 8 was consistent with hematological findings of lymphopenia (≥3 mg / kg SC). Despite the effect of ADA on the concentration of compound A, significant exposure and assessment of toxicity endpoints were achieved.

[0351] A 6-week intermittent dose (QW) SC toxicity study with an 8-week recovery period. Compound A was administered once weekly via SC to groups of 5 monkeys per animal at doses of 0 (vehicle), 2, 10, or 50 mg / kg. Scheduled necropsies were performed after 6 weeks of administration (7 doses) (3 per sex / group) and after an 8-week recovery period (2 per sex / group).

[0352] At all doses, near-complete inhibition (>95%) of IL-7-induced pSTAT5 in CD3+ T cells (PD activity) and near-complete inhibition (≧98%) of IL-7 RO in CD3+ T cells (target binding) were observed 4 hours after administration from day 1 to day 8. Recovery of pSTAT5 and loss of IL-7 RO were observed on day 22 and correlated with reduced systemic exposure to compound A, with the presence of higher levels of ADA. Overall, target involvement and PD activity correlated with sustained systemic exposure in monkeys with minimal or no ADA response.

[0353] At all doses, there was a decrease in total T cells (0.51 times lower than the mean of the vehicle control group), helper T cells (CD4+, 0.51 times lower), and cytotoxic T cells (CD8+, 0.48 times lower) on day 8, consistent with the hematological findings of a decrease in total lymphocyte count on day 8, but no decrease in B cells or NK cells. Due to the reduction in systemic exposure to compound A as a result of ADA, T cell subset numbers returned to baseline levels by day 22, except in the highest dose group of 50 mg / kg where drug levels remained in circulation. In the 50 mg / kg dose group, the decrease in T cell subsets persisted until the end of the recovery period (days 97 / 99), consistent with the drug remaining in circulation.

[0354] Following doses up to 50 mg / kg, no compound A-related changes were observed in qualitative and quantitative electrocardiogram assessments. Additional tests did not show compound A-related changes in neurological endpoints, respiratory rate, body temperature, lung sounds and mucous membrane color assessment, or determination of arterial oxygen saturation.

[0355] Overall, compound A was well-tolerated in monkeys at once-weekly doses of 2, 10, or 50 mg / kg in saturated steroids (SCs) for 6 weeks. The toxicological profile of SCs was approximately dose-proportional on day 1 across the dose range tested, but thereafter affected by the development of ADA in most animals. Animals at higher doses maintained but variable exposure, allowing for meaningful interpretation of toxicological endpoints and the definition of NOAELs. At all doses, PD activity (inhibition of IL-7-induced pSTAT5) generally correlated with IL-7 RO (target binding) and compound A exposure, and decreased due to high levels of ADA (more pronounced at lower doses).

[0356] A 3-month repeated-dose toxicity study of compound A by subcutaneous injection with a 6-month recovery period. Compound A was administered once weekly via SC to groups of 5 monkeys per sex at doses of 0 (vehicle), 10, 50, and 150 mg / kg.

[0357] During the first week of ECG evaluation, seven ventricular premature contractions (VPCs) were observed at 10-second intervals in one male monkey in the high-dose group (150 mg / kg QW). The relationship between VPCs and compound A is unclear for the following reasons: In this study, VPCs were observed in only one of 30 animals; VPCs may be an incidental finding in naive cynomolgus monkeys (approximately 8% of animals that underwent 1-minute ECGs at 2-week intervals); spontaneous occurrence over time can be highly variable when evaluated over 24 hours; and their occurrence can be sporadic. Furthermore, there were no histological correlations or changes in the QT / QTc interval to predispose this animal to ventricular arrhythmias. The presence of VPCs in this animal was not associated with any clinical signs, and therefore the arrhythmia was not considered harmful.

[0358] Overall, administration of compound A to cynomolgus monkeys by weekly subcutaneous injection for 3 months was well tolerated at doses of 10, 50, and 150 mg / kg. A return on oxygen (RO) of >95% was maintained throughout the 3-month administration phase at 50 mg / kg (7 / 10 animals) and 150 mg / kg (10 / 10 animals). Losses of >95% RO at 10 mg / kg (9 / 10 animals) and 50 mg / kg (3 / 10 animals) were observed on day 29 or day 57 and correlated with detectable ADA. Declinations in CD4+ and CD8+ T cell populations were observed, with compound A ≥50 mg / kg doses having the greatest impact on naive and CM T cells. These declines were related to the pharmacological effects of compound A and generally correlated with the observed RO. Total T lymphocyte count, T cell subsets, and B cells partially or completely recovered to baseline in males, while the decrease in T lymphocyte count, T cell subsets, and B cells remained with minimal or no recovery in female monkeys, which generally correlates with the observed durability of the RO. There was no histological correlation with lymphocyte decrease at the end of the recovery period.

[0359] Exposure to compound A remained at ≥60.800 μg*h / mL throughout the treatment phase in 7 out of 10 animals at 50 mg / kg and 10 out of 10 animals at 150 mg / kg, despite detectable ADA. Based on the non-adverse effects of compound A-related pathological changes at the end of the treatment period, the no-observed-adverse-effect level (NOAEL) was considered to be 150 mg / kg, the highest dose tested. At the phase NOAEL, mean C max The combined AUC values ​​for sex were 3570 μg / mL and 314.000 μg*h / mL, respectively.

[0360] A 6-month study of compound A administered by subcutaneous injection in sexually mature and immature cynomolgus monkeys with an 8-month recovery period (ongoing). Compound A was administered weekly via SC to groups of sexually mature and immature monkeys at doses of 0 (vehicle), 25, 50, and 150 mg / kg. Scheduled necropsies were performed after 6 months of administration (27 doses) (2-4 monkeys / sex / group) and after an 8-month recovery period (1-2 monkeys / sex / group). All doses were administered at 1 mL / kg in a vehicle / carrier consisting of 20 mM histidine, 260 mM sucrose, and 0.05% PS80, pH 6, which is consistent with the clinical formulation.

[0361] At the administration stage, low signaling of anti-compound A antibodies (less than 10,000 of the mean signal response) was detected in 10 out of 12 control animals observed in the 3-month GLP study. Similarly, the cross-linking method used to detect the presence of ADA in serum was determined to be able to detect ADA against all parts of the compound A molecule, including the human IgG1 Fc region and complementarity-determining region. No adverse clinical signs were observed in the 10 control animals with detectable ADA. Furthermore, compound A exposure was not detected in the control animals. Therefore, the reason for the detection of ADA in the control animals was determined to be unlikely to be related to compound A exposure, but likely due to a general reactivity to IgG of unknown etiology.

[0362] In the thymus, decreased lymphocyte solidity was observed in the thymus of sexually mature males administered ≥25 mg / kg (mild to moderate), and in sexually mature and immature females and women administered ≥50 mg / kg (moderate to significant), correlated with decreased thymic weight (absolute and relative to body weight and brain weight), macroscopically observed smaller thymic size, and decreased lymphocyte count. The decrease in lymphocyte solidity associated with compound A and the associated hematological effects observed in the thymus during the administration phase of the study were considered non-harmful due to the low incidence and severity of the changes. There were no significant differences between the findings noted in sexually immature and sexually mature animals. To date, no effects on the genitals have been observed in general toxicity studies.

[0363] Systemic exposure to compound A was largely independent of sex or sexual maturity. At 25 mg / kg, differences in exposure were observed, which are likely due to the presence of ADA. Systemic exposure (AUC) 0-t and C max The values ​​generally increased approximately proportionally with increasing dose, and the mean accumulation ratio ranged from approximately 2 to 5 times from day 1 to the end of the administration phase. Exposure to compound A was maintained at ≥50 mg / kg and ≥141.000 μg*h / mL on day 92 in 22 out of 28 animals throughout the administration phase, despite detectable ADA.

[0364] In summary, weekly SC administration of compound A to sexually mature and non-sexually mature cynomolgus monkeys for 6 months at dose levels of 25, 50, and 150 mg / kg was well tolerated. The NOAEL was considered to be 150 mg / kg / week. The NOAEL was the mean C of sexually mature animals (matched for sex). max and AUC 0-tThe values ​​were 3260 μg / mL and 466.125 μg*h / mL, and the mean Cmax and AUC0-t values ​​in sexually mature animals (matched for sex) were 3780 μg / mL and 540.200 μg*h / mL, respectively, 6 months after weekly SC administration. This is the dosing interval (AUC0-t) of the proposed dose of 200 mg of compound A in the Phase II trial. tau,ss This corresponds to a value >50 times greater than the predicted steady-state Cmax (Cmax,ss) and steady-state AUC over the specified period. The preliminary data reported herein are considered sufficient to support the dosing frequency, duration, and dosing regimen in the Phase II trial.

[0365] Example 7 Phase I, double-blind, placebo-controlled, single-dose and multiple-dose escalation studies to evaluate the safety, pharmacokinetic (PK), and PD of compound A after SC administration in healthy subjects. The examples demonstrate that compound A is safe and well-tolerated in healthy human subjects (men and women aged 18–50 years). Furthermore, PK studies based on data collected in these healthy human subjects show that compound A can achieve a high receptor occupancy (RO) with respect to the IL-7R receptor at relatively low doses. Additional analyses revealed that administration of compound A in healthy human subjects resulted in a minimal ADA host response, due to the low immunogenicity of compound A.

[0366] For the Part 1 SAD (single escalating dose) trial, the protocol defined up to six cohorts of eight subjects per cohort. In each cohort, subjects were randomly assigned in a 3:1 ratio to receive either compound A or the corresponding placebo. SAD cohort 6 was optional and was not utilized. At least seven subjects evaluable for safety were required for each cohort to escalate to subsequent cohorts. The five SAD cohorts administered in the trial received doses of 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, 2 mg / kg, and 4 mg / kg.

[0367] For Part 2, the MAD (Multiple Dose Escalation) trial, the protocol defined up to three cohorts of eight participants per cohort. Within each cohort, participants were randomly assigned in a 3:1 ratio to receive either compound A or the corresponding placebo. Ultimately, MAD cohorts 2 and 3 were not enrolled due to the discontinuation decision, as the study was being conducted due to the COVID-19 case burden and operational concerns regarding COVID-19.

[0368] Overall, PD analysis showed that compound treatment at doses achieving ≥95% RO also demonstrated >90% inhibition of IL-7 signaling via STAT5 phosphorylation (Figure 6A-6B). A single dose of compound A administered at ≥2 mg / kg showed sustained complete RO for at least two weeks. Repeated doses of compound A at 1 mg / kg showed sustained saturation of IL-7Rα after the second dose (Figure 6A). Furthermore, assessments of lymphocyte subsets and T cell immune function showed moderate, dose-dependent effects on these biomarkers, consistent with the expected pharmacology when compound A completely occupies IL-7Rα. Attenuation of T-dependent antibody responses to novel antigens was observed at dose levels where complete RO was maintained over the evaluation period, while the effect of compound A treatment on delayed-type hypersensitivity (DTH) responses to recall antigens was more variable.

[0369] The presence of ADA against compound A in human serum samples from Phase I treated subjects was measured using a validated assay with a three-layer approach (screening, confirmation, and titer). Generally, there was an increasing incidence of positive ADA over time in all cohorts after single and multiple SC administrations of compound A. Most ADA-positive samples had low titers close to the assay's sensitivity threshold, but showed an increase in ADA titer with time and dose levels. Based on surrogate antibody-positive controls for the ADA assay, a titer ≤80 ng / mL reflects detectable ADA below the US FDA recommended susceptibility guideline of 100 ng / mL. Of those with ADA levels below this 100 ng / mL threshold, only 8 out of 34 subjects (23.5%) had a titer >80 ng / mL during the observation period.

[0370] In the context of repeated administration of compound A in MAD Cohort 1, all six subjects treated with the active agent developed a therapeutically occurring ADA response with increased titers of 40, 80, or 320 by day 85, but a decrease in ADA titer was subsequently observed when the end of the study (day 127) was reached.

[0371] Given its ability to detect low-titer antibodies in the presence of circulating drugs, the assay can detect both clinically relevant and unrelated anti-compound A antibodies. The effects of ADA on compound A exposure and PD activity were also evaluated.

[0372] Where evaluable, no effect of ADA on PK or RO was observed, as the PK and RO profiles expressed under treatment in ADA-positive subjects were within the range of the observed PK profile in ADA-negative subjects. Where the effect of ADA on the PK profile compared to ADA-negative subjects could not be evaluated, concentrations were generally within the expected exposure range, and no accelerated clearance was observed.

[0373] Across the entire cohort, there was no association between the timing of positivity and the observation of the PK profile. Many ADA-positive samples developed under treatment after day 50, when compound A concentration was BLQ.

[0374] Similarly, there was no apparent effect on RO, and the low titer of ADA observed in the study was demonstrated to be unlikely to be clinically relevant. No ADA-related adverse events were observed in this study. Immunogenicity and its potential clinical impact will continue to be evaluated throughout the clinical development of compound A.

[0375] In the Part 1 trial (SAD), 85.7% of subjects receiving compound 1 and 70% of subjects receiving placebo reported treatment-induced adverse events (TEAEs), but all TEAEs were mild or moderate. 57.1% of subjects receiving compound A and 30.0% of subjects receiving placebo reported at least one injection site reaction (ISR) event, none of which were reported as severe. At the highest doses (2 mg / kg and 4 mg / kg), C max A temporary decrease in lymphocyte count was observed after treatment with compound A, primarily around the third day. None of the lymphocyte-reducing events were considered clinically significant.

[0376] In the Part 2 trial (MAD), similarly, all TEAEs were mild or moderate, and no ISRs were reported as severe. A decrease in absolute lymphocyte count was observed after compound A treatment, but none of the lymphocyte count events were considered clinically significant. The pre-specified protocol discontinuation criteria were <0.5 × 10⁶. 9 The total lymphocyte count for / L was not observed in any of the subjects during the study. There were no cases of lymphopenia as defined by CTCAE guidance.

[0377] Overall, the Phase I data indicate that doses up to 4 mg / kg were well-tolerated in healthy human volunteers, and there were no observable pharmacological or safety effects from ADA. We observed that doses of less than 3 mg / kg of SC achieved complete RO, and multiple administrations of compound A at 1 mg / kg SC every two weeks also achieved complete RO and pSTAT5 inhibition. The pre-specified protocol discontinuation criteria of <0.5 × 10⁻⁶ were met. 9 The total lymphocyte count for / L was not observed in any of the study subjects during the trial. There were no cases of lymphopenia as defined by the Common Terminology Criteria for Adverse Events (CTCAE) guidance.

[0378] Example 8: Preliminary Phase II clinical data in humans Evaluation of antibody serum concentration and receptor occupancy (RO) with compound A in AD patients. Serum PK and blood IL-7Rα RO of compound A were evaluated in subjects who received subcutaneous doses of 2 mg / kg (N=5) or 3 mg / kg (N=5). The intermediate cutoff date for this analysis resulted in data being included for at least 98 days (14 weeks) or 14 days (2 weeks) after PK administration. The analysis was performed by an external, open-label pharmacologist, and the results were re-blinded for the sponsor. To maintain blinding, time points or groups with N<2 were not included in the analysis. Nominal doses, as well as nominal times of administration and sampling, were used in the analysis.

[0379] The preliminary mean serum concentration versus time profiles of free compound A after 2 and 3 mg / kg SC administration every two weeks are shown in Figure 7A. Following SC administration of 2 or 3 mg / kg of compound A, a clear dose-dependent exposure was observed over the first two weeks post-administration, with trough concentrations (pre-administration) of 5.8 and 7.76 μg / mL, respectively, at day 15. With continued administration of 2 mg / kg every two weeks, 60%–80% of patients maintained trough concentrations above the target therapeutic concentration of 5 μg / mL. Trough serum concentrations generally showed high variability, with CV% ranging from approximately 40% to 90%.

[0380] The preliminary RO (mean ± standard deviation [SD]) versus time profiles after administration of compound A SC Q2W at 2 mg / kg and 3 mg / kg are shown in Figure 7B. The primary mean IL-7Rα RO on circulating CD3+ T cells reached full saturation (i.e., >90% RO) by day 3 after doses of compound A at 2 or 3 mg / kg. With repeated subcutaneous administration of compound A Q2W at 2 mg / kg, 100% of patients maintained full RO at all time points, with the exception of one outlier at day 43. After administration of compound A SC Q2W at 3 mg / kg, 100% of patients maintained full RO above 90% until day 15 of the study.

[0381] Compound A has an acceptable safety profile in subjects with AD at a maximum dose of 3 mg / kg every two weeks. All adverse events (AEs) were assessed as mild or moderate, and no severe AEs were reported. Consistent with the mechanism of action, a decrease in lymphocyte count to <1000 / μL was observed in 5 subjects across both cohorts. The lowest lymphocyte count observed was 750 / μL, and no adverse follow-on cells were reported. Subjects' lymphocyte counts returned to normal levels upon retesting approximately one week later, and no further decrease below normal levels was observed. No dose-dependent decrease in lymphocyte count was observed. The decrease in lymphocyte count was generally transient and trended back to normal levels at follow-up visits. No lymphopenia or associated AEs or infections of CTCAE grade ≥3 were observed. Administration was not delayed because the subject's TEAE or absolute lymphocyte count (ALC) was <800 / μL (as per protocol). No other clinically significant findings were observed in the clinical laboratory values ​​associated with compound A.

[0382] In summary, both the 2 mg / kg and 3 mg / kg dose groups maintained a target PK threshold >5 μg / mL (the doses were expected to achieve complete target binding in tissues). Maximum RO (>90%) was achieved in all subjects in both dose groups by day 3 and maintained throughout the entire treatment period. The safety profile was acceptable for both doses, and notably, no lymphopenia-related adverse events, including viral infections, were observed.

[0383] Based on these preliminary data, a 200 mg (approximately 2.7 mg / kg) dose of SC Q2W is expected to result in a steady-state serum trough concentration >5 μg / mL and maximum RO on circulating T cells in >90% of subjects throughout the administration, while causing little to no lymphopenia. In patients with AA, 200 mg of C after 0 mg of Q2W SC administration is expected. max,ss and AUCtau,ss The predicted median values ​​were >100 times and >50 times lower, respectively, than exposure to 150 mg / kg / week in cynomolgus monkeys from a 6-month GLP toxicity study (Example 6).

[0384] Further embodiments can be provided by combining the various embodiments described above. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the application data sheets are incorporated herein by reference in their entirety. The aspects of the embodiments may be modified as necessary to adopt concepts from various patents, applications, and publications in order to provide further embodiments.

[0385] These and other modifications may be made to embodiments in consideration of the above description. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed herein and herein, but rather as encompassing all possible embodiments, along with the entire scope of equivalents to which such claims are entitled. Thus, the claims are not limited by this disclosure.

Claims

1. A method for treating alopecia areata (AA) or reducing hair loss associated with AA in a mammal (e.g., human) subject that requires it, comprising administering an effective amount of a composition containing an antibody against IL-7Rα, A method wherein the antibody comprises (a) a heavy chain variable region (HCVR) including the VH CDR1 sequence of SEQ ID NO: 1 (DHAMH, one of SEQ ID NOs from 7 to 22, etc.), the VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and the VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) including the VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), the VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and the VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT), thereby treating AA and / or reducing alopecia in mammalian subjects.

2. A method for inducing hair growth in a mammal (e.g., human) that requires it, the method comprising administering an effective amount of a composition containing an antibody against IL-7Rα, wherein the antibody (a) A heavy chain variable region (HCVR) including VH CDR1 containing the sequence of SEQ ID NO: 1 (such as one of SEQ ID NOs from 7 to 22), the VH CDR2 sequence of SEQ ID NO: 2, and the VH CDR3 sequence of SEQ ID NO: 3, and (b) A light chain variable region (LCVR) comprising the VL CDR1 sequence of SEQ ID NO: 4, the VL CDR2 sequence of SEQ ID NO: 5, and the VL CDR3 sequence of SEQ ID NO: 6, A method comprising, thereby, inducing hair growth in the aforementioned mammal.

3. The method according to claim 2, wherein the subject is suffering from a hair loss disorder such as alopecia areata.

4. The method according to claim 1 or 3, wherein the hair loss disorder is alopecia areata.

5. The method according to claim 4, wherein the alopecia areata is alopecia totalis or alopecia universalis.

6. The method according to any one of claims 1 to 5, wherein the antibody is administered subcutaneously (s.c.) to the subject.

7. The method according to any one of claims 1 to 6, wherein the antibody is administered once a week (Q1W), once every two weeks (Q2W), once every three weeks (Q3W), once every four weeks (Q4W), once every eight weeks (Q8W), once every twelve weeks (Q12W), or any intermittent pre-rinse (as needed).

8. The method according to any one of claims 1 to 7, wherein the antibody is administered in 10 to 15 consecutive doses (for example, 11, 12, or 13 doses).

9. The method according to any one of claims 1 to 8, wherein the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO:

24.

10. The method according to any one of claims 1 to 9, wherein the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24, and is administered subcutaneously (s.c.) to a mammal (e.g., human) subject once every two weeks (Q2W) for 10 to 15 consecutive doses (e.g., 11, 12, or 13 doses) at a dose of approximately 200 mg.

11. The method according to any one of claims 1 to 10, wherein the subject has moderate to severe AA, or severe to very severe AA (for example, having overall hair loss of ≥50% of the scalp as defined by the SALT score).

12. The method according to any one of claims 1 to 11, wherein hair loss in the subject is evaluated by the mean relative change in the Alopecia Severity Tool (SALT) score at 12 weeks (week 12), 18 weeks (week 18), and / or 24 weeks (week 24) of the first dose at week 0, compared to the baseline SALT score obtained immediately before the first dose.

13. The method according to claim 12, wherein a relative reduction of ≥50% of the SALT score is achieved in the subject 12 weeks (week 12), 18 weeks (week 18), and / or 24 weeks (week 24) after the first dose in week 0.

14. The method according to claim 12 or 13, wherein the absolute SALT score of the subject is ≤ 5, 10, 20, 30, or 50 at 12 weeks (week 12), 18 weeks (week 18), and / or 24 weeks (week 24) of the first dose in week 0.

15. The method according to any one of claims 12 to 14, wherein the subject has a relative reduction of ≥50% in SALT score at 36 weeks and / or 24 weeks from baseline.

16. The method according to any one of claims 12 to 15, wherein the absolute SALT score of the subject is ≤5, ≤10, ≤20, ≤30, or ≤50 at week 36.

17. The method according to any one of claims 1 to 16, wherein hair loss includes eyelash loss and / or eyebrow hair loss.

18. The method according to claim 17, wherein the reduction in eyebrow hair loss is based on a ClinRO scale score (total coverage or minimum gap) for eyebrow alopecia of 0 or 1, with an improvement of ≥2 points from baseline at 24 weeks and / or 36 weeks.

19. The method according to claim 17, based on a ClinRO scale score (total coverage or minimum gap) for 0 or 1 trichiasis, wherein the reduction in eyelash loss has an improvement of ≥2 points from baseline at 24 weeks and / or 36 weeks.

20. The method according to any one of claims 1 to 19, wherein the patient has an improved quality of life (QOL) after treatment, as measured by improvement in SKINDEX-16 AA compared to baseline improvement at weeks 12, 18, 24, and / or 36.

21. The method according to any one of claims 1 to 20, wherein the antibody is formulated at pH 6.0 as a 100 mg / mL solution in 20 mM histidine, 260 mM sucrose, and 0.05% (w / v) polysorbate 80, optionally together with 0.05 mM pentetic acid.

22. The method according to any one of claims 1 to 21, wherein the antibody is administered to the subject in a dose of approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.2 mg / kg to approximately 8 mg / kg, approximately 0.5 mg / kg to approximately 5 mg / kg, approximately 1 mg / kg to approximately 4 mg / kg, approximately 2 mg / kg to approximately 3 mg / kg, approximately 0.2 mg / kg, approximately 0.5 mg / kg, approximately 1 mg / kg, approximately 2 mg / kg, approximately 3 mg / kg, approximately 4 mg / kg, approximately 5 mg / kg, approximately 6 mg / kg, approximately 7 mg / kg, approximately 8 mg / kg, approximately 9 mg / kg, or approximately 10 mg / kg per dose.

23. The method according to any one of claims 1 to 22, wherein the antibody is administered to the subject in doses of approximately 10 mg to approximately 800 mg, approximately 20 mg to approximately 500 mg, approximately 50 mg to approximately 300 mg, approximately 100 mg to approximately 250 mg, approximately 25 mg, approximately 50 mg, approximately 100 mg, approximately 150 mg, approximately 200 mg, approximately 250 mg, approximately 300 mg, or approximately 350 mg.

24. The method according to any one of claims 1 to 23, wherein the subject has renal impairment (for example, mild renal impairment (e.g., presence of glomerular filtration rate < 60 mL / min and / or albuminuria > 30 mg / day), moderate renal impairment (e.g., glomerular filtration rate < 45 mL / min), or severe renal impairment (e.g., glomerular filtration rate < 30 mL / min)).

25. The method according to any one of claims 1 to 24, wherein the subject is currently being treated with or has been treated with a second therapeutic agent effective for treating alopecia areata.

26. The method according to claim 25, wherein the second therapeutic agent comprises a glucocorticoid, minoxidil, anthraline, bimatoprost, methotrexate, DMARD, dupilumab, and / or a JAK inhibitor (e.g., tofacitinib, ruxolitinib, upadacitinib, abrocitinib, and baricitinib).

27. The method according to any one of claims 1 to 26, wherein the subject had alopecia areata for more than 6 months but less than 10 years prior to the administration of the antibody.

28. A method for treating alopecia areata (AA) in a mammal (e.g., human) that requires it, comprising administering an effective amount of a composition containing an antibody against IL-7Rα, The antibody comprises (a) a heavy chain variable region (HCVR) including the VH CDR1 sequence of SEQ ID NO: 1 (DHAMH, one of SEQ ID NOs from 7 to 22, etc.), the VH CDR2 sequence of SEQ ID NO: 2 (GISWNSRGIGYADSVKG), and the VH CDR3 sequence of SEQ ID NO: 3 (DEYSRGYYVLDV), and (b) a light chain variable region (LCVR) including the VL CDR1 sequence of SEQ ID NO: 4 (RASQGISSALA), the VL CDR2 sequence of SEQ ID NO: 5 (DASSLES), and the VL CDR3 sequence of SEQ ID NO: 6 (QQFNSYPLWIT), The subject has overall scalp hair loss of ≥50% as defined by the baseline SALT score, and the baseline SALT score is determined up to 24 hours before the administration of the antibody. The antibody comprises IgG1Fc without effector function, A method comprising administering 200 mg of the antibody subcutaneously every two weeks.

29. The method according to claim 28, wherein the VH CDR1 includes sequence number 7 (GFTFDDHAMH).

30. The method according to claim 28 or 29, wherein the subject had alopecia areata for more than 6 months but less than 10 years prior to the administration of the antibody.

31. The method according to any one of claims 28 to 30, wherein the composition optionally contains 20 mM histidine, 260 mM sucrose, and 100 mg / ml of the antibody in 0.05% (w / v) polysorbate 80 together with 0.05 mM pentetic acid at pH 6.

0.

32. The method according to any one of claims 28 to 31, wherein the subject has an absolute lymphocyte count (ALC) of ≥ 800 per microL.

33. The method according to any one of claims 28 to 32, wherein the administration of the antibody is stopped when the ALC of the target is < 500 per microL.

34. The method according to any one of claims 28 to 33, wherein the composition containing the antibody is administered to the subject and the serum concentration of the antibody is maintained at ≥ 5 μg / mL throughout the administration.

35. The method according to any one of claims 1 to 34, wherein the antibody is administered to the subject at a dose of approximately 200 mg.

36. The method according to any one of claims 1 to 35, wherein the antibody is administered once every two weeks (Q2W).

37. The method according to any one of claims 1 to 36, wherein the antibody is administered for at least about 13 doses.

38. The method according to any one of claims 1 to 37, wherein the antibody comprises IgG1 Fc without effector function.

39. The method according to any one of claims 1 to 38, wherein the antibody has the heavy chain sequence of SEQ ID NO: 23 and the light chain sequence of SEQ ID NO: 24, and is administered subcutaneously (s.c.) to a mammal (e.g., human) subject once every two weeks (Q2W) for 10 to 15 consecutive doses (e.g., 11, 12, or 13 doses) at a dose of approximately 200 mg.

40. A pharmaceutical composition comprising an antibody having two heavy chains and two light chains, wherein each of the heavy chains has the amino acid sequence of SEQ ID NO: 23, and each of the light chains has the amino acid sequence of SEQ ID NO: 24, and the antibody is formulated as a pharmaceutically acceptable solution having 50 to 200 mg / mL (e.g., about 100 mg / mL) of antibody in about 50 to 200 mg / mL (e.g., about 15 to 25 mM (e.g., about 20 mM)) histidine, 200 to 300 mM (e.g., about 260 mM) sucrose, and about 0.03 to 0.07% (w / v) (e.g., about 0.05% (w / v)) polysorbate 80 at pH 6.

0.

41. The pharmaceutical composition according to claim 40, wherein the solution further comprises about 0.01 to 0.10 mM (for example, about 0.05 mM) of pentetic acid.

42. A pharmaceutical composition according to claim 40, comprising essentially the same, or comprising the same, 20 mM histidine, 260 mM sucrose, and the antibody formulated as 100 mg / mL of the antibody in 0.05% (w / v) polysorbate 80 at pH 6.

0.

43. The pharmaceutical composition according to claim 40, comprising, essentially consisting of, or consisting of, 20 mM histidine, 260 mM sucrose, 0.05 mM pentetic acid, and the antibody formulated as 100 mg / mL of the antibody in 0.05% (w / v) polysorbate 80 at pH 6.

0.

44. A container (e.g., a polycarbonate bottle) containing the pharmaceutical composition according to claim 42 or 43, with an extractable volume of approximately 2 mL.

45. A container according to claim 44, comprising approximately 2.26 mL of the pharmaceutical composition according to claim 42 or 43.

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