Predicting response to IL-6 antagonists

Abstract

The present invention relates to a method for predicting response to an IL-6 antagonist, such as an anti-IL-6 antibody, by determining the concentration of IL-6 in human aqueous humor. The present invention also relates to an IL-6 antagonist for use in treating uveitis or uveitic macular edema.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to interleukin-6 (IL-6) antagonists, such as anti-IL-6 or anti-IL-6 receptor (IL-6R) antibodies, for use in treating patients with ocular diseases with IL-6-related pathophysiology characterized by increased aqueous humor (AH) IL-6 levels. The present invention also relates to biomarkers for predicting the response of patients with ocular diseases to interleukin-6 (IL-6) antagonists, such as anti-IL-6 or anti-IL-6 receptor (IL-6R) monoclonal antibodies. Provided herein are methods for identifying patients with ocular diseases that respond to IL-6 or IL-6R antagonists by measuring the level of IL-6 in aqueous humor (AH IL-6). The present invention further relates to IL-6 antagonists for use in treating uveitis or uveitic macular edema (UME). [Background technology]

[0002] Background of the Invention Interleukin-6 (IL-6) is one of several inflammatory mediators that are elevated in the eyes of patients with retinal diseases such as diabetic macular edema (DME), diabetic retinopathy (DR), age-related macular degeneration (AMD), retinal vein occlusion (RVO), and uveitis / uveitic macular edema (UME). IL-6 has been shown to cause loss of blood-retinal barrier function in human retinal endothelial cells in vitro, and inhibition of IL-6 transsignaling using sgp130Fc prevents endothelial barrier disruption in retinal endothelial cells (Valle ML et al., Inhibition of interleukin-6 transsignaling prevents inflammation and endothelial barrier disruption in retinal endothelial cells. Exp Eye Res. 2019 Jan). Therefore, intraocular inhibitors of IL-6 administered locally (e.g., via intravitreal injection) may be an attractive novel therapeutic strategy for treating DME, DR, AMD, RVO, or UME. However, there are currently no effective biomarkers to predict response to treatment with anti-IL-6 antagonists.

[0003] Therefore, there is a need for methods to predict which patients will respond particularly well to treatment with IL-6 antagonists. There is also a need to provide effective treatments for uveitis and uveitic macular edema. Summary of the Invention

[0004] Summary of the Invention Here, we develop an in vitro diagnostic immunoassay to quantify IL-6 in the aqueous humor of patients with retinal diseases (DME, DR, AMD, RVO, and UME) to predict whether patients are likely to respond to intraocular inhibition of IL-6 by compounds delivered by intravitreal injection.

[0005] Also provided is an IL-6 antagonist for use in treating uveitis or uveitic macular edema.

[0006] The following numbered paragraphs define some embodiments of the present invention.

[0007] 1'. An in vitro method for identifying a patient having an ocular disease that is likely to respond to treatment comprising an effective amount of an interleukin-6 (IL-6) antagonist, the method comprising determining the level of IL-6 in aqueous humor (AH IL-6) in a sample obtained from the patient, wherein an elevated level of AH IL-6 compared to the baseline level indicates that the patient is likely to respond to the treatment.

[0008] 2'. The method of 1', wherein the treatment is suitable for administration to the eye of a patient.

[0009] 3'. The method of 1' or 2', wherein the treatment is suitable for intravitreal, intraocular, or subconjunctival administration.

[0010] 4'. The method according to any one of 1' to 3', wherein the level of IL-6 is determined in an aqueous humor sample collected by anterior chamber paracentesis.

[0011] 5'. The method of any one of 1' to 4', wherein the ocular disease is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy (DR), dry eye (e.g., dry eye disease or dry eye syndrome), allergic conjunctivitis, uveitis, uveitic macular edema (UME), age-related macular degeneration (AMD) (e.g., wet AMD or dry AMD), proliferative diabetic retinopathy (PDR), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), macular edema secondary to RVO, neuromyelitis optica spectrum disorder (NMOSD), myopic choroidal neovascularization, eye cancer, corneal transplant, corneal abrasion, or physical injury to the eye.

[0012] 6'. The method according to 5', wherein the eye disease is diabetic macular edema (DME).

[0013] 7'. The method according to any one of 1' to 6', wherein the IL-6 antagonist is an anti-IL-6 or anti-IL-6 receptor (IL-6R) antibody or an antigen-binding fragment thereof.

[0014] 8'. The method of 7', wherein the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment thereof.

[0015] 9'.Anti-IL-6 antibody, i) a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, and a VH CDR3 comprising the sequence of SEQ ID NO: 3, and ii) a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6 The method according to 7' or 8', comprising:

[0016] 10'. The method of 9', wherein the anti-IL-6 antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:8.

[0017] 11'. The method of 10', wherein the IL-6 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9 and a light chain comprising the sequence of SEQ ID NO: 10.

[0018] 12'. The method according to any one of 1' to 11', wherein the sample is a sample obtained from the patient before treatment with an IL-6 antagonist.

[0019] 13'. The method of any one of claims 1'-12', wherein the treatment further comprises an effective amount of a second therapeutic agent.

[0020] 14'. The method of 13', wherein the second therapeutic agent is a VEGF antagonist.

[0021] 15'. The method of claim 14', wherein the VEGF antagonist is an anti-VEGF antibody.

[0022] 16'. A pharmaceutical composition comprising an IL-6 antagonist for use in treating a patient with an eye disease, wherein the patient has been determined to be likely to respond to treatment comprising an effective amount of an IL-6 antagonist by a method according to any one of 1' to 15'.

[0023] 1''. An interleukin-6 (IL-6) antagonist for use in treating a patient having an ocular disease, wherein the patient has been determined to have an elevated level of IL-6 in aqueous humor (AH IL-6) in a sample obtained from the patient compared to a baseline level.

[0024] 2''. An IL-6 antagonist for use according to 1'', formulated as a pharmaceutical composition suitable for administration to the eye of a patient.

[0025] 3''. An IL-6 antagonist for use according to 2'', wherein the pharmaceutical composition is suitable for intravitreal, intraocular, or subconjunctival administration.

[0026] 4''. An IL-6 antagonist for use according to any one of 1'' to 3'', wherein the level of IL-6 is determined in an aqueous humor sample collected by anterior chamber paracentesis.

[0027] 5''. The IL-6 antagonist for use according to any one of 1'' to 4'', wherein the ocular disease is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy (DR), dry eye (e.g., dry eye disease or dry eye syndrome), allergic conjunctivitis, uveitis, uveitic macular edema (UME), age-related macular degeneration (AMD) (e.g., wet AMD or dry AMD), proliferative diabetic retinopathy (PDR), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), macular edema secondary to RVO, neuromyelitis optica spectrum disorder (NMOSD), myopic choroidal neovascularization, eye cancer, corneal transplant, corneal abrasion, or physical injury to the eye.

[0028] 6''. The IL-6 antagonist of the use according to 5'', wherein the eye disease is diabetic macular edema (DME).

[0029] 7''. The IL-6 antagonist of any one of the uses described in 1'' to 6'', which is an anti-IL-6 or anti-IL-6 receptor (IL-6R) antibody or an antigen-binding fragment thereof.

[0030] 8''. The IL-6 antagonist for use according to 7'', wherein the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment thereof.

[0031] 9''.Anti-IL-6 antibody, i) a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, and a VH CDR3 comprising the sequence of SEQ ID NO: 3, and ii) a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6 IL-6 antagonist for use according to 7'' or 8'', comprising:

[0032] 10''. An IL-6 antagonist for use according to 9'', wherein the anti-IL-6 antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8.

[0033] 11''. An IL-6 antagonist for use according to 10'', wherein the IL-6 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9 and a light chain comprising the sequence of SEQ ID NO: 10.

[0034] 12''. An IL-6 antagonist for use according to any one of claims 1'' to 11'', wherein the sample is a sample obtained from the patient before treatment with the IL-6 antagonist.

[0035] 13''. The IL-6 antagonist for use according to any one of claims 1'' to 12'', wherein the use further comprises an effective amount of a second therapeutic agent.

[0036] 14''. The IL-6 antagonist for use according to 13'', wherein the second therapeutic agent is a VEGF antagonist.

[0037] 15''. The IL-6 antagonist for use according to 14'', wherein the VEGF antagonist is an anti-VEGF antibody.

[0038] 16''. An IL-6 antagonist for use in treating patients with uveitis or uveitic macular edema.

[0039] 17''. An IL-6 antagonist for use according to 16'', formulated as a pharmaceutical composition suitable for administration to the eye of a patient.

[0040] 18''. An IL-6 antagonist for use according to 17'', wherein the pharmaceutical composition is suitable for intravitreal, intraocular, or subconjunctival administration.

[0041] 19''. The IL-6 antagonist of any one of claims 16'' to 18'', which is an anti-IL-6 or anti-IL-6 receptor (IL-6R) antibody or an antigen-binding fragment thereof.

[0042] 20''. The IL-6 antagonist for use according to claim 19'', which is an anti-IL-6 antibody or an antigen-binding fragment thereof.

[0043] 21''.Anti-IL-6 antibody, i) a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, and a VH CDR3 comprising the sequence of SEQ ID NO: 3, and ii) a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6 19'' or 20'', comprising an IL-6 antagonist for use according to the above paragraph 19'' or 20''.

[0044] 22''. An IL-6 antagonist for use according to 21'', wherein the anti-IL-6 antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO: 7 and a light chain variable region comprising the sequence of SEQ ID NO: 8.

[0045] 23''. An IL-6 antagonist for use according to 22'', wherein the IL-6 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9 and a light chain comprising the sequence of SEQ ID NO: 10.

[0046] 24''. The IL-6 antagonist of any one of 16'' to 23'' administered intravitreally (IVT) at a dose of 0.25 mg, 1.0 mg or 2.5 mg every four weeks (Q4W).

[0047] In one aspect, the present invention relates to a method for determining whether a patient having an ocular disease is suitable for treatment with a therapy comprising an effective amount of an IL-6 antagonist, the method comprising determining the level of AH IL-6 in a sample obtained from the patient, wherein an elevated level of AH IL-6 compared to a baseline level indicates that the patient is likely to respond to the treatment.

[0048] In one aspect, the present invention relates to a method for improving the therapeutic effect of a therapy comprising an effective amount of an IL-6 antagonist in a patient having an ocular disease, the method comprising determining the level of AH IL-6 in a sample obtained from the patient, wherein an elevated level of AH IL-6 compared to a baseline level indicates that the patient is likely to respond to the therapy.

[0049] In one aspect, the present invention relates to a method of treating a patient with an ocular disease, the method comprising administering to the patient a treatment comprising an effective amount of an IL-6 antagonist, the method comprising determining the level of AH IL-6 in a sample obtained from the patient, wherein an elevated level of AH IL-6 compared to a baseline level indicates that the patient is likely to respond to the treatment.

[0050] In one aspect, the invention relates to an in vitro diagnostic immunoassay comprising an anti-IL-6 antibody for use in any of the above methods.

[0051] In one aspect, the present invention relates to the use of an IL-6 antagonist for the manufacture of a medicament for treating a patient with an ocular disease, wherein the patient has been determined by any of the methods described above to be likely to respond to a treatment comprising an effective amount of an IL-6 antagonist.

[0052] These and other embodiments are further described in the detailed description that follows. [Brief explanation of the drawings]

[0053] [Figure 1] Absolute BCVA over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean BCVA by dose group (2.5 mg, 1 mg, or 0.5 mg of RO7200220). RO7200220 was administered intravitreally (IVT) three times (Days 1, 28, and 56) four weeks apart. [Figure 2] Change in BCVA from baseline (Day 1) over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean change from baseline by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. [Figure 3] Absolute CST over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean CST by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. [Figure 4]CST change from baseline (Day 1) over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean change from baseline by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. [Figure 5] Absolute subretinal fluid (SRF) volume over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are shown as the mean and standard error for the UME population (n=23). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart, as indicated by the gray dotted lines. SRF volume was measured on SD-OCT images at a 3.0 mm radius ring of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. [Figure 6] Absolute intraretinal fluid (IRF) volume over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are shown as the mean and standard error for the UME population (n=23). RO7200220 was administered intravenously three times (Days 1, 28, and 56) 4 weeks apart, as indicated by the gray dotted lines. IRF volume was measured on SD-OCT images at a 3.0 mm radius ring of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. [Figure 7] Baseline AH IL-6 versus change from baseline in CST in patients with DME participating in Part 1 of the DOVETAIL trial. Change in CST as mean CST at Days 70 and 98 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements was available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, before RO7200220 injection) AH IL-6 concentrations. [Figure 8]Baseline AH IL-6 versus change from baseline in BCVA in patients with DME participating in Part 1 of the DOVETAIL trial. Change in BCVA as mean BCVA at days 70 and 98 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements is available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, before RO7200220 injection) AH IL-6 concentration. [Figure 9] Change from baseline in baseline AH IL-6 versus CST in patients with UME participating in Part 4 of the DOVETAIL trial. Change in CST as mean CST on days 56 and 84 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements was available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, before RO7200220 injection) AH IL-6 concentrations. [Figure 10] Change from baseline in baseline AH IL-6 versus BCVA in patients with UME participating in Part 4 of the DOVETAIL trial. Change in BCVA as mean BCVA at days 56 and 84 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements is available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, before RO7200220 injection) AH IL-6 concentrations. [Figure 11]Absolute BCVA over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean BCVA by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered intravitreally (IVT) three times (Days 1, 28, and 56) 4 weeks apart. Error bars represent standard error. Baseline is the patient's last observation before initiation of study medication. One patient in the 1 mg group postponed the Day 28 / 56 dose. This patient subsequently received doses at the Day 42 and Day 84 visits. Windowing was applied to this patient, mapping visits on Days 42, 59, 81, 123, 150, and 207 to visits on Days 42, 56, 84, 112, 140, and 196, respectively. [Figure 12] Change in BCVA from baseline (Day 1) over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as the mean change from baseline by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. Error bars represent standard deviation. Baseline is the patient's last observation before initiation of study drug. One patient in the 1 mg group postponed the Day 28 / 56 dose. This patient subsequently received doses at visits 42 and 84. Windowing was applied to this patient, mapping visits on Days 42, 59, 81, 123, 150, and 207 to visits on Days 42, 56, 84, 112, 140, and 196, respectively. [Figure 13]Figure 1 shows absolute CST over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as mean CST by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. Error bars represent standard deviation. Baseline is the patient's last observation before initiation of study drug. One patient in the 1 mg group postponed the Day 28 / 56 dose. This patient subsequently received doses at visits 42 and 84. Windowing was applied to this patient, mapping visits on Days 42, 59, 81, 123, 150, and 207 to visits on Days 42, 56, 84, 112, 140, and 196, respectively. [Figure 14] CST change from baseline (Day 1) over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are presented as the mean change from baseline by dose group (2.5 mg, 1 mg, or 0.5 mg RO7200220). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart. Error bars represent standard deviation. Baseline is the patient's last observation before initiation of study drug. One patient in the 1 mg group postponed dosing on Days 28 / 56. This patient subsequently received dosing at visits on Days 42 and 84. Windowing was applied to this patient, mapping visits on Days 42, 59, 81, 123, 150, and 207 to visits on Days 42, 56, 84, 112, 140, and 196, respectively. [Figure 15] Absolute subretinal fluid (SRF) volume over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are shown as the mean and standard error for the UME population (n=37). RO7200220 was administered IVT three times (Days 1, 28, and 56) 4 weeks apart, as indicated by the gray dotted lines. SRF volume was measured on SD-OCT images at a 3.0 mm radius ring of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. [Figure 16]Absolute intraretinal fluid (IRF) volume over time in patients with UME participating in Part 4 of the DOVETAIL study. Data are shown as the mean and standard error for the UME population (n=37). RO7200220 was administered intravenously three times (Days 1, 28, and 56) 4 weeks apart, as indicated by the gray dotted lines. IRF volume was measured on SD-OCT images at a 3.0 mm radius ring of the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. [Figure 17] Baseline AH IL-6 versus change from baseline in CST for DME patients participating in Part 1 of the DOVETAIL trial. Change in CST as mean CST at Days 70 and 98 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements was available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, pre-RO7200220 injection) AH IL-6 concentration. One patient in the 1 mg group had their Day 28 / 56 doses deferred (dosed on Days 42 and 84). Visits for this patient on Days 81 and 101 of the study were mapped to visits on Days 84 and 112. [Figure 18] Baseline AH IL-6 versus change from baseline in BCVA for DME patients participating in Part 1 of the DOVETAIL trial. Change in BCVA as mean BCVA at Days 70 and 98 relative to baseline (i.e., Day 1, day of first RO7200220 injection), assuming at least one of these measurements is available. The x-axis is the common logarithm (base 10 logarithm) of baseline (Day 1, pre-RO7200220 injection) AH IL-6 concentration. One patient in the 1 mg group had their Day 28 / 56 dosing deferred (dosed on Days 42 and 84). Visits for this patient on Days 81 and 101 of the study were mapped to visits on Days 84 and 112. DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description of the Embodiments Provided herein are interleukin-6 (IL-6) antagonists, such as anti-IL-6 or anti-IL-6 receptor (IL-6R) antibodies, for use in treating patients with ocular diseases with IL-6-mediated pathophysiology, characterized by increased aqueous humor (AH) IL-6 levels. Also provided herein are methods for identifying patients with ocular diseases that are responsive to treatment comprising an effective amount of an IL-6 antagonist by determining AH IL-6 levels.

[0055] Although molecular mediators of BRB degradation exert their activity within the retina, clinical investigation of the relevance of these molecules in patients with retinal diseases such as UME and DME relies on analysis of surrogate specimens such as vitreous and aqueous humor. Patient retinal samples cannot be collected due to the invasiveness of the sampling procedure and the adverse consequences for the patient. The vitreous is in close contact with the retina, and its molecular composition is thought to contain factors released by retinal cells. However, collection of vitreous samples is typically only possible during a qualified vitrectomy, limiting its use for analytical purposes. Aqueous humor is the fluid that fills the anterior chamber of the eye and is more easily collected than the vitreous.

[0056] definition IL-6 antagonist The term "IL-6 antagonist (IL-6a)" refers to a molecule capable of binding to IL-6 or IL-6R and inhibiting or reducing at least one IL-6 activity. IL-6 activity may include one or more of the following: binding to gp130, activation of the IL-6 signaling pathway, activation of JAK kinases, e.g., phosphorylation of JAK kinase targets, activation of STAT proteins, e.g., phosphorylation of STAT proteins, and / or expression of STAT target genes.

[0057] In one aspect, the IL-6a described herein specifically binds to site II (site 2) of IL-6 and is useful for treating IL-6-associated diseases, such as IL-6-associated eye diseases and certain other diseases described herein.

[0058] In one aspect, IL-6a is characterized by one or more of the following properties: it has a high affinity for either free IL-6 (e.g., soluble IL-6) or bound IL-6 (e.g., IL-6 bound to the IL-6 receptor), or for both free and bound IL-6; it is relatively stable in an organism; it can inhibit the binding of IL-6 bound to IL-6R (referred to herein as the IL-6 / IL-6R complex or IL-6 / IL-6R) to gp130; and / or it can have a therapeutic effect.

[0059] In one embodiment, the IL-6a is an antibody or an antibody-derived fragment. For example, the IL-6a is a high-affinity humanized Fab that can specifically bind to site II of IL-6 and potently block both cis-IL-6 signaling and trans-IL-6 signaling. In another example, the IL-6a is a full-length antibody, such as an IgG1 or IgG2 antibody.

[0060] In one aspect, IL-6a selectively binds to site II of IL-6, and such molecules can inhibit gp130 binding to IL-6, regardless of whether IL-6 is free or bound to membrane IL-6R or sIL-6R, resulting in broad inhibition of IL-6 signaling. Furthermore, by targeting the ligand (IL-6) as opposed to the IL-6 receptor, receptor-mediated clearance and ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity can be avoided.

[0061] Because IL-6 plays both pathological and protective roles in disease, the use of IL-6a to treat diseases associated with elevated IL-6 can improve certain aspects of symptoms but can also cause significant adverse effects, e.g., systemic effects. This duality of the IL-6 pathway (i.e., its ability to have desirable and / or undesirable effects) can make treating IL-6-associated disorders with systemic inhibitors undesirable. Thus, the compositions and methods provided herein can be useful for treatments that inhibit at least one IL-6 activity but do not unduly affect the beneficial activities of IL-6, in part because the compositions can be formulated for local delivery, e.g., to the eye. For example, in one aspect, IL-6a is engineered to be sized appropriately for delivery to a specific site. In some embodiments, the IL-6a is a full-length antibody. In one aspect, the IL-6a is derived from an antibody and is in a format that can have longer retention in specific compartments of the eye, e.g., the vitreous, and limited systemic leakage. In one aspect, the IL-6a is a modified antibody (e.g., an antibody with a modified Fc domain) that has longer retention in the vitreous of the eye and / or more limited systemic leakage compared to the corresponding unmodified antibody. In some embodiments, the IL-6a is an IgG2 antibody.

[0062] In one aspect, the IL-6a is a relatively small IL-6a, such as a fragment of an IL-6 antibody or other derivative of an antibody that is less than a full-length antibody, e.g., a Fab derived from an IL-6 antibody. In one aspect, the IL-6a is in a format that allows it to pass from one tissue part to another with increased kinetics compared to a corresponding full-length IL-6 antibody. In some embodiments, the IL-6a is a Fab engineered to be a larger molecule, which is more likely to have increased retention at the delivered location compared to the Fab alone, e.g., the IL-6a is dimerized via the Fc domain. In one aspect, the Fc domain is engineered to have abolished or reduced FcRn binding, which can reduce systemic accumulation compared to the same IL-6-binding entity containing a wild-type Fc. The engineered Fc domain can be, for example, an IgG1 domain or an IgG2 domain.

[0063] Typically, the IL-6 antagonists described herein have sufficiently high affinity for their target, IL-6 or IL-6R, to be effective in ameliorating at least one undesirable effect of IL-6, and are sufficiently stable to be useful as therapeutic agents.

[0064] Generally, IL-6a PKs suitable for ocular use have a sufficiently long half-life at the delivery site, e.g., the vitreous, to provide a therapeutic effect, e.g., the PK may have a half-life of at least 8 days, 10 days, 14 days, 21 days, 28 days, or 30 days. Identification of IL-6 antagonists that bind to site II

[0065] Generally, any method known in the art can be used to generate molecules capable of binding to IL-6; for example, a polypeptide library or molecule library can be screened for candidate compounds in an assay for the ability of the polypeptide or compound to bind to IL-6. Once such candidate compounds are identified, the binding site of the compound can be identified using methods known in the art. For example, the molecule can be tested for its ability to bind to wild-type IL-6 and its binding compared to the ability of the compound to bind to IL-6 mutated at site I, site II, or site III. In one aspect, the IL-6a described herein retains the ability to bind to the IL-6 / IL-6Rα complex and IL-6 and interferes with the binding of IL-6 / IL-6Rα to gp130. In one aspect, the IL-6a described herein can compete with gp130 for binding to the IL-6 / IL-6Rα complex, e.g., by binding to site II of IL-6. Such binding activity can be assayed using methods known in the art.

[0066] IL-6a candidates can be tested, for example, using the HEK-Blue™ IL-6 Assay System (InvivoGen, San Diego). HEK-Blue™ IL-6 cells are HEK293 cells stably transfected with the human IL-6R and a STAT3-inducible SEAP reporter gene. In the presence of IL-6, STAT3 is activated and SEAP is secreted. SEAP is assessed, for example, using QUANTI-Blue™ (InvivoGen, San Diego). Addition of IL-6a to cells inhibits both free and soluble receptor-bound IL-6, thereby preventing secretion or reducing SEAP levels.

[0067] K Drefers to the binding affinity equilibrium constant of a particular antibody-antigen or antibody fragment-antigen interaction. In one aspect, the antibodies or antigen-binding fragments described herein have a K of 250 pM or less, e.g., 225 pM, 220 pM, 210 pM, 205 pM, 150 pM, 100 pM, 50 pM, 20 pM, 10 pM, or 1 pM or less. D It binds to IL-6 or IL-6R. D can be determined using methods known in the art, for example, using surface plasmon resonance, for example, using a BiaCore™ system.

[0068] K off refers to the dissociation rate constant of a particular antibody-antigen interaction or antibody fragment-antigen complex. Dissociation rate constants can be determined using surface plasmon resonance, for example, using a BiaCore™ system. A relatively slow K off may contribute to desirable characteristics of the therapeutic agent, for example, allowing for less frequent administration of the inhibitor to a subject in need of such treatment.

[0069] specificity In one aspect, the IL-6a described herein specifically binds to a target, e.g., IL-6. Generally, as used herein, "specific binding" indicates that a molecule binds preferentially to a selected molecule and exhibits much lower binding affinity to one or more other molecules. In embodiments, the binding affinity to another molecule is one, two, three, or more orders of magnitude lower than the binding affinity to the target.

[0070] As discussed above, IL-6 can exist as free IL-6 and as IL-6 bound to soluble IL-6Rα. Site II of IL-6 is the optimal target for IL-6 antagonists compared to inhibitors that bind to site I of IL-6. Site I inhibitors can inhibit the binding of free IL-6 to IL-6Rα. However, such inhibitors inhibit the kappa of the complex. offExcept for substitutions limited by the IL-6 receptor, inhibitors that bind to IL-6Rα are unable to prevent activity initiated by the existing IL-6 / IL-6R complex. Another alternative, inhibitors that bind to IL-6Rα, are less suitable because their ability to prevent IL-6 activity may be limited unless present at saturating concentrations. Because the amount of IL-6 receptor is generally very large compared to the amount of IL-6, this approach may require the administration of unnecessarily large amounts of a composition that inhibits IL-6 activity by binding to the receptor. In one aspect, the IL-6a described herein can block the activity of IL-6 even when IL-6 is bound to the IL-6R. Thus, an advantage of the IL-6a described herein is that relatively less of the composition may be required to achieve a therapeutic effect compared to inhibitors that target the IL-6 receptor. Anti-receptor antibodies have been reported to be rapidly eliminated by receptor-mediated clearance, significantly limiting their PK, thus requiring larger doses, more frequent administration, or both. Furthermore, both anti-receptor IL-6 antibodies and anti-site I IL-6 antibodies pose the problem of significantly increasing tissue concentrations of IL-6 by disrupting the normal receptor-mediated clearance pathway of the ligand, thereby exposing subjects to potentially undesirable levels of IL-6 in tissues. Furthermore, the use of inhibitors targeting IL-6Rα may require the inhibitor to be present in the vicinity of both the site where inhibition is sought and the site where it is undesired, e.g., systemic treatment. The use of IL-6a, which binds to site II, the site where gp130 binds, allows for inhibition of free IL-6 as well as IL-6 that binds to IL-6R but has not yet activated the IL-6 pathway via gp130. Therefore, without wishing to be bound by theory, the IL-6 antagonists described herein are designed to bind to both forms of IL-6 (soluble and receptor-bound); specifically, the IL-6 antagonists bind to site II of IL-6, which is accessible in both forms. The IL-6a-containing compositions described herein can inhibit both cis- and trans-mediated signaling by IL-6.

[0071] In one aspect, the compositions and methods provided herein are designed to provide effective IL-6 blockade sufficient to treat at least one sign or symptom of an IL-6-associated disorder, e.g., sufficient to inhibit angiogenesis and / or inflammation.

[0072] The compositions described herein are useful, for example, for treating ocular diseases characterized by undesirably high levels of IL-6 in the vitreous (see Yuuki et al., J Diabetes Compl 15:257 (2001); Funatsu et al., Ophthalmology 110:1690, (2003); Oh et al., Curr Eye Res 35:1116 (2010); Noma et al., Eye 22:42 (2008); Kawashima et al., Jpn J Ophthalmol 51:100 (2007); Kauffman et al., Invest Ophthalmol Vis Sci 35:900 (1994); Miao et al., Molec Vis 18:574 (2012)).

[0073] Generally, the IL-6a described herein is a potent antagonist of IL-6 signaling. In one embodiment, the IL-6a described herein has a high affinity for IL-6, for example, an IC50 of 100 pM or less in a HEK-Blue IL-6 assay using 10 pM of IL-6. The high affinity of IL-6a is determined by the K D , for example, 1 nM or less, 500 pM or less, 400 pM or less, 300 pM or less, 240 pM or less, or 200 pM or less. D The determination can be made based on the following:

[0074] To produce biological IL-6a (e.g., a protein or polypeptide such as an antibody, fragment, or derivative thereof) useful for treating disorders associated with increased IL-6 expression or activity, it is typically desirable for the biological IL-6a to have a high productivity. For example, a suitable productivity is 1 g / L or more (e.g., 2 g / L or more, 5 g / L or more, or 10 g / L or more).

[0075] To effectively administer an IL-6 antagonist, the inhibitor must have a solubility compatible with the concentration at which it is administered, such as 20 mg / ml or more, 10 mg / ml or more, 5 mg / ml or more, or 1 mg / ml or more for the full-length antibody IL-6a.

[0076] Furthermore, to be a viable treatment, the inhibitor must have high stability at the body temperature of the delivery and active sites, as well as storage stability. In one aspect, the inhibitor has a T of 60° C. or higher (e.g., 60° C. or higher, 62.5° C. or higher, 65° C. or higher, 70° C. or higher, 73° C. or higher, or 75° C. or higher). m In one aspect, the inhibitor has a T of 45° C. or higher, e.g., 50° C. or higher, 51° C. or higher, 55° C. or higher, or 60° C. or higher. onset T m and T onset can be determined using methods known in the art.

[0077] Antagonists with desired characteristics can be selected from suitable types of molecules known in the art, such as antibodies, including fragments and derivatives of IL-6 site II-targeting antibodies that generally retain or maintain sufficient characteristics (e.g., desired binding properties) of the parent IL-6 antibody. Such antagonists include F ab fragments, scFv, F engineered to contain an Fc portion ab These include fragments and full-length antibodies that have been engineered to have a different framework than the parent IL-6 site II-targeting antibody.

[0078] In one aspect, the IL-6a disclosed herein comprises a human antibody antigen-binding site that can compete or cross-compete with an antibody or fragment thereof that can bind to site II of IL-6. For example, the antibody or fragment thereof can be composed of a VH domain and a VL domain disclosed herein, wherein the VH domain and the VL domain comprise a set of CDRs of an IL-6 / site II-binding antibody disclosed herein.

[0079] Any suitable method can be used to determine the domain and / or epitope bound by IL-6a, for example, by mutating various sites on IL-6. Sites whose mutations prevent or reduce binding of IL-6a and IL-6 ligand may be directly involved in binding to IL-6a or may indirectly affect the binding site, for example, by affecting the conformation of IL-6. Other methods can be used to identify the amino acids bound by IL-6a. For example, peptide binding scans, such as PEPSCAN-based enzyme-linked immunosorbent assays (ELISAs), can be used. In this type of peptide binding scan, short, overlapping peptides derived from the antigen are systematically screened for binding to binding members. Peptides can be covalently attached to a support surface to form a peptide array. Peptides can be linear or in a constrained conformation. Constrained conformations can be created using peptides with terminal cysteine ​​(cys) residues at each end of the peptide sequence. The cys residues can be covalently attached directly or indirectly to the support surface so that the peptide is held in a looped conformation. Thus, the peptides used in this method can have a cys residue added to each end of the peptide sequence corresponding to a fragment of the antigen. Double-loop peptides can also be used, in which a cys residue is additionally located at or near the center of the peptide sequence. The cys residue can be directly or indirectly covalently attached to the support surface so that the peptide forms a double-loop conformation with one loop on each side of the central cys residue. The peptides can be synthetically produced, and thus, cys residues can be engineered into desired positions, even though they do not naturally occur in the IL-6 site II sequence. Optionally, both linear and constrained peptides can be screened in peptide-binding assays.Peptide binding scanning involves identifying (e.g., using ELISA) a set of peptides to which a binding member binds, where the peptides have amino acid sequences corresponding to fragments of IL-6a (e.g., peptides comprising about 5, 10, or 15 consecutive residues of IL-6a), and aligning the peptides to identify a footprint of residues to which the binding member binds, where the footprints include residues common to the overlapping peptides. Alternatively, or additionally, peptide binding scanning methods can be used to identify peptides to which IL-6a binds with at least a selected signal:noise ratio.

[0080] Other methods known in the art, such as site-directed mutagenesis (e.g., as described herein), deuterium exchange, mass spectrometry, NMR, and X-ray crystallography, can be used to identify residues bound by the antibody and / or to confirm the results of peptide binding scans.

[0081] Typically, the useful IL-6a described herein is a human antibody molecule, a humanized antibody molecule, or a binding fragment thereof. Generally, the antibody is a monoclonal antibody. Such antibodies may be of human, mouse, rat, camelid, rabbit, sheep, pig, or bovine origin and may be produced according to methods known to those skilled in the art.

[0082] As used herein, the term "antibody molecule" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen. An antibody molecule can be a full-length antibody or a fragment thereof, e.g., an antigen-binding fragment thereof. An antibody can be polyclonal or monoclonal, multi-chain or single-chain, or an intact immunoglobulin, and can be derived from natural or recombinant sources. An antibody can be a tetramer of immunoglobulin molecules. An antibody fragment or antigen-binding fragment refers to at least a portion of an intact antibody or a recombinant variant thereof, and refers to the antigen-binding domain, e.g., the antigen-determining variable region of an intact antibody, which is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv antibody fragments, linear antibodies, single domain antibodies, e.g., sdAb (either VL or VH), camelid VHH domains, and multispecific antibodies formed from antibody fragments, e.g., bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region, and isolated CDR or other epitope-binding fragments of antibodies. Antigen-binding fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see US Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).

[0083] Exemplary IL-6 Antibodies Generally, IL-6a comprises at least the CDRs of an antibody capable of specifically binding to IL-6 (e.g., human IL-6), e.g., to site II of IL-6. A structure carrying a CDR or set of CDRs of the present invention can be an antibody heavy or light chain sequence, or a substantial portion thereof, in which the CDR or set of CDRs is located at a position corresponding to the CDR or set of CDRs of naturally occurring VH and VL antibody variable domains encoded by a rearranged immunoglobulin gene. The structure and location of immunoglobulin variable domains can be determined by reference to Kabat, et al., 1983 (National Institutes of Health), updates of which can be found by searching for "Kabat" using any internet search engine.

[0084] As disclosed herein, IL-6a is typically an antibody molecule generally comprising an antibody VH domain and / or an antibody VL domain. The VH domain comprises a set of heavy chain CDRs (VH CDRs), and the VL domain comprises a set of light chain CDRs (VL CDRs). Examples of such CDRs are provided in the Examples section of the present specification. The antibody molecule may comprise an antibody VH domain comprising VH CDR1, VH CDR2, and VH CDR3, as well as a framework. It may also comprise an antibody VL domain comprising VL CDR1, VL CDR2, and VL CDR3, as well as a framework.

[0085] Disclosed herein are IL-6 antagonists comprising a VH CDR1, a VH CDR2, and a VH CDR3, such as those disclosed herein, and a VL CDR1, a VL CDR2, and a VL CDR3, such as those disclosed herein. The CDRs can be derived from one or more antibodies. For example, the VL CDR can be derived from the same or a different antibody as the VH CDR.

[0086] In one embodiment, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the sequence of SEQ ID NO:1, a VH CDR2 comprising the sequence of SEQ ID NO:2, and a VH CDR3 comprising the sequence of SEQ ID NO:3.

[0087] In one aspect, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a VL CDR1 comprising the sequence of SEQ ID NO:4, a VL CDR2 comprising the sequence of SEQ ID NO:5, and a VL CDR3 comprising the sequence of SEQ ID NO:6.

[0088] In one embodiment, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, a VH CDR3 comprising the sequence of SEQ ID NO: 3, a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6.

[0089] In one aspect, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:7.

[0090] In one aspect, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a light chain variable region comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:8.

[0091] In one aspect, the anti-IL-6 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:8.

[0092] In one aspect, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a heavy chain comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:9.

[0093] In one aspect, the anti-IL-6 antibody or antigen-binding fragment thereof comprises a light chain comprising a sequence at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO:10.

[0094] In one aspect, the anti-IL-6 antibody, or antigen-binding fragment thereof, comprises a heavy chain comprising the sequence of SEQ ID NO:9 and a light chain comprising the sequence of SEQ ID NO:10.

[0095] In one embodiment, the anti-IL-6 antibody is RO7200220, comprising a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, a VH CDR3 comprising the sequence of SEQ ID NO: 3, a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 and the light chain variable region comprises the sequence of SEQ ID NO: 8, or wherein the heavy chain comprises the sequence of SEQ ID NO: 9 and the light chain comprises the sequence of SEQ ID NO: 10.

[0096] In embodiments, the antibody molecule, e.g., antibody or antigen-binding fragment, has increased affinity and / or increased potency for human IL-6 compared to an antibody or antigen-binding fragment comprising one or more corresponding sequences of EBI-029 or the sequences of the antibodies described in WO 2014 / 074905, the entire contents of which are incorporated herein by reference. In one aspect, the antibody or antigen-binding fragment has increased affinity and / or increased potency for human IL-6 compared to tocilizumab.

[0097] The IL-6a described herein can comprise an antibody constant region or a portion thereof, e.g., a human antibody constant region or a portion thereof. For example, a VL domain can bind at its C-terminus to an antibody light chain constant domain, including a human CK or CL chain. Similarly, a VH domain-based IL-6a can bind at its C-terminus to all or a portion (e.g., the CH1 domain) of an immunoglobulin heavy chain from any antibody isotype, e.g., IgG, IgA, IgE, and IgM, and any isotype subclass, particularly IgG1, IgG2, IgG3, and IgG4. In embodiments, the antibody or antigen-binding fragment is engineered to reduce or eliminate ADCC activity.

[0098] In one embodiment, an antibody of the invention is an IgG2 antibody. In one embodiment, an antibody of the invention comprises an IgG2 framework, an IgG2 constant region, or an IgG2 Fc region.

[0099] IgG2 antibodies can exist as three major structural isoforms: IgG2-A, IgG2-B, and IgG2-A / B (Wypych J. et al. Journal of Biological Chemistry. 2008, 283:16194-16205). This structural heterogeneity is due to different structures of the disulfide bond linking the Fab arm to the heavy chain hinge region. In the IgG2-A isoform, there is no disulfide bond linking the Fab arm to the hinge region. In the IgG2-B isoform, both Fab arms have disulfide bonds linking the heavy and light chains to the hinge region. The IgG2-A / B isoform is a hybrid between the IgG2-A and IgG2-B isoforms, and has only one Fab arm with a disulfide bond linking the heavy and light chains of one Fab arm to the hinge region. Conversion of IgG2 antibodies between two or all of their different structural isoforms, also known as disulfide shuffling, naturally occurs in vivo and in vitro for both naturally occurring and recombinant antibodies. As a result, IgG2 antibody preparations in the art contain heterogeneous mixtures of IgG2-A, IgG2-B, and IgG2-A / B isoforms. Different IgG2 isoforms may have unique and distinct functional properties, such as differences in stability, aggregation, viscosity, Fc receptor binding, or potency. The presence of multiple isoforms or elevated levels of a particular isoform in an IgG2 antibody preparation may adversely affect stability, aggregation, or potency. Several fragments of IgG2 antibodies that may still undergo disulfide shuffling and exist in any of the structural isoforms A, A / B, and / or B can be readily envisioned, e.g., fragments that retain residues involved in shuffling disulfide bonds, e.g., fragments that include at least the IgG2 hinge region.

[0100] Antibody fragments containing an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab', Fab'-SH, scFv, Fv, dAb, Fd, and disulfide-stabilized variable region (dsFv). A variety of other antibody molecules containing one or more antibody antigen-binding sites can be engineered, such as F(ab')2, F(ab)3, diabodies, triabodies, tetrabodies, and minibodies. Examples of antibody molecules and methods for their construction and use are described in Holliger and Hudson, 2005, Nat Biotechnol 23:1126-1136. Non-limiting examples of binding fragments include Fab fragments composed of the VL, VH, constant light domain (CL), and constant heavy domain 1 (CH1) domains, Fd fragments composed of the VH and CH1 domains, Fv fragments composed of the VL and VH domains of a single antibody, dAb fragments composed of the VH or VL domain, isolated CDR regions, F(ab')2 fragments, which are bivalent fragments containing two linked Fab fragments, single-chain Fv molecules (scFv) in which the VH and VL domains are linked by a peptide linker that allows the two domains to associate to form an antigen-binding site, bispecific single-chain Fv dimers (e.g., as disclosed in WO 1993 / 011161), and diabodies, which are multivalent or multispecific fragments constructed using gene fusion (e.g., as disclosed in WO 94 / 13804). Fv, scFv, or diabody molecules can be stabilized by the incorporation of disulfide bridges linking the VH and VL domains. Minibodies containing scFv linked to the CH3 domain can also be used as IL-6a antibodies. Other antibody fragments and derivatives that can be used as IL-6a antibodies include Fab' fragments, which differ from Fab fragments in that they have several additional amino acid residues at the carboxyl terminus of the heavy chain CH1 domain and contain one or more cysteines from the antibody hinge region, and Fab'-SH fragments, in which the cysteine ​​residues in the constant domains have free thiol groups.

[0101] In one embodiment, the antibody fragment, IL-6a, is chemically modified to improve or introduce desired properties, for example, PEGylation to increase half-life or uptake.

[0102] A dAb (domain antibody) is a small monomeric antigen-binding fragment of an antibody (the variable region of an antibody heavy or light chain). VH dAbs occur naturally in camelids (e.g., camels and llamas) and can be produced by immunizing camelids with a target antigen, isolating antigen-specific B cells, and directly cloning dAb genes from individual B cells.

[0103] In one embodiment, IL-6a can be incorporated as part of a bispecific antibody prepared using methods known in the art, e.g., chemically or from a hybrid hybridoma. Such molecules can be bispecific antibody fragments of the type discussed above. One non-limiting example of a method for generating bispecific antibodies is BiTE™ technology, which allows the binding domains of two antibodies with different specificities to be directly linked via a short, flexible peptide. This combines two antibodies on a short, single polypeptide chain. Diabodies and scFvs can be constructed without an Fc region, using only the variable domains, potentially reducing the effects of anti-idiotypic reaction. Bispecific antibodies can be constructed as whole IgG, as bispecific Fab'2s, as Fab'PEGs, as diabodies, or as bispecific scFvs. Furthermore, two bispecific antibodies can be linked to form tetravalent antibodies using routine methods known in the art.

[0104] Bispecific diabodies, in contrast to bispecific whole antibodies, are useful in part because they can be constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) of appropriate binding specificity can be readily selected from libraries using phage display (WO 1994 / 13804). If one arm of the diabody is held constant, e.g., with specificity for site II of IL-6, the other arm can be varied to generate libraries from which antibodies of appropriate specificity can be selected.

[0105] Bispecific whole antibodies can be produced by alternative technical approaches described in WO 1996 / 27011, WO 1998 / 50431 and WO 2006 / 028936.

[0106] In one embodiment, IL-6a can comprise an antigen-binding site within a non-antibody molecule, for example, by incorporating one or more CDRs, e.g., a set of CDRs, into a non-antibody protein scaffold, as discussed in detail below. In one embodiment, the CDRs are incorporated into a non-antibody scaffold. IL-6 site II binding sites can be generated by arranging CDRs on a non-antibody protein scaffold, such as fibronectin or cytochrome B, or by randomizing or mutating amino acid residues in loops within the protein scaffold to confer binding specificity for IL-6 site II. Scaffolds for engineering novel binding sites in proteins are known in the art. For example, protein scaffolds for antibody mimetics are disclosed in International Publication No. 200034784, which describes proteins (antibody mimetics) containing a fibronectin type III domain with at least one randomized loop. A suitable scaffold for grafting one or more CDRs, e.g., a set of HCDRs, can be provided by any domain member of the immunoglobulin gene superfamily. The scaffold can be a human or non-human protein. An advantage of non-antibody protein scaffolds is that they can provide the scaffold molecule with an antigen-binding site that is smaller and / or easier to manufacture than at least some antibody molecules. The small size of the binding member can confer useful physiological properties, such as the ability to enter cells, penetrate deep into tissues, or reach targets within other structures, or the ability to bind within the protein cavity of the target antigen. Typically, these are proteins with a stable scaffold and one or more variable loops, in which the amino acid sequence of one or more loops is specifically or randomly mutated to create an antigen-binding site that binds to the target antigen. Such proteins include the IgG-binding domain of protein A from Staphylococcus aureus, transferrin, tetranectin, fibronectin (e.g., using the 10th fibronectin type III domain), lipocalin, and gamma crystalline and other Affilin scaffolds™ (Scil Proteins, Halle, Germany).Other exemplary approaches include synthetic microbodies based on cyclic compounds—small proteins with intramolecular disulfide bonds, microproteins (e.g., Versabodies™, Amunix Inc., Mountain View, CA) and ankyrin repeat proteins (DARPins, e.g., Molecular Partners AG, Zurich-Schlieren, Switzerland). Such proteins also include small engineered protein domains, such as immunodomains (see, e.g., U.S. Patent Application Publication Nos. 2003 / 082630 and 2003 / 157561). An immunodomain comprises at least one complementarity-determining region (CDR) of an antibody.

[0107] In one aspect, the antibodies disclosed herein can be modified to reduce their ability to fix complement and participate in complement-dependent cytotoxicity (CDC). In one aspect, the antibodies are modified to reduce their ability to activate effector cells and participate in antibody-dependent cellular cytotoxicity (ADCC). In one aspect, the antibodies disclosed herein can be modified to reduce their ability to activate effector cells and participate in antibody-dependent cellular cytotoxicity (ADCC), and to reduce their ability to fix complement and participate in complement-dependent cytotoxicity (CDC).

[0108] formulation IL-6a, such as IL-6 antibody, is 0.1mg / ml~100mg / ml, 0.1~80mg / ml, 0.1~50mg / ml, 0.1mg / ml~20mg / ml, 0.1mg / ml~5mg / ml, 0.1mg / ml~1mg / ml, 1mg / ml~100mg / ml, 5mg / ml~100mg / ml, 5mg / ml~30mg / ml, 10mg / ml~100mg / ml, 10mg / ml~30mg / ml, 20mg / ml~100mg / ml, 30mg / ml~100mg / ml, 40mg / ml~100mg / ml, 50mg / ml~100mg / m It can be formulated at a concentration of 1, 60mg / ml to 100mg / ml, 1mg / ml to 80mg / ml, 5mg / ml to 80mg / ml, 10mg / ml to 80mg / ml, 20mg / ml to 80mg / ml, 40mg / ml to 80mg / ml, 50mg / ml to 80mg / ml, 60mg / ml to 80mg / ml, 1mg / ml to 60mg / ml, 5mg / ml to 60mg / ml, 10mg / ml to 60mg / ml, 20mg / ml to 60mg / ml, 30mg / ml to 60mg / ml, 40mg / ml to 60mg / ml, or 50mg / ml to 60mg / ml. For example, formulations contain about 1 mg / ml, 2 mg / ml, 5 mg / ml, 10 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 40 mg / ml, 50 mg / ml or 55 mg / ml.

[0109] The IL-6a formulation may include other pharmaceutically acceptable excipients. In one embodiment, the IL-6a, e.g., IL-6 antibody, is formulated with one or more or all of a buffer, a surfactant, and an isotonicity agent (e.g., a sugar and / or a salt). In one embodiment, the formulation further includes one or more chelating agents, one or more preservatives, one or more antioxidants, and / or one or more amino acids. In one embodiment, the formulation further includes one or more additional therapeutic agents, e.g., a second therapeutic agent.

[0110] Pharmaceutical Compositions and Formulations The pharmaceutical compositions and formulations described herein can be formulated in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, such as nanoparticles and liposomes. The form generally depends on the intended mode of administration and therapeutic application. The pharmaceutical compositions described herein are typically in the form of an injectable or infusible solution, or are formulated for topical delivery, for example, topical ocular delivery.

[0111] In one aspect, the pharmaceutical compositions described herein are sterile and stable under the conditions of manufacture and storage. Pharmaceutical compositions can also be tested to ensure compliance with regulatory and industry standards for administration. The compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug (e.g., biologic) concentrations. Sterile injectable solutions can be prepared by incorporating the agents described herein in the required amount in an appropriate solvent containing one or a combination of the ingredients listed above, as needed, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the agents described herein into a sterile vehicle containing a basic dispersion medium and any other required ingredients. For sterile powders for the preparation of sterile injectable solutions, exemplary preparation methods include vacuum drying and freeze-drying, which yield a powder of the agents described herein and any additional desired ingredients from a previously sterile-filtered solution thereof. The proper fluidity of the solution can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prolonged absorption of an injectable composition can be manipulated by including an agent that delays absorption, such as monostearate salts and gelatin. Such agents may be particularly useful in low-dose formulations. In one embodiment, the formulation contains 1 mg / ml or less of a therapeutic protein (e.g., IL-6a, e.g., an IL-6 antibody or antigen-binding fragment thereof described herein), and gelatin is included in the formulation.

[0112] In one aspect, pharmaceutical compositions or formulations are prepared using carriers.For example, the formulations can be delivered as controlled release formulations and delivered by implants or microencapsulated delivery systems.Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used.See, for example, "Sustained and Controlled Release Drug Delivery Systems," JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0113] Ophthalmic packs can be used to provide prolonged contact of ophthalmic formulations with the eye. A cotton pledget is soaked with the formulation and inserted into the upper or lower eyelid. The formulation can also be administered by iontophoresis. This procedure involves keeping the solution in contact with the cornea within an eyecup containing electrodes. Diffusion of the drug is mediated by an electrical potential difference. Iontophoresis systems that have been used include Ocuphor® 1 (Iomed Inc., USA), Eyegate® II Delivery System 1 (EyeGate Pharma, USA), and Visulex® 1 (Aciont Inc., USA). See Amo and Urtti, Drug Discovery Today, 13:143 (2008).

[0114] Another method for sustained ocular delivery is the use of gelling agents. These materials can be delivered in liquid form, such as eye drops or intraocular injections. After injection, the polymer undergoes a phase change, forming a semi-solid or solid matrix that releases the drug over an extended period of time. The phase transition can be triggered by changes in temperature, ionic concentration, or pH.

[0115] For topical ophthalmic use, gel-forming solutions such as Timoptic®-XE1 (Merck, USA) containing Gelrite® (an anionic heteropolysaccharide purified from gellan gum), Pilogel® 1 (Alcon, Inc., Switzerland) eye drops containing poly(acrylic acid), and Azasite® 1 (Insite Vision, USA) are being clinically tested. These materials enhance drug retention compared to conventional eye drops, resulting in increased drug absorption into the eye and reduced dosing frequency. See Amo and Urtti, Drug Discovery Today, 13:135-143 (2008).

[0116] Administration The therapeutic or pharmaceutical compositions described herein can be delivered by injection, for example, intravitreal, periocular, or subconjunctival injection. The therapeutic can be injected under the conjunctiva to facilitate transfer through the sclera into the eye by simple diffusion. The therapeutic can also be injected under Tenon's capsule, beneath the conjunctiva and the more posterior part of the eye, to deliver the drug to the ciliary body, choroid, and retina. The therapeutic can also be administered by retrobulbar injection.

[0117] Generally, the pharmaceutical compositions or treatments described herein can be administered to a subject by any suitable method, for example, intravenous administration as a bolus, or by continuous infusion over a period of time via intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intrasynovial, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural injection, intrasternal injection and infusion. Other suitable modes of administration include topical routes (e.g., skin or mucosa) or inhalation routes (e.g., intranasal or pulmonary). In certain applications, the administration route is one of intravenous injection or infusion, subcutaneous injection, or intramuscular injection. For administration to the eye, the administration mode is topical administration to the eye, for example, in the form of drops. Examples of devices that may contain and / or be used to administer the formulation include simple eye droppers, squeeze bottles with or without metering capabilities, and blow / fill / seal (BFS) devices such as those manufactured by Catalent (Somerset, NJ), multi-use devices using, for example, tip seal technology, silver / oligodynamic technology, sterile filters, disintegration of primary containers, etc.

[0118] An additional consideration for the container is that it provides an acceptable shelf life once filled, e.g., that evaporation is acceptably low, and / or that the formulation meets release assay specifications, e.g., those described herein. In one aspect, the container is suitable for providing a shelf life of at least 2 years, e.g., at least 3 years, at least 4 years, or at least 5 years, e.g., at 5°C. In one aspect, the container is suitable for providing a shelf life of at least 3 years at 5°C. In one aspect, the container is suitable for providing a shelf life of at least 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 months at room temperature. In one aspect, the container is suitable for providing a shelf life of at least 5 months at room temperature. A variety of suitable container materials are known in the art, e.g., certain plastics, such as low-density polyethylene (LDPE), high-density polyethylene (HDPE), or polypropylene.

[0119] The pharmaceutical composition or treatment can be prepared for single-use application in a container, or can be prepared for use in a multi-use container.

[0120] The pharmaceutical compositions or treatments described herein can be delivered intravitreally, e.g., to treat disorders associated with the posterior segment of the eye. Methods of intravitreal administration are known in the art and include, e.g., intraocular injection, implantable devices.

[0121] In one embodiment, the pharmaceutical composition or treatment is administered intravitreally using an implantable device. In one embodiment, the pharmaceutical composition includes a thermal stabilizer, e.g., sorbitol. In one embodiment, the sorbitol is present at a concentration of 5% w / v or greater.

[0122] Implantable devices can be, for example, non-biodegradable devices such as polyvinyl alcohol-ethylene vinyl acetate polymer and polysulfone capillary fibers, biodegradable devices such as polylactic acid, polyglycolic acid and polylactic-co-glycolic acid, polycaprolactone, and polyanhydrides. These devices can be delivered in forms such as nanoparticles, liposomes, or microspheres.

[0123] dosage The pharmaceutical compositions or treatments described herein can be administered as a fixed dose, a weight-based dose (e.g., mg / kg), or an age-based dose. The pharmaceutical compositions or treatments described herein can be administered, for example, four times a day, three times a day, twice a day, once a day, every other day, every third, fourth, or fifth day, weekly, every two weeks, every three weeks, every four weeks, every five weeks, monthly, every two months, every three months, every four months, every six months, or as needed (discretionary).

[0124] Therapeutic or pharmaceutical compositions can include a "therapeutically effective amount" of an agent described herein. A therapeutically effective amount of an agent can vary depending on factors such as the individual's disease state, age, sex, and weight, as well as the ability of the compound to elicit a desired response in the individual, e.g., improvement in at least one disorder parameter (e.g., symptom), or improvement in at least one symptom of the disorder (and, optionally, the effects of any additional agents administered). A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. In one aspect, a "therapeutically effective amount" is determined on a population of individuals, the amount being effective to ameliorate at least one symptom or sign of a cytokine-associated disorder, e.g., an IL-6-associated disorder, in at least 5%, 10%, 25%, 50%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the affected population. A pharmaceutical composition or treatment is typically administered in a therapeutically effective amount.

[0125] The pharmaceutical composition can be administered using medical devices known in the art, such as implants, infusion pumps, hypodermic needles, and needleless hypodermic injection devices. The device can, for example, include one or more housings for storing the pharmaceutical composition and can be configured to deliver a unit dose of IL-6a, such as an IL-6 antibody or fragment thereof described herein, and optionally a second therapeutic agent. The dose can be a fixed dose, i.e., a physically discrete unit suitable as a unit dose for the subject to be treated, each unit containing a predetermined amount of IL-6a, such as an IL-6 antibody or fragment thereof described herein, calculated to produce the desired therapeutic effect, together with a pharmaceutical carrier, and optionally with another agent, such as one available as a commercially available or prescription product.

[0126] In one aspect, to treat a disorder described herein, a pharmaceutical composition or therapy is administered to a subject with the disorder in an amount and for a time sufficient to induce a sustained improvement in at least one sign or symptom of the disorder. An improvement is considered "sustained" if the subject shows improvement over an extended period of time, e.g., on at least two occasions separated by 1-4 weeks. The degree of improvement can be determined based on the signs or symptoms, and can also be determined using questionnaires administered to the subject, such as quality of life questionnaires.

[0127] Improvement can be induced by repeatedly administering a dose of the formulation until the subject shows improvement over baseline for the selected signs and / or symptoms. In treating chronic conditions, the amount of improvement can be assessed by repeated administration over a period of at least one month or more, for example, one, two, or three months or more, or indefinitely. In treating acute conditions, the agent can be administered over a period of one to six weeks, or even as a single dose.

[0128] Although the degree of the disorder after initial or intermittent treatment appears to be improved in light of one or more signs or symptoms, treatment can be continued indefinitely at the same level or at a reduced dose or frequency. Treatment can also be discontinued, for example, upon improvement or disappearance of signs or symptoms. Once treatment is reduced or discontinued, it can be resumed if symptoms reappear.

[0129] In one embodiment, IL-6a, preferably IL-6 antibody, is administered intravitreally (IVT) at a dose of 0.25 mg, 1.0 mg, or 2.5 mg every four weeks (Q4W) for the treatment of uveitis or UME, more particularly UME. In another embodiment, IL-6 antibody is administered intravitreally (IVT) at a dose of 0.25 mg every four weeks (Q4W) for the treatment of uveitis or UME, more particularly UME. In one embodiment, IL-6 antibody is administered intravitreally (IVT) at a dose of 1.0 mg every four weeks (Q4W) for the treatment of uveitis or UME, more particularly UME.

[0130] disease "Ocular diseases" that can be treated with the IL-6a of the present invention include diseases in which IL-6 expression, e.g., elevated IL-6 expression, is associated with or is a prerequisite for the disease state. Such diseases include those in which IL-6-induced angiogenesis and inflammation contribute to the pathology of the disease. This includes diseases in which IL-6 levels are elevated compared to normal levels, such as diseases in which IL-6 is elevated in the vitreous (e.g., diabetic macular edema, diabetic retinopathy, and uveitis) or in ocular tissues. As described in International Publication No. 2014 / 074905, the entire contents of which are incorporated herein by reference, it has previously been shown that blocking the IL-6 pathway by administering an IL-6 antibody in a mouse and rat choroidal neovascularization model that reproduces the pathological processes underlying many IL-6-related diseases, such as DME, reduces angiogenesis to levels similar to those of an anti-VEGF positive control. These in vivo results indicate that local inhibition of IL-6 may be useful in treating ocular diseases associated with IL-6 expression and vascular leakage, such as macular edema.

[0131] Examples of IL-6-related diseases include, but are not limited to, certain eye diseases such as dry eye (e.g., dry eye disease or dry eye syndrome), allergic conjunctivitis, uveitis, age-related macular degeneration (AMD) (wet (exudative) AMD or dry (atrophic) AMD), proliferative diabetic retinopathy (PDR), diabetic macular edema (DME), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), neuromyelitis optica (NMO), or myopic choroidal neovascularization. Other eye disorders that can be treated include those caused by trauma, such as corneal transplantation, corneal abrasion, or other such physical damage to the eye. Other eye disorders that can be treated include eye cancers, such as cancers affecting the eye and the vicinity of the eye, such as the orbit or eyelid.

[0132] As used herein, the term "treating" refers to administering an agent described herein to a subject, e.g., a patient, in an amount, manner, and / or manner effective to ameliorate a condition, symptom, or parameter associated with a disorder, e.g., a disorder described herein, or to prevent the onset or progression of the disorder, to a statistically significant degree or to a degree detectable by one of ordinary skill in the art. Treatment can be to cure, cure, alleviate, relieve, alter, correct, improve, alleviate, ameliorate, or affect the disorder, the symptoms of the disorder, or the predisposition to the disorder. The effective amount, manner, or manner can vary depending on the subject and can be tailored to the subject. Exemplary subjects include humans, primates, and other non-human mammals. The pharmaceutical compositions or treatments described herein can also be given prophylactically to reduce the risk of developing a disorder or its symptoms or signs.

[0133] In one embodiment, the IL-6-related disease is an inflammatory disease. In one embodiment, the disease is glaucoma.

[0134] In one aspect, the condition is ocular pain, eg, pain associated with an ocular disease or disorder.

[0135] In one embodiment, treating a subject also includes determining whether the subject has an IL-6-associated disorder, and optionally, whether the subject is resistant to other non-IL-6 inhibitory treatments, such as steroids or anti-VEGF agents.

[0136] The pharmaceutical compositions or treatments described herein can be administered to subjects having or at risk of such IL-6-related disorders.

[0137] The therapeutic or pharmaceutical compositions described herein are particularly suitable for use in ocular disorders, e.g., in which it is desirable to administer an IL-6 antagonist described herein, e.g., an IL-6 antibody or fragment thereof, directly to the eye or topically to the ocular region.

[0138] A subject with dry eye disorder may exhibit ocular inflammation, which may include scratching, stinging, itching, burning or pressure, irritation, pain, and redness. Dry eye disorder may be associated with excessive tearing and insufficient tear production. The pharmaceutical compositions or treatments described herein can be administered to such subjects to improve or prevent the onset or worsening of one or more of these symptoms. The pharmaceutical compositions or treatments described herein can also be used to alleviate pain in a subject, such as eye pain, for example, pain caused by neuroinflammation.

[0139] The pharmaceutical compositions or treatments described herein can be administered to subjects with allergic reactions affecting the eye, such as subjects experiencing severe allergic (atopic) eye diseases, such as allergic conjunctivitis. See, e.g., Keane-Myers et al. (1999) Invest Ophthalmol Vis Sci, 40(12):3041-6.

[0140] The pharmaceutical compositions or treatments described herein can be administered to subjects having or at risk of diabetic retinopathy. See, e.g., Demircan et al. (2006) Eye 20:1366-1369 and Doganay et al. (2006) Eye, 16:163-170.

[0141] Uveitis. Uveitis includes acute and chronic forms and involves inflammation of one or more of the iris, ciliary body, and choroid. Chronic forms may be associated with systemic autoimmune diseases, such as Behçet's syndrome, ankylosing spondylitis, juvenile rheumatoid arthritis, Reiter's syndrome, and inflammatory bowel disease. In anterior uveitis, inflammation is primarily in the iris (also iritis). Anterior uveitis can affect subjects with systemic autoimmune diseases as well as those without. Intermediate uveitis involves inflammation of the anterior vitreous, peripheral retina, and ciliary body, often with little anterior or chorioretinal inflammation. Panpars planitis results from inflammation of the pars plana between the iris and choroid. Posterior uveitis involves the uvea and primarily the choroid and is also called choroiditis. Posterior uveitis may be associated with systemic infection or autoimmune disease. It may persist for months or even years. The pharmaceutical compositions or treatments described herein can be administered to a subject to treat any of the aforementioned forms of uveitis, including complications caused by uveitis, such as uveitis macular edema (UME), which is characterized by the accumulation of fluid in the retinal layers and / or subretinal space.UME is the main cause of visual impairment in cases of uveitis.See, for example, Tsai et al. (2009) Mol Vis 15:1542-1552 and Trittibach et al. (2008) Gene Ther.15(22):1478-88. IL-6 signaling is thought to regulate and amplify intraocular inflammation and immune responses by inhibiting T cell apoptosis and mediating the differentiation of Th1 cells into Th17 cells, and is thought to be responsible for the pathogenesis of autoimmune disorders such as uveitis (Amadi-Obi et al., Th17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27 / STAT1. Nat Med 2007;13(6):711-8).Systemic inhibition of IL-6R with tocilizumab has been demonstrated to improve vision and reduce macular thickness in multiple forms of uveitis, including patients with refractory UME, and there is now solid supporting evidence, including retrospective case series and prospective, randomized, investigator-initiated trials (Sepah et al., Primary (Month-6) outcomes of the STOP-Uveitis study: evaluating the safety, tolerability, and efficacy of tocilizumab in patients with non-infectious uveitis. Am J Ophthalmol 2017;183:71-80). Nonclinically, in a mouse model of experimental autoimmune uveitis, IL-6 inhibition by intravenous administration of anti-IL-6 mAb attenuated vascular leakage and macular edema, reduced ocular production of IL-17, and improved overall uveitis scores, suggesting that local targeting of IL-6 may be sufficient for therapeutic efficacy (Tode J et al., Intravitreal injection of anti-Interleukin (IL)-6 antibody attenuates experimental autoimmune uveitis in mice. Cytokine 2017;96:8-15).

[0142] In one aspect, the pharmaceutical compositions or treatments described herein can be used to treat subjects with or at risk of age-related macular degeneration (AMD), such as wet (exudative) AMD or dry (atrophic) AMD.The pharmaceutical compositions or treatments described herein can be applied locally to the eye, injected (e.g., intravitreally), or provided systemically.See, for example, Olson et al. (2009) Ocul Immunol Inflamm 17(3):195-200.

[0143] Diabetic macular edema (DME) involves blockage and leakage of retinal blood vessels, leading to decreased visual acuity and potentially blindness. Standard treatment for DME includes topical administration of steroids or anti-VEGF antibodies. However, many patients are resistant to these treatments. The pathogenesis of diabetic macular edema involves components of angiogenesis, inflammation, and oxidative stress. IL-6 is induced by hypoxia and hyperglycemia and can increase vascular inflammation, vascular permeability, and pathological angiogenesis. IL-6 can directly induce VEGF expression and promote choroidal neovascularization in animal models. In DME patients, ocular IL-6 levels are positively correlated with macular thickness and disease severity. IL-6 levels have been reported to be elevated in patients who fail anti-VEGF treatment and decreased in patients who respond to anti-VEGF treatment. Thus, the administration of IL-6a described herein is useful for treating diabetic patients in combination with anti-VEGF therapy, or as an alternative to anti-VEGF therapy, for example, for patients who do not respond to anti-VEGF therapy. Treatment of macular edema with IL-6a may also improve safety by eliminating the need to completely inhibit either mechanism to inhibit pathology, thus maintaining some of the desired physiological roles of each cytokine. Thus, local IL-6a treatment in combination with VEGF inhibition can reduce dose frequency and reduce adverse effects of treatment.

[0144] In DME, there is a positive correlation between vitreous IL-6 levels and both disease severity and VEGF-refractory subjects. Thus, IL-6a as described herein can be used to treat DME subjects who are refractive to steroid therapy, anti-VEGF therapy, or both. Subjects who are refractive to a given treatment, e.g., steroid therapy or anti-VEGF therapy, or both, do not show improvement, reduction, or reversal of selected symptoms. In one embodiment, IL-6a, e.g., an IL-6 antibody or fragment thereof described herein, is used in combination with anti-VEGF therapy or steroid therapy, e.g., to treat DME. Thus, in one embodiment, the pharmaceutical composition or treatment described herein can include an anti-VEGF agent or a steroid.

[0145] The pharmaceutical compositions or treatments described herein can be administered by any mode for treating ocular diseases. The pharmaceutical compositions or treatments described herein can be delivered parenterally. Alternatively or additionally, the pharmaceutical compositions or treatments described herein can be delivered directly to the eye or near the eye. For example, the pharmaceutical compositions or treatments described herein can be administered topically, intraocularly, intravitreally, or subconjunctivally, for example, by intravitreal injection.

[0146] Assay Aqueous humor samples can be collected, for example, by anterior chamber paracentesis using a thin needle and syringe or pipette (Van der Lelij A et al., Diagnostic anterior chamber paracentesis in uveitis: a safe procedure? Br J Ophthalmol 1997; 81(11):976-9; Trivedi D et al., Safety profile of anterior chamber paracentesis performed at the slit lamp. Clin Exp Ophthalmol 2011; 39(8):725-8; Kitazawa K et al., Safety of anterior chamber paracentesis using a 30-gauge needle integrated with a specially designed disposable pipette. Br J Ophthalmol 2017; 101(5):548-50).

[0147] In some embodiments, the biomarkers are detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, immunodetection methods, mass spectrometry, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, nanostring, SAGE, MassARRAY technology, and FISH, and combinations thereof. In some embodiments, the biomarkers are detected in the sample by protein expression. In some embodiments, protein expression is confirmed by immunohistochemistry (IHC).

[0148] In one embodiment, the biomarkers are detected in the sample by mRNA expression. In one embodiment, mRNA expression is confirmed using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, NanoString, SAGE, MassARRAY technology, or FISH.

[0149] In one embodiment, the sample is an AH sample.

[0150] In one embodiment, the sample is obtained prior to treatment with an IL-6 antagonist, hi one embodiment, the AH sample is fresh or frozen-thawed.

[0151] The presence and / or level / amount / concentration of various biomarkers in a sample can be analyzed by several methodologies, many of which are known in the art and understood by those of skill in the art, including, but not limited to, immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), MassARRAY, proteomics, quantitative blood-based assays (e.g., serum ELISA), biochemical enzyme activity assays, in situ hybridization, Southern analysis, Northern analysis, whole genome sequencing, polymerase chain reaction ("PCR") (including quantitative real-time PCR ("qRT-PCR")) and other amplification-based detection methods, e.g., branched DNA, SISBA, TMA, etc.), RNA-Seq, FISH, microarray analysis, gene expression profiling, and / or serial analysis of gene expression ("SAGE"), as well as any of a wide variety of assays that can be performed by protein, gene, and / or tissue array analysis. Exemplary protocols for assessing the status of genes and gene products are found, for example, in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern blotting), 4 (Southern blotting), 15 (immunoblotting), and 18 (PCR analysis). Multiplexed immunoassays, such as those available from Rules Based Medicine or Meso Scale Discovery ("MSD"), can also be used.

[0152] In one embodiment, the sample is a clinical sample. In one embodiment, the sample is used in a diagnostic assay.

[0153] In one aspect, a "reference sample" or "control sample" is a single sample or a combined plurality of samples from the same subject or individual obtained at one or more time points different from when the test sample was obtained. In one aspect, a reference sample or control sample is a combined plurality of samples from one or more healthy individuals other than the subject or individual. In one aspect, a reference sample or control sample is a combined plurality of samples from one or more individuals with a disease or disorder (e.g., DME) other than the subject or individual.

[0154] The term "detection" includes any means of detection, including direct and indirect detection.

[0155] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, e.g., a predictive, diagnostic, and / or prognostic indicator. A biomarker can serve as an indicator of a particular subtype of a disease or disorder (e.g., DME) characterized by particular molecular, pathological, histological, and / or clinical features. In one aspect, a predictive biomarker can serve as an indicator of a better or worse response to a particular treatment.

[0156] The "amount," "concentration," or "level" of a biomarker can be measured by methods known to those of skill in the art. The assessed level, concentration, or amount of the biomarker can be used to predict response to treatment.

[0157] The term "level" is used to refer to the amount or concentration of a biomarker in a biological sample.

[0158] The term "reference level" can refer to a predetermined value. In one embodiment, the reference level can be obtained by measuring the amount or concentration of a biomarker (e.g., AH IL-6) in a control sample. In one embodiment, the level of AH IL-6 in the sample from the patient is increased or elevated compared to the reference level, indicating that the patient is likely to respond to treatment with an IL-6 antagonist. In one embodiment, the level of AH IL-6 in the sample from the patient is decreased compared to the reference level, indicating that the patient is unlikely to respond to treatment with an IL-6 antagonist.

[0159] Both the "level" and the "basal level" can be expressed in terms of the concentration of IL-6 in the aqueous humor.

[0160] In one aspect, the terms "increase," "increased," "elevated," or "above" refer to a level above a reference level. In one aspect, an increase in level refers to an overall increase of about any 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the amount or concentration of a biomarker compared to the reference level. In one aspect, an increased level refers to an increase in the amount or concentration of a biomarker in a sample, the increase being at least about any of 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold above the reference level. In one aspect, elevated levels refer to an overall increase of greater than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to baseline levels.

[0161] The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological condition, disease, or condition (e.g., DME). For example, "diagnosis" can refer to the identification of a particular type of eye disease. "Diagnosis" can also refer to the classification of a particular subtype of cancer, for example, by histopathological criteria or by molecular features (e.g., a subtype characterized by the expression of one or a combination of biomarkers, such as particular genes or proteins encoded by said genes).

[0162] "Response" or whether a patient is "likely to respond" can be assessed using any endpoint that indicates benefit to an individual, such as, but not limited to, a clinically relevant improvement in best-corrected visual acuity (BCVA), a reduction in central foveal thickness (CST) or central foveal thickness (CFT), a reduction in fluid accumulation (e.g., retinal fluid), and / or a reduction in the severity of diabetic retinopathy. Change in BCVA from baseline is a clinical endpoint widely accepted by health organizations. In clinical practice, anatomical biomarkers of retinal thickness (e.g., CST or CFT) or fluid presence are more commonly used as efficacy biomarkers for treatment decisions. Retinal thickness can be measured by OCT imaging, such as spectral-domain optical coherence tomography (SD-OCT). Retinal fluid can be defined as intraretinal or subretinal (IRF and SRF, respectively), based on its location within the neurosensory retinal layer for IRF and between the neurosensory retina and the underlying retinal pigment epithelium for SRF.

[0163] Combination therapy The IL-6 antagonist can be used alone or in combination with other drugs in treatment. For example, the IL-6 antagonist can be co-administered with at least one additional therapeutic agent. In one embodiment, the additional therapeutic agent is a VEGF antagonist. In one embodiment, the VEGF antagonist is an anti-VEGF antibody, such as bevacizumab, ranibizumab, or brolucizumab. In one embodiment, the VEGF antagonist is a bispecific antibody, such as faricimab. In one embodiment, the VEGF antagonist is a soluble VEGF receptor, such as aflibercept.

[0164] Such combination therapy as described above includes combined administration (where two or more therapeutic agents are contained in the same or separate formulations) and separate administration, where administration of the IL-6 antagonist may occur prior to, simultaneously with, and / or after administration of the additional therapeutic agent.

[0165] Diagnostic Kits, Assays, and Products Also disclosed herein are diagnostic kits that include one or more reagents for determining the presence of a biomarker in a sample from a patient with an ophthalmic disease.

[0166] Also disclosed herein is an assay for identifying a patient with an ocular disease who should be administered an IL-6 antagonist, the method comprising determining the level of IL-6 in aqueous humor in a sample obtained from the patient.

[0167] Also disclosed herein is a packaged product comprising an IL-6 antagonist in a pharmaceutically acceptable carrier and a package insert indicating that the IL-6 antagonist is for treating a patient with an ocular disease based on elevated IL-6 concentrations in aqueous humor. The method of treatment includes any of the methods of treatment disclosed herein.

[0168] Additionally, a method for producing a product containing, in a single package, a pharmaceutical composition containing an IL-6 antagonist and a package insert indicating that the pharmaceutical composition is for treating patients with ocular diseases based on high IL-6 concentrations in aqueous humor is disclosed.

[0169] The article of manufacture comprises a container and a label or package insert affixed to or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds or contains a composition comprising a cancer medication as an active agent and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0170] The article of manufacture may further include a second container comprising a pharmaceutically acceptable diluent buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0171] The articles of manufacture of the invention also include information, for example in the form of a package insert, indicating that the composition is used to treat cancer based on the level of the biomarker(s) herein. The package insert or label can be in any form, such as paper or on an electronic medium such as a magnetic recording medium (e.g., a floppy disk) or a CD-ROM. The label or package insert can also include other information regarding the pharmaceutical compositions and dosage forms in the kit or article of manufacture.

[0172] The singular forms "a," "an," and "the" are understood to include plural referents unless otherwise indicated.

[0173] The invention will now be further described with reference to the following non-limiting figures and examples. [Example]

[0174] Example 1: DOVETAIL (BP40899) Clinical Trial DOVETAIL (BP40899) is the first study in which RO7200220 will be administered to humans. This Phase 1, multicenter, non-randomized, open-label, multiple-ascending-dose study of RO7200220 with unilateral intravitreal (IVT) administration is designed to evaluate a range of IVT doses expected to be safe and potentially effective in patients with DME and UME. The overall study will provide data for safety and tolerability, as well as characterization of pharmacokinetics (PK), systemic anti-drug antibodies (ADAs), duration of target engagement (PD) in aqueous humor, and early signals of biological activity. The study is being conducted in adult male and female participants with centrally-onset DME (Parts 1, 2, and 3) or UME (Part 4). The treatment benefit for participants enrolled in this first-in-human study is unknown. The primary objective of DOVETAIL is to evaluate the safety and tolerability of RO7200220 as monotherapy (DME and UME populations) and in combination with ranibizumab (DME population only). Secondary objectives will be to investigate the systemic PK of RO7200220. Efficacy variables such as BCVA and ocular anatomical outcomes (e.g., central subfield thickness by OCT) will be exploratory analyzed. Aqueous humor samples will be collected by anterior chamber paracentesis in all patients participating in DOVETAIL. IL-6 levels in the aqueous humor of study participants will be exploratory analyzed.

[0175] In Part 1, RO7200220 was administered as monotherapy via IVT injection twice, 6 weeks apart, to eligible DME study eyes. Six interim escalating dose levels were administered sequentially to six different cohorts. Each participant within a given cohort received RO7200220 at the following assigned dose level in a fixed volume of 50 μL in a single designated study eye: Cohort 1: 0.01 mg RO7200220 (starting dose) Cohort 2: 0.05 mg RO7200220 Cohort 3: 0.25 mg RO7200220 Cohort 4: 1 mg RO7200220 Cohort 5: 2.5 mg RO7200220 Cohort 6: 5 mg RO7200220. Each cohort consisted of a minimum of three individuals (i.e., A minimum number of participants (required for a decision to increase to the next dose level) and a maximum of six participants were enrolled.

[0176] In Part 4, RO7200220 was evaluated as monotherapy in the UME population. Three different doses (0.25 mg, 1 mg, and 2.5 mg administered intravenously three times, 4 weeks apart) were investigated in participants with UME. A total of 37 participants with UME received RO7200220 at doses ranging from 0.25 mg to 2.5 mg every 4 weeks (Q4W). Data from 28 participants were evaluated at the first cutoff point (Figures 1-6 and 9-10), and data from all 37 participants were evaluated at the second cutoff point (Figures 11-18). Clinically meaningful improvements in BCVA and CST were observed in Phase 1 DOVETAIL participants at the 0.25, 1, and 2.5 mg dose levels with an acceptable safety profile. The 2.5 mg dose was identified as the maximum tolerated dose.

[0177] Signs of clinical efficacy were consistently observed across cohorts in Part 4 of the DOVETAIL trial. Promising numerical improvements in BCVA (increase, Figures 1, 2, 11, and 12) and CST (decrease, Figures 3, 4, 13, and 14) were detected in the overall UME population (Part 4), without apparent return to baseline at the end of the study. In particular, the mean change from baseline in BCVA was 9.9 [9.4] letters (SD 8.9 [9.3]) at Day 84 (4 weeks after the third and final study treatment administration, n = 36

[23] ). The mean change from baseline in CST was -169.3 μm [-170.5 μm] (SD 147.5 [143.8]) at Day 84 (4 weeks after the third and final study treatment administration, n = 34

[21] ). Numbers in brackets indicate the number of the first cutoff point.

[0178] Furthermore, analysis of SD-OCT images showed resolution of both subretinal and intraretinal fluid (SRF and IRF, Figures 5, 6, 15, and 16) in UME patients treated with RO7200220, further supporting the beneficial clinical effect of inhibiting IL-6 in the treatment of UME.

[0179] A preliminary analysis of available data examined whether baseline levels of aqueous humor IL-6 were related to central foveal thickness (CST), a variable commonly used to assess anatomical responses in retinal diseases such as DME and UME (Schmidt-Erfurth U et al.: Guidelines for the Management of Diabetic Macular Edema by the European Society of Retina Specialists (EURETINA). Ophthalmologica 2017;237:185-222) (Figures 7, 9, and 17), and best-corrected visual acuity (BCVA), a typical primary clinical efficacy endpoint for regulatory approval of drugs to treat this disease (Figures 8, 10, and 18). We observed an association between baseline AH IL-6 concentrations (logarithmic scale) and a decrease in CST for both DME and UME. Furthermore, we observed an association between baseline AH IL-6 concentrations (logarithmic scale) and an increase in BCVA for DME and UME. The direction of the association between AH IL-6 and CST and BCVA is consistent with IL-6 inhibition being more effective in patients with high AH IL-6.

Claims

1. An interleukin-6 (IL-6) antagonist for use in treating a patient with an ocular disease, wherein the patient has been determined to have an elevated level of IL-6 in aqueous humor (AH IL-6) in a sample obtained from the patient compared to a baseline level.

2. 10. The IL-6 antagonist for use according to claim 1, formulated as a pharmaceutical composition suitable for administration to the eye of said patient.

3. 3. The IL-6 antagonist for use according to claim 2, wherein the pharmaceutical composition is suitable for intravitreal, intraocular, or subconjunctival administration.

4. The IL-6 antagonist for use according to any one of claims 1 to 3, wherein the level of IL-6 is determined in an aqueous humor sample collected by anterior chamber paracentesis.

5. 5. The IL-6 antagonist for use according to any one of claims 1 to 4, wherein the ocular disease is selected from the group consisting of diabetic macular edema (DME), diabetic retinopathy, dry eye (e.g., dry eye disease or dry eye syndrome), allergic conjunctivitis, uveitis, uveitic macular edema (UME), age-related macular degeneration (AMD) (e.g., wet AMD or dry AMD), proliferative diabetic retinopathy (PDR), rhegmatogenous retinal detachment (RRD), retinal vein occlusion (RVO), neuromyelitis optica (NMO), myopic choroidal neovascularization, ocular cancer, corneal transplant, corneal abrasion, or physical injury to the eye.

6. 6. The IL-6 antagonist for use according to claim 5, wherein the eye disease is diabetic macular edema (DME).

7. The IL-6 antagonist for use according to any one of claims 1 to 6, which is an anti-IL-6 or anti-IL-6 receptor (IL-6R) antibody or an antigen-binding fragment thereof.

8. The IL-6 antagonist for use according to claim 7, which is an anti-IL-6 antibody or an antigen-binding fragment thereof.

9. The anti-IL-6 antibody i) a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, and a VH CDR3 comprising the sequence of SEQ ID NO: 3; and ii) a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6; 9. The IL-6 antagonist for use according to claim 7 or 8, comprising:

10. 10. The IL-6 antagonist for use according to claim 9, wherein the anti-IL-6 antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:

8.

11. 11. The IL-6 antagonist for use according to claim 10, wherein said IL-6 antibody comprises a heavy chain comprising the sequence of SEQ ID NO:9 and a light chain comprising the sequence of SEQ ID NO:

10.

12. The IL-6 antagonist for use according to any one of claims 1 to 11, wherein the sample is a sample obtained from the patient before treatment with the IL-6 antagonist.

13. The IL-6 antagonist for use according to any one of claims 1 to 12, wherein said use further comprises an effective amount of a second therapeutic agent.

14. 14. The IL-6 antagonist for use according to claim 13, wherein the second therapeutic agent is a VEGF antagonist.

15. The IL-6 antagonist for use according to claim 14, wherein the VEGF antagonist is an anti-VEGF antibody.

16. An IL-6 antagonist for use in treating patients with uveitis or uveitic macular edema.

17. 17. The IL-6 antagonist for use according to claim 16, formulated as a pharmaceutical composition suitable for administration to the eye of said patient.

18. 18. The IL-6 antagonist for use according to claim 17, wherein the pharmaceutical composition is suitable for intravitreal, intraocular, or subconjunctival administration.

19. The IL-6 antagonist for use according to any one of claims 16 to 18, which is an anti-IL-6 or anti-IL-6 receptor (IL-6R) antibody or an antigen-binding fragment thereof.

20. 20. The IL-6 antagonist for use according to claim 19, which is an anti-IL-6 antibody or an antigen-binding fragment thereof.

21. The anti-IL-6 antibody i) a VH CDR1 comprising the sequence of SEQ ID NO: 1, a VH CDR2 comprising the sequence of SEQ ID NO: 2, and a VH CDR3 comprising the sequence of SEQ ID NO: 3; and ii) a VL CDR1 comprising the sequence of SEQ ID NO: 4, a VL CDR2 comprising the sequence of SEQ ID NO: 5, and a VL CDR3 comprising the sequence of SEQ ID NO: 6; 21. The IL-6 antagonist for use according to claim 19 or 20, comprising:

22. 22. The IL-6 antagonist for use according to claim 21, wherein the anti-IL-6 antibody comprises a heavy chain variable region comprising the sequence of SEQ ID NO:7 and a light chain variable region comprising the sequence of SEQ ID NO:

8.

23. 23. The IL-6 antagonist for use according to claim 22, wherein said IL-6 antibody comprises a heavy chain comprising the sequence of SEQ ID NO: 9 and a light chain comprising the sequence of SEQ ID NO:

10.

24. 24. The IL-6 antagonist for use according to any one of claims 16 to 23, administered intravitreally (IVT) at a dose of 0.25 mg, 1.0 mg or 2.5 mg every four weeks (Q4W).

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