Method for treating vitiligo by using interferon-gamma antibody

Abstract

The present invention provides methods of treating vitiligo using antibodies that specifically bind to IFN-gamma.

Description

Technical Field

[0001] The present disclosure relates generally to methods for treating vitiligo, and in particular to methods using antibodies that bind interferon-γ (IFN-γ). Background Art

[0002] Vitiligo is a T cell-mediated inflammatory skin disorder that progressively destroys melanocytes in the skin, leading to epidermal melanocyte loss, patchy disfiguring depigmentation, and an unpredictable clinical course that complicates patient management. IFN-γ and its signature cytokines, including CXCL9, CXCL10, and granzyme B (GzmB), are most highly expressed in skin lesions of patients with vitiligo and are key cytokines that promote the recruitment of autoreactive cytotoxic T cells in patients with vitiligo. IFN-γ acts directly on melanocytes by reducing their viability, inducing apoptosis, and / or downregulating melanin production. IFN-γ may stimulate immune cells by activating immune receptor (CXCR3) expression during T cell activation and stimulating CXCL9 / CXCL10 expression. Therefore, reducing IFN-γ expression will reduce T cell trafficking to sites of inflammatory lesions.

[0003] IFN-γ is a key regulator of the host immune system and contributes to autoimmune pathology in many diseases. In addition to acting as an inducer of chemokine-recruited T cells, previous reports show that IFN-γ signaling maintains skin pigmentation homeostasis in a leprosy model. The direct pathological effects of IFN-γ on melanocytes in vitiligo are rarely studied. There are conflicting results in studies on the key cytotoxic effects of cytokines on melanocytes. Induced cell death, oxidative stress, and melanogenesis damage are key in the pathogenesis of vitiligo. Most studies of cell death pathways in vitiligo are limited to apoptosis. Recent advances have led to the discovery of many new regulated cell death pathways, including necroptosis, pyroptosis, and ferroptosis, which may be the cause of melanocyte loss in vitiligo. Summary of the invention

[0004] Contrary to the understanding of IFN-γ and vitiligo in the art, the present disclosure provides methods and compositions for treatment, which are based on the surprising discovery that antibodies that can block IFN-γ activity can be used to treat vitiligo. Without being bound by theory, it is proposed that cytokines highly expressed in vitiligo skin lesions cause direct toxicity to melanocytes. In vitro melanocyte studies support the direct role of IFN-γ itself in melanocyte loss, increased oxidative stress, and melanin production disruption. In vitiligo patients, it is proposed that IFN-γ signaling leads to melanocyte loss, increased oxidative stress, and melanin production disruption. Therefore, the present disclosure provides a method of treatment, wherein vitiligo patients are administered IFN-γ antibodies, wherein the antibodies neutralize the effects of IFN-γ signaling, and thus regulate cell death by ferroptosis cell death associated with oxidative stress, which can induce autoimmunity in vitiligo. In addition, in vivo toxicology results show that the administration of anti-IFN-γ antibodies is safe and has no side effects, and can therefore be used as a safer and potentially more effective option for treating vitiligo.

[0005] In some embodiments, the present invention provides a method for treating vitiligo, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier. In some embodiments, the IFN-γ antibody modulates the biological function of IFN-γ. In some embodiments, the IFN-γ antibody neutralizes IFN-γ.

[0006] In some embodiments of the method, the IFN-γ antibody inhibits, reduces and / or completely blocks the signaling activity of IFN-γ; optionally, wherein the IFN-γ antibody inhibits, reduces and / or completely blocks the IFN-γ signaling activity of original human IFN-γ, mature human IFN-γ or truncated human IFN-γ.

[0007] In some embodiments of the method, the IFN-γ antibody reduces one or more of: IFN-γ-dependent cytokine production; IFN-γ-dependent T cell dysfunction, IFN-γ-dependent immune tolerance, and IFN-γ-dependent inflammation.

[0008] In some embodiments of the method, the IFN-γ antibody reduces the expression of IFN-γ, CXCL9, CXCL10 and / or CXCL11 in melanocytes.

[0009] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0010] A VH region having a CDR-H1 amino acid sequence selected from SEQ ID NO: 120 or 123, a CDR-H2 amino acid sequence selected from SEQ ID NO: 121 or 124, and a CDR-H3 amino acid sequence selected from SEQ ID NO: 122 or 125; and

[0011] A VL region comprising a CDR-L1 amino acid sequence selected from SEQ ID NO: 132 or 135, a CDR-L2 amino acid sequence selected from SEQ ID NO: 133 or 136, and a CDR-L3 amino acid sequence selected from SEQ ID NO: 134 or 137.

[0012] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0013] (a) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 120, a CDR-H2 amino acid sequence of SEQ ID NO: 121, and a CDR-H3 amino acid sequence of SEQ ID NO: 122, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 132, a CDR-L2 amino acid sequence of SEQ ID NO: 133, and a CDR-L3 amino acid sequence of SEQ ID NO: 134;

[0014] (b) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 123, a CDR-H2 amino acid sequence of SEQ ID NO: 124, and a CDR-H3 amino acid sequence of SEQ ID NO: 125, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 135, a CDR-L2 amino acid sequence of SEQ ID NO: 136, and a CDR-L3 amino acid sequence of SEQ ID NO: 137; or

[0015] (c) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 123, a CDR-H2 amino acid sequence of SEQ ID NO: 124 and a CDR-H3 amino acid sequence of SEQ ID NO: 125, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 132, a CDR-L2 amino acid sequence of SEQ ID NO: 133 and a CDR-L3 amino acid sequence of SEQ ID NO: 134.

[0016] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0017] A VH region having a CDR-H1 amino acid sequence selected from SEQ ID NO: 120 or 123, a CDR-H2 amino acid sequence selected from SEQ ID NO: 121 or 124, and a CDR-H3 amino acid sequence selected from SEQ ID NO: 122 or 125; and a VL region comprising a CDR-L1 amino acid sequence selected from SEQ ID NO: 132 or 135, a CDR-L2 amino acid sequence selected from SEQ ID NO: 133 or 136, and a CDR-L3 amino acid sequence selected from SEQ ID NO: 134 or 137; and

[0018] a VH region comprising an amino acid sequence at least 90% identical to SEQ ID NO: 109, 110, 164 or 165; and a VL region comprising a VL region having an amino acid sequence at least 90% identical to SEQ ID NO: 113 or 114.

[0019] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0020] (a) the VH region amino acid sequence of SEQ ID NO: 109 and the VL region amino acid sequence of SEQ ID NO: 113;

[0021] (b) the VH region amino acid sequence of SEQ ID NO: 110 and the VL region amino acid sequence of SEQ ID NO: 114;

[0022] (c) the VH region amino acid sequence of SEQ ID NO: 109 and the VL region amino acid sequence of SEQ ID NO: 114;

[0023] (d) the VH region amino acid sequence of SEQ ID NO: 110 and the VL region amino acid sequence of SEQ ID NO: 113;

[0024] (e) the VH region amino acid sequence of SEQ ID NO: 164 and the VL region amino acid sequence of SEQ ID NO: 113; or

[0025] (f) the VH region amino acid sequence of SEQ ID NO:165 and the VL region amino acid sequence of SEQ ID NO:113.

[0026] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0027] A VH region having a CDR-H1 amino acid sequence selected from SEQ ID NO: 120 or 123, a CDR-H2 amino acid sequence selected from SEQ ID NO: 121 or 124, and a CDR-H3 amino acid sequence selected from SEQ ID NO: 122 or 125; and

[0028] A VL region comprising a CDR-L1 amino acid sequence selected from SEQ ID NO: 132 or 135, a CDR-L2 amino acid sequence selected from SEQ ID NO: 133 or 136, and a CDR-L3 amino acid sequence selected from SEQ ID NO: 134 or 137.

[0029] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0030] a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 183, 185, 187 or 189, and

[0031] A light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 184 or 186.

[0032] In some embodiments of the method, wherein the IFN-γ antibody comprises:

[0033] (a) the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 184;

[0034] (b) the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 186;

[0035] (c) the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 186;

[0036] (d) the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 184;

[0037] (e) the heavy chain amino acid sequence of SEQ ID NO: 187 and the light chain amino acid sequence of SEQ ID NO: 184; or

[0038] (f) the heavy chain amino acid sequence of SEQ ID NO:189 and the light chain amino acid sequence of SEQ ID NO:184.

[0039] In some embodiments of the method, the VH region of the IFN-γ antibody further comprises an amino acid substitution selected from the group consisting of N76A and N76Q.

[0040] In some embodiments of the method, the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501, and Fontuzumab. In some embodiments of the method, the IFN-γ antibody is 2A6, 2B6, 2A6A, 2A6Q, AB, or BA. In some embodiments of the method, 2A6Q does not induce infection or drug-induced side effects in in vivo toxicology assays.

[0041] In some embodiments of the method, the IFN-γ antibody is an immunoglobulin molecule, Fv, disulfide-linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, human antibody, humanized antibody, multispecific antibody, Fab, bispecific antibody, Fab' fragment, bispecific antibody, F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR) or a single chain antibody.

[0042] In some embodiments of the method, the subject is administered by at least one route selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal, and transdermal.

[0043] In some embodiments of the method, the subject in need thereof is a patient suffering from vitiligo.

[0044] In some embodiments of the method, the composition is administered to the subject more than once a day, at least once a day, at least once a week, or at least once a month.

[0045] In some embodiments of the method, it further comprises administering at least one additional therapeutic agent.

[0046] In some embodiments of the methods, the additional therapeutic agent is administered to the subject in need thereof prior to administration of the composition, after administration of the composition, and / or simultaneously with administration of the composition.

[0047] In some embodiments of the method, the IFN-γ antibody is present at 1×10-8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 M or less binding affinity to human IFN-γ; optionally, wherein the equilibrium dissociation constant (K D ) to measure binding affinity. In some embodiments, the IFN-γ antibody is also measured at 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 M or less binding affinity to rhesus / cynomolgus monkey IFN-γ, optionally wherein the equilibrium dissociation constant (K D ) to measure binding affinity.

[0048] In some embodiments of the method, the IFN-γ antibody increases IL-2 production by SEB-stimulated human PBMCs by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, or at least 2.20-fold.

[0049] In some embodiments, the present invention provides a use of a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for treating vitiligo in a subject in need thereof.

[0050] In some embodiments, the present invention provides a use of a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for preparing a medicament for treating vitiligo in a subject in need thereof.

[0051] In some embodiments of the use, the composition is used with at least one additional therapeutic agent.

[0052] In some embodiments of the use, the composition is administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal, and transdermal.

[0053] In some embodiments of the use, the composition is for use more than once a day, at least once a day, at least once a week, or at least once a month.

[0054] In some embodiments of the use, the IFN-γ antibody is an immunoglobulin molecule, Fv, disulfide-linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, human antibody, humanized antibody, multispecific antibody, Fab, bispecific antibody, Fab' fragment, bispecific antibody, F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR) or a single-chain antibody.

[0055] In some embodiments of the use, the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501 and fontuzumab.

[0056] In some embodiments, the present invention provides a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for treating vitiligo in a subject in need thereof.

[0057] In some embodiments of the composition, the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501 and fontuzumab. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Electrochemiluminescence and chemokine panel expression analysis of paired skin interstitial fluid analytes in vitiligo patients and healthy controls with a high-sensitivity mesoscale electrochemiluminescence multiplex panel (MSD) is shown. IFN-γ, CXCL9, CXCL10, CXCL11 were highly expressed in skin lesions of vitiligo patients. (*p<0.05, ns: not significant, Ctrl: healthy controls)

[0059] Figure 2A IFN-γ was shown to reduce melanocyte viability in a dose-dependent manner. Various concentrations of recombinant human IFN-γ were added to human primary melanocytes. Cells were cultured for an additional 72 h at 37 °C and cell viability was determined using Celltiter-Glo. Calculation of IC of IFN-γ that causes 50% inhibition of melanocyte viability 50 It is 8.114ng / mL.

[0060] Figure 2BNeutralization of IFN-γ by EI-001 (i.e., 2A6Q) restores cell viability. Various concentrations of EI-001 were added to melanocytes cultured with 30 ng / mL recombinant human IFN-γ. Cells were cultured for an additional 72 hours at 37°C and luciferase activity was analyzed using Celltiter Glo. Calculated EC of EI-001 50 It is 5.214ng / mL.

[0061] Figure 2C The cell viability of melanocytes after treatment with IFN-γ, CXCL9, CXCL10, and CXCL11 on days 2 and 7 is shown. A. Individual effects: IFN-γ at 10 ng / mL and 100 ng / mL both destroyed the cell viability of melanocyte B, while CXCL9, CXCL10, and CXCL11 did not. B. Synergistic effect: Melanocytes treated with IFN-γ, CXCL9, CXCL10, and CXCL11 did not show a synergistic effect on cell death in melanocytes. C. Cell morphological changes: IFN-γ induced morphological changes of melanocytes in primary melanocytes within 7 days of treatment, while IFN-α did not. Treatment dose: 2A6Q (10 μg / mL), IFN-γ (10 or 100 ng / mL), CXCL9 (10 ng / mL), CXCL10 (10 ng / mL), CXCL11 (10 ng / mL). (*p<0.01, **p<0.0001; ns: not significant)

[0062] Figure 3A Effect of IFN-γ on melanin production in human primary melanocytes is shown. Various concentrations of EI-001 were added to melanocytes cultured with 20 ng / mL recombinant human IFN-γ. Cells were cultured for an additional 7 days at 37°C and analyzed for melanin content. Calculation of IC of IFN-γ that causes 50% inhibition of melanin production in melanocytes 50 It is 0.392ng / mL.

[0063] Figure 3B Neutralization of IFN-γ by EI-001 restores melanin production in melanocytes. Various concentrations of EI-001 were added to human primary melanocytes cultured with 20 ng / mL recombinant human IFN-γ. The cells were cultured for an additional 7 days at 37°C and melanin content was determined using absorbance at 470 nm. Calculated EC of IFN-γ to restore 50% inhibition of melanin production in melanocytes 50 It is 129.1ng / mL.

[0064] Figure 3CIFN-γ is shown to disrupt melanin production in melanocytes. A. IFN-γ induces downregulation of melanin regulators (MITF, MLANA, TYRP1, DCTT, TYR, TRPM1) in primary melanocytes. (IFN-γ 100 ng / mL 24 hours vs control) B. IFN-γ affects melanin content secretion as observed by flow cytometry. C. IFN-γ causes a decrease in MITF expression in primary melanocytes. Immunoblotting of MITF in melanocytes treated with IFN-γ (10 or 100 ng / mL) for 8 hours and 24 hours. β-actin was used as a control. (*p<0.05; ns: not significant)

[0065] Figure 4 EI-001 is shown to inhibit CXCR3 expression on CD4+ and CD8+ T cells. Ability of EI-001 to inhibit CXCR3 expression on CD4+ (A, C and E) and CD8+ (B, D and F) T cells from 3 individual donors.

[0066] Figure 5 IFN-γ induces early apoptosis. A. Flow cytometry using Annexin V-7AAD shows that IFN-γ significantly upregulates Annexin V but not 7AAD. B. IFN-γ induces early apoptosis as expressed by Annexin V in a dose-dependent manner, and 2A6Q rescues apoptosis. (48 hours of IFN-γ v.s 2A6Q 10 μg / mL). (IFN-γ (10 or 100 ng / mL), *p<0.0001; ns: not significant)

[0067] Figure 6 IFN-γ induces oxidative stress and lipid peroxidation in melanocytes. A. IFN-γ induces intracellular ROS. B. IFN-γ induces lipid peroxidation in melanocytes. C. IFN-γ induces downregulation of ferroptosis regulators (SLC7A11, SLC3A2, ferritin, GPX4, TFR1) in primary melanocytes. (IFN-γ 100 ng / mL 24 hours vs control). D. Immunoblotting of SLC7A11 and SLC3A2 in melanocytes treated with IFN-γ for 2 days. GADPH was used as a loading control. (IFN-γ (10 or 100 ng / mL), *p<0.05, **p<0.0001; ns: not significant)

[0068] Figure 7 shows that human monoclonal anti-IFN-γ antibody (2A6Q) rescues IFN-γ toxicity to melanocytes. A. Cell death inhibitors cannot restore IFN-γ-induced cell death. ROS scavenger (NAC), necroptosis inhibitor (NEC-1), pyroptosis inhibitor (VX-765), apoptosis inhibitor (QVDOPH). B. Ferroptosis inhibitors rescue cell viability of melanocytes on day 7 of treatment. C. 2A6Q rescues cell viability of melanocytes in a time-dependent manner on days 2 and 7 of treatment. D. Treatment with IFN-γ, CXCL9, CXCL10 and / or CXCL11, 2A6Q rescues cell viability of melanocytes in a time-dependent manner on days 2 and 7 of treatment. Treatment dose: 2A6Q (10 μg / mL), IFN-γ (10 ng / mL), CXCL9 (10 ng / mL), CXCL10 (10 ng / mL), CXCL11 (10 ng / mL). (*p<0.05, **p<0.001). DETAILED DESCRIPTION

[0069] The present disclosure provides methods of treatment and related compositions, which are based on the surprising discovery that antibodies that can block the signaling activity of IFN-γ can be used to treat vitiligo. Therefore, the present disclosure provides a method for treating vitiligo, wherein a patient in need is administered an IFN-γ antibody. The IFN-γ antibodies that can be used in the methods and compositions can reduce, inhibit and / or block IFN-γ signaling activity. As described in more detail below, therefore, the methods of treatment and related compositions can stimulate and / or otherwise restore normal immune function, which can effectively treat vitiligo in a subject in need.

[0070] Terminology and Technology

[0071] For the description herein and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes more than one protein, and reference to "a compound" refers to more than one compound. The use of "comprising," "including," "containing," "comprising," and "containing" are interchangeable and are not intended to be limiting. It should also be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the language "consisting essentially of" or "consisting of" may be used to alternatively describe the embodiments.

[0072] Where a range of values ​​is provided, it is understood that each intervening integer of the values ​​between the upper and lower limits of the range, and each intervening integer of the values, and any other stated or intervening values ​​within the range are encompassed within the present invention unless the context clearly specifies otherwise. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also included within the present invention, subject to any specifically excluded limitations within the range. When the range includes one or two of these limitations, the range excluding (i) one or (ii) two of the included limitations is also included in the present invention. For example, "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to 10", etc.

[0073] Generally, as used herein, nomenclature and the techniques and methods described herein include those well known and commonly used by those of ordinary skill in the art, such as Sambrook et al., Molecular Cloning-A Laboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter "Sambrook"); Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (supplemented through 2011) (hereinafter "Ausubel"); Antibody Engineering, Vols. 1 and 2, R. Kontermann and S. Dubel, eds., Springer-Verlag, Berlin and Heidelberg (2010); Monoclonal Antibodies: Methods and Protocols, V. Ossipow and N. Fischer, eds., 2nd Ed., Humana Press (2014); Therapeutic Antibodies: From Bench to Clinic, Z. An, ed., J. Wiley & Sons, Hoboken, NJ (2009); and those commonly used techniques and methods described in Phage Display, Tim Clackson and Henry B. Lowman, eds., Oxford University Press, United Kingdom (2004).

[0074] All publications, patents, patent applications, and other documents cited in this disclosure are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated herein by reference for all purposes.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that the terms used herein are only used to describe specific embodiments and are not intended to be limiting. For the purpose of explaining the present disclosure, the following description of the terms will apply, and where appropriate, terms used in the singular will include the plural form, and vice versa.

[0076] As used herein, "IFN-γ", "IFN-g" or "interferon-γ" refers to a dimeric soluble cytokine, various forms of interferon γ, which are members of the type II interferon class, including but not limited to naturally occurring IFN-γ from primates (e.g., humans, rhesus monkeys and cynomolgus monkeys), rodents, various pre- and post-translational forms of IFN-γ (e.g., native human IFN-γ, mature human IFN-γ or truncated human IFN-γ) and recombinant forms of IFN-γ.

[0077] As used herein, "vitiligo" refers to a chronic autoimmune condition that causes patches of skin to lose pigment or color. Vitiligo occurs when melanocytes are attacked and destroyed, causing the skin to turn milky white. The cause of vitiligo is unknown, but may be related to changes in the immune system, genetic factors, stress, or sun exposure.

[0078] As used herein, "antibody" refers to a molecule comprising one or more polypeptide chains that specifically bind to or immunologically react with a specific antigen. Exemplary antibodies of the present disclosure include natural antibodies, whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (or heteroconjugate) antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, antigen-binding antibody fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0079] "IFN-γ antibody", "anti-IFN-γ antibody" or "antibody that binds to IFN-γ" refers to an antibody that binds to IFN-γ with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting IFN-γ. In some embodiments, the extent of binding of the IFN-γ antibody to an unrelated non-IFN-γ antigen is less than about 10% of the binding of the antibody to IFN-γ, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that binds to IFN-γ has a binding affinity of <1 μM, <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM or <0.01 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 - 13 M, for example 10-9 M to 10 -13 The dissociation constant (Kd) of M) is shown in Table 1. The specifications of PCT applications PCT / CN2018 / 085836 and PCT / US2019 / 024663 are incorporated herein by reference in their entirety.

[0080] "Full length antibody," "intact antibody" or "whole antibody" are used interchangeably herein and refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that include an Fc region as defined herein.

[0081] "Antibody fragment" or "antigen-binding fragment" refers to a portion of a full-length antibody that is capable of binding to the same antigen as the full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; dimeric antibodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0082] The "class" of an antibody refers to the type of constant domain or region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these are further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0083] "Variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, wherein each domain comprises four conserved framework regions (FR) and three hypervariable regions (HVR) (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated from antibodies that bind to an antigen using a VH or VL domain to screen libraries of complementary VL or VH domains, respectively (see, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991)).

[0084] As used herein, "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence and / or later forms structurally defined loops ("hypervariable loops"). Typically, a natural antibody comprises four chains with six HVRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). HVRs typically comprise amino acid residues from hypervariable loops and / or from "complementarity determining regions" (CDRs). Exemplary hypervariable loops occur at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). Unless otherwise indicated, HVR residues and other residues in the variable domain (e.g., FR residues) are numbered herein according to Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0085] As used herein, "complementarity determining region" or "CDR" refers to a region within the hypervariable region of a variable domain that has the highest sequence variability and / or is involved in antigen recognition. Typically, a natural antibody comprises four chains with six CDRs: three in the heavy chain variable domain, VH (H1, H2, H3), and three in the light chain variable domain, VL (L1, L2, L3). Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3) occur at amino acid residues 24-34 of L1, amino acid residues 50-56 of L2, amino acid residues 89-97 of L3, amino acid residues 31-35 of H1, amino acid residues 50-65 of H2, and amino acid residues 95-102 of H3 (KabaT et al., supra). In addition to CDR-H1 in VH, CDRs typically include amino acid residues that form hypervariable loops.

[0086] "Framework" or "FR" refers to the variable region residues other than the hypervariable region (HVR) residues. The FR of the variable region is generally composed of four FR domains: FR1, FR2, FR3 and FR4. It can be seen that the HVR and FR sequences usually appear in the following sequence in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0087] "Natural antibody" refers to naturally occurring immunoglobulin molecules. For example, a natural IgG antibody is a heterotetrameric glycoprotein of about 150 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From N- to C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2 and CH3). Similarly, from N- to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. Based on the amino acid sequence of the constant domain of the antibody, the light chain of the antibody can be dominated by one of two types (referred to as κ (κ) and λ (λ)).

[0088] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies (e.g., variant antibodies, which comprise naturally occurring or or mutations produced during the production of monoclonal antibodies, and are generally present in small amounts). Contrary to polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed to a single determinant on an antigen. Therefore, the term "monoclonal" represents the characteristics of an antibody obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibodies to be produced by any particular method. For example, the monoclonal antibody used can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of a human immunoglobulin locus, and other exemplary methods described herein for preparing monoclonal antibodies.

[0089] A "chimeric antibody" refers to an antibody in which a portion of the heavy chain and / or light chain is derived from a particular source or species, while the remainder of the heavy chain and / or light chain is derived from a different source or species.

[0090] A "humanized antibody" refers to a chimeric antibody comprising an amino acid sequence from a non-human HVR and an amino acid sequence from a human FR. In certain embodiments, a humanized antibody will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the FTVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0091] As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to IFN-γ is substantially free of antibodies that specifically bind to antigens other than IFN-γ). However, an isolated antibody that specifically binds to IFN-γ may have cross-reactivity with other antigens (e.g., IFN-γ molecules from other species). In addition, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0092] As used herein, "human antibody" is intended to include antibodies with variable regions, wherein the framework region and CDR region are derived from human germline immunoglobulin sequences. In addition, if the antibody comprises a constant region, the constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences derived from other mammalian species (e.g., mice) have been grafted onto human framework sequences.

[0093] A "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, human monoclonal antibodies are produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0094] As used herein, "recombinant human antibodies" include all human antibodies prepared, expressed, produced or isolated by recombinant methods, such as (a) antibodies isolated from transgenic or transchromosomal human immunoglobulin gene animals (such as mice) or hybridomas prepared therefrom, (b) antibodies isolated from host cells transformed to express human antibodies, such as antibodies isolated from transfectomas, (c) antibodies isolated from recombinant, combinatorial human antibody libraries, and (d) antibodies prepared, expressed, produced or isolated by any other method involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable regions in which framework regions and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using transgenic animals of human Ig sequences, in vivo somatic cell mutagenesis), so that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, may not naturally exist in the human antibody germline repertoire in vivo.

[0095] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). "Binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents. Affinity can be measured by conventional methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0096] "Specific binding" or "specific binding to" means that the antibody binds to the antigen with a specificity of no more than about 1×10 -7 The affinity value of M is bound.

[0097] "Treatment", "treatment" or "treating" refers to a clinical intervention that attempts to change the natural course of a disease in a subject being treated, and may be performed for prevention or during a clinical pathological process. The desired therapeutic outcome may include, but is not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, the treatment of HBV infection may include administering to a subject a therapeutically effective amount of a pharmaceutical preparation comprising an IFN-γ antibody to prevent, delay the development of, slow the progression of, or eradicate HBV infection.

[0098] A "pharmaceutical composition" or "composition" or "formulation" refers to a preparation in a form that permits the biological activity of the active ingredient to be effective, and which contains no additional components that would be toxic to a subject to which the formulation would be administered.

[0099] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation other than the active ingredient that is non-toxic to the subject to which it is administered. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0100] As used herein, "therapeutically effective amount" refers to the amount of an active ingredient or drug (e.g., a pharmaceutical composition) that achieves a desired treatment or prevention result (e.g., treatment or prevention of a subject's disease, disorder, or condition). In the case of a vitiligo subject, a therapeutically effective amount of a therapeutic agent is an amount that reduces, prevents, inhibits, and / or alleviates one or more symptoms associated with vitiligo to a certain extent. For therapeutic treatment of vitiligo, in vivo efficacy can be measured, for example, by assessing the duration, severity, and / or recurrence of symptoms, response rate (RR), duration of response, and / or quality of life.

[0101] "Subject" refers to mammals, including but not limited to primates (eg, humans and non-human primates such as monkeys), rodents (eg, mice and rats), rabbits, and domesticated animals (eg, cows, sheep, cats, dogs, and horses).

[0102] The "subjects in need thereof" referred to herein include patients suffering from vitiligo.

[0103] Method for treating vitiligo using IFN-γ antibody

[0104] The present invention provides a method for treating vitiligo, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier. Various IFN-γ antibody compositions and modes of administration for treatment methods are described in more detail below and the Examples illustrate. In addition, treatment methods that also include administering additional therapeutic agents are further described below and illustrated in the Examples.

[0105] IFN-γ is a dimeric soluble cytokine, which is a member of the II type interferon class. IFN-γ is an important immunostimulatory and immunomodulatory molecule, which functions as an activator of macrophages and an inducer of expression of class II major histocompatibility complex (MHC) molecules. IFN-γ is mainly produced by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response, and once antigen-specific immunity occurs, it is produced by CD4 Th1 and CD8 cytotoxic T lymphocytes (CTL) effector T cells. The abnormal expression of IFN-γ is related to many autoinflammatory and autoimmune diseases. The importance of IFN-γ in the immune system is considered to be at least partially derived from the ability to inhibit viral replication. The amino acid and nucleotide sequences and annotations of IFN-γ in human, primate and other mammalian forms are publicly available. See, for example, the complete amino acid sequence of human IFN-γ of UniProt entry number P01579. The amino acid sequence of human IFN-γ is disclosed as SEQ ID NO:166 of the attached sequence table.

[0106] Typically, IFN-γ antibodies that can be used in methods for treating vitiligo have functional properties that reduce, inhibit and / or block (partially or completely) IFN-γ signaling activity. In some embodiments, the IFN-γ antibodies used in the methods of the present disclosure are capable of regulating the biological function of IFN-γ, neutralizing IFN-γ, or reducing the binding of IFN-γ to its receptor. In some embodiments, IFN-γ antibodies can reduce, inhibit and / or block the signaling activity of original human IFN-γ, mature human IFN-γ, or truncated human IFN-γ, and / or the ability of original human IFN-γ, mature human IFN-γ, or truncated human IFN-γ to bind to its receptor; thereby reducing IFN-γ-dependent cytokine production, IFN-γ-dependent T cell dysfunction, IFN-γ-dependent immune tolerance, and / or IFN-γ-dependent inflammation.

[0107] Exemplary IFN-γ antibodies for use in the methods of the present disclosure may include, for example, IFN-γ antagonists or inhibitors that modulate at least one biological function or activity of IFN-γ. The biological functions or activities of IFN-γ include, for example, binding to IFN-γ receptors (IFN-γ-Rs), modulating (e.g., enhancing) major histocompatibility complex (MHC) class II expression on cell surfaces, modulating (e.g., reducing or inhibiting) cell proliferation, and / or modulating immune responses.

[0108] In some embodiments, the IFN-γ antibody used in the methods of the present disclosure completely or partially inhibits IFN-γ signaling activity. In some embodiments, the IFN-γ antibody completely or partially inhibits IFN-γ signaling activity by partially or completely blocking the binding of IFN-γ to the IFN-γ receptor. However, it is expected that the IFN-γ antibody can also completely or partially inhibit IFN-γ signaling by a mechanism that does not involve direct inhibition of IFN-γ binding to the IFN-γ receptor.

[0109] In some embodiments, the IFN-γ antibodies used in the methods and compositions of the present disclosure can be characterized by various well-known immunoglobulin features (e.g., CDRs, HVRs, FRs, V H and V L The amino acid sequences and encoding nucleotide sequences of the IFN-γ domains are described below. Table 1 below provides an overview of the sequences and sequence identifiers of exemplary IFN-γ antibodies used in the methods of the present disclosure (including IFN-γ antibodies described elsewhere herein as "2A6", "2B6", "2A6_Q" (or "2A6Q") and "2A6_A" (or "2A6A")). The sequences are contained in the attached sequence listing.

[0110] Table 1: IFN-γ antibody sequences

[0111]

[0112]

[0113]

[0114]

[0115] In addition, the IFN-γ antibodies used in the methods and compositions of the present disclosure include any antibodies known in the art that specifically bind to IFN-γ and neutralize, inhibit, reduce and / or otherwise block IFN-γ signaling activity. Exemplary antibodies known in the art include the following IFN-γ antibodies: AMG-811 (described in, e.g., U.S. Patent No. 7,335,743B2), NI-0501 (described in, e.g., U.S. Patent No. 7,700,098B2), HuZAF (described in, e.g., U.S. Patent No. 6,329,511B1). The disclosure of each of U.S. Patent Nos. 7,335,743B2, 7,700,098B2 and 6,329,511B1 is incorporated herein by reference in its entirety.

[0116] Typically, the IFN-γ antibodies useful in the therapeutic methods of the present disclosure exhibit high affinity binding to IFN-γ. In some embodiments, the anti-IFN-γ antibodies provided herein have a high affinity binding to IFN-γ of <100 nM, <10 nM, <1 nM, <0.1 nM, <0.01 nM, or <0.01 nM (e.g., 10 -8 M or smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M) for IFN-γ binding dissociation constant (Kd).

[0117] In addition, the anti-IFN-γ antibodies that can be used in the methods of the present disclosure include antibodies that can bind to human IFN-γ and cynomolgus monkey IFN-γ with high affinity, and in some embodiments, bind to human IFN-γ and cynomolgus monkey IFN-γ with high affinity. More specifically, in some embodiments, the IFN-γ antibodies that can be used in the methods of the present disclosure are 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 M or less binding affinity to human IFN-γ. In some embodiments, the anti-IFN-γ antibodies of the present disclosure bind to human IFN-γ with an affinity of 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11M or less binding affinity to cynomolgus monkey IFN-γ. In some embodiments, the anti-IFN-γ antibodies of the present disclosure are at 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 It binds to human IFN-γ and cynomolgus monkey IFN-γ with a binding affinity of M or less.

[0118] Typically, the binding affinity of IFN-γ antibodies can be measured using any of a variety of tests and represented by a variety of quantitative values. Specific IFN-γ binding assays that can be used to measure antibody affinity are disclosed in the embodiments herein. In addition, antigen binding assays are known in the art and can be used herein, including but not limited to any direct or competitive binding assays using technology, such as Western blot, radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), "sandwich" immunoassay, surface plasmon resonance-based assays (BIAcore assays as described in WO2005 / 012359), immunoprecipitation assays, fluorescent immunoassays, and protein A immunoassays.

[0119] In some embodiments, the anti-IFN-γ antibodies useful in the methods of the present disclosure reduce, inhibit and / or completely block the binding of IFN-γ to its cognate receptor IFN-γ-R, thereby modulating IFN-γ-mediated immunomodulation and / or immune signaling. The ability of IFN-γ antibodies to inhibit these immunomodulatory and immune signaling pathways mediated by IFN-γ binding can be tested in vitro using known cell-based assays (including various cell-based assays described in the examples of the present disclosure).

[0120] Thus, in some embodiments, the IFN-γ antibodies useful in the methods of the present disclosure are characterized by the ability to increase the measurable immune response in human PBMCs stimulated by Staphylococcal enterotoxin B (SEB) by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, or at least 2.20-fold. For example, in some embodiments, the IFN-γ antibodies increase the IL-2 production and / or cell proliferation of human PBMCs stimulated by SEB by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, or at least 2.20-fold. Additional functional properties of exemplary IFN-γ antibodies useful in the methods of the present disclosure are further described below and in the Examples.

[0121] As described above, it is expected that the IFN-γ antibodies that can be used in the methods for treating vitiligo disclosed herein may include any immunoglobulin comprising one or more polypeptide chains that specifically bind to IFN-γ or immunologically react with IFN-γ. It can be seen that the IFN-γ antibodies that can be used in the methods disclosed herein include natural antibodies, whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific (or heteroconjugate) antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, antigen-binding antibody fragments (e.g., Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments), antibody fusions, and synthetic antibodies (or antibody mimetics).

[0122] In some embodiments of the present invention, the IFN-γ antibody is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. 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 CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0123] In some embodiments according to the present disclosure, the IFN-γ antibody can be an immunoglobulin molecule, Fv, disulfide-linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, double antibody, human antibody, humanized antibody, multispecific antibody, Fab, dual-specific antibody, Fab' fragment, bispecific antibody, F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementary determining region (CDR) or a single-chain antibody. Specifically, the IFN-γ antibody can be a human protein or a humanized binding protein.

[0124] In some embodiments of the present disclosure, the IFN-γ antibody is an "antigen binding fragment", for example, a diabody, Fab, Fab', F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide-stabilized diabody (dsdiabody), a single-chain antibody molecule (scFv), a scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody comprising one or more CDRs, or any other antibody fragment that binds to an antigen but does not comprise a complete antibody structure. In such embodiments, the antigen binding fragment is capable of binding to the same IFN-γ antigen bound by a parent antibody or parent antibody fragment (e.g., a parent scFv). In certain embodiments, the antigen binding fragment may comprise one or more CDRs from a particular human antibody grafted to a framework region from one or more different human antibodies.

[0125] Among the above antigen-binding fragments, Fab, which has a structure of light chain and heavy chain variable regions, light chain constant regions and the first constant region (CH1) of the heavy chain, has one antigen-binding site. Fab' is different from Fab in that Fab' has a hinge region that contains at least one cysteine ​​residue at the C-terminus of the heavy chain CH1 domain. When the cysteine ​​residues in the hinge region of Fab' are linked by disulfide bonds, F(ab')2 is generated.

[0126] Fv is the smallest antibody fragment with only heavy chain variable region and light chain variable region, and the recombinant technology of producing Fv fragment is well known in the art. "Single-chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region connected to each other directly or through a peptide linker sequence. Double-chain Fv can have a structure in which the heavy chain variable region is connected to the light chain variable region by a non-covalent bond, and single-chain Fv can generally form a dimer structure such as in double-chain Fv, wherein the heavy chain variable region is covalently bound to the light chain variable region by a peptide linker, or the heavy chain and light chain variable regions are directly connected to each other at their C-terminal ends. The linker can be a peptide linker comprising 1 to 100 or 2 to 50 any amino acids, and its suitable sequence is known in the art.

[0127] Antigen-binding fragments can be obtained using proteases (for example, whole antibodies can be digested with papain to obtain Fab fragments, and with pepsin to obtain F(ab')2 fragments), or can be prepared by genetic recombination techniques.

[0128] In some embodiments of the methods of the present disclosure, the IFN-γ antibody may be a multispecific antibody, such as a bispecific antibody. In some embodiments, the multispecific antibody is a monoclonal antibody having at least two different binding sites, each binding site having binding specificity for a different antigen, at least one of which specifically binds to IFN-γ. In some embodiments, the multispecific antibody is a bispecific antibody comprising specificity for IFN-γ and specificity for another antigen that mediates immunomodulation and / or immune signaling. In some embodiments of the bispecific antibody, another specificity is for an antigen, which is an immune checkpoint molecule selected from the following PD1, PD-L1, CTLA-4, TIGIT, LAG3, PVRIG, KIR, TIM-3, CRTAM, BTLA, CD244, CD160, LIGHT, GITR, 4-1BB, OX40, CD27, TMIGD2, ICOS, CD40, CD47, SIRPa, NKG2D, NKG2A, TNFRSF25, CD33, CEA, Epcam, GPC3, CD200, CD200R, CD73, CD83, CD39, TRAIL, CD226 and VISTA. In some embodiments of the anti-IFN-γ bispecific antibody, the other antigen to which the antibody is specific is selected from PD1, PD-L1 and CTLA-4.

[0129] Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see, e.g., Milstein and Cuello, Nature 305:537 (1983), WO 93 / 08829, and Traunecker et al., EMBO J. 10:3655 (1991)). "Knob-in-hole" engineering can also be used to generate bispecific antibodies that can be used for the anti-IFN-γ antibodies of the present disclosure. Techniques for Knob-in-hole engineering are known in the art and are described, e.g., in U.S. Pat. No. 5,731,168.

[0130] In some embodiments, the IFN-γ antibodies that can be used in the methods of the present disclosure can be modified to increase or decrease the degree of antibody glycosylation. Addition or deletion of antibody glycosylation sites can be performed by changing the amino acid sequence to create or remove one or more glycosylation sites. For example, the exemplary IFN-γ antibodies 2A6_A and 2A6_Q provided in the present disclosure have sequences modified to remove V H N-linked glycosylation site at position 76 in the region.

[0131] It is also expected that the carbohydrate connected to the Fc region can be changed. Typically, the natural antibody produced by mammalian cells comprises a branched biantennary oligosaccharide connected to the asparagine of about position 297 ("N297") of the CH2 domain of the Fc region by N-connection (see, e.g., Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose connected to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the modification of the oligosaccharide in the Fc region of the antibody may produce variants with certain improved properties. In some embodiments, the IFN-γ antibody of the present invention may be a variant of a parent antibody, wherein the variant comprises a carbohydrate structure lacking the fucose connected to the Fc region (directly or indirectly). For example, the amount of fucose in such an antibody can be about 1% to about 80%, about 1% to about 65%, about 5% to about 65%, or about 20% to about 40%. The amount of fucose can be determined by calculating the average amount of fucose within the sugar chain at N297 relative to the sum of all carbohydrate structures (e.g., complex, mixed and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry (see, e.g., WO2008 / 077546). N297 refers to the asparagine residue at about position 297 (Eu numbering of Fc region residues) in the Fc region; however, due to minor sequence variations in antibodies, N297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300.

[0132] In some embodiments, the fucosylation variant may have improved ADCC function. See, for example, US Patent Publication Nos. US2003 / 0157108 or US2004 / 0093621. Examples of "defucosylated" or "fucose-deficient" antibodies and methods of making the same are disclosed in, e.g., US2003 / 0157108; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO2000 / 61739; WO2001 / 29246; WO2003 / 085119; WO2003 / 084570; WO2005 / 035586; WO2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).

[0133] Cell lines that can be used to produce defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (see, e.g., Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US2003 / 015710 and WO2004 / 056312), and knockout cell lines, such as, for example, α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003 / 085107).

[0134] In some embodiments, the IFN-γ antibodies useful in the methods of the present disclosure may comprise one or more amino acid modifications (i.e., Fc region variants) in the Fc region. The Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid substitution at one or more amino acid residue positions. The following describes a variety of Fc region variants known in the art that can be used for anti-IFNγ antibodies of the present disclosure.

[0135] In some embodiments, it is expected that IFN-γ antibodies are Fc region variants with altered effector functions. In some embodiments, antibodies with altered effector functions have some (but not all) effector functions of parent antibodies, reduced effector functions or no effector functions (e.g., no effector). For certain applications where effector functions are unnecessary or harmful (e.g., ADCC) and / or the half-life of antibodies in vivo is important, no effector Fc region variants are more desirable. Fc region variant antibodies with reduced effector functions or no effector can include amino acid substitutions at one or more of the following Fc region positions: 238, 265, 269, 270, 297, 327, and 329. (See, e.g., U.S. Patent No. 6,737,056). Such Fc region variants can include amino acid substitutions at two or more of 265, 269, 270, 297, and 327 positions. Such Fc region variants can also include replacing residues 265 and 297 with alanine (see, e.g., U.S. Patent No. 7,332,581).

[0136] Therefore, in one embodiment, the present invention provides a method for treating vitiligo, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier.

[0137] Thus, the exemplary cell-based model studies support a mechanism by which IFN-γ antibodies can neutralize cell-based pathways associated with the pathogenesis of vitiligo. That is, the present invention demonstrates that IFN-γ antibodies can be used to provide improved methods for treating vitiligo.

[0138] Mode of administration of IFN-γ antibody composition

[0139] Administering a composition or formulation comprising an IFN-γ antibody to a subject in need thereof according to a method of treatment provides a therapeutic effect of protecting the subject from vitiligo and / or treating vitiligo.

[0140] In some embodiments of the methods of treatment disclosed herein, an IFN-γ antibody composition or formulation comprising an IFN-γ antibody is administered to a subject by any administration method for systemic drug delivery or delivery to a desired target tissue. Systemic administration generally refers to administering an antibody to a subject at a site other than directly into a desired target site, tissue, or organ, so that the antibody or its formulation enters the subject's circulatory system and thus undergoes any mode of metabolism and other similar processes.

[0141] Therefore, the modes of administration that can be used for the vitiligo treatment methods disclosed herein may include, but are not limited to, injection, infusion, instillation, and inhalation. Administration by injection may include intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.

[0142] In one embodiment of the method according to the present disclosure, wherein the IFN-γ antibody can be administered to the subject in need by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal, and transdermal. In one embodiment, the IFN-γ antibody can be administered intravenously to a subject in need.

[0143] In some embodiments, the formulation of the IFN-γ antibody is formulated to protect the antibody from inactivation in the intestinal tract. Thus, the method of treatment may include oral administration of the formulation.

[0144] In some embodiments, the present invention provides a composition or formulation comprising an IFN-γ antibody as a drug for treating vitiligo. In addition, in some embodiments, the present invention also provides a composition or formulation comprising an IFN-γ antibody for use in the manufacture or preparation of a drug for treating vitiligo. In another embodiment, the drug is used in a method for treating vitiligo, the method comprising administering an effective amount of the drug to a subject in need thereof. In certain embodiments, the drug further comprises an effective amount of at least one additional therapeutic agent or therapy.

[0145] In another embodiment, a medicament is used to treat vitiligo in a subject, comprising administering to the subject an effective amount of the medicament to treat vitiligo.

[0146] For the treatment of vitiligo, the appropriate dosage of the IFN-γ antibodies contained in the compositions and formulations of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on factors including the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapies administered to the patient, the patient's clinical history and response to the antibody, and the judgment of the attending physician.

[0147] Typically, the treatment regimen that can be used for the method of the present disclosure can be determined by the medical staff of the subject in need. When included in the compositions and preparations described herein, the IFN-γ antibodies of the present disclosure can be appropriately administered to the patient once or in a series of treatments. Various dosing regimens contemplated herein include, but are not limited to, single or multiple administrations, bolus administration, and pulse infusions at various time points.

[0148] In some embodiments of the methods, the composition comprising an IFN-γ antibody is administered to the subject more than once a day, at least once a day, at least once a week, or at least once a month.

[0149] Depending on the type and severity of vitiligo, about 1 μg / kg to 15 mg / kg of IFN-γ antibody in the formulation of the present disclosure is an initial candidate dose for administration to a human subject, whether, for example, by one or more separate administrations or by continuous infusion. Typically, the administration dose of the antibody will be in the range of about 0.05 mg / kg to about 10 mg / kg. In some embodiments, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg or 10 mg / kg (or any combination thereof) may be administered to the patient.

[0150] Dosage administration can be maintained for several days or longer, depending on the condition of the subject, for example, administration can be continued until vitiligo is fully treated, as determined by methods known in the art. In some embodiments, an initial higher loading dose can be administered, followed by one or more lower doses. However, other dosage regimens can be useful. The progress of the therapeutic effect of dosage administration can be monitored by conventional techniques and determination.

[0151] It can be seen that in some embodiments of the methods of the present invention, the administration of the IFN-γ antibody comprises a daily dose of about 1 mg / kg to about 100 mg / kg. In some embodiments, the dosage of the IFN-γ antibody comprises a daily dose of at least about 1 mg / kg, at least about 5 mg / kg, at least about 10 mg / kg, at least about 20 mg / kg or at least about 30 mg / kg.

[0152] Combination therapy with additional therapeutic agents

[0153] In one embodiment according to the present invention, the method may further include the step of administering at least one additional therapeutic agent. For example, an additional therapeutic agent may be administered in combination with an IFN-γ antibody composition to a subject in need—for example, simultaneously with the IFN-γ antibody composition; before administering the IFN-γ antibody composition; or after administering the IFN-γ antibody composition. In some embodiments, the additional therapeutic agent may include an additional therapy for vitiligo or a treatment for a disease or condition associated with vitiligo. It is expected that in a combined treatment method, the efficacy of the therapeutic agent may be improved by administering a combination of an IFN-γ antibody composition and an additional therapeutic agent. Without being bound by theory, it is believed that after administering a therapeutically effective amount of an IFN-γ antibody composition and an optional additional therapeutic agent, the immune response associated with vitiligo may be reduced or improved in a subject in need, even to an undetectable level. In addition, the reduced or improved immune response may include a reduction in antibodies or cytokines that further modulate the activity of the immune system.

[0154] In one embodiment, a method comprising administering at least one additional therapeutic agent is performed, wherein the additional therapeutic agent is an IFN-γ antagonist. As described elsewhere herein, an IFN-γ antagonist that can be used in a combination therapy method may include an IFN-γ antagonist that reduces the level of at least one biological activity of IFN-γ of a therapeutic agent for treating a subject already suffering from vitiligo. In some embodiments, the therapeutic agent is selected from a second IFN-γ neutralizing antibody, an IFN-γ receptor antagonist, and a small molecule inhibitor of IFN-γ or IFN-γ receptor.

[0155] In other embodiments of the present invention, the additional therapeutic agent can be selected from the following group: therapeutic agent; imaging agent; cytotoxic agent; angiogenesis inhibitor; kinase inhibitor; co-stimulatory molecule blocker; adhesion molecule blocker; anti-cytokine antibody or its functional fragment; methotrexate; cyclosporine; rapamycin; FK506; detectable label or reporter; TNF antagonist; anti-rheumatic agent; muscle relaxant; anesthetic; non-steroidal anti-inflammatory drug (NSAID); analgesic; anesthetic; sedative; local anesthetic; neuromuscular blocker; antimicrobial agent; antipsoriatic drug; corticosteroid; anabolic steroid; erythropoietin; immunization; immunoglobulin; immunosuppressant; growth hormone; hormone replacement drug; radiopharmaceutical; antidepressant; antipsychotic; stimulant; asthma medication; beta agonist; inhaled steroid; epinephrine or its analog; cytokine; and cytokine antagonist; immunomodulator.

[0156] Example

[0157] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative rather than limiting. Those skilled in the art will readily appreciate that the specific embodiments are merely illustrative of the present invention, as more fully described in the claims that follow. Each embodiment and feature described in this application should be understood to be interchangeable and combinable with each embodiment contained therein.

[0158] Example 1: IFN-γ Antibody

[0159] A. Recombinant IFN-γ Antibody Production

[0160] Under standard conditions, in a TM 293 expression medium (Gibco, 12338018) in a 250 mL flask at 1 × 10 6 FreeStyle TM 293-F cells (Thermo Scientific, R79007). Exponentially growing FreeStyle cells were transfected using linear polyethyleneimine (PEI) with an average molecular weight of 25 kDa (Polysciences, Warrington, Pa.) as a transfection reagent and a total of 88 μg of plasmid DNA. TM 293-F cells (1.5-2×10 6 After transfection, the cells were cultured for 3 days and the culture medium was harvested. The culture medium was centrifuged at 3000 rpm for 10 minutes to remove FreeStyle TM 293-F cell debris, and the resulting supernatant was collected and filtered through a 0.45 μm filter.

[0161] B. Recombinant Antibody Purification

[0162] The obtained supernatant obtained by purifying the above-mentioned with Protein A Sepharose Fast Flow beads (GE Healthcare, 17-1279-01) is then obtained to obtain recombinant antibodies. In brief, 80 μ L of Protein A Sepharose Fast Flow beads are added to 80 mL of supernatant, and evenly divided into two 50 mL tubes, which are rotated and incubated at 4 ° C for 24 hours. Then, the test tube is centrifuged at 3000 rpm for 10 minutes, then the obtained supernatant is removed, and the beads are balanced with PBS. The beads balanced with 0.1 M glycine (pH 3.0) are eluted, and the eluate is collected in a tube containing 1 M Tris (pH 8.0), and dialyzed with PBS buffer, to obtain monoclonal antibodies respectively, including 2A6, 2B6, 2A6A, 2A6Q and BA anti-INF-γmAb.

[0163] For clarity, the CDRs on the VH and VL chains of each monoclonal antibody are summarized in Table 2. The CDRs were annotated according to the Kabat (Wu, TT and Kabat, EA, 1970 J. Exp. Med. 132: 211-250) and IMGT systems (Lefranc M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212) and by Internet-based sequence analysis, such as described in, for example, www.imgt.org / IMGT_vquest / share / textes / index.html and www.ncbi.nlm.nih.gov / igblast. In the following Table 2, "K" in CDR means based on the Kabat system, "I" in CDR means based on the IMGT system, Table 2

[0164]

[0165]

[0166] Example 2: IFN-γ, CXCL9, CXCL10 and CXCL11 are highly expressed in skin lesions of patients with vitiligo

[0167] To understand the possible pathogenic pathways, we first performed cytokine analysis in skin lesions of vitiligo patients and healthy controls. We selected IFN-γ, CXCL9, CXCL10, CXCL11, IL13, IL15, IL17A, and IL18 based on previous cohorts.

[0168] Materials and methods: The high-sensitivity Meso Scale Discovery (MSD) electrochemiluminescence assay for cytokine panel studies was used to measure IFN-γCXCL9, CXCL10, CXCL11, IL13, IL15, IL17A, IL18 in skin blister interstitial fluid analytes. The MSD plate was analyzed on an MS2400 imager (MSD). (Dabitao et al., 2011) The sample volume requirement of MSD is much smaller than conventional ELISA with higher sensitivity, enabling more cytokines to be studied in a single blister interstitial fluid analyte. (Hwang et al., 2019). The MSD assay was performed according to the manufacturer's instructions. All standards and samples were measured in triplicate. Some cytokine panel results from the same patient in this study have been used in clinical biomarker studies of vitiligo activity and severity (CY Ng, 2022).

[0169] Results: Figure 1As shown, paired blister interstitial fluid from lesional (VL) and non-lesional (V-NL) skin was collected from 32 vitiligo patients and 10 healthy volunteers (control, Ctrl) and analyzed by a panel of cytokines selected by MSD immunoassay. The mean age of vitiligo patients was 44.58±15.81 years and that of healthy controls was 42.82±8.69 years. The male to female ratio of healthy and vitiligo patients was 1:1. The BSA of vitiligo was 12% (±13.1%) and the VES score was 9.02 (±9.822). The expression of IFN-γ and its downstream cytokines CXCL9, CXCL10 and CXCL11 was higher in vitiligo patients than in healthy controls. By comparison, no significant changes in IL-15, IL-17A and IL-18 cytokines were observed.

[0170] Example 3: IFN-γ directly affects the cell viability and cell morphology changes of melanocytes

[0171] Functional studies were performed to explore the pathogenic effects of high expression of cytokines IFN-γ, CXCL9, CXCL10, and CXCL11 on melanocytes.

[0172] Materials and methods: To detect the dose dependence of IFN-γ inhibition of human primary melanocyte (HEMn-DP) cell viability, HEMn-DP cells were seeded into 96-well plates at a concentration of 10,000 cells / well in 100 μL assay medium (M254 supplemented with 1x HMGS, 10 ng / mL PMA plus penicillin-streptomycin) at 37°C for 5 hours. After seeding, 100 μL of serial dilutions of IFN-γ were added and then incubated at 37°C for 72 hours. Cell proliferation was then measured using the CellTiter-Glo kit (Promega) according to the manufacturer's instructions. Luminescence was measured using an iD3 multimode reader (Molecular Devices), and data were analyzed using nonlinear regression curve fitting using GraphPad Prism7.

[0173] To determine the efficacy of 2A6Q (ie, EI-001), HEMn-DP cells were seeded as previously described and pre-incubated with various concentrations of EI-001 in 100 μL assay medium for IFN-γ for 25 minutes. After incubation, the EI-001 mixture was added to each well (final IFN-γ concentration was 30 ng / mL), and the melanocytes were cultured for another 72 hours at 37°C, and the cell proliferation of the melanocytes was determined by luciferase activity using Celltiter Glo.

[0174] To determine the cell viability of melanocytes after treatment with IFN-γ, CXCL9, CXCL10, and CXCL11, cells were collected and seeded into 96-well plates on days 2 and 7. After adhesion, cells were treated with IFN-γ (0, 10, 100 ng / mL) and different compounds: 2A6Q, CXCL9, CXCL10, CXCL11, ROS scavenger (NAC), necroptosis inhibitor (NEC-1), cell pyroptosis inhibitor (VX-765), cell apoptosis inhibitor (QVD-OPH), ferroptosis inhibitor (Liproxstaitin-1, Ferrostatin-1, DFO). Cell viability was measured using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer's protocol. The luminometer ( iD3 Multi-Mode Microplate Reader (Molecular Devices)) measured the absorbance of the final reaction product. After calculation, the viability of the control cells was 100%, and all other cells were normalized to the control cells and expressed as relative cell viability (%). Treatment dose: N-acetylcysteine ​​(NAC; 0.05mM, 1mM), necrotizing inhibitor-1 (NEC-1; 20μM, 2μM), Belnacasan (VX-765; 50μg / mL, 5μg / mL), quinoline-Val Asp-difluorophenoxymethyl ketone (QVD-OPH; 10μM, 2μM), Liproxstaitin-1 (10μM, 5μM, 1μM, 0.05μM), Ferrostatin-1 (5μM, 1μM), Desferrioxamine (DFO, 10μM, 5μM, 1μM, 0.05μM), 2A6Q (10μg / mL), CXCL9 (10ng / mL, 10pg / mL), CXCL10 (10ng / mL, 10pg / mL), CXCL11 (10ng / mL, 10pg / mL).

[0175] Results: Figure 2A As shown, IFN-γ dose-dependently reduced the viability of melanocytes. Various concentrations of recombinant human IFN-γ were added to human primary melanocytes. The IC of IFN-γ that caused 50% inhibition of melanocyte viability was calculated. 50 is 8.114ng / mL. Figure 2B As shown in Figure 2, EI-001 neutralized the proliferation inhibitory effect of IFN-γ and restored cell viability. The calculated EC 50 It is 5.214ng / mL.

[0176] like Figure 2C As shown, IFN-γ significantly reduced the cell viability of melanocytes on day 7 of treatment, while CXCL9, CXCL10 or CXCL11 did not. The synergistic effects of IFN-γ, CXCL9, CXCL10 and / or CXCL11 on the cell viability of melanocytes were subsequently studied. Specifically, CXCL10 has been shown to enhance IFN-γ-mediated cytotoxicity in melanocytes. Interestingly, no synergistic effect was found between these cytokines. Therefore, IFN-γ itself is the most critical cytokine for melanocyte viability. IFN-γ directly affects the cell viability and cell morphology changes of melanocytes.

[0177] Type I IFN and IFN-γ share overlapping but not identical pathways through the JAK / STAT pathway. In cell morphology assays, IFN-γ was found to contribute to morphological changes in melanocytes, with loss of dendrites and expansion of melanocyte cytoplasm, whereas IFN-α did not. The effects of IFN-γ were dose-dependent and at concentrations as low as 1 ng / mL, whereas IFN-α was not.

[0178] Example 4: IFN-γ disrupts melanin production in melanocytes

[0179] To investigate the effect of IFN-γ on melanogenesis, melanocytes were treated with 100 ng / mL of IFN-γ and matched controls for transcriptome analysis.

[0180] Materials and methods: To examine the dose-dependency of IFN-γ inhibition of HEMn-DP cell melanin production, HEMn-DP cells were seeded at a concentration of 200,000 cells / well into 24-well plates in 1 mL of assay medium (M254 supplemented with 1x HMGS, 10 ng / mL PMA plus penicillin-streptomycin) with serial dilutions of IFN-γ, and then incubated at 37°C for 7 days. The cell assay medium was replaced with fresh medium with serially titrated concentrations of IFN-γ on days 3 and 5. On day 7, the cells were first washed twice with PBS, lysed in 250 μL of 1N NaOH / 10% DMSO solution, and then incubated at 80°C for 1 hour. After lysis, each lysate was transferred to a 96-well plate, and the absorbance of melanin was measured at 470 nM. The relative melanin amount was standardized with the cell viability of each sample measured by the CellTiter-Glo kit, and the data were analyzed using nonlinear regression curve fitting using GraphPad Prism7. In order to determine the efficacy of EI-001, HEMn-DP cells were inoculated as previously described, and IFN-γ was pre-incubated with a specified amount of EI-001 in 1 mL of assay medium for 25 minutes. After incubation, the mixture was added to each well (final IFN-γ concentration was 20 ng / mL). The melanin content of melanocytes was determined as previously described.

[0181] Total RNA was extracted from melanocytes using an RNA purification kit (Zymo Research, r2050). After RNA samples were extracted from primary melanocytes and melanocyte cell lines (Gibco), samples were sent to Biotools Co., Ltd Taiwan for RNA sequencing. RNA quality was detected using an RNA integrity value greater than 8 by Agilent Technologies 2100 Bio-analyzer. After rRNA depletion (Epicenter), RNA fragmentation and library preparation (Illumina), the constructed library was sequenced with 150 bp paired ends using an Illumina NovaSeq6000 sequencer. The results of each sample were compared with the GRCh38 genome by HISAT2 software and analyzed by Biotools Co., Ltd Taiwan.

[0182] The cells were washed in PBS and lysed in RIPA lysis buffer with protease inhibitors and phenylmethanesulfonyl fluoride (PMSF; Sigma-Aldrich, P7626). The total cell lysate was sonicated and centrifuged at 4°C, 13000rpm for 30min. The protein concentration was quantified using Bradford assay. The total cell lysate was separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% BSA or 5% milk in TBST for 1 hour at room temperature and incubated with the primary antibody overnight at 4°C and with the HRP-conjugated secondary antibody for 1 hour at room temperature. The proteins were visualized by Thermo ibright1500. The primary antibodies were as follows: anti-human SLC7A11 (CST, 12691), anti-human SLC3A2 (CST, 13180), anti-IRF1 (CST, 8478), anti-MITF (CST, 12590) and anti-GAPDH (Abcam, ab8245).

[0183] Results: Figure 3A As shown, IFN-γ dose-dependently reduced melanin production in melanocytes. The IC value of IFN-γ that caused 50% inhibition of melanin production in melanocytes was calculated. 50 is 0.392ng / mL. Figure 3B As shown, EI-001 neutralized the melanin production inhibitory effect of IFN-γ and restored melanin production in melanocytes. The calculated EC of IFN-γ that restored 50% inhibition of melanin production in melanocytes 50 is 129.1ng / mL. Figure 3C As shown, IFN-γ induces downregulation of melanin regulators (MITF, MLANA, TYRP1, DCTT, TYR, TRPM1) in primary melanocytes. To further confirm this finding, immunoblotting of MITF (a key regulator of melanin production) was performed in melanocytes 8 hours and 24 hours after treatment with different doses of IFN-γ. The results showed that at 24 hours of treatment, IFN-γ dose-dependently downregulated the expression of MITF in melanocytes. In addition, spectrophotometry further confirmed that IFN-γ treatment reduced melanin content in a dose-dependent manner, which could be rescued by adding 2A6Q.

[0184] Example 5: EI-001 inhibits CXCR3 expression on CD4+ and CD8+ T cells

[0185] This example demonstrates that the IFN-γ neutralizing antibody EI-001 (2A6Q) inhibits CXCR3 expression on CD4+ and CD8+ T cells during T cell activation.

[0186] Materials and Methods: PBMCs from healthy adult donors were isolated from whole blood using Ficoll-Paque density gradient centrifugation. PBMCs were incubated for 24 h in a humidified 37 °C incubator in RPMI-1640 medium supplemented with 5% human serum and standard antibiotics, 2-ME, and HEPES. PBMCs were then plated at 1 × 10 6 10 cells / well and 1 mL of fresh medium / well were seeded in a 24-well plate and stimulated by mixing the cells with human T cell TransAct (1:200). The antibodies indicated in the results were then added and mixed thoroughly. After 2 days of culture, PBMCs were collected, washed 3 times with PBS, resuspended in culture medium, and cultured with the indicated antibodies for another 2 days.

[0187] PBMCs were processed as required (see above) and then harvested for flow cytometry antibody staining using anti-human CD4, anti-human CD8, anti-human CD56 and anti-human CXCR3 antibodies on ice in the dark for 30 minutes. PBMCs were then washed twice with FACS buffer and the expression of CXCR3 was determined by flow cytometry using an Attune NxT flow cytometer. Flow cytometry data analysis was performed using Flow Jo software.

[0188] Results: Figure 4 As shown, to understand the inhibitory ability of EI-001 in CXCR3 expression on T cells, EI-001 (2A6Q) was added to the culture medium during the activation of PBMCs. The expression of CXCR3 on CD4+ T cells and CD8+ T cells was determined by flow cytometry. The IC of EI-001 inhibiting CXCR3 expression on CD4+ T cells 50 The IC of CXCR3 expression on CD8+ T cells was 0.279-1.313 μg / mL. 50 1.029-6.941 μg / mL( Figure 4 ). This data supports the conclusion that neutralization of IFN-γ by EI-001(2A6Q) inhibits CXCR3 expression on CD4 and CD8+ T cells during T activation.

[0189] Example 6: IFN-γ induces early apoptosis in melanocytes

[0190] This example demonstrates that IFN-γ induces early apoptosis in melanocytes, which can be rescued by 2A6Q.

[0191] Materials and methods: Apoptosis of melanocytes was detected using Annexin V (BD, 550474) and 7AAD (1 mg / mL, Sigma A9400-1MG). Cells were isolated and resuspended in 100 mL of binding buffer (BD, 556454) containing Annexin V and 7AAD for 15 minutes at room temperature in the dark. Prior to analysis, cells were added to 300 mL of binding buffer and immediately analyzed on a flow cytometer (Verse BD).

[0192] result: Figure 5 Figure 7: IFN-γ+ induces early apoptosis in melanocytes in a dose-dependent manner, as confirmed by Annexin V, and 2A6Q is able to reverse apoptosis. However, pan-caspase inhibitors cannot rescue IFN-γ-induced cell death in melanocytes, suggesting that in addition to inducing apoptosis, there are other alternative pathways that contribute to cell loss in melanocytes. (Figure 7A)

[0193] Example 7: IFN-γ increases ROS lipid peroxidation and induces ferroptotic cell death in melanocytes

[0194] It has been reported that IFN-γ induces ferroptosis and lipid peroxidation during melanoma cancer immunotherapy, contributing to the release of damage-associated molecular patterns (DAMPs) in melanoma tumors during cancer immunotherapy. Similar studies have not been performed on normal melanocytes.

[0195] Materials and methods: For intracellular ROS measurement, melanocytes were washed with PBS and incubated with 20 μM CM-H2DCFDA (Invitrogen) at 37°C in the dark for 30 min. Cells were resuspended with PBS and detected by flow cytometry at an excitation wavelength of 488 nm and an emission wavelength of 530 nm; data were analyzed using FlowJo.

[0196] To detect lipid peroxidation, melanocytes were seeded in 6-well plates and treated with IFN-γ the next day. Melanocytes were washed with PBS and incubated with Hanks balanced salt solution (HBSS, Gibco) containing 5 μM BODIPY 581 / 591C11 (Invitrogen, D3861) and incubated at 37°C in a tissue culture incubator for 15 minutes. Cells were resuspended with fresh HBSS and detected by flow cytometry; data were analyzed using FlowJo.

[0197] Results: Figure 6As shown in A, IFN-γ significantly elevated intracellular ROS at 10 ng / mL and 100 ng / mL IFN-γ on day 7, as measured by 20 μM CMH2DCFDA. In addition, lipid peroxidation levels increased in a dose-dependent manner in melanocytes treated with IFN-γ on day 6 ( Figure 6 B). RNA sequencing analysis of melanocyte transcriptomic expression of ferroptosis markers showed downregulation of SLC7A11, SLC3A2, ferritin, GPX4, and TFR1 after 24 h of incubation with IFN-γ. ( Figure 6 C). Immunoblotting of SLC7A11 after 48 h of incubation with IFN-γ also showed that IFN-γ dose-dependently downregulated the expression of SLC7A11 in melanocytes, suggesting a ferroptotic process. ( Figure 6 D)

[0198] Example 8: Human anti-IFN-γ monoclonal antibody (2A6Q) reverses melanocyte cell death and melanin production in melanocytes

[0199] Melanocyte loss is a hallmark of vitiligo; therefore, we aimed to address the effects of ROS scavengers and other anti-death molecules to rescue IFN-γ-mediated melanocyte loss.

[0200] Results: As shown in Figure 7, N-acetylcysteine ​​(NAC) was provided to the culture medium as a ROS scavenger, but no rescue effect of NAC was found. Interestingly, 2A6Q reversed IFN-γ-induced ROS in melanocytes. To investigate the induced melanocyte cell death rather than apoptosis, cell viability studies were repeated in melanocytes treated with IFN-γ for 7 days using necroptosis inhibitors (NEC-1), pyroptosis inhibitors (VX-765), apoptosis inhibitors (QVD-OPH, a pan-caspase inhibitor), and 2A6Q. (Figure 7A). Ferropoptosis inhibitors Ferrostatin-1, DFO, and Liproxstaitin-1 were used to study IFN-γ-induced ferroptosis in melanocytes. The results showed that 1 μM Liproxstaitin-1 could partially rescue IFN-γ-induced melanocyte loss. In contrast, the rescue effect of 2A6Q was more significant than that of all other cell death inhibitors. In addition, 2A6Q also reversed the downregulation of MITF and the decrease in melanin content ( Figure 3C ), early apoptosis ( Figure 5 B), cellular oxidative stress ( Figure 6 A), lipid peroxidation ( Figure 6 B) and reversed ferroptosis confirmed by SLC7A11 in melanocytes. ( Figure 6D).

[0201] Example 9: 2A6Q is well tolerated and shows few side effects at all dose levels compared to NI-0501

[0202] This example demonstrates that the IFN-γ neutralizing antibody EI-001 (2A6Q) is well tolerated and exhibits a better side effect profile than another anti-IFN-γ antibody (NI-0501) when potential toxicity is assessed after 4 weeks of repeated intravenous infusion of EI001.

[0203] Materials and methods: A total of 40 cynomolgus monkeys (20 females and 20 males) were divided into 4 groups, with 5 animals / sex / group, and EI-001 (2A6Q) (10, 30 and 100 mg / kg) or vehicle (formulation buffer) was administered by 30-minute intravenous infusion in a volume of 10 mL / kg, once a week, for a total of 5 doses, followed by a 6-week recovery period. All animals were planned to be autopsied at the end of the main phase (day 30) and at the end of the recovery phase (day 72). During the study, the following parameters were evaluated: clinical observations, ophthalmology, body weight, food consumption, body temperature (rectal temperature), clinical pathology (hematology, coagulation, plasma chemistry, lymphocyte immunophenotype and urinalysis), gross pathology, organ weight and histopathology. Toxicokinetics, immunogenicity, local irritation assessment, cardiovascular (ECG, skin temperature, blood pressure), respiratory and central nervous system safety pharmacology assessments were performed simultaneously. Toxicokinetic samples were collected before dosing, immediately after the first and fourth doses, and at 1 hour, 4 hours, 8 hours, 24 hours, 72 hours, 120 hours, and 168 hours. Immunogenicity samples were collected before dosing on days 1, 15, and 22 and on days 57 and 71.

[0204] Results: As shown in Table 3, all animals treated with EI-001(2A6Q) survived to their scheduled necropsy. No EI-001(2A6Q)-related changes were found in this study in clinical observations, ophthalmology, body weight, food consumption, local irritation, body temperature (rectal temperature), safety pharmacology evaluations (cardiovascular parameters, respiratory and central nervous system), clinical pathology (hematology, coagulation, plasma chemistry and urinalysis), lymphocyte immunophenotype, gross pathology or organ weights. In addition, only changes associated with EI-001(2A6Q) administration were observed at the microscopic level. Compared to prior toxicology studies using the anti-IFN-γ antibody NI-0501 (NDA / BLA Multi-Disciplinary Review and Evaluation application number: 761107; www.accessdata.fda.gov / drugsatfda_docs / nda / 2018 / 761107Orig1s000MultidisciplineR.pdf), treatment with EI-001 (2A6Q) did not increase mortality or susceptibility to infection in treated monkeys. In addition, treatment with EI-001 (2A6Q) did not induce observable symptoms of infection. These findings indicate that EI-001 (2A6Q) does not induce any signs of infection. The overall results suggest that treatment with EI-001 (2A6Q) may be a safer and potentially more effective method for neutralizing IFN-γ. Therefore, EI-001 (2A6Q) has a better therapeutic effect in treating diseases induced by IFN-γ such as vitiligo.

[0205]

[0206] In summary, we demonstrated that single agents that block apoptosis, necroptosis, ferroptosis, and ROS production cannot protect melanocytes from IFN-γ-mediated cytotoxicity. These observations suggest that the cytotoxic effects of IFN-γ are mediated through multiple pathways. In contrast to blocking selected cell death pathways, our in vitro findings support the rescue effect of 2A6Q on IFN-γ-induced cell death, oxidative stress, and loss of melanocyte function by interrupting IFN-γ signaling, which is a potential therapeutic option for vitiligo. In addition, in vivo toxicology results showed that EI-001(2A6Q) treatment did not cause any significant adverse drug events. Therefore, EI-001(2A6Q) may be a safe and promising therapeutic strategy for vitiligo.

[0207] Although the foregoing disclosure of the present invention has been described in considerable detail by way of example and illustration for purposes of clarity and understanding, the present disclosure including the embodiments, descriptions and embodiments described herein is for purposes of illustration, which is intended to be exemplary and should not be construed as limiting the present disclosure. It will be clear to those skilled in the art that various modifications or changes may be made to the embodiments, descriptions and embodiments described herein, and these modifications or changes will be included in the spirit and scope of the present disclosure and the appended claims. In addition, those skilled in the art will recognize many methods and procedures comparable to those described herein. All these equivalents are understood to be within the scope of the present disclosure and are covered by the appended claims. Other embodiments of the present invention are set forth in the following claims.

Claims

1. A method for treating vitiligo, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier.

2. The method of claim 1, wherein the IFN-γ antibody modulates the biological function of IFN-γ.

3. The method of claim 1, wherein the IFN-γ antibody neutralizes IFN-γ. 4 . The method according to claim 1 , wherein the IFN-γ antibody inhibits, reduces and / or completely blocks the signaling activity of IFN-γ.

5. The method of claim 4, wherein the IFN-γ antibody reduces the IFN-γ signaling activity of native human IFN-γ, mature human IFN-γ, or truncated human IFN-γ.

6. The method of claim 1, wherein the IFN-γ antibody reduces one or more of: IFN-γ-dependent cytokine production; IFN-γ-dependent T cell dysfunction, IFN-γ-dependent immune tolerance, and IFN-γ-dependent inflammation.

7. The method of claim 1, wherein the IFN-γ antibody reduces the expression of IFN-γ, CXCL9, CXCL10 and / or CXCL11 in melanocytes.

8. The method of any one of claims 1-7, wherein the IFN-γ antibody increases IL-2 production and / or cell proliferation of SEB-stimulated human PBMCs by at least 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, at least 1.9-fold, at least 2-fold, at least 2.1-fold, or at least 2.20-fold.

9. The method according to any one of claims 1 to 9, wherein the IFN-γ antibody comprises: A VH region having a CDR-H1 amino acid sequence selected from SEQ ID NO: 120 or 123, a CDR-H2 amino acid sequence selected from SEQ ID NO: 121 or 124, and a CDR-H3 amino acid sequence selected from SEQ ID NO: 122 or 125; and A VL region comprising a CDR-L1 amino acid sequence selected from SEQ ID NO: 132 or 135, a CDR-L2 amino acid sequence selected from SEQ ID NO: 133 or 136, and a CDR-L3 amino acid sequence selected from SEQ ID NO: 134 or 137.

10. The method according to any one of claims 1-10, wherein the IFN-γ antibody comprises: (a) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 120, a CDR-H2 amino acid sequence of SEQ ID NO: 121, and a CDR-H3 amino acid sequence of SEQ ID NO: 122, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 132, a CDR-L2 amino acid sequence of SEQ ID NO: 133, and a CDR-L3 amino acid sequence of SEQ ID NO: 134; (b) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 123, a CDR-H2 amino acid sequence of SEQ ID NO: 124, and a CDR-H3 amino acid sequence of SEQ ID NO: 125, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 135, a CDR-L2 amino acid sequence of SEQ ID NO: 136, and a CDR-L3 amino acid sequence of SEQ ID NO: 137; or (c) a VH region having a CDR-H1 amino acid sequence of SEQ ID NO: 123, a CDR-H2 amino acid sequence of SEQ ID NO: 124 and a CDR-H3 amino acid sequence of SEQ ID NO: 125, and a VL region comprising a CDR-L1 amino acid sequence of SEQ ID NO: 132, a CDR-L2 amino acid sequence of SEQ ID NO: 133 and a CDR-L3 amino acid sequence of SEQ ID NO:

134.

11. The method according to any one of claims 10-11, wherein the IFN-γ antibody comprises: (a) a VH region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 109, 110, 164 or 165; and (b) a VL region comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 113 or 114.

12. The method according to any one of claims 1-12, wherein the IFN-γ antibody comprises: (a) the VH region amino acid sequence of SEQ ID NO: 109 and the VL region amino acid sequence of SEQ ID NO: 113; (b) the VH region amino acid sequence of SEQ ID NO: 110 and the VL region amino acid sequence of SEQ ID NO: 114; (c) the VH region amino acid sequence of SEQ ID NO: 109 and the VL region amino acid sequence of SEQ ID NO: 114; (d) the VH region amino acid sequence of SEQ ID NO: 110 and the VL region amino acid sequence of SEQ ID NO: 113; (e) the VH region amino acid sequence of SEQ ID NO: 164 and the VL region amino acid sequence of SEQ ID NO: 113; or (f) the VH region amino acid sequence of SEQ ID NO:165 and the VL region amino acid sequence of SEQ ID NO:

113.

13. The method according to any one of claims 10-13, wherein the IFN-γ antibody comprises: (a) a heavy chain amino acid sequence that is at least 90% identical to SEQ ID NO: 183, 185, 187 or 189; and (b) a light chain amino acid sequence that is at least 90% identical to SEQ ID NO: 184 or 186.

14. The method according to any one of claims 1-14, wherein the IFN-γ antibody comprises: (a) the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 184; (b) the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 186; (c) the heavy chain amino acid sequence of SEQ ID NO: 183 and the light chain amino acid sequence of SEQ ID NO: 186; (d) the heavy chain amino acid sequence of SEQ ID NO: 185 and the light chain amino acid sequence of SEQ ID NO: 184; (e) the heavy chain amino acid sequence of SEQ ID NO: 187 and the light chain amino acid sequence of SEQ ID NO: 184; or (f) the heavy chain amino acid sequence of SEQ ID NO:189 and the light chain amino acid sequence of SEQ ID NO:

184.

15. The method of any one of claims 10-15, wherein the VH region of the IFN-γ antibody further comprises an amino acid substitution selected from the group consisting of N76A and N76Q.

16. The method of any one of claims 1-16, wherein the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501, and fontuzumab.

17. The method of claim 17, wherein infection or drug-induced side effects are not induced by 2A6Q in an in vivo toxicology assay.

18. The method of any one of claims 1-18, wherein the IFN-γ antibody is an immunoglobulin molecule, Fv, disulfide-linked Fv, monoclonal antibody, scFv, chimeric antibody, single domain antibody, CDR-grafted antibody, diabody, human antibody, humanized antibody, multispecific antibody, Fab, bispecific antibody, Fab' fragment, bispecific antibody, F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR), or a single chain antibody.

19. The method of any one of claims 1-18, wherein the subject is administered by at least one route selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal, and transdermal.

20. The method of any one of claims 1-19, wherein the subject in need thereof is a patient suffering from vitiligo.

21. The method of any one of claims 1-20, wherein the composition is administered to the subject more than once a day, at least once a day, at least once a week, or at least once a month.

22. The method of any one of claims 1-20, further comprising administering at least one additional therapeutic agent.

23. The method of claim 22, wherein the additional therapeutic agent is administered to the subject in need thereof prior to, after, and / or simultaneously with the administration of the composition.

24. The method according to any one of claims 1 to 23, wherein the IFN-γ antibody is present at 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 Binds to human IFN-γ with an affinity of M or less.

25. The method according to any one of claims 1 to 24, wherein the IFN-γ antibody is present in an amount of 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 M or less binding affinity to rhesus IFN-γ or cynomolgus IFN-γ.

26. The method according to any one of claims 1 to 25, wherein the IFN-γ antibody is present at 1×10 -8 M or smaller, 1×10 -9 M or smaller, 1×10 -10 M or smaller or 1×10 -11 It binds to human IFN-γ and cynomolgus monkey IFN-γ with a binding affinity of M or less.

27. Use of a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for treating vitiligo in a subject in need thereof.

28. Use of a composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for preparing a medicament for treating vitiligo in a subject in need thereof.

29. The use according to claim 27 or 28, wherein the composition is used together with at least one additional therapeutic agent.

30. The use according to claim 27 or 28, wherein the composition is administered by at least one mode selected from the group consisting of parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, oral, sublingual, intranasal, and transdermal.

31. The use according to claim 27 or 28, wherein the composition is for use more than once a day, at least once a day, at least once a week or at least once a month.

32. The method of claim 27 or 28, wherein the IFN-γ antibody is an immunoglobulin molecule, an Fv, a disulfide-linked Fv, a monoclonal antibody, a scFv, a chimeric antibody, a single domain antibody, a CDR-grafted antibody, a diabody, a human antibody, a humanized antibody, a multispecific antibody, a Fab, a bispecific antibody, a Fab' fragment, a bispecific antibody, a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; a Fd fragment consisting of a VH and CH1 domain; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, a dAb fragment, an isolated complementarity determining region (CDR) or a single-chain antibody.

33. The use according to claim 27 or 28, wherein the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501 and fontuzumab.

34. A composition comprising a therapeutically effective amount of an IFN-γ antibody and a pharmaceutically acceptable carrier for use in treating vitiligo in a subject in need thereof.

35. The composition of claim 34, wherein the IFN-γ antibody is an antibody selected from the group consisting of 2A6, 2B6, 2A6A, 2A6Q, AB, BA, AMG811, NI-0501 and fontuzumab.

Images