Methods of treating vitiligo

Abstract

The instant disclosure relates to the use of an anti-CXCL-10 antibody for the treatment of vitiligo. Also provided are specific doses and administration protocols as well as criteria for patient selection and endpoints of efficacy of these methods of treatment.

Description

Attorney Docket No.: 96555819 / EDBI-015 / 001 WOMETHODS OF TREATING VITILIGOCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Provisional Patent Application No. 63 / 685,040 filed August 20, 2024, which is incorporated herein by reference in its entirety.INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing XML associated with this application is provided electronically in XML file format and is hereby incorporated by reference into the specification. The name of the XML file containing the Sequence Listing XML is “EDBI-015_001WO_SeqList.xml”. The XML file is 10,333 bytes, created on August 19, 2025, and is being submitted electronically via USPTO Patent Center.FIELD

[0003] The instant disclosure relates to the use of an anti-CXCL-10 antibody for the treatment of vitiligo. Also provided are specific doses and administration protocols as well as criteria for patient selection and endpoints of efficacy of these methods of treatment.BACKGROUND

[0004] Vitiligo is an autoimmune disease characterized by patches of depigmented skin (lesions) as a result of the destruction of melanocytes which are essential for melanin production. With the global prevalence ranging between 0.5 - 2% of the population, this disease has a high psychological impact on patients due to its presence on visible regions of the body such as the face and hands and the unpredictability of disease progression (Bibeau et al. 2022; Kruger and Schallreuter 2012; Nguyen et al. 2016). Vitiligo can be classified into two forms: non segmental vitiligo accounts for 90% of the cases and is characterized by symmetrical and bilateral lesions whereas segmental vitiligo is much less common and characterized by unilateral lesions (Ezzedine et al. 2015; Seneschal et al. 2021).

[0005] Current treatment options available for vitiligo remain far from ideal, often are not universally effective in all patient populations, and do not repigment all locations. Immunosuppression via topical corticosteroids and calcineurin inhibitors as well as systemic corticosteroid treatment help stabilize active disease, while phototherapy is often used for1305304127repigmentation (Frisoli et al. 2020; Seneschal and Boniface 2023). Currently only one treatment has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of vitiligo, Opzelura (ruxolitinib), a topical Janus Kinase (JAK)-inhibitor. Due to the long-term safety concerns of the currently available therapeutics, a novel systemic, safe, and effective targeted treatment option for vitiligo patients remains a critical unmet medical need.

[0006] It has been well established that the immune system plays a crucial role in targeting melanocytes for destruction in vitiligo (Migayron et al. 2020). Genome-wide association studies clearly demonstrated the association of vitiligo with genes related to both the innate and adaptive immune response (Tulic et al. 2019). Furthermore, both human cohort studies and animal models support the essential role the immune system plays in vitiligo pathogenesis (Frisoli and Harris 2017). Specifically, the IFN-y / CXCLIO pathway has been implicated in both the innate and adaptive immune response in vitiligo; therefore, inhibition of CXCL10 represents an intriguing potential therapeutic avenue to target both arms of the immune response in vitiligo.

[0007] Evidence suggests that the innate immune response is activated early on in vitiligo due to stress signals released from melanocytes and keratinocytes followed by subsequent activation of the adaptive immune response (Bergqvist and Ezzedine 2021). Studies have suggested that due to endogenous or exogenous stress, NK and ILC1 cells produce IFN-y which in turn induces the production of CXCL10 by keratinocytes and melanocytes (Richmond et al. 2017a; Tulic et al. 2019). CXCL10 in turn binds to CXCR3B on melanocytes and induces the initial apoptosis of melanocytes demonstrating the importance of the innate immune system, and particularly CXCL10, in the initiation of vitiligo.

[0008] Through examination of the adaptive immune response in vitiligo, it has been well established that cytotoxic CD8+ T cells play a crucial role in the destruction of melanocytes. These cytotoxic CD8+ T cells infiltrate the perilesional skin and localize to the epidermis and dermis adjacent to melanocytes (Badri et al. 1993; Bergqvist and Ezzedine 2021; Le Poole et al. 1996; Ogg et al. 1998; Wankowicz-Kalinska et al. 2003). The IFN-y pathway is crucial to recruitment of these autoreactive CXCR3+CD8+ T cells to the skin (Harris et al. 2012).CXCL10 creates a chemoattractant gradient that is essential in the trafficking of CD8+ T cells to the skin. More importantly, in mice where depigmentation was already established, neutralization of CXCL10 (using an anti-CXCLIO antibody) led to regimentation indicating that CXCL10 not only plays a role in the progression of vitiligo but also its maintenance (Rashighi et2305304127al. 2014). Overall, multiple studies clearly demonstrate the importance of targeting CXCL10 signaling through CXCR3 to prevent the recruitment of cytotoxic CD8+ T cells to skin and the destruction of melanocytes.

[0009] EB06 (formerly known as NI-0801) is a fully human IgG I Z2 monoclonal antibody (mAh) that binds to the human chemokine CXCL10 (C-X-C motif chemokine ligand 10) with high affinity.

[0010] EB06 is being developed for the treatment of autoimmune conditions including vitiligo. Evidence from both animals and humans has highlighted CXCL10 as a key mediator in both the initiation and progression of vitiligo. As a potent inhibitor of CXCL10, EB06 may inhibit both mechanisms of melanocyte cell death mediated by CXCL10. Edesa is evaluating whether CXCL10 inhibition by EB06 can reduce melanocyte cell death and thus prevent and reverse depigmentation in vitiligo patients.SUMMARY

[0011] In one aspect, provided herein is a method of treating vitiligo in a subject in need thereof, the method comprising administering to the subject 2 mg / kg to 25 mg / kg of an anti-CXCL-10 antibody comprising a variable heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3 and a variable light chain comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO 5 and a CDR3 of SEQ ID NO: 6.

[0012] . In some embodiments, the anti-CXCL-10 antibody is administered at a dose of 2.5 mg / kg. In some embodiments, the anti-CXCL-10 antibody is administered at a dose of 5 mg / kg. In some embodiments, the anti-CXCL-10 antibody is administered at a dose of 10 mg / kg. In some embodiments, the anti-CXCL-10 antibody is administered at a dose of 20 mg / kg. In some embodiments, the anti-CXCL-10 antibody is administered for a period of 24 weeks. In some embodiments, the anti-CXCL-10 antibody is administered for a period of 52 weeks. In some embodiments, the anti-CXCL-10 antibody is administered every other week. In some embodiments, the anti-CXCL-10 antibody is administered by intravenous infusion.

[0013] In some embodiments, the subject achieves a 50% improvement in the facial Vitiligo Area Scoring Index at week 24. In some embodiments, the subject achieves a 50% improvement in the total Vitiligo Area Scoring Index at week 24. In some embodiments, the subject achieves a score of 4 or 5 on the Vitiligo Noticeability Scale at Week 24. In some embodiments, the subject has at least a 25% improvement in Vitiligo Extent Score from prior to the administration within330530412724 weeks. In some embodiments, the body surface area affected by vitiligo decreases by at least 25% from prior to the administration within 24 weeks. In some embodiments, the vitiligo is nonsegmented vitiligo.BRIEF DESCRIPTION OF DRAWINGS

[0014] FIG. 1 shows inhibition of CXCLIO-induced calcium flux by EB06 at different concentrations of EB06 in the presence of 10 nM of CXCL10 in LI.2 cells expressing hCXCR3 previously loaded with the calcium sensitive fluorescent dye Fura 2 / AM. The calcium flux kinetics were recorded by measuring the fluorescence at 514 nm upon excitation at 340 or 380 nm.

[0015] FIG. 2 shows blocking of CXCLIO-induced cells chemotaxis by EB06 in LI.2 cells. L1.2-hCXCR3 cells were allowed to migrate through a 96-well chemotaxis filter plate in response to 1 nM of recombinant human CXCL10 and in the presence of varying concentrations ofEB06.

[0016] FIG. 3 shows activity of EB06 on CXCL10. EB06 and A2 were captured in ELISA plates with anti-human Fc. Fluorescein isothiocyanate (FITC)-labeled heparin was used as a prototypic GAG and was incubated with hCXCLIO before addition to the captured anti-hCXCLIO antibodies. Binding was revealed using a horseradish peroxidase (HRP)-coupled anti-FITC Fab and a fluorescent substrate.

[0017] FIG. 4 shows inhibition of lymphocyte transendothelial migration across human sinusoidal endothelial cells by EB06 under conditions of physiological flow.

[0018] FIG. 5 shows specificity of EB06 for human CXCL10 in a panel of human chemokines expressed as fusion proteins with N-Utilization Substance Protein A (NusA). EB06 was immobilized and different amounts of the chemokine fusion protein, as well as NusA control, were added. Binding was revealed with an anti-NusA antibody and an HRP-conjugated secondary antibody, using a fluorescent substrate.

[0019] FIG. 6 shows EB06 binding to mutated variants of rabbit CXCL10. Four variants of rabbit CXCL10 were expressed as NusA fusion proteins. The proteins were coated into Maxisorb plates and different concentrations of EB06 added to the wells. The signal was revealed with a secondary anti-human Fc antibody and a chromogenic substrate.

[0020] FIG. 7 shows a schematic summarizing the clinical trial protocol described in Example 3.4305304127DETAILED DESCRIPTION

[0021] Provided herein are methods of treating vitiligo in a subject in need thereof comprising the administration of an anti-CXCLIO antibody.Anti-CXCLIO Antibodies

[0022] The methods described herein comprise the administration of an antibody targeting CXCL-10 (also known as IP-10). In some embodiments, the anti-CXCLIO antibody is an antibody described in International Patent Application Publication No. WO 2008 / 106200, which is incorporated herein by reference in its entirety for examples of anti-CXCLIO antibodies that may be used in the methods described herein. In some embodiments, an anti-CXCLIO antibody used in the methods disclosed herein is EB06, also known as NI-0801. The CDR sequences of EB06 are set forth in Table 1.Table 1: EB06 CDR Sequences0023] In some embodiments, the anti-CXCLIO antibody comprising a variable heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3 and a variable light chain comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO 5 and a CDR3 of SEQ ID NO: 6.

[0024] In some embodiments, the antibody comprises a VH sequence of SEQ ID NO: 7: QVQLVESGGGVVQPGRSLRLSCAASGFTFSNSGIHWVRQAPGKGLEWVAVISYDGSNKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLRDNAEYTDYWGQGTLVTVSS (SEQ ID NO: 7).

[0025] In some embodiments, the antibody comprises a VL sequence of SEQ ID NO: 8: NFMLTQPHSVSESPGKTVTISCTGSGGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGS IDSSSNSASLTISGLKTEDEADYYCQSYDPLPVWVFGGGTKLTVLGQP (SEQ ID NO: 8).

[0026] In some embodiments, the antibody comprises a light chain sequence of SEQ ID NO: 9: NFMLTQPHSVSESPGKTVTISCTGSGGSIASNYVQWYQQRPGSSPTTVIYEDNQRPSGVPDRFSGS IDSSSNSASLTISGLKTEDEADYYCQSYDPLPVWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQA NKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSY SCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 9)5305304127

[0027] In some embodiments, the antibody comprises a heavy chain sequence of SEQ ID NO: 10: QVQLVESGGGVVQPGRSLRLSCAASGFTFSNSGIHWVRQAPGKGLEWVAVISYDGSNKYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLRDNAEYTDYWGQGTLVTVSSASTKGPS VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWE SNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC2 (SEQ ID NO: 10).

[0028] The anti-CXCLIO antibody used in the methods disclosed herein may be formualted in any suitable pharmaceutically acceptable carrier. The antibodies may be provided in any suitable unit dosage compositions.Patients and Indications

[0029] The methods described herein are useful for the treatment of vitiligo. Vitiligo is an autoimmune disease characterized by patches of depigmented skin (lesions) as a result of the destruction of melanocytes which are essential for melanin production. In some embodiment, a subject treated in accordance with a method described herein has non-segmental vitiligo. Non- segmental vitiligo may be diagnosed by depigmented area including > 0.5% body surface area on the face or > 5% total body surface area, a Facial Vitiligo Area Scoring Index (F-VASI) of 0.5 or more, or a Total Vitiligo Area Scoring Index (T-VASI) of 4 or more.

[0030] The terms “subject” and “patient” are used interchangeably herein. In preferred embodiments, the patient is human.Doses and Administration

[0031] In certain embodiments, the anti-CXCLIO antibody is administered at a dose of about 0.5 mg / kg body weight to about 25 mg / kg body weight. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 0.5 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 1.5 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 2 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 2.5 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 5 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 10 mg / kg. In some embodiments, the anti-CXCLIO6305304127antibody is administered at a dose of about 15 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 20 mg / kg. In some embodiments, the anti-CXCLIO antibody is administered at a dose of about 25 mg / kg.

[0032] In some aspect, a subject is administered a total amount of 125 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 150 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 175 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 200 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 225 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 250 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 300 mg of the anti- CXCLIO antibody. In some aspect, a subject is administered a total amount of 450 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 600 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 900 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 1200 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 1500 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 1800 mg of the anti-CXCLIO antibody. In some aspect, a subject is administered a total amount of 2100 mg of the anti-CXCLIO antibody.

[0033] In some aspects, the anti-CXCLIO antibody may be administered for a period of about 12 to 64 weeks or longer, for example up to 2 years. In some embodiments, the anti-CXCLIO antibody is administered for a period of 12 weeks. In some embodiments, the anti-CXCLIO antibody is administered for a period of 24 weeks. In some embodiments, the anti-CXCLIO antibody is administered for a period of 36 weeks. In some embodiments, the anti-CXCLIO antibody is administered for a period of 52 weeks. In some embodiments, the anti-CXCLIO antibody is administered for a period of 64 weeks. In some embodiments, the anti-CXCLIO antibody is administered for a period of 3 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 6 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 9 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 12 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 15 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 18 months. In some embodiments, the anti-CXCLIO7305304127antibody is administered for a period of 21 months. In some embodiments, the anti-CXCLIO antibody is administered for a period of 24 months.

[0034] The frequency of the administration may be varied depending on the patient’s progress and disease severity. In some embodiments, the anti-CXCLIO antibody is administered twice per week. In some embodiments, the anti-CXCLIO antibody is administered weekly. In some embodiments, the anti-CXCLIO antibody is administered every other week. In some embodiments, the anti-CXCLIO antibody is administered once a month.

[0035] The anti-CXCLIO antibody may be administered intravenously, preferably by intravenous infusion. In some embodiments, the anti-CXCLIO antibody is administered by intravenous infusion over the course of about 1 hour. In some embodiments, the anti-CXCLIO antibody is administered by intravenous infusion over the course of about 2 hours. In some embodiments, the anti-CXCLIO antibody is administered by intravenous infusion over the course of about 3 hours.Endpoints of Efficacy

[0036] Any suitable measurement may be used to monitor a patient’s progress and the efficacy of treatment. Examples of such endpoints include improvements in the area affected by vitiligo and the noticeability of the vitiligo, as well as quality of life.Vitiligo Area Scoring Index

[0037] One example of an endpoint to measure the efficacy of a method of treatment disclosed herein is the Vitiligo Area Scoring Index (VASI), which measures re-pigmentation (see, e.g., Kawakami and Hashimoto, Dermatol Res Pract. 2011; 2011 : 750342). VASI can be applied to the face (facial VASI or F-VASI) or the total body (total VASI or T-VASI).

[0038] In some embodiments, a method disclosed herein results in an improvement in the F- VASI of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% compared to the F- VASI before the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement in the F-VASI of at least about 25%, at least about 50%, at least about 75%, or at least about 90% compared to the F-VASI before the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement in the F-VASI of about 25%, about 50%, about 75%, or about 90%8305304127compared to the F-VASI before the administration of the anti-CXCLIO antibody. The F-VASI may be assessed at any suitable point after the administration. For example, F-VASI may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.

[0039] In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 50% improvement in F- VASI compared to the F-VASI before the administration of the anti-CXCLIO antibody. In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 75% improvement in F-VASI compared to the F-VASI before the administration of the anti-CXCLIO antibody. In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 90% improvement in F-VASI compared to the F-VASI before the administration of the anti-CXCLIO antibody. The F-VASI may be assessed at any suitable point after the administration. For example, F-VASI may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.

[0040] In some embodiments, a method disclosed herein results in an improvement in the T- VASI of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% compared to the T- VASI before the administration of the anti-CXCL-10 antibody. In some embodiments, a method disclosed herein results in an improvement in the T-VASI of at least about 25%, at least about 50%, or at least about 75% compared to the T-VASI before the administration of the anti- CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement in the T-VASI of about 25%, about 50%, or about 75% compared to the T-VASI before the administration of the anti-CXCLIO antibody. The T-VASI may be assessed at any suitable point after the administration. For example, T-VASI may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.

[0041] In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 50% improvement in T- VASI compared to the T-VASI before the administration of the anti-CXCLIO antibody. In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 75% improvement in T-VASI compared to9305304127the T-VASI before the administration of the anti-CXCLIO antibody. In some embodiments, at least about 25%, about 50%, about 75%, or about 90% of subjects treated in accordance with a method disclosed herein achieve a 90% improvement in T-VASI compared to the T-VASI before the administration of the anti-CXCLIO antibody. The T-VASI may be assessed at any suitable point after the administration. For example, T-VASI may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.Vitiligo Noticeability Scale

[0042] Another endpoint of efficacy for the treatment of vitiligo is the Vitiligo Notability Scale (VNS), which is a patient-rated outcome measure of vitiligo treatment response that assigns a score of 1-5, with 1 being more noticeable, 2 being as noticeable, 3 being slightly less noticeable, 4 being a lot less noticeable and 5 being no longer noticeable (see, e.g., Batchelor et al., Br J Dermatol. 2016 Feb; 174(2): 386-394).

[0043] In some embodiments, a method disclosed herein results in an improvement of 1 point on the VNS compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 2 points on the VNS compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 3 points on the VNS compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 4 points on the VNS compared to the score prior to the administration of the anti-CXCLIO antibody. The VNS may be assessed at any suitable point after the administration. For example, VNS may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.

[0044] In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 4 on the VNS within 24 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 5 on the VNS within 24 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 4 on the VNS within 36 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 5 on the VNS within 36 weeks of administration of the anti-CXCLIO10305304127antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 4 on the VNS within 52 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 5 on the VNS within 52 weeks of administration of the anti-CXCLIO antibody.Vitiligo Extent Score

[0045] The Vitiligo Extent Score (VES) is a system to score the amount of body surface area affected by vitiligo (see Van Geel et al., Melanocytes / Melanoma 136(5): 978-984, May 2016).

[0046] In some embodiments, a method disclosed herein results in an improvement in the VES of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% compared to the VES before the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement in the VES of at least about 25%, at least about 50%, or at least about 75% compared to the VES before the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement in the VES of about 25%, about 50%, or about 75% compared to the VES before the administration of the anti- CXCLIO antibody. The VES may be assessed at any suitable point after the administration. For example, VES may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.Body Surface Area

[0047] The body surface area affected with depigmentation may also be measured by taking photographs of the affected area and calculating the percentage area affected.

[0048] In some embodiments, a method disclosed herein results in a decrease in the Body Surface Area (BSA) affected with depigmentation of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% compared to body surface area affected with depigmentation before the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in a decrease in the body surface area affected with depigmentation of at least about 25%, at least about 50%, or at least about 75% compared to the body surface area affected with depigmentation the administration of the anti-CXCLIO antibody. In some embodiments, a11305304127method disclosed herein results in a decrease in the body surface area affected with depigmentation of about 25%, about 50%, or about 75% compared to the body surface area affected with depigmentation before the administration of the anti-CXCLIO antibody. The body surface area affected with depigmentation may be measured at any suitable point after the administration, e.g., 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.Static Investigator Global Assessment (sIGA) Outcome Measure

[0049] The Static Investigator Global Assessment (sIGA) Outcome Measure is a global score of vitiligo severity for the entire body. The score ranges from 0 (clear) to 5 (severe vitiligo). It incorporates location, distribution, size, depigmentation within lesions and presence or absence of signs of activity.

[0050] In some embodiments, a method disclosed herein results in an improvement of 1 point on the sIGA scale compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 2 points on the sIGA scale compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 3 points on the sIGA scale compared to the score prior to the administration of the anti-CXCLIO antibody. In some embodiments, a method disclosed herein results in an improvement of 4 points on the sIGA scale compared to the score prior to the administration of the anti-CXCLIO antibody. The sIGA scale score may be assessed at any suitable point after the administration. For example, sIGA scale score may be measured 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.

[0051] In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 0 on the sIGA scale within 24 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 1 on the sIGA scale within 24 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 0 on the sIGA scale within 36 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 1 on the sIGA scale within 36 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein12305304127achieves a score of 0 on the sIGA scale within 52 weeks of administration of the anti-CXCLIO antibody. In some embodiments, a subject treated in accordance with a method disclosed herein achieves a score of 1 on the sIGA scale within 52 weeks of administration of the anti-CXCLIO antibody.Vitiligo-specific Quality-of-Life Instrument

[0052] Vitiligo-specific Quality-of-Life Instrument (VitiQoL) may be used as the specific index for assessment of quality of life in vitiligo patients. The questionnaire consists of 16 questions with a seven-point Likert scale (0-6). The final scores could range from 0 to 90, in which, patients with higher scores showed poorer quality of life.

[0053] In some embodiments, a method disclosed herein results in a decrease in the score on the VitiQoL of about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, or about 70% to about 80% compared to the score before the administration. In some embodiments, a method disclosed herein results in a decrease in the score on the VitiQoL of at least about 25%, at least about 50%, or at least about 75% compared to the score prior to the administration. In some embodiments, a method disclosed herein results in the score on the VitiQoL of about 25%, about 50%, or about 75% compared to the body surface area affected with depigmentation before the administration. The VitiQoL may be measured at any suitable point after the administration, e.g., 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or 64 weeks after the administration of the anti-CXCLIO antibody.EXAMPLESExample 1: Non-Clinical Studies with EB06Non-Clinical Pharmacology: In Vitro PharmacologyAffinity and binding kinetics ofEB06 to human CXCL10

[0054] The affinity and kinetics of EB06 binding to its target, human CXCL10 were characterized using a Biacore 2000 instrument (Biacore AB, Uppsala, Sweden). EB06 demonstrated an affinity constant (KD) of 0.14 ± 0.04 nM.13305304127Inhibition of human CXCL10 biological activity in vitro EB06 inhibits human CXCLIO-induced calcium flux

[0055] Having demonstrated a high affinity target-drug interaction, the ability of EB06 to neutralize the biological activity of CXCL10 was tested in vitro. Upon binding of CXCL10 to its receptor CXCR3, a signaling cascade leads to a rise in intracellular calcium concentration. Therefore, the ability of EB06 to inhibit the induction of calcium flux was evaluated in vitro, using a mouse Pre-B cell line (LI.2 cells) expressing human (h) CXCR3. When these cells were exposed to CXCL10, an immediate and transient increase in intracellular calcium was detected using the calcium sensitive dye Fura 2 / AM and real time fluorescence measurements. Using this system, EB06 neutralized CXCL10 signaling with a half-maximal inhibitory concentration (ICso) of 2.2 ± 0.05 nM (FIG. lError! Reference source not found.).EB06 inhibits human CXCLIO-induced chemotaxis

[0056] The physiological response of the CXCLIO-induced signaling cascade is chemotaxis of CXCR3 -expressing lymphocytes towards the source of CXCL10. Thus, the most relevant assay for testing CXCL10 biological activity in vitro is cell chemotaxis. LI.2 cells expressing hCXCR3 were placed on the top of a chemotaxis filter plate and allowed to migrate (over a 3- hour period) through the filter in response to 1 nM of recombinant hCXCLIO in the presence of various concentrations of EB06. The results demonstrated that EB06 neutralizes the chemotactic activity of CXCL10 in a dose-dependent and saturable manner with an ICso of 0.35 ± 0.2 nM (FIG. 2).

[0057] Furthermore, having demonstrated a potent blocking capacity for recombinant protein, the ability of EB06 to neutralize endogenous CXCL10 (i.e. produced by the human cell line THP-1 stimulated with IFN-y) was also tested. Using the cell chemotaxis assay, it was demonstrated that EB06 also neutralizes the activity of native human CXCL10.EB06 neutralizes CXCL10 in the presence of GAGs

[0058] CXCL10 forms oligomers and binds to GAGs expressed at the surface of endothelial cells. EB06 and A2, another anti-CXCLIO neutralizing antibody, were compared in an ELISA for their capacity to bind CXCL10 when bound to heparin, a prototypic GAG. Antibodies were immobilized and incubated with labelled heparin alone or CXCL10 bound to labelled heparin. The results demonstrated that the epitope recognized by EB06 is accessible when CXCL10 is14305304127bound to heparin. In contrast, the control A2, was unable to bind to CXCL10 in this context (FIG. 3). In conclusion, EB06 can bind to CXCL10 sequestered on GAG and neutralize this chemokine in its physiological context.

[0059] Taken together, these results demonstrate that EB06 binds to human CXCL10 with high affinity, can neutralize the biological function of either the recombinant or native form of CXCL10, and that it neutralizes CXCL10 in its physiological context.EB06 inhibits recruitment of T-cells into inflamed human tissue

[0060] Using flow-based adhesion assays, it has been demonstrated that CXCR3 is the dominant receptor promoting entry of lymphocytes into specific tissues, as blocking of CXCR3 in these assays significantly reduced transendothelial migration (see Curbishley et al., Am J Pathol. 167(3):887-899y et al. 2005). The same approach was used to assess the ex vivo impact of CXCL10 inhibition by EB06 on transendothelial migration (FIG. 4).

[0061] Confirming that transendothelial migration is predominantly CXCR3 mediated, an anti- CXCR3 antibody was highly efficient at inhibiting this process. Comparable to CXCR3 blockade, EB06 neutralization of CXCL10 led to a 60% reduction in transendothelial migration. Importantly, the addition of mAbs targeting CXCL9 and CXCL11 did not result in further reduction in transendothelial migration. Altogether, this demonstrates the specificity of the critical role that CXCL10 plays in transendothelial migration into human tissues, and that the other CXCR3 ligands are not redundant in this process. This experiment supports the use of EB06 in vitiligo, as transendothelial migration is required for CD8+T-cell movement from the bloodstream through to the skin.Capacity ofEB06 for binding to other human chemokines

[0062] As chemokines are a family of proteins that share similar three-dimensional structure, the specificity of EB06 for human CXCL10 was tested against a panel of human chemokines of CXC and CC families. The chemokines CXCL9, CXCL11, CCL2, CCL3, CCL4, CCL5 and CCL8 were expressed as fusion proteins and tested in an ELISA. The results demonstrated that EB06 only binds to human CXCL10 (FIG. 5). Further, the neutralizing capacity of EB06 was tested against the two other main CXCR3 ligands, CXCL9 and CXCL11, in an in vitro chemotaxis assay and no inhibition was observed.15305304127Characterization of the EB06 epitope

[0063] The amino acid sequence alignments from human and rabbit CXCL10 show that there are 11 amino acids that are different between the two species. As EB06 does not bind to rabbit CXCL10, amino acids in the rabbit sequence were replaced, by site-directed mutagenesis, with the human residues found at the same position. In this way, the contribution of these amino acids to EB06 binding was established. Four variants of rabbit CXCL10 were expressed and tested in an ELISA for EB06 binding. The two residues, S13 and Q17, present in the human CXCL10 sequence confer binding to the mutated form of the rabbit protein and thus, appear to be critical for EB06 binding (FIG. 6). These residues are not conserved in any of the CXCL10 sequences except for cynomolgus and rhesus monkeys indicating that these are the only species that are relevant for toxicology studies.Cross-reactivity ofEB06 to cynomolgus monkey CXCL10

[0064] Recombinant cynomolgus monkey CXCL10 was generated. Using this protein in an ELISA and in vitro chemotaxis assays, it was demonstrated that EB06 can bind and neutralize the activity of cynomolgus CXCL10. The affinity and kinetics of EB06 binding to cynomolgus CXCL10 were measured on a Biacore 2000 system and an affinity constant (KD) of 0.54 nM was found. In contrast, the ELISA showed that EB06 does not bind to commercially available rat and mouse CXCL10.Antibody dependent cellular cytotoxicity and complement dependent cytotoxicity

[0065] EB06 is an intact human IgGl antibody and thus retains the full characteristics of this isotype, including the capacity to engage Fey receptors and to bind complement.

[0066] However, as the target of EB06 is a soluble protein, no antibody dependent cell cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) activities are anticipated. This is supported by the lack of toxicity of EB06 observed both in humans and non-human primates. In addition, no change in the pattern of circulating pro-inflammatory cytokines was detected during preclinical and clinical studies indicating that EB06 did not induce abnormal lymphocyte activation or cytokine release both of which are features of ADCC / CDC related activity.16305304127EB06 cross-reactivity in healthy human tissue

[0067] A GLP human tissues cross-reactivity study was performed at Histologix, Nottingham, UK using biotinylated EB06 and hlgGl 1 isotype control antibodies. The purpose of this study was to investigate the extent of binding of fluorescein-5-isothiocyanate (FITC) conjugated EB06 in a panel of normal human tissues from both male and female tissue donors. A control hlgGl antibody was used to aid in the discrimination between specific and non-specific binding of EB06.

[0068] In summary, the pattern of EB06 cross-reactivity in human tissue was consistent with previous literature on the distribution of CXCL10 in multiple human tissues. There was no unexpected staining in major organ systems (heart, lungs, kidney, and gastrointestinal tract) responsible for vital functions.ToxicologyStudies in Cynomolgus Monkeys

[0069] A Dose Range Finding Study was performed to evaluate the toxicity and tolerability of EB06 in cynomolgus monkeys following weekly intravenous infusions at doses up to 100 mg / kg for 4 weeks. There were four study groups. Group 1 was dosed intravenously on Day 1 at 0.1 mg / kg and on Day 8 at 10 mg / kg. Group 2 was dosed intravenously on Day 4 at 1 mg / kg. Groups 3 and 4 were dosed weekly at 10 mg / kg or 100 mg / kg respectively for 4 weeks. A minimum number of primates were used (4 males and 4 females). They were acclimatized for a minimum of 13 days prior to the commencement of dosing and housed separately throughout the study.

[0070] EB06 administration was associated with mild hematological effects which included small decreases in peripheral blood neutrophils and mild increases in neutrophils in bone marrow, but no test article- or study procedure-related changes were noted in clinical observations, food consumption, parameters associated with serum chemistry, gross pathology, or organ weights. Thus, in conclusion, once weekly intravenous administration of EB06 for four consecutive weeks was well tolerated by cynomolgus monkeys at a dosage of 100 mg / kg under the conditions of the study. From these results it was considered that appropriate dose levels for the subsequent 13-week study would be 25, 75, and 150 mg / kg. The half-life was estimated to be between 5-7 days, thus supporting weekly dosing in the repeat dose toxicity study.17305304127

[0071] Based on the results of the Dose Range Finding Study, a 13-Week Intravenous Infusion Toxicity Study in Cynomolgus Monkeys Followed by an 11 -Week Recovery Period was conducted. The purpose of this study was to characterize EB06 toxicity and toxicokinetics in cynomolgus monkeys given weekly intravenous administration for 13 consecutive weeks. Reversibility or the de novo onset of toxicity following cessation of treatment was also examined during an 11 -week recovery period. Dose administration was conducted once weekly for 13 weeks. Test or control articles were administered via short intravenous infusion over an administration time of 10-15 minutes to cynomolgus monkeys at dosages of 0 (control), 25, 75, and 150 mg / kg (Groups 1, 2, 3, and 4, respectively).

[0072] No test article-related changes were noted in parameters associated with clinical observations, food consumption, blood pressure, rectal temperature, respiratory rate, ophthalmologic examinations, clinical pathology (fecal occult blood, urinalysis, hematology, coagulation, and serum chemistry). In summary, EB06 treatment was not associated with gross, or histopathologic changes or changes in organ weights at the terminal necropsy. Reduced bodyweight gain was noted in all test article-treatment groups. The reduced body weight gain was accounted for by a few animals losing weight. However, the magnitude of loss was very small and not associated with apparent toxic effects or increasing doses of EB06. Since there were no abnormalities in any of the other parameters examined, and because all test article treatment groups experienced overall net gains in body weight after 13 weeks of treatment, this change was considered not to be adverse. In conclusion, EB06 was well tolerated after treatment up to 150 mg / kg up to 13 weeks and a NOAEL of 150 mg / kg / week was defined for the study.

[0073] During the 13-Week Toxicity Study, samples were collected for anti-drug antibody analysis. Due to the long half-life of the product, residual EB06 levels remained high and above the level which interfered with anti-drug antibody analysis, even throughout the recovery period. Therefore, the immunogenicity assay could not be performed. The pharmacokinetic profile of the drug in cynomolgus monkeys shows that all the animals were exposed to the drug and as no changes in clearance were observed in any animal, it is assumed that no neutralizing antibodies against EB06 were developed in this study.

[0074] The evaluation of the humoral response in EB06 treated cynomolgus monkeys was performed by assessing the ability of the animals to produce antigen specific antibodies (IgM and IgG), after two Keyhole Limpet Hemocyanin (KLH) immunizations. All EB06 treated animals18305304127developed primary and secondary anti-KLH IgM and IgG endpoint titer responses similar to the placebo control group. Collectively, these results indicate that EB06 treatment presents no deleterious or suppressive effect on immune cell function in cynomolgus monkeys.

[0075] No adverse events at the injection site were observed in the 13-Week Toxicity Study despite repetitive administrations of EB06.Non-Clinical Pharmacokinetics

[0076] A 13-week GLP Toxicity Study was conducted in cynomolgus monkeys to evaluate the toxicity of EB06 following 25 mg / kg, 75 mg / kg and 150 mg / kg once weekly IV administration for a total of 13 doses. The pharmacokinetics of EB06 was assessed as part of this study.

[0077] Overall, EB06 showed a high level of systemic exposure after intravenous administration over the 13-week dosing period and pharmacokinetics were approximately dose proportionate, in terms of exposure based on maximum concentration (Cmax) and area under the plasma concentration versus time curve (AUCo-ies). For 150 mg / kg at Day 85, the mean value of Cmax was 8107 pg / mL and AUCo-ies was 918551 pg h / mL (both genders, 0-168 h data set).

[0078] EB06 demonstrated a long ti / 2 of approximately 415 h (17.3 days) with a MRT of approximately 560 h (23.3 days). The clearance (CL) of EB06 was low and yielded an overall value of approximately 0.04 (mL / h) / kg as estimated from the 0-2016 h data sets. The volume of distribution, the overall Vss value as estimated from the 0-2016 h data sets was approximately 25 mL / kg. No apparent differences in serum concentrations or pharmacokinetics (PK) parameters of EB06 were observed between male and female cynomolgus monkeys.Non-Clinical PharmacodynamicsFACS characterization of circulating inflammatory cells in EB06 treated cynomolgus monkeys

[0079] Fluorescent activated cell sorting (FACS) was used to measure absolute numbers of B cells, T cell subsets, NK cells, and CXCR3 expression on T-cell subsets in whole blood samples from cynomolgus monkeys, 6- and 13 -weeks post treatment, as compared to pretreatment. Thirteen weekly doses of EB06 did not alter the capacity of the cynomolgus monkeys to maintain significant levels of circulating T cells, B cells and NK cells.19305304127

[0080] EB06 treatment resulted in a significant increase of circulating T cells expressing CXCR3, the receptor for CXCL10. This increase was observed more consistently in CD8+T cells than in CD4+T cells. These findings suggest that EB06 may disrupt the CXCL10 chemoattractant gradient and thus may reduce anti-melanocytic T cells at the epidermis in vitiligo patients.

[0081] These effects were observed towards the end of the active treatment period and during the recovery phase. Given the small number of animals in each group, no conclusions regarding a dose response relationship could be drawn.Circulating CXCL10 levels in EB06 treated cynomolgus monkeys

[0082] The serum levels of CXCL10 were assessed during the 13-Week Toxicity Study, wherein cynomolgus monkeys were exposed to 13 weekly injections of drug.

[0083] The basal serum level of free CXCL10 in the cynomolgus monkeys was 47 ± 14 pg / mL. Following the first administration of EB06, serum CXCL10 levels rose to reach a steady state that remained relatively constant over the course of treatment (291 ± 122 pg / mL,253 ± 135 pg / mL and 175 ± 101 pg / mL in the 25 mg / kg, 75 mg / kg and 150 mg / kg dose groups, respectively). This CXCL10 level was inversely correlated to the antibody dose, reflecting that, as the dose of EB06 increases, more CXCL10 is bound by EB06 and therefore becomes undetectable in the ELISA which only detected free CXCL10. During the recovery phase, an increase in CXCL10 levels relative to baseline occurred that correlated with the dose of EB06 to which the animals were exposed (a maximum of 51 ± 15 ng / mL and 83 ± 27 ng / mL in the 25 mg / kg and 150 mg / kg dose groups, respectively).

[0084] Throughout the recovery period, EB06 was present in a large molar excess over measured CXCL10 levels. These serum samples were assessed for CXCL10 biological activity in an in vitro chemotaxis assay. It was found that the sera of animals exposed to EB06 did not contain biologically active CXCL10 indicating it remains complexed with EB06. Importantly, despite the increased levels of plasma CXCL10 measured during the recovery phase, no toxicity was observed in cynomolgus monkeys.

[0085] In conclusion, EB06 was well tolerated after treatment with up to 150 mg / kg for up to 13 weeks (NOAEL of 150 mg / kg / week). There were no changes in any vital organ system (heart and other cardiovascular, respiratory, renal or CNS function) that indicated a risk to patients up to the top dose of 150 mg / kg / week. None of the above findings were linked to any clinical20305304127adverse event (AE) or any abnormal laboratory values and were therefore not considered to have any safety implications for the administration to humans.Example 2: Clinical Experience with EB06

[0086] EB06 has been evaluated in three different clinical trials:

[0087] The NI-0801-01 Study is a randomized, double-blinded, placebo-controlled, single center, phase I study of the safety of escalating single IV doses of EB06 in 30 healthy volunteers. Each of the following dose levels, 0.1, 1.0, 3.0, 10 and 20 mg / kg, were administered to a cohort of six subjects using a 2: 1 active to placebo ratio.

[0088] The NI-0801-02 Study is a randomized, parallel-group, placebo-controlled, double-blind study evaluating the effect of single doses of EB06 on the severity of nickel induced allergic contact dermatitis (ACD) in otherwise healthy volunteer subjects with proven nickel sensitivity. Two different fixed doses of EB06 (180 mg, n = 8 and 720 mg, n = 8) and matching placebo (n = 6) were tested.

[0089] The NI-0801-03 Study is an open label, proof-of-concept study in Primary Biliary Cirrhosis (PBC) patients with an incomplete response to ursodeoxycholic acid (UDCA) designed to provide efficacy and safety data on the use of EB06 in PBC. The objective of this study was to assess the efficacy of multiple doses of EB06 on the improvement of hepatic function in PBC and to evaluate the safety and tolerability of multiple doses of EB06. A total of 29 patients were enrolled into a single cohort to receive 6 consecutive administrations of 10 mg / kg of EB06.Human pharmacokinetics of EB06

[0090] Pharmacokinetic analysis from the three studies to date suggests that the nonlinear kinetics of EB06 in healthy volunteers indicate that the clearance is mediated by binding to the drug target CXCL10. In patients with PBC, EB06 clearance was higher than expected, which could be explained by patients presenting with 5-fold higher levels of CXCL10 production when compared to healthy volunteers and requiring higher EB06 doses to maintain inhibition of CXCL10 over time. Patients with vitiligo are expected to present with CXCL10 levels about 2- 3x higher than healthy volunteers and thus fall between those of PBC patients and healthy volunteers. Overall, considering all the data available in EB06 pharmacokinetic profile, EB06 is compatible with sustainable administration in a chronic disease such as vitiligo.21305304127Safety data

[0091] Safety experience from the three studies testing a single or multiple infusions of the drug in adult healthy volunteers, subjects with nickel sensitivity or patients with PBC (total of 65) was similar and demonstrated that administration of EB06 was safe and well tolerated up to a dose of 20 mg / kg.

[0092] No serious adverse reactions were reported from the two single-dose studies.

[0093] Safety results from the multiple infusion study reported one SAE which occurred in a patient in the follow-up phase of the study. The patient had an accident at home during the night, three weeks after the last study drug infusion leading to fractured ribs with severe left side hemopneumothorax which resulted in hospitalization but no cause for the fall was identified. This event was considered to be unrelated to the study drug.Clinical Pharmacodynamics

[0094] With the exception of serum CXCL10 levels, none of the other PD parameters measured revealed systematic changes over time or between different treatment groups in the NI-0801-01 Study. These findings suggest that treatment with EB06 may disrupt the CXCL10 chemoattractant gradient and thus can potentially disrupt anti-melanocytic T cells from being trafficked to the epidermis. Importantly, the safety data obtained in the NI-0801-01 Study suggest that the rise in complexed serum CXCL10 levels is not associated with any adverse effects.

[0095] While a dose-dependent increase in serum CXCL10 levels was observed in the NI-0801- 02 Study, no chemotactic activity could be measured ex vivo using the patients’ sera, demonstrating that the CXCL10 was bound to EB06 rendering it biologically inactive. For other inflammatory cytokines and chemokines measured, no significant differences were detected between placebo- and drug-treated groups in this study, indicating that EB06 infusion did not lead to cytokine release.

[0096] In The NI-0801-02 Study, EB06 reached the site of inflammation as it significantly modified mRNA expression of local pro-inflammatory markers relevant to skin inflammation including CCR6 and IL-22. The immunohistochemical analysis demonstrated no major effects on cellular migration, although a decrease in the relevant receptor-positive cell types (CXCR3) was observed in the cohort receiving 180 mg of EB06. Overall, the favorable safety and22305304127tolerability of EB06, its pharmacokinetic profile as well as the PD effects demonstrated in the inflamed tissue support further development of EB06 in chronic inflammatory indications.

[0097] PD results from The NI-0801-03 Study suggest that EB06 treatment prevents the translocation of CXCR3+ cells from circulation to the peripheral tissues suggesting treatment may be efficacious for vitiligo patients. The study demonstrated favorable safety and tolerability profile for multiple EB06 infusions.Example 3: A Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Safety and Efficacy of EB06 versus Placebo in Adults with Moderate to Severe Non-Segmental Vitiligo.Study Design

[0098] This is a multicenter, randomized, double-blind, placebo-controlled, study to evaluate the safety and efficacy of EB06 (2.5 mg / kg, 5 mg / kg or 10 mg / kg) versus placebo in adults with moderate to severe non-segmental vitiligo.

[0099] Subjects for whom informed consent has been obtained, fulfill all the inclusion criteria and none of the exclusion criteria will be accepted into the study. After providing informed consent, subjects may undergo a screening period of up to 30 days prior to Day 1 (0 to 30 days). The purpose of the optional screening period is to washout from any current treatments when required.

[0100] Following enrollment in the study, eligible subjects will be randomized at a ratio of 1 : 1 : 1 : 1 at Baseline to receive an infusion of either one of the following: EB06 2.5 mg / kg, EB06 5 mg / kg; EB06 10 mg / kg; or Placebo for every two weeks up to 24 weeks. The ceiling (maximum) for each dose level is as follows:2.5 mg / kg: 225mg; 5 mg / kg: maximum dose of 450 mg; 10 mg / kg: maximum dose of 900 mg;.

[0101] Participants will be stratified by site and baseline skin type (Fitzpatrick scale Type I [pale white] and II [white] versus Type III [light brown], IV [moderate brown], V [dark brown], and VI [deeply pigmented dark brown to black]).

[0102] Subjects will come to the study site for scheduled visits at screening, and every two weeks for intravenous (IV) infusion of study drug and assessments for up to 24 weeks. A followup visit will occur 36 weeks after treatment initiation.23305304127

[0103] A total of 38 evaluable patients will be considered in each arm of the study (EB06 2.5 mg / kg; EB06 5 mg / kg; EB06 10 mg / kg; and Placebo) for a total of 152 evaluable. Allowing for 20% attrition, a total of 184 vitiligo patients are planned for enrollment (up to 46 per group ) in the study.

[0104] The Study Period will have 12 EB06 IV infusions of one of the following doses:2.5 mg / kg (maximum dose 225mg), 5 mg / kg (maximum dose 450 mg), or 10 mg / kg (maximum dose 900 mg). EB06 has been formulated for intravenous (IV) administration, as a sterile, isotonic, clear, colorless to slightly yellow solution. The product is presented in 10 mL single-use glass vials containing 9 mL of EB06 at the concentration of 10.0 mg / mL.A placebo solution will be administered via IV infusion every two weeks for up to 24 weeks (12 IV infusions in total) in adults with moderate to severe non-segmental vitiligo.Objectives:

[0105] Primary Objective: To evaluate the clinical efficacy of EB06 vs. Placebo in adults with moderate to severe non-segmental vitiligo.

[0106] Secondary Objective: To evaluate the safety and tolerability of EB06 in adults with moderate to severe non-segmental vitiligo.

[0107] Exploratory Objective: To evaluate descriptively the dose-relationship between 3 different strengths of EB06: 2.5 mg / kg (max dose 225 mg), 5mg / kg (max dose 450mg), and lOmg / kg (max dose 900mg).Study Endpoints:

[0108] The primary Efficacy Endpoint for the Main Study is the proportion of patients achieving 50% improvement in Facial Vitiligo Area Scoring Index (F-VASI50) at Week 24.

[0109] Secondary Efficacy Endpoints for the Main Study include:• Proportion of patients achieving F-VASI50 at Week 36.• Proportion of patients achieving F-VASI75 at Week 24 and Week 36.• Proportion of patients achieving F-VASI90 at Week 24 and Week 36.• Proportion of patients achieving T-VASI50 at Week 24 and Week 36.• Mean percent change from baseline in F-VASI at Week 24 and Week 36.• Mean percent change from baseline in the T-VASI at Week 24 and Week 36.24305304127• Proportion of subjects achieving a score of 4 (a lot less noticeable) or 5 (no longer noticeable) on the Vitiligo Noticeability Scale (VNS) at Week 24 and Week 36.• Percent change in the Vitiligo Extent Score from baseline at Week 24 and Week 36.• Proportion of subjects achieving a static investigator global assessment (sIGA) of 0 or 1, with at least a 2-point reduction from baseline at Week 24 and Week 36.• Mean percent change from baseline in Body Surface Area (BSA) affected with depigmentation at Week 24 and Week 36.• Change from baseline in the quality-of-life score measured by vitiligo-specific quality-of- life instrument (VitiQoL) at Week 24 and Week 36.• Percent change from baseline in extent of depigmentation in target lesions at Week 24.

[0110] Exploratory Endpoints: The dose-relationship of the different strengths of EB06 (2.5 mg / kg, 5mg / kg, lOmg / kg) will be compared descriptively based on the efficacy (F-VASI, T- VASI, VNS, VES, sIGA, and BSA) and quality of life (VitiQoL) measurements. The descriptive characterisation of PK profiles of multiple doses will be compared at the different strengths of EB06 (2.5mg / kg, 5mg / kg, lOmg / kg).

[0111] Safety endpoint:

[0112] Safety and tolerability will be assessed by the number of treatment-emergent adverse events (TEAEs) and serious adverse events (SAEs).Inclusion Criteria

[0113] All the following criteria need to be met for inclusion:1. Clinical diagnosis of non-segmental vitiligo with depigmented area including > 0.5% BSA on the face, > 0.5 F-VASI, > 5% total BSA , > 4 T-VASI.2. Men and women > 18 years of age at the time of consent.3. Subjects who agree to discontinue all agents used to treat vitiligo from screening through to the end of the treatment period at Week 24. Over the counter preparations deemed acceptable by the investigator and camouflage makeups are permitted.4. Female subjects of childbearing potential and at risk for pregnancy: negative pregnancy test and agreement to use at least one method of effective contraception throughout the study and for at least 12 weeks after the last dose of assigned treatment.253053041275. Male subjects with female partners of childbearing potential agree to use at least one method of effective contraception by yourself or your partner, as applicable, during study duration and for at least 12 weeks after the last dose of assigned treatment.Exclusion Criteria

[0114] Subjects will be excluded if any of the following criteria are met:1. Subjects with more than 30% of poliosis within facial vitiligo areas.2. Other forms of vitiligo (e.g., segmental) or other hypopigmentation disorders (including but not limited to tinea versicolor, piebaldism, pityriasis alba, post-inflammatory hypopigmentation, nevus anemicus, nevus depigmentosus, senile leukoderma, chemical / drug-induced leukoderma, tuberous sclerosis, etc.).3. Subjects who have used permanent depigmentation treatments, such as monobenzone, for past treatment of vitiligo. Note: prior use of hydroquinone is not prohibited (as it is a bleaching agent, not a depigmentation treatment).4. Subject has used topical treatments applied on vitiligo affected areas such as corticosteroids, calcineurin inhibitors, phosphodiesterase-4 inhibitor, retinoids or JAK inhibitors within 1 week prior to Day 1.5. Subject has used systemic immunomodulators (other than biologies, e.g., methotrexate, cyclosporin, corticosteroids, JAK inhibitors), and / or melanocyte stimulating pharmaceuticals (e.g. afamelanotide) that could affect vitiligo, or treatments that would increase skin sensitivity to ultraviolet (UV) light (e.g. 8-methoxypsoralen) within 4 weeks prior to Day 1.6. Subject has used laser or light-based vitiligo treatments within 4 weeks prior to Day 1.7. Subject has used any biological agent or experimental procedure for vitiligo within 12 weeks or 5 half-lives (whichever is longest) prior to Day 1.8. Pregnant subjects; breastfeeding subjects; and subjects of childbearing potential who are unwilling or unable to use one method of contraception as outlined in this protocol for the duration of the study and for at least 28 days after the last dose of investigational product.9. Subjects who have a known history of being inadequately treated for chronic infectious disease (e.g. hepatitis B or C, human immunodeficiency virus).10. Current or recent history (< 30 days) of a clinically meaningful bacterial, fungal, or parasitic infection.2630530412711. Subjects with a history of malignancy in the past 5 years, except for the following adequately treated, nonmetastatic malignancies: basal cell skin cancer not involving areas with vitiligo, squamous cell carcinomas of the skin not involving areas with vitiligo, or in situ cervical cancer.12. Subjects with other active coexisting skin and non-skin autoimmune diseases (e.g. psoriasis, multiple sclerosis, etc.).13. Clinically significant findings or conditions which might, in the opinion of the Principal Investigator, interfere with study evaluations, pose a risk to subject safety during the study, or require treatment with an interfering therapy.14. Subjects with known allergy or reaction to any component of the study formulation.Statistical Methods

[0115] The sample size calculation is done based on the primary endpoint and comparison EB06 vs. Placebo. The sample size calculation considered a therapeutic success of 50% in the EB06 groups versus 20% in the placebo group, a power of 80%, an alpha risk of 5%, and a two-tailed hypothesis test. In total, 38 evaluable subjects will be considered for each arm of the study. Considering a 20% attrition, an additional 8 subjects will be added in each arm of the study. Therefore, up to 184 vitiligo subjects are planned for enrollment, 46 treated with EB06 2.5 mg / kg, 46 treated with EB06 5mg / kg, 46 treated with EB06 10 mg / kg, and 46 treated with placebo.

[0116] The primary analysis will be based on the per-protocol (PP) population. The primary alternative hypothesis will be tested using exact logistic regression. The between-group testing will be performed with a significance level of 0.05, unless otherwise specified in the SAP. Odds ratio and corresponding 95% confidence interval will be provided for comparing all three dosage arms of EB06 and placebo at Week 24. Sensitivity and supportive analyses of the efficacy endpoints will also be assessed on the intent-to-treat (ITT) populations.

[0117] Safety will be assessed descriptively with the incidence of treatment emergent adverse events. All adverse events will be coded with the MedDRA dictionary of terms and severity will be graded according to the Common Terminology Criteria for Adverse Events (CTCAE). The incidence of adverse events will be described by System Organ Class and Preferred Terms, Description of adverse events will include seriousness, severity, and causality in relation to the study treatments.27305304127

Claims

CLAIMSWhat is claimed is:

1. A method of treating vitiligo in a subject in need thereof, the method comprising administering to the subject 3 mg / kg to 25 mg / kg of an anti-CXCL-10 antibody comprising a variable heavy chain comprising a CDR1 of SEQ ID NO: 1, a CDR2 of SEQ ID NO: 2, and a CDR3 of SEQ ID NO: 3 and a variable light chain comprising a CDR1 of SEQ ID NO: 4, a CDR2 of SEQ ID NO 5 and a CDR3 of SEQ ID NO: 6.

2. The method of claim 1, wherein the anti-CXCL-10 antibody is administered at a dose of 2.5 mg / kg.

3. The method of claim 1, wherein the anti-CXCL-10 antibody is administered at a dose of 5 mg / kg.

4. The method of claim 1, wherein the anti-CXCL-10 antibody is administered at a dose of 10 mg / kg.

5. The method of claim 1, wherein the anti-CXCL-10 antibody is administered at a dose of 20 mg / kg.

6. The method of any one of claims 1-5, wherein the anti-CXCL-10 antibody is administered for a period of 24 weeks.

7. The method of any one of claims 1-5, wherein the anti-CXCL-10 antibody is administered for a period of 52 weeks.

8. The metho of any one of claims 1-6, wherein the anti-CXCL-10 antibody is administered every other week.

9. The method of any one of claims 1-8, wherein the anti-CXCL-10 antibody is administered by intravenous infusion.

10. The method of any one of claims 1-9, wherein the subject achieves a 50% improvement in the facial Vitiligo Area Scoring Index at week 24.2830530412711. The method of any one of claims 1-10, wherein the subject achieves a 50% improvement in the total Vitiligo Area Scoring Index at week 24.

12. The method of any one of claims 1-11, wherein the subject achieves a score of 4 or 5 on the Vitiligo Noticeability Scale at Week 24.

13. The method of any one of claims 1-12, wherein the subject has at least a 25% improvement in Vitiligo Extent Score from prior to the administration within 24 weeks.

14. The method of any one of claims 1-13, wherein the body surface area affected by vitiligo decreases by at least 25% from prior to the administration within 24 weeks.

15. The method of any one of claims 1-14, wherein the vitiligo is non-segmented vitiligo.29305304127

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