HK1243138B - Therapeutic and diagnostic methods for il-33-mediated disorders - Google Patents

Description

Treatment and diagnostic methods for IL-33-mediated diseases

Technical Field

This invention relates to methods for treating patients with interleukin-33 (IL-33) mediated diseases and methods for determining whether a patient faces an increased risk of IL-33 mediated diseases.

Background Technology

Interleukin-33 (IL-33) is a member of the interleukin-1 (IL-1) cytokine family encoded by the IL33 gene and constitutively expressed in structural cells such as smooth muscle cells, epithelial cells, and endothelial cells. IL-33 can be induced by inflammatory factors in macrophages and dendritic cells. Environmental triggers such as cellular stress caused by allergens, toxins, and pathogens can lead to IL-33 release. Bioavailable IL-33 associates with a heterodimeric IL-33 receptor complex consisting of the inhibitory tumorigenicity 2 (ST2) protein and the interleukin-1 receptor accessory protein (IL-1RAcP) to activate the AP-1 pathway and NF-κB pathway via the adaptor protein myeloid differentiation primary response 88 (MyD88) and possibly via the MyD88-adaptor head-like (Mal) protein. IL-33 stimulates numerous cell types, including innate type II (ILC2) cells, mast cells, basophils, eosinophils, and dendritic cells, to promote type II immunity.

It has been proposed that the IL-33 pathway is involved in a variety of diseases, including allergy-related diseases. For these diseases, there is still a need to develop improved methods to identify patient groups best suited for treatment options.

Overview

This invention relates to methods for treating patients with interleukin-33 (IL-33) mediated diseases and methods for determining whether a patient faces an increased risk of IL-33 mediated diseases.

In one aspect, the present invention relates to a method for treating a patient with an interleukin-33 (IL-33)-mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain a G allele at polymorphism rs4988956 (SEQ ID NO:1) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4988956 (SEQ ID NO:1).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism of rs4988956 (SEQ ID NO:1) in a sample derived from the patient, wherein if the patient's genotype contains a G allele at rs4988956 (SEQ ID NO:1) or an equivalent allele at a polymorphism of rs4988956 (SEQ ID NO:1), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs4988956 (SEQ ID NO:1) or at a polymorphism linked to polymorphism rs4988956 (SEQ ID NO:1) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each G allele at polymorphism rs4988956 (SEQ ID NO:1) or each equivalent allele at a polymorphism linked to polymorphism rs4988956 (SEQ ID NO:1) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of periostin in a patient-derived sample. In some embodiments, if the periostin level in the sample is at or below a periostin reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the periostin level in the sample is at or above a periostin reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism linked to rs4988956 (SEQ ID NO:1) in non-alignment has a D’ value greater than or equal to 0.6 relative to rs4988956 (SEQ ID NO:1). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to rs4988956 (SEQ ID NO:1) in non-alignment is the polymorphism listed in Table 3. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to rs4988956 (SEQ ID NO:1). In some embodiments, the equivalent allele is the major allele of the polymorphism linked to rs4988956 (SEQ ID NO:1).

In another aspect, the present invention relates to a method for treating a patient with an interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain an A allele at polymorphism rs10204137 (SEQ ID NO:2) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10204137 (SEQ ID NO:2).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism of rs10204137 (SEQ ID NO:2) in a sample derived from the patient, wherein if the patient's genotype contains the A allele at the polymorphism of rs10204137 (SEQ ID NO:2) or an equivalent allele at the polymorphism of rs10204137 (SEQ ID NO:2), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs10204137 (SEQ ID NO:2) or at a polymorphism linked to polymorphism rs10204137 (SEQ ID NO:2) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each A allele at polymorphism rs10204137 (SEQ ID NO:2) or each equivalent allele at a polymorphism linked to polymorphism rs10204137 (SEQ ID NO:2) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism linked to the rs10204137 (SEQ ID NO:2) in disequilibrium has a D’ value greater than or equal to 0.6 relative to the rs10204137 (SEQ ID NO:2). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to the rs10204137 (SEQ ID NO:2) in disequilibrium is the polymorphism listed in Table 3. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to the rs10204137 (SEQ ID NO:2) in disequilibrium. In other embodiments, the equivalent allele is the major allele of the polymorphism linked to the rs10204137 (SEQ ID NO:2) in disequilibrium.

In another aspect, the present invention relates to a method for treating a patient with an interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain a C allele at polymorphism rs10192036 (SEQ ID NO:3) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192036 (SEQ ID NO:3).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism in linkage disequilibrium with rs10192036 (SEQ ID NO:3) in a sample derived from the patient, wherein if the patient's genotype contains a C allele at rs10192036 (SEQ ID NO:3) or an equivalent allele at a polymorphism in linkage disequilibrium with rs10192036 (SEQ ID NO:3), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs10192036 (SEQ ID NO:3) or at a polymorphism linked to polymorphism rs10192036 (SEQ ID NO:3) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each C allele at polymorphism rs10192036 (SEQ ID NO:3) or each equivalent allele at a polymorphism linked to polymorphism rs10192036 (SEQ ID NO:3) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism linked to the rs10192036 (SEQ ID NO:3) in linkage disequilibrium has a D’ value greater than or equal to 0.6 relative to the rs10192036 (SEQ ID NO:3). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to the rs10192036 (SEQ ID NO:3) in linkage disequilibrium is the polymorphism listed in Table 3. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to the rs10192036 (SEQ ID NO:3) in linkage disequilibrium. In other embodiments, the equivalent allele is the major allele of the polymorphism linked to the rs10192036 (SEQ ID NO:3) in linkage disequilibrium.

In another aspect, the present invention relates to a method for treating a patient with IL-33-mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain a C allele at polymorphism rs10192157 (SEQ ID NO:4) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192157 (SEQ ID NO:4).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism of rs10192157 (SEQ ID NO:4) in a sample derived from the patient, wherein if the patient's genotype contains a C allele at rs10192157 (SEQ ID NO:4) or an equivalent allele at a polymorphism of rs10192157 (SEQ ID NO:4), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs10192157 (SEQ ID NO:4) or at a polymorphism linked to polymorphism rs10192157 (SEQ ID NO:4) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each C allele at polymorphism rs10192157 (SEQ ID NO:4) or each equivalent allele at a polymorphism linked to polymorphism rs10192157 (SEQ ID NO:4) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism linked to the rs10192157 (SEQ ID NO:4) in disequilibrium has a D’ value greater than or equal to 0.6 relative to the rs10192157 (SEQ ID NO:4). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to the rs10192157 (SEQ ID NO:4) in disequilibrium is the polymorphism listed in Table 3. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to the rs10192157 (SEQ ID NO:4) in disequilibrium. In other embodiments, the equivalent allele is the major allele of the polymorphism linked to the rs10192157 (SEQ ID NO:4) in disequilibrium.

In another aspect, the present invention relates to a method for treating a patient with an interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain a T allele at polymorphism rs10206753 (SEQ ID NO:5) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10206753 (SEQ ID NO:5).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism of rs10206753 (SEQ ID NO:5) in a sample derived from the patient, wherein if the patient's genotype contains a T allele at or at an equivalent allele at a polymorphism of rs10206753 (SEQ ID NO:5), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs10206753 (SEQ ID NO:5) or at a polymorphism linked to polymorphism rs10206753 (SEQ ID NO:5) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each T allele at polymorphism rs10206753 (SEQ ID NO:5) or each equivalent allele at a polymorphism linked to polymorphism rs10206753 (SEQ ID NO:5) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism linked to the rs10206753 (SEQ ID NO:5) in disequilibrium has a D’ value greater than or equal to 0.6 relative to the rs10206753 (SEQ ID NO:5). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to the rs10206753 (SEQ ID NO:5) in disequilibrium is the polymorphism listed in Table 3. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to the rs10206753 (SEQ ID NO:5) in disequilibrium. In other embodiments, the equivalent allele is the major allele of the polymorphism linked to the rs10206753 (SEQ ID NO:5) in disequilibrium.

In another aspect, the present invention relates to a method for treating a patient with an interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient’s genotype has been determined to contain a T allele at polymorphism rs4742165 (SEQ ID NO:6) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising determining a genotype at or at a polymorphism of rs4742165 (SEQ ID NO:6) in a sample derived from the patient, wherein if the patient's genotype contains a T allele at rs4742165 (SEQ ID NO:6) or an equivalent allele at a polymorphism of rs4742165 (SEQ ID NO:6), the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the genotype at polymorphism rs4742165 (SEQ ID NO:6) or at a polymorphism linked to polymorphism rs4742165 (SEQ ID NO:6) in a sample derived from a patient with IL-33-mediated disease; and (b) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the genotype, wherein the presence of each T allele at polymorphism rs4742165 (SEQ ID NO:6) or each equivalent allele at a polymorphism linked to polymorphism rs4742165 (SEQ ID NO:6) indicates an increased likelihood that the patient will respond to treatment comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the polymorphism linked to the rs4742165 (SEQ ID NO:6) in non-alignment has a D’ value greater than or equal to 0.6 relative to the rs4742165 (SEQ ID NO:6). In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphism linked to the rs4742165 (SEQ ID NO:6) in non-alignment is the polymorphism listed in Table 4. In some embodiments, the equivalent allele is the minor allele of the polymorphism linked to the rs4742165 (SEQ ID NO:6). In other embodiments, the equivalent allele is the major allele of the polymorphism linked to the rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to a method for treating a patient with interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the patient's genotype has been determined to contain two or more of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the [missing information - likely a genotype or genotype] polymorphism. The T allele at s10206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium selected from polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising identifying samples derived from the patient containing rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), rs4742165 (SEQ ID NO:6), and those containing rs4988956 (SEQ ID NO:6). The genotype at two or more polymorphisms at linkage disequilibrium in polymorphisms of rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) is considered to have an increased risk of IL-33-mediated disease if: (a) the patient's genotype is included in polymorphism rs498895. (a) The patient's genotype contains the G allele at polymorphism rs10204137 (SEQ ID NO:2); (b) The patient's genotype contains the A allele at polymorphism rs10192036 (SEQ ID NO:3); (d) The patient's genotype contains the C allele at polymorphism rs10192157 (SEQ ID NO:4); (e) The patient's genotype contains the T allele at polymorphism rs10206753 (SEQ ID NO:5); (f) The patient's genotype The method includes a T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or (g) the patient's genotype includes an equivalent allele at a linkage-disequilibrium polymorphism selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6). In some embodiments, the method further includes administering an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) identifying in a sample derived from a patient with IL-33-mediated disease a drug selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs474216. 5 (SEQ ID NO:6) and genotypes at two or more polymorphisms at polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6); and (b) based on genotype, identify patients as potentially responsive to IL-33 axis-binding antagonists. Treatment, wherein: (i) each G allele at polymorphism rs4988956 (SEQ ID NO:1); (ii) each A allele at polymorphism rs10204137 (SEQ ID NO:2); (iii) each C allele at polymorphism rs10192036 (SEQ ID NO:3); (iv) each C allele at polymorphism rs10192157 (SEQ ID NO:4); (v) each T allele at polymorphism rs10206753 (SEQ ID NO:5); (vi) each T allele at polymorphism rs4742165 (SEQ ID NO:1); The presence of each T allele at polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) indicates an increased likelihood of the patient responding to treatment containing an IL-33 axis-binding antagonist. In some embodiments of this aspect, the patient's genotype has been identified to include the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the A allele at polymorphism rs10204137 (SEQ ID NO:2). In some embodiments of this aspect, the patient's genotype has been identified as containing the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10192036 (SEQ ID NO:3). In some embodiments of this aspect, the patient's genotype has been identified as also containing the C allele at polymorphism rs10192157 (SEQ ID NO:4) or the T allele at polymorphism rs10206753 (SEQ ID NO:5). In some embodiments of this aspect, the patient's genotype has been identified as also containing the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs10206753 (SEQ ID NO:5). In some embodiments of this invention, the patient's genotype has been identified to include: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the T allele at polymorphism rs4742165 (SEQ ID NO:6). In some embodiments, the method further includes administering an IL-33 axis-binding antagonist to the patient.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the polymorphism with polymorphic linkage disequilibrium selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) has a D’ value greater than or equal to 0.6 relative to the selected polymorphism. In some embodiments, the D’ value is greater than or equal to 0.8. In some embodiments, the polymorphisms in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) are the polymorphisms listed in Table 3 or Table 4. In some embodiments, the equivalent allele is the minor allele of the polymorphism rs4742165 (SEQ ID NO:6). In other embodiments, the equivalent allele is the major allele of the polymorphism rs10206753 (SEQ ID NO:5).

In another aspect, the present invention relates to a method for selecting a therapy for a patient suffering from IL-33-mediated disease, the method comprising: (a) determining the level of a periosteal protein in a sample derived from the patient; (b) comparing the level of the periosteal protein in the patient-derived sample to a reference level of a periosteal protein; and (c) if the level of the periosteal protein in the sample is at or below the reference level, selecting a therapy comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the therapy comprising an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for treating a patient with an interleukin-33 mediated disease, the method comprising administering to the patient a therapy comprising an IL-33 axis-binding antagonist, wherein the level of soluble ST2 (sST2) in a sample derived from the patient has been determined to be at or above the sST2 reference level.

In another aspect, the present invention relates to a method for determining whether a patient faces an increased risk of IL-33-mediated disease, the method comprising: (a) determining the level of sST2 in a sample derived from the patient; and (b) comparing the level of sST2 in the patient-derived sample to a reference level of sST2, wherein if the level of sST2 in the patient-derived sample is at or above the reference level, the patient faces an increased risk of IL-33-mediated disease. In some embodiments, the method further comprises administering to the patient a therapy comprising an IL-33 axis-binding antagonist.

In another aspect, the present invention relates to a method for selecting a therapy for a patient with IL-33-mediated disease, the method comprising: (a) determining the level of sST2 in a sample derived from the patient; (b) comparing the level of sST2 in the sample derived from the patient with a reference level of sST2; and (c) if the level of sST2 in the sample is at or above the reference level, selecting a therapy comprising an IL-33 axis-binding antagonist. In some embodiments, the method further comprises administering the therapy comprising an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment comprising an IL-33 axis-binding antagonist, the method comprising: (a) determining the level of sST2 in a sample derived from the patient; (b) comparing the level of sST2 in the sample derived from the patient to a reference level of sST2; and (c) identifying the patient as likely to respond to treatment comprising an IL-33 axis-binding antagonist based on the level of sST2 in the sample derived from the patient, wherein if the level of sST2 in the sample is at or above the reference level, the likelihood of the patient responding to treatment comprising an IL-33 axis-binding antagonist increases. In some embodiments, the method further comprises administering the therapy comprising an IL-33 axis-binding antagonist to the patient.

In another aspect, the present invention relates to a method for evaluating the treatment response of a patient treated with an IL-33 axis-binding antagonist, the method comprising: (a) determining the level of sST2 in a patient-derived sample at time points during or after administration of the IL-33 axis-binding antagonist; and (b) maintaining, adjusting, or discontinuing the patient's treatment based on a comparison of the level of sST2 in the patient-derived sample with a reference level of sST2. Wherein, a change in the level of sST2 in the patient-derived sample compared with a reference level indicates a response to IL-33 axis-binding antagonist treatment. In some embodiments of this aspect, the change is an increase in the level of sST2 and treatment is maintained. In other embodiments of this aspect, the change is a decrease in the level of sST2 and treatment is discontinued.

In another aspect, the present invention relates to a method for monitoring the response of a patient treated with an IL-33 axis-binding antagonist, the method comprising (a) determining the level of sST2 in a patient-derived sample at a time point during or after administration of the IL-33 axis-binding antagonist; and (b) comparing the level of sST2 in the patient-derived sample with a reference level of sST2, thereby monitoring the response in a patient receiving IL-33 axis-binding antagonist treatment.

In some embodiments of any of the foregoing aspects, the method further includes determining the level of a periosteal protein in a sample derived from the patient. In some embodiments, if the level of a periosteal protein in the sample is at or below a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases. In other embodiments, if the level of a periosteal protein in the sample is at or above a periosteal protein reference level, the likelihood of the patient responding to IL-33 axis binding antagonist therapy increases.

In some embodiments of any of the foregoing aspects, the sST2 level is the level of the sST2 protein. In some embodiments, the patient-derived sample is a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some embodiments, the patient-derived sample is a serum sample. In some embodiments, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In some embodiments, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some embodiments, the individual group has asthma. In some embodiments, the sST2 reference level is the median level. In some embodiments, the individual group is a female individual group and the patient is female. In some embodiments, the individual group is a male individual group and the patient is male. In some implementations, reference levels are measured in individuals at an earlier time point (e.g., before treatment with an IL-33 axis binding antagonist) or at an earlier time point during treatment with an IL-33 axis binding antagonist.

In some embodiments of any of the foregoing aspects, the IL-33 axis binding antagonist is administered in combination with a trypsin-β binding antagonist, a chemoattractant receptor homolog (CRTH2) binding antagonist expressed on Th2 cells, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a JAK1 antagonist, and/or an interleukin-5 (IL-5) binding antagonist. In some embodiments, the IL-33 axis binding antagonist is an IL-33 binding antagonist, an ST2 binding antagonist, or an IL-1RAcP binding antagonist. In some embodiments, (a) the IL-33 binding antagonist is an anti-IL33 antibody or its antigen-binding fragment; (b) the ST2 binding antagonist is the ST2-Fc protein, an anti-ST2 antibody, or its antigen-binding fragment; or (c) the IL-1RAcP binding antagonist is an anti-IL-1RAcP antibody.

In some embodiments of any of the foregoing aspects, IL-33-mediated diseases are selected from inflammatory diseases, immune diseases, fibrotic diseases, eosinophilic diseases, infections, pain, central nervous system diseases, solid tumors, and eye diseases. In some embodiments, inflammatory diseases are selected from asthma, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, and chronic obstructive pulmonary disease (COPD). In some embodiments, immune diseases are selected from asthma, rheumatoid arthritis, allergic reactions, anaphylactic reactions, anaphylactic shock, allergic rhinitis, psoriasis, inflammatory bowel disease (IBD), segmental ileitis, diabetes, and liver diseases. In some embodiments, the fibrotic disease is idiopathic pulmonary fibrosis (IPF). In some embodiments, the eosinophilic disease is eosinophil-associated gastrointestinal disease (EGID). In some embodiments, EGID is eosinophilic esophagitis. In some embodiments, the infection is a helmintic infection, a protozoan infection, or a viral infection. In some embodiments, the protozoan infection is *Leishmania major* infection. In some embodiments, the viral infection is respiratory syncytial virus (RSV) infection or influenza infection. In some embodiments, the pain is inflammatory pain. In some embodiments, the central nervous system disease is Alzheimer's disease. In some embodiments, the solid tumor is selected from breast tumors, colon tumors, prostate tumors, lung tumors, kidney tumors, liver tumors, pancreatic tumors, gastric tumors, intestinal tumors, brain tumors, bone tumors, and skin tumors. In some embodiments, the eye disease is age-related macular degeneration (AMD) or retinopathy.

In some embodiments of any of the above aspects, the reference level of periosteal protein is between about 23 ng/ml and about 50 ng/ml.

In some implementations of any of the foregoing aspects, the sample derived from the patient is a whole blood sample, a serum sample, a plasma sample, or a combination thereof.

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having a G allele at polymorphism rs4988956 (SEQ ID NO:1) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4988956 (SEQ ID NO:1).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as containing a G allele at polymorphism rs4988956 (SEQ ID NO:1) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4988956 (SEQ ID NO:1).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified to contain a G allele at polymorphism rs4988956 (SEQ ID NO:1) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4988956 (SEQ ID NO:1).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having an A allele at polymorphism rs10204137 (SEQ ID NO:2) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10204137 (SEQ ID NO:2).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as having an A allele at polymorphism rs10204137 (SEQ ID NO:2) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10204137 (SEQ ID NO:2).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified as containing an A allele at polymorphism rs10204137 (SEQ ID NO:2) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10204137 (SEQ ID NO:2).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having a C allele at polymorphism rs10192036 (SEQ ID NO:3) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192036 (SEQ ID NO:3).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as having a C allele at polymorphism rs10192036 (SEQ ID NO:3) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192036 (SEQ ID NO:3).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified as containing a C allele at polymorphism rs10192036 (SEQ ID NO:3) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192036 (SEQ ID NO:3).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having a C allele at polymorphism rs10192157 (SEQ ID NO:4) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192157 (SEQ ID NO:4).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as having a C allele at polymorphism rs10192157 (SEQ ID NO:4) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192157 (SEQ ID NO:4).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified as containing a C allele at polymorphism rs10192157 (SEQ ID NO:4) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10192157 (SEQ ID NO:4).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having a T allele at polymorphism rs10206753 (SEQ ID NO:5) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10206753 (SEQ ID NO:5).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as having a T allele at polymorphism rs10206753 (SEQ ID NO:5) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10206753 (SEQ ID NO:5).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified to contain a T allele at polymorphism rs10206753 (SEQ ID NO:5) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs10206753 (SEQ ID NO:5).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having a T allele at polymorphism rs4742165 (SEQ ID NO:6) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patient has been identified as having a T allele at polymorphism rs4742165 (SEQ ID NO:6) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified to contain a T allele at polymorphism rs4742165 (SEQ ID NO:6) or an equivalent allele at a polymorphism in linkage disequilibrium with polymorphism rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as containing two or more of the following alleles: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the G allele at polymorphism rs10192157 (SEQ ID NO:4). The T allele at rs10206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium selected from polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patients have been identified as containing two or more of the following alleles: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the G allele at polymorphism rs10204137 (SEQ ID NO:2). The T allele at 206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist used in a method of treating a patient with IL-33-mediated disease, wherein the patient has been identified as containing two or more of the following alleles: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the IL-33 axis-binding antagonist used in a method of treating a patient with IL-33-mediated disease. The T allele at 10206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium selected from polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In another aspect, the present invention relates to an IL-33 axis-binding antagonist for treating patients with IL-33-mediated diseases, wherein prior to any administration of the IL-33 axis-binding antagonist, the patient has been identified as having sST2 levels at or above a reference level in a sample derived from the patient.

In another aspect, the present invention relates to the use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated diseases, wherein the patients have been identified as having sST2 levels at or above a reference level in samples derived from the patients.

In another aspect, the present invention relates to a composition comprising an effective amount of an IL-33 axis-binding antagonist in a method for treating a patient with IL-33-mediated disease, wherein the patient has been identified as having sST2 levels at or above a reference level in a sample derived from the patient.

Brief description of the attached diagram

Figure 1A shows a schematic diagram of the interleukin-33 (IL-33) receptor complex. The red line indicates the location of the protective ST2 variant shown. MAPKK, mitogen-activated protein kinase kinase.

Figure 1B is a schematic diagram of the crystal structure of the TLR10Toll/interleukin-1 receptor (TIR) domain, showing the tagged positions of the A433T and Q501R variants of ST2.

Figures 1C and 1D show the results of reporter molecular analysis experiments that determined the IL-33 response in common ST2 variants or protective ST2 variants (A433T Q501R T549I L551S). HEK-BLUE ^™ cells expressing the variants shown were stimulated for 20 h with progressively increasing concentrations of recombinant human IL-33 (Figure 1C) or IL-1β (Figure 1D). Cytokine activity was measured by the induction of the NF-κB/AP-1 secreted alkaline phosphatase (SEAP) reporter gene. Each figure shows mean ± SEM values for three single clonal cell lines. * indicates p < 0.05. Data represent three independent experiments. The table shows the maximum half-maximal effective concentration ( _EC50 ) of IL-33 (Figure 1C) or IL-1β (Figure 1D) for the variants shown.

Figures 2A and 2B show the results of reporter molecular analysis experiments that determined the IL-33 response in common or protective ST2 variants. Batch clones of HEK-BLUE ^™ cells expressing the ST2 variants shown were stimulated for 20 h with progressively increasing concentrations of recombinant human IL-33 (Figure 2A) or IL-1β (Figure 2B). Cytokine activity was measured by the induction of the NF-κB/AP-1SEAP reporter gene. The graphs show the mean ± SD of three replicates. Data represent three independent experiments. The table shows the _EC50 for IL-33 (Figure 2A) or IL-1β (Figure 2B) of the variants shown.

Figure 3A is a histogram showing the results of flow cytometry experiments that compared the surface expression levels of the IL1RL1 variant shown in HEK-BLUE ^™ cells.

Figure 3B is a histogram showing the results of flow cytometry experiments that compared the surface expression levels of IL-1RAcP in HEK-BLUE ^™ cells expressing the indicated IL1RL1 variant.

Figure 3C is a graph showing the average fluorescence intensity (MFI) of ST2 surface expression from the graph shown in Figure 3A.

Figure 3D shows the results of quantitative reverse transcription polymerase chain reaction (RT-PCR) measurements of IL1RL1 (left inset) and IL1RAcP (right inset). The expression levels are shown relative to the housekeeping gene RPL19 (encoding the ribosomal protein L19).

Figure 4 is a graph showing the interleukin-8 (IL-8) secretion level of purified blood eosinophils as assessed by enzyme-linked immunosorbent assay (ELISA), said blood eosinophils obtained from human donors carrying protective or common IL1RL1 variants treated with purified IL-33 at the concentration shown.

Figure 5A is a graph showing the sST2 mRNA levels from purified blood eosinophils and basophils, as assessed by ELISA, obtained from human donors carrying either a protective or common IL1RL1 variant, by quantitative RT-PCR.

Figure 5B is a graph showing plasma sST2 levels from human donors carrying protective or common IL1RL1 variants, as assessed by ELISA. The figure shows the mean ± SEM for 3 single clones or 4 independent donors in each group. * indicates p < 0.05, as determined by paired t-test.

Figure 6 is a table showing the correlation between protective IL1RL1 variants and peristin levels. CHR, chromosome; P, p-value; OR, odds ratio; and MAF, minor allele frequency.

Figure 7A is a box plot showing the observed distribution of soluble ST2 in asthma serum under log2 transformation based on genotype and sex (female ♀, male ♂) at rs3771166.

Figure 7B is a box plot showing the observed distribution of soluble ST2 in asthma serum (sST2) under log2 transformation based on genotype and sex (female ♀, male ♂) at rs4742165.

Figure 7C is a graph showing the least squares mean and standard error of asthma sST2 levels after log-2 transformation, based on the rs4742165 (x-axis) and rs3771166 genotypes (series). The right axis corresponds to the untransformed sST2 level (ng/mL).

Figure 8 is a series of plots showing the paired correlations of baseline biomarker levels. Paired analysis is presented for baseline biomarker levels. The lower half of the plot is a scatter plot, and the upper half is a count of Spearman ρ estimates and paired data. The x and y axes are defined by the intersection of rows and columns along with the diagonal (biomarker and unit) and the plot boundaries (scale). The lines represent the LOWESS fit of the data.

Detailed description of the embodiments of the present invention

I. Definition

The term “application” refers to administering a pharmaceutical composition (e.g., containing interleukin-33 (IL-33 axis-binding antagonist)) to a patient (e.g., a patient with asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)).

As used herein, “antagonist” refers to a compound or drug that inhibits or reduces the biological activity of a molecule it binds to. Antagonists include antibodies that bind to, for example, IL-33 axis proteins, synthetic peptides or naturally occurring peptide sequences, immunoadhesins, and small molecule antagonists, optionally conjugated or fused to another molecule. A “blocking antibody” or “antibody antagonist” is an antibody that inhibits or reduces the biological activity of an antigen it binds to.

The term “asthma” in this article refers to a condition characterized by variable and recurrent symptoms that may or may not be related to underlying inflammation, reversible airflow obstruction (e.g., reversible by bronchodilators), and bronchial hyperresponsiveness. Asthma can therefore be inflammatory/inflammatory asthma or non-inflammatory/non-inflammatory asthma. Examples of asthma include allergic asthma, exercise-induced asthma, aspirin-sensitive/exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, asthma untreated by corticosteroids, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed and untreated asthma, smoking-induced asthma, asthma uncontrolled by corticosteroids, and other asthma, as mentioned by Bousquet et al. (J. Allergy Clin. Immunol. 126(5):926-938, 2010).

The terms “biomarker” and “marker” are used interchangeably herein to refer to a molecular marker based on DNA, RNA, protein, sugar, or glycolipid, whose expression or presence in a subject or patient sample is detectable by standard methods (or the methods disclosed herein) and, for example, can be used to identify a subject’s risk profile for a disease or condition and/or the likelihood of a mammalian subject’s responsiveness or sensitivity to a treatment (e.g., a treatment containing an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist)). In samples obtained from patients with the likelihood of an increased or decreased response to an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), the expression of such a biomarker can be determined to be higher or lower than a reference level (e.g., including the median expression level of the biomarker in samples from a group/population of patients (e.g., asthma patients); the level of the biomarker in samples from a group/population of control individuals (e.g., healthy individuals); or the level in samples previously obtained from the individual at an earlier time). In some implementations, individuals with higher or lower expression levels than a reference expression level for at least one gene, such as periostein or ST2 (e.g., sST2), may be identified as potentially responsive to treatment containing an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist).

"Symptom" or "disease" is any condition that would benefit from treatment or diagnosis (e.g., determining the risk of IL-33-mediated disease) using the methods of the present invention. This includes both chronic and acute symptoms or diseases, including those pathological conditions that make mammals susceptible to the symptom discussed. Examples of symptoms to be treated herein include IL-33-mediated diseases (e.g., asthma, allergic rhinitis, atopic dermatitis, and fibrosis (e.g., pulmonary fibrosis, such as idiopathic pulmonary fibrosis)).

The term "effective amount" refers to the amount of medicine that is effective in treating a disease or condition in a subject or patient (such as a mammal, for example, a human).

The term "genotype" refers to a description of the alleles of a gene contained in an individual or sample. In the context of this invention, no distinction is made between the genotype of an individual and the genotype of a sample derived from that individual. Although genotypes are generally determined from diploid cell samples, they can also be determined from haploid cell (e.g., sperm cell) samples.

Unless otherwise stated, the terms “interleukin-1 receptor-like 1 (IL1RL1)” and “ST2” used interchangeably herein refer to any natural ST2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). ST2 is also referred to in the art as DER4, T1, and FIT-1. This terminology encompasses “full-length”, unprocessed ST2, and any form of ST2 produced by cellular processing. At least four isoforms of ST2 are known in the art, including soluble (sST2, also referred to as IL1RL1-a) and transmembrane (ST2L, also referred to as IL1RL1-b) (which arises from differential mRNA expression in a dual-promoter system) and (produced by alternative splicing) ST2V and ST2LV, as described below. The domain structure of ST2L includes three extracellular immunoglobulin-like C2 domains, one transmembrane domain, and one cytoplasmic Toll/interleukin-1 receptor (TIR) domain. sST2 lacks the transmembrane and cytoplasmic domains contained within ST2L and includes a unique 9-amino acid (a.a.) C-terminal sequence (see, e.g., Kakkar et al., Nat. Rev. Drug Disc. 7:827-840, 2008). sST2 can function as a decoy receptor for inhibiting soluble IL-33. This term also covers naturally occurring variants of ST2, such as splice variants (e.g., ST2V, which lacks the third immunoglobulin motif and has a unique hydrophobic tail, and ST2LV, which lacks the transmembrane domain of ST2L) or allelic variants (e.g., variants that mitigate or induce asthma risk, as described herein). An exemplary amino acid sequence of human ST2 can be found, for example, under UniProtKB accession number Q01638. ST2 is part of the IL-33 receptor along with the co-receptor protein IL-1RAcP. The binding of IL-33 to ST2 and the co-receptor interleukin-1 receptor co-protein (IL-1RAcP) forms a 1:1:1 ternary signal transduction complex that promotes downstream signal transduction, as shown in Figure 1A (see, for example, Lingel et al., Structure 17(10):1398-1410, 2009 and Liu et al., Proc. Natl. Acad. Sci. 110(37):14918-14924, 2013).

Unless otherwise stated, as used herein, the term “interleukin-33 (IL-33)” means any natural IL-33 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). IL-33 is also referred to in the art as nuclear factor of high endothelial microvessels (NF-HEV; see, e.g., Baekkevold et al., Am. J. Pathol. 163(1):69-79, 2003), DVS27, C9orf26, and interleukin-1 family member 11 (IL-1F11). This term encompasses “full-length”, unprocessed IL-33, as well as any form of IL-33 produced by cellular processing. Full-length, unprocessed human IL-33 contains 270 amino acids and may also be referred to as IL-33 _1-270 . Processed forms of human IL-33 include, for example, IL-33 _95-270 , IL-33 _99-270 , IL-33 _109-270 , IL-33 _112-270 , IL-33 _1-178 , and IL-33 _179-270 (et al., Proc. Natl. Acad. Sci. 109(5):1673-1678, 2012 and Martin, Semin. Immunol. 25:449-457, 2013). In some embodiments, processed forms of human IL-33, such as IL-33 _95-270 , IL-33 99-270, IL-33 _109-270 , or other forms processed by proteases such as calcium-activated protease, protease 3, neutrophil elastase, and cathepsin G, may have increased biological activity compared to full- _length IL-33. This terminology also covers naturally occurring variants of IL-33, such as splice variants (e.g., splice variant spIL-33 lacking constitutive activity of exon 3, Hong et al., J. Biol. Chem. 286(22):20078-20086, 2011) or alleles. IL-33 can be present intracellularly (e.g., within the nucleus) or as a secreted cytokine. The full-length IL-33 protein contains a helical-turn-helical DNA-binding motif, including a nuclear localization sequence (amino acids 1-75 of human IL-33), which includes a chromatin-binding motif (amino acids 40-58 of human IL-33). Processed and secreted forms of IL-33 lack these N-terminal motifs. An exemplary amino acid sequence of human IL-33 can be found, for example, under UniProtKB accession number O95760.

The term "IL-33 axis" refers to nucleic acids (e.g., genes or mRNA transcribed from such genes) or polypeptides involved in IL-33 signal transduction. For example, the IL-33 axis may include ligand IL-33, receptors (e.g., ST2 and/or IL-1RAcP), adaptor molecules (e.g., MyD88), or proteins associated with receptor molecules and/or adaptor molecules (e.g., kinases such as interleukin-1 receptor-associated kinase 1 (IRAK1) and interleukin-1 receptor-associated kinase 4 (IRAK4) or E3 ubiquitin ligases such as TNF receptor-associated factor 6 (TRAF6)).

"IL-33 axis binding antagonist" refers to a molecule that inhibits the interaction between an IL-33 axis binding partner and one or more binding partners. As used herein, IL-33 axis binding antagonists include IL-33 binding antagonists, ST2 binding antagonists, and IL1RAcP binding antagonists. Exemplary IL-33 axis-binding antagonists include anti-IL-33 antibodies and their antigen-binding fragments (e.g., anti-IL-33 antibodies such as ANB-020 (AnaptysBio Inc.) or any antibody described in EP1725261, US8187596, WO2011031600, WO2014164959, WO2015099175 or WO2015106080, each of which is incorporated herein by reference in its entirety); peptides that bind IL-33 and/or its receptors (ST2 and/or IL-1RAcP) and block ligand-receptor interactions (e.g., ST2-Fc protein; immunoadhesives, peptibody, and soluble ST2, or derivatives thereof); and anti-IL-33 receptor antibodies (e.g., anti-ST2 antibodies, such as A...). MG-282 (Amgen) or STLM15 (Janssen) or any anti-ST2 antibody described in WO 2013/173761 or WO 2013/165894, each of which is incorporated herein by reference in its entirety; or ST2-Fc proteins, such as those described in WO 2013/173761; WO 2013/165894; or WO 2014/152195, each of which is incorporated herein by reference in its entirety; and IL-33 receptor antagonists, such as small molecule inhibitors, aptamers that bind to IL-33, and nucleic acids that hybridize to IL-33 axis nucleic acid sequences under stringent conditions (e.g., short interfering RNA (siRNA) or clustered regularly spaced short palindromic repeat RNA sequences (CRISPR-RNA or crRNA)).

The term "ST2 binding antagonist" refers to a molecule that inhibits the interaction of ST2 with IL-33, IL1RAcP and/or a second ST2 molecule. ST2 binding antagonists can be proteins such as “ST2-Fc proteins” that comprise an IL-33 binding domain (e.g., all or part of an ST2 or IL1RAcP protein) and a polymerizing domain (e.g., the Fc portion of an immunoglobulin, such as the Fc domain of an isotype of IgG1, IgG2, IgG3, and IgG4, and any allotype of IgG within each isotype group), said domains being directly linked to each other or indirectly linked through adapters (e.g., serine-glycine (SG) adapters, glycine-glycine (GG) adapters, or variants thereof (e.g., SGG, GGS, SGS, or GSG adapters)), and include, but are not limited to, the ST2-Fc proteins and their variants described in WO 2013/173761, WO 2013/165894, and WO 2014/152195, each of which is incorporated herein by reference in its entirety.

As used herein, the term "IL-33-mediated disease" refers to any disease or symptom mediated by or associated with the IL-33 axis. In some embodiments, IL-33-mediated disease is associated with excessively high levels or activity of IL-33, wherein atypical symptoms may manifest locally and/or systemically in vivo due to IL-33 levels or activity. Exemplary IL-33-mediated diseases include inflammatory diseases, immune diseases, fibrotic diseases, eosinophilic diseases, infections, pain, central nervous system diseases, solid tumors, and eye diseases. IL-33-mediated diseases are described, for example, in Nature Reviews Immunology 10:103-110, 2010, which is incorporated herein by reference in its entirety.

Exemplary inflammatory diseases include asthma (e.g., allergic asthma, exercise-induced asthma, aspirin-sensitive/exacerbating asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, asthma untreated with corticosteroids, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed and untreated asthma, smoking-related asthma, asthma uncontrolled by corticosteroids, etc.), airway inflammation, airway hyperresponsiveness, airway hyperresponsiveness, sinusitis, polypoid sinusitis, nasal polyposis, arthritis (e.g., osteoarthritis, rheumatoid arthritis, collagen-induced arthritis, arthritic joint inflammation due to injury, etc.), eosinophilic inflammation, mast cell-mediated inflammatory diseases, sepsis, septic shock, seronegative myocolopathy (SMI). Erosive enthesopathy and SEA syndrome, osteoporosis, eosinophilic esophagitis, scleroderma, dermatitis, atopic dermatitis, allergic rhinitis, bullous pemphigoid, chronic urticaria, chondritis, polymyalgia rheumatica, polyarteritis nodosa, Wegener's granulomatosis, Behcet's disease, myositis, polymyositis, dermatomyositis, vasculitis, arteritis, diabetic nephropathy, interstitial cystitis, graft-versus-host disease (GVHD), inflammatory gastrointestinal diseases (e.g., inflammatory bowel disease (IBD), ulcerative colitis (UC), segmental ileitis (CD), colitis (e.g., environmental damage (e.g., caused by treatment regimens such as chemotherapy), Colitis caused by or related to radiation therapy, including infectious colitis, ischemic colitis, collagenous or lymphocytic colitis, necrotizing enterocolitis, colitis in various diseases such as chronic granulomatous disease or celiac disease, food allergies, gastritis, infectious gastritis or enterocolitis (e.g., chronic active gastritis caused by Helicobacter pylori infection) and other forms of gastrointestinal inflammation caused by infectious agents, and inflammatory lung diseases (e.g., chronic obstructive pulmonary disease (COPD), eosinophilic lung inflammation), infection-induced lung diseases (including those associated with viruses such as influenza, parainfluenza, rotavirus, human metapneumovirus, and respiratory syncytial virus). Bacterial infections, fungal infections (e.g., Aspergillus), parasitic infections or prions, allergen-induced lung diseases, pollutant-induced lung diseases (e.g., asbestosis, silicosis or beryllosis), gastric aspiration-induced lung diseases, immune dysregulation, inflammatory diseases with genetic predispositions such as cystic fibrosis, physical trauma-induced lung diseases (e.g., ventilator injury), emphysema, bronchitis, sarcoidosis, histiocytosis, lymphangioleiomyomatosis, acute lung injury, acute respiratory distress syndrome, chronic lung disease, bronchopulmonary dysplasia, pneumonia (e.g., community-acquired pneumonia, nosocomial pneumonia, ventilator-associated pneumonia, viral pneumonia, bacterial pneumonia, and severe pneumonia), airway deterioration, and acute respiratory distress syndrome (ARDS).

Exemplary immune diseases include those at least partially mediated by mast cells, such as asthma (e.g., allergic asthma), eczema, pruritus, allergies, atopic hypersensitivity, anaphylactic reactions, anaphylactic shock, allergic bronchopulmonary aspergillosis, allergic rhinitis, allergic conjunctivitis, and autoimmune diseases, including rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis, pancreatitis, psoriasis, plaque psoriasis, guttate psoriasis, skinfold psoriasis, pustular psoriasis, erythrodermic psoriasis, and paraneoplastic autoimmune diseases. Autoimmune hepatitis, bullous pemphigoid, myasthenia gravis, inflammatory bowel disease, segmental ileitis, ulcerative colitis, celiac disease, thyroiditis (e.g., Graves' disease), Sjögren's syndrome, Guillain-Barre disease, Raynaud's phenomenon, Addison's disease, liver diseases (e.g., primary biliary cirrhosis, primary sclerosing cholangitis, non-alcoholic fatty liver disease and non-alcoholic steatohepatitis), and diabetes (e.g., type 1 diabetes).

As used herein, the term "fibrotic disease" or "fibrosis" refers to a disease involving the formation of excessive fibrous connective tissue in an organ or tissue. Exemplary fibrotic diseases include pulmonary fibrosis, liver fibrosis (e.g., cirrhosis-related fibrosis (e.g., alcohol-induced cirrhosis, virus-induced cirrhosis, post-hepatitis C cirrhosis, and primary biliary cirrhosis), schistosomiasis, cholangitis (e.g., sclerosing cholangitis), and autoimmune-induced hepatitis), renal fibrosis (e.g., tubulointerstitial fibrosis, scleroderma, diabetic nephritis, and glomerulonephritis), and dermal fibrosis (e.g., scleroderma, hypertrophic and keloid scarring, renal fibrotic scleroderma). Fibrosis can be organ-specific or systemic (e.g., myelofibrosis, neurofibromatosis, fibroma, intestinal fibrosis, and surgically induced fibrotic adhesions), cardiac fibrosis (e.g., fibrosis associated with myocardial infarction), vascular fibrosis (e.g., fibrosis associated with restenosis and atherosclerosis after angioplasty), ocular fibrosis (e.g., fibrosis associated with cataract surgery, proliferative vitreoretinopathy, and retroorbital fibrosis), and myelofibrosis (e.g., idiopathic myelofibrosis and drug-induced myelofibrosis). Fibrosis can be organ-specific or systemic (e.g., systemic sclerosis and fibrosis associated with GVHD).

Examples of pulmonary fibrosis include, for example, lung or pulmonary fibrosis associated with idiopathic pulmonary fibrosis, fibrosis with collagen vascular disease, Hermansky-Pudlak syndrome, adult respiratory distress syndrome, nonspecific interstitial pneumonia, respiratory bronchiolitis, sarcoidosis, histiocytosis X, obliterative bronchiolitis, and cryptogenic organizing pneumonia. In one implementation, the pulmonary fibrosis is idiopathic pulmonary fibrosis.

As used in this article, "eosinophilic disease" is a disease associated with an excessively high eosinophil count, in which atypical symptoms can manifest locally or systemically in the body depending on the level or activity of eosinophils. Eosinophilic disorders include, but are not limited to, asthma (including aspirin-sensitive asthma, atopic asthma, and severe asthma), eosinophilic inflammation, atopic dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (periarteritis nodosa with atopic), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema reactions, including occasional hemorrhage edema, helminth infection (in which eosinophils may have a protective role), onchocerciasis dermatitis, eosinophil-associated gastrointestinal disorders (EGIDs), including but not limited to eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic colitis and eosinophilic colitis, nasal micropolyps and polyps, aspirin intolerance, and obstructive sleep apnea. Eosinophil-derived secretions are also associated with promoting angiogenesis and connective tissue formation in tumors and conditions such as chronic asthma, segmental ileitis, scleroderma, and endocardial myocardial fibrosis (Munitz et al., Allergy 59:268-275, 2004; Adamko et al., Allergy 60:13-22, 2005; Oldhoff et al., Allergy 60:693-696, 2005). Other examples include cancers (e.g., glioblastoma (such as glioblastoma multiforme) and non-Hodgkin's lymphoma (NHL)), atopic dermatitis, allergic rhinitis, inflammatory bowel disease, fibrosis (e.g., pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF) and fibrosis secondary to sclerosis) and liver fibrosis) and COPD.

Examples of infections include worm infections (e.g., nematode infections, such as Trichuris muris infection in mice, which is an infection model of the human parasite Trichuris trichiura), protozoan infections (e.g., Leishmania major infection), and viral infections (e.g., respiratory syncytial virus infection and influenza virus infection).

Examples of pain include inflammatory pain, hyperalgesia (e.g., mechanical hyperalgesia), atypical pain, and hypernociception (e.g., hypernociception of the skin and joints, which may or may not be antigen-induced).

Examples of central nervous system diseases include subarachnoid hemorrhage, inflammatory diseases of the central nervous system, neurodegenerative diseases (e.g., Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease), bipolar disorder, and central nervous system infections (e.g., viral infections).

Examples of solid tumors include tumors of the colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, gastrointestinal tract, anus, gallbladder, lips, nasopharynx, skin, uterus, male reproductive organs, urinary organs, bladder, and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.

Examples of eye diseases include age-related macular degeneration (AMD), including wet or dry AMD, geographic atrophy (GA), retinopathy (e.g., diabetic retinopathy (DR) and retinopathy of prematurity (ROP)), polypoid choroidal angiopathy (PCV), diabetic macular edema, dry eye disease, Bechet's disease, and retinal detachment.

The list above is not exhaustive, and professionals understand that diseases or conditions can fall into multiple categories. For example, asthma can be classified as an inflammatory disease and an immune disease in some cases, and some clinicians consider it an autoimmune disease.

Unless otherwise stated, as used herein, "chemokine homologous molecule expressed on Th2 cells (CRTH2)" refers to any naturally occurring CRTH2 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). CRTH2 is also known as G protein-coupled receptor 44 (GPR44), differentiation antigen cluster 294 (CD294), DL1R, and DP2. This term encompasses "full-length," unprocessed CRTH2, and any form of CRTH2 produced through cellular processing. An exemplary amino acid sequence of human CRTH2 can be found, for example, under UniProtKB accession number Q9Y5Y4.

The term "CRTH2 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of CRTH2 with one or more binding partners (such as prostaglandin D2). Exemplary CRTH2 binding antagonists known in the art include AMG-853, AP768, AP-761, MLN6095, and ACT129968.

Unless otherwise stated, as used herein, the term "interleukin-5 (IL-5)" refers to any naturally occurring IL-5 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term covers "full-length," unprocessed IL-5, as well as any form of IL-5 produced through cellular processing. This term also covers naturally occurring variants of IL-5, such as splice variants or allelic variants. Exemplary amino acid sequences of IL-5 can be found, for example, under UniProtKB accession number P05113.

The term "IL-5 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of IL-5 with one or more binding partners, such as IL-5 receptor α (IL5RA) . Exemplary IL-5 binding antagonists that can be used in the methods of the present invention include, for example, anti-IL-5 antibodies (e.g., mepolizumab and reslizumab) and anti-IL-5R antibodies.

Unless otherwise stated, as used herein, the term "interleukin-13 (IL-13)" refers to any naturally occurring IL-13 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). IL-13 is a cytokine secreted by many cell types, including helper T cell type 2 (Th2) cells. This term encompasses "full-length," unprocessed IL-13, and any form of IL-13 produced through cellular processing. An exemplary amino acid sequence of human IL-13 can be found, for example, under UniProtKB accession number P35225.

"IL-13 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of IL-13 with one or more binding partners, such as IL-4 receptor α (IL4Rα), IL-13 receptor α1 (IL13RA1), and IL-13 receptor α2 (IL13RA2)). IL-13 binding antagonists include anti-IL-13 antibodies, such as lebrikizumab, 228B/C-1, 228A-4, 227-26, and 227-43 (see, for example, U.S. Patent Nos. 7,674,459; 8,067,199; 8,088,618; 8,318,160; and 8,734,797).

Unless otherwise stated, as used herein, the term "interleukin-17 (IL-17)" means any naturally occurring IL-17 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats), and includes family members IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. This term encompasses "full-length," unprocessed IL-17, and any form of IL-17 produced through cellular processing. The amino acid sequence of exemplary human IL-17A can be found, for example, under UniProtKB accession number Q16552. The amino acid sequence of exemplary human IL-17B can be found, for example, under UniProtKB accession number Q9UHF5. The amino acid sequence of exemplary human IL-17C can be found, for example, under UniProtKB accession number Q9P0M4. The amino acid sequence of an exemplary human IL-17D can be found, for example, under UniProtKB accession number Q8TAD2. The amino acid sequence of an exemplary human IL-17E can be found, for example, under UniProtKB accession number Q9H293. The amino acid sequence of an exemplary human IL-17F can be found, for example, under UniProtKB accession number Q96PD4.

"IL-17 binding antagonist" refers to a molecule that reduces, blocks, inhibits, eliminates, or interferes with signal transduction resulting from the interaction of IL-17 with one or more binding partners, such as interleukin-17 receptor A (IL17RA), interleukin-17 receptor B (IL17RB), interleukin-17 receptor C (IL17RC), interleukin-17 receptor D (IL17RD), interleukin-17 receptor E (IL17RE), and interleukin-17 receptor E-like (IL17REL)). Exemplary IL-17 binding antagonists include, for example, anti-IL-17 antibodies (e.g., ixekizumab (LY2439821)) and anti-IL-17R antibodies (e.g., brodalumab (AMG-827)).

Unless otherwise stated, as used herein, the term "Janus kinase 1 (JAK1)" refers to any naturally occurring JAK1 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). This term encompasses "full-length," unprocessed JAK1, and any form of JAK1 produced through cellular processing. This term also encompasses naturally occurring JAK1 variants, such as splice variants or allelic variants. Exemplary amino acid sequences of JAK1 can be found, for example, under UniProtKB accession number P23458.

As used herein, the term "JAK1 antagonist" refers to a compound or drug that inhibits or reduces the biological activity of JAK1. Exemplary JAK1 antagonists include small molecule inhibitors (e.g., ruxolitinib, GLPG0634, and GSK2586184).

Unless otherwise stated, as used herein, the term "trypsin-β" refers to any naturally occurring trypsin-β from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). As used herein, this term encompasses trypsin-β-1 (encoded by the TPSAB1 gene, which also encodes trypsin-β-1) and trypsin-β-2 (encoded by the TPSB2 gene). This term encompasses "full-length," unprocessed trypsin-β, and any form of trypsin-β produced by cellular processing. An exemplary amino acid sequence of human trypsin-β-2 can be found, for example, under UniProtKB accession number P20231.

As used herein, the term "trypsin-β antagonist" refers to a compound or drug that inhibits or reduces the biological activity of trypsin β.

As used herein, the phrase “inform the patient” in the context of treatment means, using generated information or data relating to the level or presence of at least one biomarker (e.g., periosteal protein) in a patient’s sample as described herein, to identify whether the patient is suitable for or unsuitable for a particular therapy (e.g., a therapy containing an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist)). In some embodiments, the recommendation may include identifying a patient who needs to adjust the effective amount of the therapy being administered (e.g., an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist)). In some embodiments, the recommended treatment includes recommending an adjustment to the amount of the therapy being administered (e.g., an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist)). As used herein, the phrase “inform the patient” or “provide advice” in the context of treatment may also mean, using generated data, to propose or select a therapy (e.g., a therapy containing an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist)) for a patient who has been identified as or selected as more or less likely to respond to a particular therapy. The information or data used or generated can be in any form, written, oral, or electronic. In some embodiments, using the generated information or data includes notification, presentation, reporting, storage, transmission, transfer, supply, transmission, distribution, or a combination thereof. In some embodiments, notification, presentation, reporting, storage, transmission, transfer, supply, transmission, distribution, or a combination thereof is performed by a computing device, an analytical device, or a combination thereof. In some other embodiments, notification, presentation, reporting, storage, transmission, transfer, supply, transmission, distribution, or a combination thereof is performed by a laboratory or medical practitioner. In some embodiments, the information or data includes a comparison of biomarker (e.g., periosteal protein) levels with a reference level. In some embodiments, the information or data includes an indication of the presence or absence of a biomarker (e.g., periosteal protein) in a sample. In some embodiments, the information or data includes an indication of whether a patient is suitable or unsuitable for treatment with a particular therapy (e.g., a therapy containing an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist)).

A “kit” is any article (e.g., packaging or container) that contains at least one reagent, such as a probe for determining genotype polymorphism as described herein and/or a drug for treating IL-33-mediated diseases (e.g., an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist)). This article is preferably marketed, distributed, or sold as a unit for performing the methods of the present invention.

The terms “level,” “level of expression,” or “expression level” are used interchangeably and generally refer to the amount of polynucleotide or amino acid products or proteins in a biological sample. “Expression” generally refers to the process by which information encoding a gene is converted into a structure present and functioning in the cell. Therefore, according to the present invention, “expression” of a gene can refer to transcription into a polynucleotide, translation into a protein, or even post-translational modification of a protein. Fragments of transcribed polynucleotides, translated proteins, or post-translational modified proteins should also be considered as expressed, regardless of whether they originate from transcripts produced by alternative splicing, degraded transcripts, or post-translational processing of proteins (e.g., by means of proteolytic digestion). “Expressed genes” include those transcribed into polynucleotides such as mRNA and subsequently translated into proteins, and also include those transcribed into RNA but not translated into proteins (e.g., transfer RNA and ribosomal RNA).

The terms "oligonucleotide" and "polynucleotide" are used interchangeably and refer to a molecule composed of two or more, preferably more than three, deoxyribonucleotides or ribonucleotides. Its exact size will depend on many factors, which in turn depend on the final function or use of the oligonucleotide. Oligonucleotides can be synthesized or derived by cloning. Chimeras of deoxyribonucleotides and ribonucleotides may also be within the scope of this invention.

The term "patient" refers to any single animal requiring diagnosis or treatment, more specifically mammals (including non-human animals such as dogs, cats, horses, rabbits, zoo animals, cows, pigs, sheep, and non-human primates). Even more specifically, in this document, a patient is a human. In the context of this invention, a patient can be a subject of any suitable population group (e.g., any population group described in Example 4). In some embodiments, the patient may belong to a population group of African, Asian, and/or European ancestry, such as a patient of Nordic ancestry. The patient can be a clinical patient, a clinical trial volunteer, an experimental animal, etc. The patient may be suspected of having an IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)), at risk of having the condition, or diagnosed with the condition.

The term “patient with…” refers to a patient who exhibits clinical signs of a disease (e.g., IL-33-mediated disease (e.g., asthma) or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)).

As used herein, the term "periosteal protein" refers to a protein in humans encoded by the POSTN gene (including any known isoforms or variants thereof). Periosteal protein is also referred to in the art as osteoblast-specific factor or OSF-2. According to the NCBI database, human periosteal protein isoforms 1, 2, 3, and 4 are known in the art to contain the following amino acid sequences: NP_006466.2, NP_001129406.1, NP_001129407.1, and NP_001129408.1, respectively. An additional form of periosteal protein is described in U.S. Patent Publication 2012/0156194. This isoform is referred to herein as "isoform 5" and has been partially sequenced. Isoform 5 contains the amino acid sequence of SEQ ID NO:23 of U.S. Patent Publication 2012/0156194, which is incorporated herein by reference in its entirety. In some implementations, the periosteal protein is serum periosteal protein or plasma periosteal protein (i.e., periosteal protein derived from serum samples or plasma samples obtained from whole blood, or from whole blood obtained from a patient, respectively).

The term "pharmaceutical composition" refers to a sterile product in such a form that the biological activity of the drug is effective and that it does not contain any additional components that would be unacceptably toxic to the subject to which the preparation will be administered.

In this paper, such nucleotide positions in the genome are referred to as “polymorphisms” or “polymorphic sites,” where more than one sequence may exist in the population at these positions. Polymorphic sites can be, for example, nucleotide sequences of two or more nucleotides, inserted nucleotides or nucleotide sequences, deleted nucleotides or nucleotide sequences, or microsatellites. Polymorphic sites of two or more nucleotide lengths can be 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more, 20 or more, 30 or more, 50 or more, 75 or more, 100 or more, 500 or more, or about 1000 nucleotides in length, wherein all or some of the nucleotide sequences differ within the region. As described below, polymorphic sites of single nucleotide length are referred to herein as single nucleotide polymorphisms (SNPs). When two, three, or four alternative nucleotide sequences are present at a polymorphic site, each nucleotide sequence is called a “polymorphic variant” or “nucleic acid variant.” Each possible variant in the DNA sequence is called an “allele.” Generally, the first identified allele is arbitrarily designated as the reference allele, and the other alleles are designated as alternative or variant alleles. A “common” allele is the dominant allele in a given population; for example, the allele is present in multiple members of the population at a generally accepted frequency greater than about 2%. In the presence of two polymorphic variants, the polymorphic variant represented by the majority of samples from the population is called the “dominant allele” or “major allele,” and the less dominant polymorphic variant in the population is called the “uncommon allele” or “minor allele.” For this polymorphism, an individual carrying two dominant alleles or two uncommon alleles is “homozygous.” For this polymorphism, an individual carrying one dominant allele and one uncommon allele is “heterozygous.” In the case of C/G or A/T SNPs, the alleles are indeterminate and depend on the strand used to extract data from the genotyping platform. In these C/G or A/T SNP cases, the C or G nucleotide or the A or T nucleotide may be risk alleles and depend on the correlation of allele frequencies.

Alleles that are associated with an increased risk of a disease or condition (e.g., IL-33-mediated diseases such as asthma) or with an odds ratio or relative risk >1 are called “risk alleles” or “effect alleles”. A “risk allele” or “effect allele” can be a minor allele or a major allele.

As used herein, “equivalent allele” or “surrogate allele” refers to an allele expected to express in a manner similar to that of a risk allele and selected based on allele frequency and/or a high ^r² value (greater than or equal to 0.6) and/or a high D' value (≥0.6), while risk alleles and/or selected SNPs are as defined herein. In one embodiment, a high ^r² value is ≥0.6, ≥0.7, ≥0.8, ≥0.9, or 1.0. In one embodiment, a high D' value is ≥0.6, ≥0.7, ≥0.8, ≥0.9, or 1.0.

As used in this article, "linkage disequilibrium" or "LD" refers to alleles that are not randomly correlated at different loci; that is, alleles that are not proportionally correlated with their frequencies at different loci. If an allele is in positive linkage disequilibrium, the alleles occur together more frequently than expected to be statistically independent. Conversely, if an allele is in negative linkage disequilibrium, the alleles occur together less frequently than expected to be statistically independent.

When used in this article, “odds ratio” or “OR” refers to the ratio of the probability of disease in an individual with a biomarker (allele or polymorphism) to the probability of disease in an individual without that biomarker (allele or polymorphism).

When used in this article, “haplotype” refers to a group of alleles that are sufficiently closely linked on a single chromosome to be inherited as a unit.

In some embodiments, the term "reference level" herein refers to a predetermined value. As those skilled in the art will appreciate, reference levels are predetermined and set to meet requirements, for example, specificity and/or sensitivity. These requirements may vary, for example, among different regulatory bodies. For instance, it may be necessary to set analytical sensitivity or specificity to certain limits, such as 80%, 90%, or 95%. These requirements may also be defined in terms of positive or negative predictive values. However, based on the teachings given in this invention, achieving a reference level that meets those requirements will always be possible. In one embodiment, the reference level is determined in healthy individuals. In one embodiment, a reference value has been predetermined for the disease entity to which the patient belongs (e.g., an IL-33-mediated disease, such as asthma). In some embodiments, the reference level may be set to any percentage between, for example, 25% and 75% of the total distribution of values in the disease entity under study. In other embodiments, the reference level may be set, for example, to the median, tertiary, quartile, or quintile as determined from the total distribution of values in the disease entity under study or a given population. In one implementation, the reference level can be set, for example, to the median value determined from the overall distribution of values across the disease entities under study. In one implementation, the reference level can be dependent on the patient's gender; for example, males may have a different reference level than females.

In some embodiments, the terms “increased” or “above” mean that the level is at the reference level or that the overall level of the marker (e.g., periosteal protein or sST2) detected by the methods described herein is increased by 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100% or more compared to the level from the reference sample.

In some embodiments, the terms “reduced” or “below” as used herein refer to a level below a reference level or to an overall reduction of 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of a marker (e.g., periosteal protein or sST2) detected by the methods described herein compared to the level from a reference sample.

In some implementations, the term "at reference level" means that the level of a marker (e.g., periosteal protein or sST2) is the same as the level detected from a reference sample by the methods described herein.

The term "response" or "responsiveness" to treatment or therapy (e.g., treatment containing an IL-33 axis-binding antagonist, such as an ST2-binding antagonist) refers to a clinical or therapeutic benefit conferred on a patient by or as a result of treatment, wherein the patient is at risk of or has IL-33-mediated disease (e.g., asthma or pulmonary fibrosis, such as idiopathic pulmonary fibrosis). Such benefits may include a cellular or biological response to or caused by the antagonist treatment, a complete response, a partial response, stable disease (no progression or relapse), or a response with subsequent relapse. It is readily apparent to a technician whether a patient is in a responsive state. For example, an asthma patient responding to treatment containing an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) may exhibit an observable and/or measurable reduction or absence of one or more of the following exemplary symptoms: recurrent wheezing, cough, dyspnea, chest tightness, symptoms that appear or worsen at night, symptoms triggered by cold air, exercise, or exposure to allergens.

The term "single nucleotide polymorphism" or "SNP" refers to a single base substitution within a DNA sequence that results in genetic variation. SNPs can occur in any region of a gene. In some cases, polymorphisms can lead to changes in protein sequence. These changes in protein sequence may or may not affect protein function.

When used herein, the term “selected SNP” refers to an SNP selected from polymorphisms rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); and rs4742165 (SEQ ID NO:6).

As used herein, the term "alternative SNP" refers to an SNP that is expected to behave in a manner similar to that of the selected SNP and is selected based on similar allele frequencies and/or has linkage disequilibrium with the selected SNP as measured by ^r² ≥ 0.6 and/or D' ≥ 0.6. Alternative SNPs include the SNPs listed in Tables 3 and 4 that are linkage disequilibrium with the SNPs described herein (including polymorphisms rs4988956 (SEQ ID NO: 1); rs10204137 (SEQ ID NO: 2); rs10192036 (SEQ ID NO: 3); rs10192157 (SEQ ID NO: 4); rs10206753 (SEQ ID NO: 5); and rs4742165 (SEQ ID NO: 6)). In some embodiments, alternative SNPs are listed in Table 3. In other embodiments, alternative SNPs are listed in Table 4.

The terms "sample" and "biological sample" are used interchangeably to refer to any biological sample obtained from an individual, including body fluids, body tissues (e.g., lung samples), nasal samples (including nasal swabs or nasal polyps), sputum, cells, or other sources. Body fluids include, for example, lymph, serum, fresh whole blood, frozen whole blood, plasma (including fresh or frozen plasma), peripheral blood mononuclear cells, urine, saliva, semen, synovial fluid, and cerebrospinal fluid. Methods for obtaining tissue biopsy samples and body fluids from mammals are well known in the art.

As used herein, “therapeutic” or “treatment” refers to a clinical intervention intended to alter the natural processes of an individual or cell being treated, and may be performed for prevention or during a clinicopathological process. Desired therapeutic effects include preventing the onset or recurrence of disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, slowing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improved prognosis. Subjects requiring treatment may include those who already have the condition, those at risk of developing the condition, or those seeking to prevent the condition. For example, if, after receiving asthma therapy, a patient exhibits an observable and/or measurable reduction or absence of one or more of the following: recurrent wheezing, cough, shortness of breath, chest tightness, symptoms that occur or worsen at night, symptoms triggered by cold air, exercise, or exposure to allergens, the patient's asthma may have been successfully “treated.”

As used herein, “tumor” refers to all tumorous cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. As mentioned herein, the terms “cancer,” “carcinoma,” “proliferative disorder,” “proliferative disease,” and “tumor” are not mutually exclusive.

II. Treatment methods

This invention provides a method for treating patients with IL-33-mediated diseases (e.g., asthma or pulmonary fibrosis, such as idiopathic pulmonary fibrosis). In some embodiments, the method of this invention includes a method based on the biomarkers of this invention (e.g., polymorphisms (e.g., polymorphisms selected from rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); rs4742165 (SEQ ID NO:6)); and related... Therapy was administered to patients for the presence and/or expression levels of rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and/or rs4742165 (SEQ ID NO:6) as linkage-disequilibrium SNPs, periosteal protein and/or sST2. In some cases, Table 3 lists polymorphisms that are chain-disequilibrium relative to rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), and/or rs10206753 (SEQ ID NO:5). In some cases, Table 4 lists polymorphisms that are chain-disequilibrium relative to rs4742165 (SEQ ID NO:6).

In some embodiments, the method of the present invention includes administering to the patient a therapy comprising one or more of (e.g., 1, 2, 3, 4, 5, 6, or 7) of an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a Janus kinase 1 (JAK1) antagonist, and/or an interleukin-5 (IL-5) binding antagonist, wherein the patient’s genotype has been determined to contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, or more) alleles selected from: the G allele at polymorphism rs4988956 (SEQ ID NO: 1); the A allele at polymorphism rs10204137 (SEQ ID NO: 2); and the G allele at polymorphism rs4988956 (SEQ ID NO: 1). The C allele at rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium selected from polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6). In some implementations, Table 3 lists SNPs that are linked out of balance with polymorphisms rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), and/or rs10206753 (SEQ ID NO:5). In some implementations, Table 4 lists SNPs that are linked out of balance with polymorphism rs4742165 (SEQ ID NO:6).

In some embodiments, the method of the present invention includes administering to the patient a therapy comprising one or more of (e.g., 1, 2, 3, 4, 5, 6, or 7) of an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a Janus kinase 1 (JAK1) antagonist, and/or an interleukin-5 (IL-5) binding antagonist, wherein the patient’s genotype has been determined to contain at least one allele listed in Table 2 (e.g., IL-33 axis binding antagonist, ST2 binding antagonist, ST2 binding antagonist, ST3 ... For example, 1, 2, 3, 4, or 5 alleles (e.g., the patient's genotype may include the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and/or the T allele at polymorphism rs10206753 (SEQ ID NO:5)).

In some embodiments, the method of the present invention includes administering a therapy to the patient comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a Janus kinase 1 (JAK1) antagonist, and/or an interleukin-5 (IL-5) binding antagonist, wherein the patient’s genotype has been determined to include the T allele at polymorphism rs4742165 (SEQ ID NO: 6).

In any of the foregoing methods, the equivalent allele can be located at an alternative SNP that is in linkage disequilibrium with one or more of the selected SNPs described herein. In some embodiments, linkage disequilibrium is a D' value or ^r² value. In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95 or higher). In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the D' value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95, or higher). In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the alternative SNP is the SNP described in Table 3 or Table 4. In any of the foregoing methods, the equivalent allele can be a minor allele or a major allele. In some cases, the equivalent allele is a minor allele. In other cases, the equivalent allele is a major allele.

The genotype of a patient can be determined using any method or assay described herein (e.g., in Part IV of the detailed description of the invention or in Example 1) or known in the art. In some embodiments, these methods involve or further include determining the level of a biomarker (e.g., periosteal protein or sST2) in a sample derived from the patient. The level of a biomarker (e.g., periosteal protein or sST2) can be determined using any assay or method described herein or known in the art. For example, the level of periosteal protein can be determined using the periosteal protein assay described in WO2012/083132, which is incorporated herein by reference in its entirety. In another example, the level of sST2 can be determined using any assay or method described herein or known in the art (e.g., in Example 3).

In other embodiments, the method of the present invention includes administering a therapy to the patient comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a Janus kinase 1 (JAK1) antagonist, and/or an interleukin-5 (IL-5) binding antagonist, wherein prior to any administration of the IL-33 axis binding antagonist, the patient has been determined to have sST2 levels at or above a reference level in a patient-derived sample. In some cases, the sST2 level is the level of the sST2 protein. In some cases, the patient-derived sample is a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some cases, the patient-derived sample is a serum sample. In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, where each member of the group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, where each member of the group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some cases, this individual group has asthma. In some cases, the sST2 reference level is the median level. In some cases, this individual group is a female individual group and the patients are female. In some cases, this individual group is a male individual group and the patients are male.

In some implementations, IL-33-mediated diseases can be inflammatory diseases, immune diseases, fibrotic diseases, eosinophilic diseases, infections, pain, central nervous system diseases, solid tumors, or eye diseases. For example, in some cases, inflammatory diseases can be asthma, airway hyperresponsiveness, airway inflammation, sepsis, septic shock, atopic dermatitis, allergic rhinitis, rheumatoid arthritis, or chronic obstructive pulmonary disease (COPD). In some cases, immune diseases can be asthma, rheumatoid arthritis, allergies, atopic hypersensitivity, anaphylactic reactions, anaphylactic shock, allergic rhinitis, psoriasis, inflammatory bowel disease (IBD), segmental ileitis, diabetes, or liver disease. In some cases, fibrotic diseases can be idiopathic pulmonary fibrosis (IPF). In some cases, eosinophilic diseases can be eosinophil-associated gastrointestinal diseases (EGID). In some cases, EGID can be eosinophilic esophagitis. In some cases, infections can be helmintic infections, protozoan infections, or viral infections. In some cases, protozoan infections can be caused by *Leishmania major*. In some cases, viral infections can be caused by respiratory syncytial virus (RSV) or influenza. In some cases, pain can be inflammatory pain. In some cases, central nervous system disorders can be Alzheimer's disease. In some cases, solid tumors can be breast tumors, colon tumors, prostate tumors, lung tumors, kidney tumors, liver tumors, pancreatic tumors, gastric tumors, intestinal tumors, brain tumors, bone tumors, or skin tumors. In some implementations, eye diseases can be age-related macular degeneration (AMD) or retinopathy. In specific cases, IL-33-mediated diseases can be asthma, allergic rhinitis, atopic dermatitis, COPD, eosinophilic esophagitis, or pulmonary fibrosis (e.g., IPF). For example, in some cases, IL-33-mediated disease is asthma. In other cases, IL-33-mediated disease is pulmonary fibrosis (e.g., IPF).

In some cases, the method of the present invention includes administering a therapy to a patient comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. For example, the method may include administering a therapy comprising an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, or an IL-5 binding antagonist. In other cases, the method may include administering a therapy comprising at least two drugs, such as an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a trypsin-β binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a CRTH2 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-13 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-17 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a JAK1 antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-5 binding antagonist, a trypsin-β binding antagonist and a CRTH2 binding antagonist, or a trypsin-β binding antagonist... Anti-inflammatory drugs and IL-13 binding antagonists, trypsin-β binding antagonists and IL-17 binding antagonists, trypsin-β binding antagonists and JAK1 antagonists, trypsin-β binding antagonists and IL-5 binding antagonists, CRTH2 binding antagonists and IL-13 binding antagonists, CRTH2 binding antagonists and IL-17 binding antagonists, CRTH2 binding antagonists and JAK1 antagonists, CRTH2 binding antagonists and IL-5 binding antagonists, IL-13 binding antagonists and IL-17 binding antagonists, IL-13 binding antagonists and JAK1 antagonists, IL-13 binding antagonists and IL-5 binding antagonists, IL-17 binding antagonists and JAK1 antagonists, IL-17 binding antagonists and IL-5 binding antagonists, or JAK1 antagonists and IL-5 binding antagonists. In other cases, the method may include administering a therapy comprising at least three drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may include administering a therapy comprising at least four drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may include administering a therapy comprising at least five drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In still other cases, the method may include administering a therapy comprising at least six drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may include administration of a therapy comprising an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and an IL-5 binding antagonist.

In some cases, the present invention includes an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) for treating patients with IL-33-mediated disease, wherein prior to any administration of the IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist), the patient has been identified as containing one, two, three, four, five, six, seven, or more than seven alleles of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); and the C allele at polymorphism rs10192157 (SEQ ID NO:2). The C allele at rs10206753 (SEQ ID NO:4); the T allele at rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In some cases, the present invention includes the use of an effective amount of an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) in the manufacture of a medicament for treating patients with IL-33-mediated disease, wherein the patient has been identified to contain one, two, three, four, five, six, seven, or more than seven alleles of the following alleles: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); and the allele at polymorphism rs10192157 (SEQ ID NO:4). C allele; T allele at polymorphism rs10206753 (SEQ ID NO:5); T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In some cases, the present invention includes a composition comprising an effective amount of an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) used in methods for treating patients with IL-33-mediated disease, wherein the patient has been identified to contain one, two, three, four, five, six, or seven or more of the following alleles: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); and the allele at polymorphism rs10192157 (SEQ ID NO:4). The C allele; the T allele at polymorphism rs10206753 (SEQ ID NO:5); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or an equivalent allele at a polymorphism selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6). The composition may be a pharmaceutical composition.

In some cases, the present invention includes an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) for treating a patient with IL-33-mediated disease, wherein prior to any administration of the IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist), the patient has been determined to have sST2 levels at or above a reference level in a patient-derived sample. In some cases, the sST2 level is the level of the sST2 protein. In some cases, the patient-derived sample is a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some cases, the patient-derived sample is a serum sample. In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of said group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, where each member of the group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some cases, this individual group has asthma. In some cases, the sST2 reference level is the median level. In some cases, the individual group is a female group and the patients are female. In some cases, the individual group is a male group and the patients are male.

In other instances, the invention includes the use of an effective amount of an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) in the manufacture of a medicament for treating patients with IL-33-mediated disease, wherein the patient has been identified as having sST2 levels at or above a reference level in a patient-derived sample. In some instances, the sST2 level is the level of the sST2 protein. In some instances, the patient-derived sample is a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some instances, the patient-derived sample is a serum sample. In some instances, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of said group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In some instances, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of said group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some instances, this group of individuals has asthma. In some cases, the sST2 reference level was the median level. In some cases, the individual group was a female individual group and the patient was female. In some cases, the individual group was a male individual group and the patient was male.

In some cases, the present invention includes a composition comprising an effective amount of an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) used in methods for treating patients with IL-33-mediated disease, wherein the patient has been identified as having sST2 levels at or above a reference level in a patient-derived sample. In some cases, the sST2 level is the level of the sST2 protein. In some cases, the patient-derived sample is a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some cases, the patient-derived sample is a serum sample. In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In some cases, the sST2 reference level is the sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some cases, this group of individuals has asthma. In some cases, the sST2 reference level was the median level. In some cases, the individual group was a female individual group and the patient was female. In some cases, the individual group was a male individual group and the patient was male.

Exemplary IL-33 axis-binding antagonists include anti-IL-33 antibodies such as ANB-020 (Anaptyx Bio Inc.) or any antibody described in WO2014164959, EP1725261, US8187569, WO2011031600, WO2015099175 or WO2015106080, each of which is incorporated herein by reference in its entirety; or anti-ST2 antibodies such as AMG-282 (Amgen) or STLM15 (Janssen) or any antibody described in WO2013173761 or WO2013165894, each of which is incorporated herein by reference in its entirety.

Exemplary ST2 binding antagonists include the ST2-Fc protein and its variants described in WO 2013/173761, WO 2013/165894 and WO 2014/152195, each of which is incorporated herein by reference in its entirety.

Exemplary CRTH2 binding antagonists that can be used in the methods of the present invention include, for example, AMG-853, AP768, AP-761, MLN6095 and ACT129968.

Exemplary IL-13 binding antagonists that can be used in the methods of the present invention include, for example, anti-IL-13 antibodies, including lebrikizumab, 228B/C-1, 228A-4, 227-26, and 227-43. Additional anti-IL-13 antibodies are described, for example, in U.S. Patent Nos. 7,674,459; 8,067,199; 8,088,618; 8,318,160; and 8,734,797.

Exemplary IL-17 binding antagonists that can be used in the methods of the present invention include, for example, anti-IL-17 antibodies (e.g., ixekizumab (LY2439821) and anti-IL-17R antibodies (e.g., brodalumab (AMG-827)).

Exemplary JAK1 antagonists that can be used in the methods of the present invention include, for example, small molecule inhibitors (e.g., ruxolitinib, GLPG0634, and GSK2586184).

Exemplary IL-5 binding antagonists that can be used in the methods of the present invention include, for example, anti-IL-5 antibodies (e.g., mepolizumab and relizumab) and anti-IL-5R antibodies.

In some implementations, the patient's genotype includes one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, or more than 7): the G allele at polymorphism rs4988956 (SEQ ID NO: 1); the A allele at polymorphism rs10204137 (SEQ ID NO: 2); the C allele at polymorphism rs10192036 (SEQ ID NO: 3); the C allele at polymorphism rs10192157 (SEQ ID NO: 4); the A allele at polymorphism rs10206753 (SEQ ID NO: 4); and the C allele at polymorphism rs10206753 (SEQ ID NO: 5). The T allele at rs4742165 (SEQ ID NO:6); the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In other implementations, the patient's genotype includes at least two of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the T allele at polymorphism rs10206753 (SEQ ID NO:5). The T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

For example, in some cases, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the A allele at polymorphism rs10204137 (SEQ ID NO:2); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10192036 (SEQ ID NO:3); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10192157 (SEQ ID NO:4); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10206753 (SEQ ID NO:2). 5) T allele at position; G allele at polymorphism rs4988956 (SEQ ID NO:1) and T allele at polymorphism rs4742165 (SEQ ID NO:6); G allele at polymorphism rs4988956 (SEQ ID NO:1) and equivalent alleles at polymorphisms in linkage disequilibrium selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6); in polymorphism r The A allele at s10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192036 (SEQ ID NO:3); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192157 (SEQ ID NO:4); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the T allele at polymorphic rs10206753 (SEQ ID NO:5); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the T allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192036 (SEQ ID NO:3); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:3 ... The A allele at rs104137 (SEQ ID NO:2) and the equivalent allele at the polymorphism selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6); the C allele at polymorphism rs10192036 (SEQ ID NO:3) and the C allele at polymorphism rs10192157 (SEQ ID NO:4); the equivalent allele at polymorphism rs10192036 (SEQ ID NO:6). The C allele at rs10206753 (SEQ ID NO:3) and the T allele at rs10206753 (SEQ ID NO:5); the C allele at rs10192036 (SEQ ID NO:3) and the T allele at rs4742165 (SEQ ID NO:6); the C allele at rs10192036 (SEQ ID NO:3) and the T allele at rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6). The polymorphism at linkage disequilibrium of 65 (SEQ ID NO:6) is represented by the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs10206753 (SEQ ID NO:5); the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs4742165 (SEQ ID NO:6); the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), and rs10192036 (SEQ ID NO:3). The polymorphisms selected from rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) are alleles at the linkage disequilibrium polymorphisms; the T alleles at polymorphism rs10206753 (SEQ ID NO:5) and at polymorphism rs4742165 (SEQ ID NO:6); the T alleles at polymorphism rs10206753 (SEQ ID NO:5) and at polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), and rs10192157 (SEQ ID NO:6). The equivalent alleles at the linkage disequilibrium polymorphisms of rs4742165 (SEQ ID NO:6), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6); or the T allele at polymorphism rs4742165 (SEQ ID NO:6) and the equivalent alleles at the linkage disequilibrium polymorphisms of rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6).

In other implementations, the patient's genotype includes at least three of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the T allele at polymorphism rs10206753 (SEQ ID NO:5). The T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In other implementations, the patient's genotype includes at least four of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and so on. The T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In some implementations, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the T allele at polymorphism rs10206753 (SEQ ID NO:5). In some implementations, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the T allele at polymorphism rs4742165 (SEQ ID NO:6). In some implementations, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the G allele at polymorphism rs10206753 (SEQ ID NO:5). The T allele at rs4742165 (SEQ ID NO:6); and the equivalent allele at the polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In any of the foregoing embodiments, the equivalent allele may be located in an alternative SNP that is in linkage disequilibrium with one or more of the selected SNPs described herein. In some embodiments, linkage disequilibrium is a D' value or ^r² value. In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95 or higher). In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the D' value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95, or higher). In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the alternative SNP is the SNP described in Table 3 or Table 4. In any of the foregoing methods, the equivalent allele can be a minor allele or a major allele. In some cases, the equivalent allele is a minor allele. In other cases, the equivalent allele is a major allele.

A therapy may be administered by any suitable means, including parenteral, local, subcutaneous, intraperitoneal, intrapulmonary, intranasal, and/or intralesional administration (e.g., a therapy comprising one or more of the following antagonists: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, interleukin-13 (IL-13) binding antagonists, interleukin-17 (IL-17) binding antagonists, Janus kinase 1 (JAK1) antagonists, and/or interleukin-5 (IL-5) binding antagonists). Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Intrathecal administration is also conceived. Furthermore, the antagonist may be appropriately administered via pulsatile infusion, e.g., with a reduced dose of the antagonist.

As a general recommendation, the effective amount of the antagonist administered parenterally at each dose is in the range of about 20 mg to about 5000 mg, administered in one or more doses. Exemplary dosing regimens for antibodies such as anti-IL-33 antibodies include 100 or 400 mg every 1, 2, 3, or 4 weeks, or doses of about 1, 3, 5, 10, 15, or 20 mg/kg every 1, 2, 3, or 4 weeks. Dosages may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses) (e.g., by infusion).

However, as shown above, the amount of these proposed antagonists is influenced by many treatment decisions. As indicated above, key factors in selecting the appropriate dosage and regimen are the outcomes. In some implementations, the antagonist is administered as close as possible to the first sign, diagnosis, appearance, or occurrence of IL-33-mediated disease. Pharmaceutical compositions containing antagonists will be formulated, dosed, and administered in accordance with good medical practice. Factors considered in this context include the specific type of IL-33-mediated disease being treated, the specific mammal being treated, the individual patient's clinical condition, the cause of the IL-33-mediated disease, the site of drug delivery, possible side effects, the type of antagonist, the method of administration, the administration regimen, and other factors known to the medical practitioner. The effective amount of the antagonist to be administered will be governed by these considerations.

An antagonist, such as an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an interleukin-13 (IL-13) binding antagonist, an interleukin-17 (IL-17) binding antagonist, a JAK1 antagonist, or an interleukin-5 (IL-5) binding antagonist, may be used in combination with at least one additional compound having anti-asthmatic, anti-inflammatory, anti-autoimmune, anti-fibrotic, and/or anticancer properties in a combination drug formulation or as a dosing regimen of combination therapy. The at least one additional compound in the combination drug formulation or dosing regimen preferably has an activity complementary to the antagonist composition, so that they do not adversely affect each other.

The appropriate dose of any of the drugs used in combination is the dose currently in use and may be reduced due to the combined effect (synergistic effect) of newly identified drugs and other chemotherapeutic agents or treatments.

Combination therapy can provide and is proven to be "synergistic," meaning that the effect achieved when the active ingredients are used together is greater than the sum of the effects of using the compounds separately. A synergistic effect can be achieved when the active ingredients are disposed of as follows: (1) co-formulated and administered or delivered simultaneously in combined unit dose formulations; (2) delivered alternately or simultaneously as discrete formulations; or (3) administered via some other regimen. Combination administration can include co-administration using individual formulations or single drug formulations, and sequential administration in any order, wherein preferably there is a period of time during which two (or all) active substances exert their biological activity simultaneously. When delivered in alternating therapy, a synergistic effect can be achieved when the compounds are administered or delivered sequentially (e.g., by different injections in separate syringes). Typically, in alternating therapy, each active ingredient in an effective dose is administered sequentially, i.e., sequentially, while in combination therapy, two or more active ingredients in an effective dose are administered together. When administered sequentially, the combination can be administered in two or more administrations.

In the context of this invention, a therapy (e.g., a therapy comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7) can be administered additionally or as a co-therapy or combination therapy with other asthma therapies: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists), as described below or known in the art. Moderate asthma is currently treated with daily inhaled anti-inflammatory corticosteroids or mast cell inhibitors such as sodium cromoglycate or nedolome, with the addition of an inhaled β2-agonist (3-4 times daily) as needed to relieve breakthrough symptoms or allergen-induced or exercise-induced asthma. Exemplary inhaled corticosteroids include, and additional asthma therapies include, long-acting bronchodilators (LABDs). In some implementations, the LABD is a long-acting β-2 agonist (LABA), a leukotriene receptor antagonist (LTRA), a long-acting muscarinic antagonist (LAMA), theophylline, or an oral corticosteroid (OCS). Exemplary LABDs include PERFOROMIST ^™ and

In the context of this invention, the therapy can be administered additionally or as a co-therapy or combination therapy with one or more chemotherapeutic agents that are part of a standard chemotherapy regimen for solid tumors known in the art (e.g., a therapy comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) antagonists: IL-33 axis binding antagonists (e.g., ST2 binding antagonists, such as ST2-Fc protein), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists). Examples of drugs included in such standard chemotherapy regimens include 5-fluorouracil, leucovovin, irinotecan, gemcitabine, erlotinib, capecitabine, taxanes such as docetaxel and paclitaxel, interferon-alpha, vinorelbine, and platinum-based chemotherapeutic agents such as paclitaxel, carboplatin, cisplatin, and oxaliplatin. Examples of combination therapy for metastatic pancreatic cancer include gemcitabine-erlotinib plus bevacizumab, administered at doses of 5 mg/kg or 10 mg/kg body weight every two weeks, or 7.5 mg/kg or 15 mg/kg body weight every three weeks. Examples of combination therapy for renal cell carcinoma include interferon-alpha plus bevacizumab at a dose of 10 mg/kg body weight every two weeks. Additionally, patients can be treated with a combination of irinotecan, 5-fluorouracil, and leucovovin (also known as IFL, for example, bolus-IFL), oxaliplatin, leucovovin, and 5-fluorouracil (also known as the FOLFOX4 regimen), or capecitabine and oxaliplatin (also known as XELOX). Therefore, in another embodiment of the invention, a patient with IL-33-mediated disease is treated with one or more chemotherapeutic agents such as 5-fluorouracil, leucovorin, irinotecan, gemcitabine-erlotinib, capecitabine, and/or platinum-based chemotherapeutic agents such as paclitaxel, carboplatin, and oxaliplatin. Additionally, the treatment to be administered may be given as a co-therapy or combination therapy with radiotherapy.

At least one additional compound may be a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an anti-hormonal agent, or a combination thereof. Such molecules are appropriately combined in amounts effective for the intended purpose. Pharmaceutical compositions containing an IL-33 axis binding antagonist (e.g., an anti-IL-33 antibody or an ST2 binding antagonist, such as ST2-Fc protein) may also contain therapeutically effective amounts of an antitumor agent, a chemotherapeutic agent, a growth inhibitor, a cytotoxic agent, or a combination thereof.

III. Diagnostic Methods

This invention provides methods for identifying and/or monitoring patients facing an increased risk (i.e., increased susceptibility) to have or develop one or more IL-33-mediated diseases (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)). These methods are particularly useful for increasing the likelihood that administering a therapy (e.g., a therapy comprising one or more of the following antagonists: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists) to patients with IL-33-mediated diseases is effective. In several embodiments, the method includes determining the genotype at one or more polymorphisms (e.g., IL1RL1 polymorphisms and/or IL33 polymorphisms) in a biological sample from the patient (e.g., polymorphisms listed in Tables 1, 2, 3, and 4). In some embodiments, the present invention provides based on the biomarkers of the present invention (e.g., polymorphisms (e.g., selected from rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); rs4742165 (SEQ ID NO:6)); and polymorphisms relative to rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:6). Methods for identifying and/or monitoring patients who face an increased risk (i.e., increased susceptibility) to have or develop one or more IL-33-mediated diseases (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)). This includes methods for identifying and/or monitoring the presence and/or expression levels of EQ ID NO:2, rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and/or rs4742165 (SEQ ID NO:6) as linkage non-equilibrium polymorphisms, periosteal protein and/or sST2). In some cases, Table 3 lists polymorphisms that are linkage disequilibrium relative to rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4) and/or rs10206753 (SEQ ID NO:5). In some cases, Table 4 lists polymorphisms that are linkage disequilibrium relative to rs4742165 (SEQ ID NO:6). In some embodiments, the method includes determining the levels of one or more biomarkers (e.g., periosteal protein and/or sST2) in a sample derived from a patient.

The methods and assays of the present invention provide a convenient, efficient, and potentially cost-effective means of obtaining data and information that can be used to evaluate appropriate or effective therapies for treating patients (e.g., patients with IL-33-mediated disease). For example, patients may provide biological samples (e.g., blood samples, nasal samples, lung samples, sputum, etc.) before treatment (e.g., administration of a therapy comprising one or more of the following antagonists: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists) and these samples can be examined by various in vitro assays to determine whether the patient's cells are likely to respond to treatment.

The method can be implemented in various testing modalities, including assays for detecting genetic information (e.g., DNA or RNA sequencing), gene or protein expression (e.g., polymerase chain reaction (PCR) and enzyme immunoassay), and biochemical analyses for detecting appropriate activities, as described below. The presence of one or more alleles of the polymorphisms listed in Table 2 in samples from patients is associated with a risk of IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)). Example 1 shows that the presence of one or more alleles of the polymorphisms listed in Table 2 suggests this risk, and therefore, in several embodiments, the invention includes determining the genotype at one or more of these polymorphisms in the methods described herein.

In one embodiment, the present invention provides a method for determining whether a patient faces an increased risk of IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)), the method comprising determining the genotype of at least one allele (e.g., 1, 2, 3, 4, 5, 6, 7 or more alleles) listed in Tables 1, 2, 3 or 4 in a sample derived from the patient, wherein if the patient's genotype contains alleles listed in Table 2; the T allele at polymorphism rs4742165 (SEQ ID NO: 6) If a patient has an IL-33-mediated disease risk, and/or an allele in a SNP that is linkage disequilibrium relative to rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and/or rs4742165 (SEQ ID NO:6), the patient faces an increased risk of IL-33-mediated disease.

For example, in some embodiments, the method includes identifying one or more polymorphisms (e.g., 1, 2, 3, 4, 5, 6, 7) selected from rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); rs4742165 (SEQ ID NO:6) in a sample derived from a patient. (or more than 7 polymorphisms); and genotypes at polymorphisms linked to non-equilibrium at polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6), wherein if the patient's genotype contains (a) at polymorphism rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6), wherein ... (a) G allele at polymorphism rs10204137 (SEQ ID NO:2); (b) A allele at polymorphism rs10192036 (SEQ ID NO:3); (c) C allele at polymorphism rs10192157 (SEQ ID NO:4); (d) C allele at polymorphism rs10206753 (SEQ ID NO:5); (e) T allele at polymorphism rs4742165 (SEQ ID NO:1); If the patient has an T allele at rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6), then the patient faces an increased risk of asthma.

In some cases, if a patient's genotype contains one or more of the following (e.g., 1, 2, 3, 4, 5, 6, 7, or more than 7): the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); or the T allele at polymorphism rs10206753 (SEQ ID NO:5); Patients with an equivalent allele at the T allele of polymorphism rs4742165 (SEQ ID NO:6); and/or at a polymorphism of linkage disequilibrium selected from polymorphisms of rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6) face an increased risk of IL-33-mediated disease. In some cases, patients at increased risk of IL-33-mediated disease have genotypes containing the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the T allele at polymorphism rs10206753 (SEQ ID NO:5). Gene; the T allele at polymorphism rs4742165 (SEQ ID NO:6); or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In other embodiments, the genotype of patients facing an increased risk of IL-33-mediated disease includes at least two of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the G allele at polymorphism rs10206753 (SEQ ID NO:5). The T allele; the T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

For example, in some cases, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the A allele at polymorphism rs10204137 (SEQ ID NO:2); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10192036 (SEQ ID NO:3); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10192157 (SEQ ID NO:4); the G allele at polymorphism rs4988956 (SEQ ID NO:1) and the C allele at polymorphism rs10206753 (SEQ ID NO:2). 5) T allele at position; G allele at polymorphism rs4988956 (SEQ ID NO:1) and T allele at polymorphism rs4742165 (SEQ ID NO:6); G allele at polymorphism rs4988956 (SEQ ID NO:1) and equivalent alleles at polymorphisms in linkage disequilibrium selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6); in polymorphism r The A allele at s10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192036 (SEQ ID NO:3); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192157 (SEQ ID NO:4); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the T allele at polymorphic rs10206753 (SEQ ID NO:5); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the T allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs10192036 (SEQ ID NO:3); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:6); the A allele at polymorphic rs10204137 (SEQ ID NO:2) and the C allele at polymorphic rs4742165 (SEQ ID NO:3 ... The A allele at rs104137 (SEQ ID NO:2) and the equivalent allele at the polymorphism selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6); the C allele at polymorphism rs10192036 (SEQ ID NO:3) and the C allele at polymorphism rs10192157 (SEQ ID NO:4); the equivalent allele at polymorphism rs10192036 (SEQ ID NO:6). The C allele at rs10206753 (SEQ ID NO:3) and the T allele at rs10206753 (SEQ ID NO:5); the C allele at rs10192036 (SEQ ID NO:3) and the T allele at rs4742165 (SEQ ID NO:6); the C allele at rs10192036 (SEQ ID NO:3) and the T allele at rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6). The polymorphism at linkage disequilibrium of 65 (SEQ ID NO:6) is represented by the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs10206753 (SEQ ID NO:5); the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs4742165 (SEQ ID NO:6); the C allele at polymorphism rs10192157 (SEQ ID NO:4) and the T allele at polymorphism rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), and rs10192036 (SEQ ID NO:3). The polymorphisms selected from rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) are alleles at the linkage disequilibrium polymorphisms; the T alleles at polymorphism rs10206753 (SEQ ID NO:5) and at polymorphism rs4742165 (SEQ ID NO:6); the T alleles at polymorphism rs10206753 (SEQ ID NO:5) and at polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), and rs10192157 (SEQ ID NO:6). The equivalent alleles at the linkage disequilibrium polymorphisms of rs4742165 (SEQ ID NO:6), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6); or the T allele at polymorphism rs4742165 (SEQ ID NO:6) and the equivalent alleles at the linkage disequilibrium polymorphisms of rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6).

In other implementations, the genotype of patients at risk of IL-33-mediated disease includes at least three of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the genotype at polymorphism rs10206753 (SEQ ID NO:5). T allele; T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In other embodiments, the patient's genotype includes at least four of the following: the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the G allele at polymorphism rs10206753 (SEQ ID NO:5). The T allele at rs4742165 (SEQ ID NO:6); and/or the equivalent allele at a polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In other embodiments, the genotype of patients facing IL-33-mediated disease risk includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); and the T allele at polymorphism rs10206753 (SEQ ID NO:5). In some implementations, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the T allele at polymorphism rs4742165 (SEQ ID NO:6). In some implementations, the patient's genotype includes the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); the T allele at polymorphism rs10206753 (SEQ ID NO:5); and the T allele at polymorphism rs4988956 (SEQ ID NO:1). The T allele at rs4742165 (SEQ ID NO:6); and the equivalent allele at the polymorphism in linkage disequilibrium with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6).

In another embodiment, the present invention provides a method for determining whether a patient with IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)) is likely to respond to treatment comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) of the following antagonists: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists, the method comprising: (a) identifying in a sample derived from a patient with IL-33-mediated disease a substance selected from rs4988956 (SEQ ID NO: 1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); rs4742165 (SEQ ID NO:6) at one or more polymorphisms (e.g., 1, 2, 3, 4, 5, 6, 7 or more polymorphisms) and at the polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs1 (a) Genotypes at linkage disequilibrium polymorphisms of rs4988956 (SEQ ID NO:1); (b) genotypes based on linkage disequilibrium polymorphisms of rs4988956 (SEQ ID NO:1); (ii) each A allele at ... The presence of each T allele at polymorphism rs4742165 (SEQ ID NO: 5); (vi) each T allele at polymorphism rs4742165 (SEQ ID NO: 6); and/or (vii) the presence of each equivalent allele at a polymorphism of linkage disequilibrium selected from rs4988956 (SEQ ID NO: 1), rs10204137 (SEQ ID NO: 2), rs10192036 (SEQ ID NO: 3), rs10192157 (SEQ ID NO: 4), rs10206753 (SEQ ID NO: 5), and rs4742165 (SEQ ID NO: 6) indicates an increased likelihood of the patient responding to the treatment. In some cases, the method also includes determining the level of periosteal proteins in samples derived from the patient. In these cases, the method may further include informing the patient that if the level of periosteal protein in the sample is at, below, or above a periosteal protein reference level, their likelihood of responding to treatment containing one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists.

In another embodiment, the present invention provides a method for improving the therapeutic efficacy of IL-33-mediated disease therapy for patients suffering from IL-33-mediated disease, the method comprising: (a) identifying in a sample derived from an asthma patient one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or more) polymorphisms selected from rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); rs10206753 (SEQ ID NO:5); rs4742165 (SEQ ID NO:6); and (b) a polymorphism selected from rs4988956 (SEQ ID NO:6). (a) Genotypes at linkage disequilibrium polymorphisms of rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6); and (b) based on genotype, identifying patients more suitable for treatment by adding one or more of the following (e.g., 1, 2, 3, 4, 5, 6, or 7) antagonists to the treatment of IL-33-mediated disease: IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists. Antagonists, JAK1 antagonists, and/or IL-5 binding antagonists, wherein: (i) each G allele at polymorphism rs4988956 (SEQ ID NO:1); (ii) each A allele at polymorphism rs10204137 (SEQ ID NO:2); (iii) each C allele at polymorphism rs10192036 (SEQ ID NO:3); (iv) each C allele at polymorphism rs10192157 (SEQ ID NO:4); (v) each T allele at polymorphism rs10206753 (SEQ ID NO:5); (vi) each T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or (vii) an antagonist selected from rs4988956 (SEQ ID NO:1); The presence of each allele at the linkage disequilibrium polymorphism of SEQ ID NO:1, rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6) indicates an increased potential for improved therapeutic efficacy of therapies for IL-33-mediated disease by the addition of IL-33 axis-binding antagonists (e.g., ST2-binding antagonists), trypsin-β-binding antagonists, CRTH2 antagonists, IL-13-binding antagonists, IL-17-binding antagonists, JAK1 antagonists, and/or IL-5-binding antagonists. In some cases, the method also includes determining the level of periosteal proteins in samples derived from the patient. In these cases, the method may also include informing the patient that if the level of periosteal proteins in the sample is at, below, or above a periosteal protein reference level, they have an increased likelihood of responding to treatment with an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist.

In any of the foregoing embodiments, the equivalent allele may be located in an alternative SNP that is in linkage disequilibrium with one or more of the selected SNPs described herein. In some embodiments, linkage disequilibrium is a D' value or ^r² value. In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95 or higher). In some embodiments, the D' value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the D' value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.60 (e.g., ≥0.60, ≥0.65, ≥0.7, ≥0.75, ≥0.8, ≥0.85, ≥0.9, ≥0.95, or higher). In some embodiments, the ^r² value between the selected SNP and the alternative SNP is ≥0.70, ≥0.80, or ≥0.90. In some embodiments, the ^r² value between the selected SNP and the alternative SNP is 1.0. In some embodiments, the alternative SNP is the SNP described in Table 3 or Table 4. In any of the foregoing methods, the equivalent allele can be a minor allele or a major allele. In some cases, the equivalent allele is a minor allele. In other cases, the equivalent allele is a major allele.

In any of the foregoing diagnostic methods, the method may further include administering a therapy to a patient comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) antagonists: an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. For example, the method may further comprise administering a therapy comprising an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, or an IL-5 binding antagonist. In other cases, the method may also include administering a therapy comprising at least two drugs, such as an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a trypsin-β binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a CRTH2 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-13 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-17 binding antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and a JAK1 antagonist, an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist) and an IL-5 binding antagonist, a trypsin-β binding antagonist and a CRTH2 binding antagonist, or a trypsin-β binding antagonist... Anti-inflammatory drugs and IL-13 binding antagonists, trypsin-β binding antagonists and IL-17 binding antagonists, trypsin-β binding antagonists and JAK1 antagonists, trypsin-β binding antagonists and IL-5 binding antagonists, CRTH2 binding antagonists and IL-13 binding antagonists, CRTH2 binding antagonists and IL-17 binding antagonists, CRTH2 binding antagonists and JAK1 antagonists, CRTH2 binding antagonists and IL-5 binding antagonists, IL-13 binding antagonists and IL-17 binding antagonists, IL-13 binding antagonists and JAK1 antagonists, IL-13 binding antagonists and IL-5 binding antagonists, IL-17 binding antagonists and JAK1 antagonists, IL-17 binding antagonists and IL-5 binding antagonists, or JAK1 antagonists and IL-5 binding antagonists. In other cases, the method may also include administering a therapy comprising at least three drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may also include administering a therapy comprising at least four drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may also include administering a therapy comprising at least five drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In still other cases, the method may also include administering a therapy comprising at least six drugs selected from IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 binding antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and IL-5 binding antagonists. In other cases, the method may include administration of a therapy comprising an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 binding antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and an IL-5 binding antagonist.

In other instances, the present invention provides a method for selecting a therapy for a patient with IL-33-mediated disease, the method comprising: (a) determining the level of a periosteal protein in a sample derived from the patient; (b) comparing the level of the periosteal protein in the sample derived from the patient to a periosteal protein reference level, wherein a periosteal protein level in the sample at or below the periosteal protein reference level identifies a patient who may respond to treatment comprising an IL-33 axial binding antagonist (e.g., an ST2 binding antagonist); and (c) if the patient is identified as potentially responding to treatment comprising an IL-33 axial binding antagonist (e.g., an ST2 binding antagonist), selecting a therapy comprising an IL-33 axial binding antagonist (e.g., an ST2 binding antagonist) and recommending the selected therapy comprising an IL-33 axial binding antagonist (e.g., an ST2 binding antagonist) to the patient. In some embodiments, the method further includes administering an asthma therapy comprising one or more antagonists selected from IL-33 binding antagonists (e.g., anti-IL-33 antibody or its antigen-binding fragment), ST2 binding antagonists (e.g., ST2-Fc protein, anti-ST2 antibody or its antigen-binding fragment), and/or IL-1RAcP binding antagonists (e.g., anti-IL1RAcP antibody). For example, in some embodiments, the method further includes administering an IL-33 binding antagonist, an ST2 binding antagonist, or an IL1RAcP binding antagonist. In some embodiments, the method further includes administering at least two of the following antagonists: an IL-33 binding antagonist, an ST2 binding antagonist, and an IL1RAcP binding antagonist (e.g., an IL-33 binding antagonist and an ST2 binding antagonist (e.g., ST2-Fc protein); an IL-33 binding antagonist and an IL1RAcP binding antagonist; or an ST2 binding antagonist (e.g., ST2-Fc protein) and an IL1RAcP binding antagonist). In some implementations, the method further includes the administration of an IL-33 binding antagonist, an ST2 binding antagonist (e.g., ST2-Fc protein), and an IL1RAcP binding antagonist.

In other instances, the present invention provides a method for determining whether a patient faces an increased risk of IL-33-mediated disease (e.g., asthma) or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis), the method comprising: (a) determining the level of sST2 in a sample derived from the patient; and (b) comparing the level of sST2 in the patient-derived sample to a reference level of sST2, wherein if the level of sST2 in the patient-derived sample is at or above the reference level, the patient faces an increased risk of IL-33-mediated disease. In some instances, the method further comprises administering a therapy comprising treatment containing one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) antagonists: IL-33 axis-binding antagonists (e.g., ST2... The method may include IL-33 axis binding antagonists (e.g., ST2 binding antagonists), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists. In some cases, the method may also include determining the level of periosteal proteins in a patient-derived sample. In these cases, the method may further include informing the patient that if the level of periosteal proteins in the sample is at, below, or above a periosteal protein reference level, they have an increased likelihood of responding to treatment with an IL-33 axis binding antagonist (e.g., ST2 binding antagonist), trypsin-β binding antagonist, CRTH2 antagonist, IL-13 binding antagonist, IL-17 binding antagonist, JAK1 antagonist, and/or IL-5 binding antagonist.

In another instance, the present invention provides a method for selecting a therapy for a patient with IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)), the method comprising: (a) determining the level of sST2 in a sample derived from the patient; (b) comparing the level of sST2 in the sample derived from the patient with a reference level of sST2; and (c) if the level of sST2 in the sample is at or above the reference level, selecting a therapy comprising an IL-33 axis-binding antagonist. In some instances, the method further comprises administering a therapy comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or 7) antagonists: an IL-33 axis-binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. In some instances, the method further comprises determining the level of a periosteum protein in a sample derived from the patient. In these cases, the method may also include informing the patient that if the level of periosteal proteins in the sample is at, below, or above a periosteal protein reference level, they have an increased likelihood of responding to treatment with an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist.

In another embodiment, the present invention provides a method for determining whether a patient with IL-33-mediated disease is likely to respond to treatment containing an IL-33 axis-binding antagonist, the method comprising: (a) determining the level of sST2 in a sample derived from the patient; (b) comparing the level of sST2 in the sample derived from the patient to a reference level of sST2; and (c) identifying the patient as likely to respond to treatment containing an IL-33 axis-binding antagonist based on the level of sST2 in the sample derived from the patient, wherein if the level of sST2 in the sample is at or above the reference level, the likelihood of the patient responding to treatment containing an IL-33 axis-binding antagonist increases. In some cases, the method further includes administering a therapy comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. In some cases, the method further includes determining the level of periosteal proteins in a sample derived from the patient. In these cases, the method may also include informing the patient that if the level of periosteal proteins in the sample is at, below, or above a periosteal protein reference level, they have an increased likelihood of responding to treatment with an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist.

In another embodiment, the present invention provides a method for evaluating the treatment response of a patient treated with an IL-33 axis-binding antagonist, the method comprising: (a) determining the level of sST2 in a patient-derived sample at time points during or after administration of the IL-33 axis-binding antagonist; and (b) maintaining, adjusting, or discontinuing the patient's treatment based on a comparison of the level of sST2 in the patient-derived sample with a reference level of sST2, wherein a change in the level of sST2 in the patient-derived sample compared to the reference level represents a response to IL-33 axis-binding antagonist treatment. In some embodiments, the change is an increase in the level of sST2 and maintenance of treatment. In some embodiments, the change is an increase in the level of sST2 and a change in treatment to a different therapeutic agent (e.g., a different IL-33 axis-binding antagonist). In some embodiments, the change is an increase in the level of sST2 and a change in treatment by increasing the dose of the IL-33 binding antagonist or increasing the frequency of administration of the IL-33 binding antagonist. In other embodiments, the change is a decrease in the level of sST2 and maintenance of treatment. In other embodiments, the change is a decrease in the level of sST2 and a reduction in treatment. In other embodiments, the change is a decrease in sST2 levels and a change in treatment by reducing the dose of the IL-33 binding antagonist or reducing the frequency of IL-33 binding antagonist administration. In other embodiments, the change is a decrease in sST2 levels and discontinuation of treatment. In some cases, the method also includes determining the level of periosteal proteins in a sample derived from the patient. In these cases, the method may also include informing the patient that if the level of periosteal proteins in the sample is at, below, or above a periosteal protein reference level, they have an increased likelihood of responding to treatment with an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), trypsin-β binding antagonist, CRTH2 antagonist, IL-13 binding antagonist, IL-17 binding antagonist, JAK1 antagonist, and/or IL-5 binding antagonist.

In another embodiment, the present invention provides a method for monitoring the response of a patient treated with an IL-33 axis-binding antagonist, the method comprising (a) determining the level of sST2 in a patient-derived sample at a time point during or after administration of the IL-33 axis-binding antagonist; and (b) comparing the level of sST2 in the patient-derived sample with a reference level of sST2, thereby monitoring the response in a patient receiving IL-33 axis-binding antagonist treatment. In some embodiments, the method further comprises determining the level of periosteal proteins in a patient-derived sample. In these embodiments, the method may further comprise informing the patient that if the level of periosteal proteins in the sample is at, below, or above a reference level of periosteal proteins, they have an increased likelihood of responding to treatment with an IL-33 axis-binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist.

In any of the foregoing methods, the level of sST2 can be, for example, the level of the sST2 protein or the level of the sST2 nucleic acid (e.g., mRNA). In some embodiments, the level is the level of the sST2 protein.

In any of the foregoing methods, the patient-derived sample can be a whole blood sample, a serum sample, a plasma sample, or a combination thereof. In some embodiments, the patient-derived sample can be a serum sample.

In any of the foregoing methods, in some embodiments, the level of sST2 in the patient-derived sample may be at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 15, or 20 times higher than the reference level. In any of the foregoing methods, in some embodiments, the level of sST2 in samples derived from patients may be at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 9, 10, 15, or 20 times lower than the reference level.

Any suitable sST2 reference level can be used in any of the foregoing methods. In any of the foregoing methods, the reference level can be an sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs4742165 (SEQ ID NO: 6). In any of the foregoing methods, the sST2 reference level can be an sST2 level determined from a group of individuals, wherein each member of the group has a genotype containing two G alleles at polymorphism rs3771166 (SEQ ID NO: 8). In some cases, the individual group has asthma. In some embodiments, the sST2 reference level can be the mean, median, or any suitable value. In some cases, the reference level is the median level. In some cases, the individual group is a female individual group and the patients are female. In other cases, the individual group is a male individual group and the patients are male. In some implementations, sST2 reference levels are measured in individuals at an earlier time point (e.g., before administration of an IL-33 binding antagonist) or at an earlier time point during treatment with an IL-33 binding antagonist.

In some cases, any of the foregoing methods also includes administering a therapy comprising one or more of the following antagonists (e.g., 1, 2, 3, 4, 5, 6, or 7): an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), a trypsin-β binding antagonist, a CRTH2 antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. In some embodiments, the IL-33 axis binding antagonist is an IL-33 binding antagonist, an ST2 binding antagonist, or an IL-1RAcP binding antagonist.

In some cases, any of the foregoing methods further includes administering an antagonist selected from one or more of the following, based on the fact that the level of sST2 in a patient-derived sample is at or above a reference level: an IL-33 binding antagonist (e.g., an anti-IL-33 antibody or its antigen-binding fragment), an ST2 binding antagonist (e.g., ST2-Fc protein, an anti-ST2 antibody or its antigen-binding fragment), and/or an IL-1RAcP binding antagonist (e.g., an anti-IL1RAcP antibody). For example, in some embodiments, the method further includes administering an IL-33 binding antagonist, an ST2 binding antagonist, or an IL1RAcP binding antagonist. In some embodiments, the method further includes administering at least two of the following antagonists: an IL-33 binding antagonist, an ST2 binding antagonist, and an IL1RAcP binding antagonist (e.g., an IL-33 binding antagonist and an ST2 binding antagonist (e.g., ST2-Fc protein); an IL-33 binding antagonist and an IL1RAcP binding antagonist; or an ST2 binding antagonist (e.g., ST2-Fc protein) and an IL1RAcP binding antagonist). In some embodiments, the method further includes administering an IL-33 binding antagonist, an ST2 binding antagonist (e.g., ST2-Fc protein), and an IL1RAcP binding antagonist.

Those in the medical field, particularly those skilled in applying diagnostic testing and therapeutic medication, recognize that biological systems vary to varying degrees and are not always entirely predictable, and therefore many good diagnostic tests or therapeutics occasionally prove ineffective. Thus, ultimately, it relies on the attending physician's judgment to determine the most appropriate course of treatment for an individual patient based on test results, the patient's condition and history, and their own experience. There may even be situations where, for example, a patient does not face an increased risk of IL-33-mediated disease (e.g., asthma and/or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)) based on data from diagnostic testing or other standards, especially if all or most other apparent treatment options have failed or if some synergistic effect is expected when administered with another treatment, the physician will choose to treat the patient with an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist).

The present invention also provides a method for identifying a biomarker that can be used to monitor sensitivity or responsiveness to an IL-33 axis-binding antagonist, such as an anti-IL-33 antibody or an ST2-binding antagonist, the method comprising: (a) measuring the level of a candidate biomarker in a sample obtained from a patient with IL-33-mediated disease, the sample being obtained prior to administration of any dose of an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist) to the patient, wherein a change in the expression of the candidate biomarker relative to a control (i.e., an increase or decrease) indicates that the biomarker determines that the IL-33-mediated disease is more effectively treated with an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist). In some embodiments, the biomarker is a genetic biomarker and its expression is analyzed.

IV. Detection of nucleic acid polymorphism

In several embodiments, the therapeutic and diagnostic methods provided by this invention involve determining a patient's genotype at one or more polymorphisms (e.g., polymorphisms in IL1RL1 described in Tables 1 and 2). Detection techniques for nucleic acids to evaluate the presence of SNPs include operations well known in the field of molecular genetics. Many, but not all, methods involve the amplification of nucleic acids. Amplification guidelines for performing amplification are provided in the art. Exemplary references include manuals such as Erlich, ed., *PCR Technology: Principles and Applications for DNA Amplification*, *Freeman Press*, 1992; Innis et al., ed., *PCR Protocols: A Guide to Methods and Applications*, *Academic Press*, 1990; Ausubel, ed., *Current Protocols in Molecular Biology*, 1994–1999, including appendices updated to April 2004; and Sambrook et al., ed., *Molecular Cloning*, *A Laboratory Manual*, 2001. A general method for detecting single nucleotide polymorphisms is disclosed in Kwok's book, Single Nucleotide Polymorphisms: Methods and Protocols, Humana Press, 2003.

Although these methods generally employ PCR steps, other amplification protocols can also be used. Suitable amplification methods include ligase chain reaction (see, for example, Wu et al., Genomics 4:560-569, 1988); strand substitution analysis (see, for example, Walker et al., Proc. Natl. Acad. Sci. USA 89:392-396, 1992; U.S. Patent No. 5,455,166); and several transcription-based amplification systems, including those described in U.S. Patent Nos. 5,437,990, 5,409,818, and 5,399,491; transcription amplification systems (TAS) (Kwoh et al., Proc. Natl. Acad. Sci. USA 86:1173-1177, 1989); and self-sustaining sequence replication (3SR) (Guatelli et al., Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990; WO1992/08800). Alternatively, methods for amplifying the probe to detectable levels can be used, such as Qβ-replicaase amplification (Kramer et al., Nature 339:401-402, 1989; Lomeli et al., Clin. Chem. 35:1826-1831, 1989). A review of known amplification methods is provided, for example, by Abramson et al., Curr. Opin. Biotech. 4:41-47, 1993.

Individual genotypes, haplotypes, SNPs, microsatellites, or other polymorphisms can be detected using oligonucleotide primers and/or probes. Oligonucleotides can be prepared by any suitable method, typically through chemical synthesis. Oligonucleotides can be synthesized using commercially available reagents and instruments. Alternatively, they can be purchased from commercial sources. Methods for synthesizing oligonucleotides are well known in the art (see, for example, Narang et al., Meth. Enzymol. 68:90-99, 1979; Brown et al., Meth. Enzymol. 68:109-151, 1979; Beaucage et al., Tetra. Lett. 22:1859-1862, 1981; and the solid-phase support method in U.S. Patent No. 4,458,066). Furthermore, modifications to the above-described synthetic methods can be used to desiredly influence enzyme behavior relative to the synthesized oligonucleotides. For example, incorporating modified phosphodiester bonds (e.g., thiophosphates, methylphosphates, phosphoramidides, or boron phosphates) or bonds other than phosphate derivatives into oligonucleotides can prevent cleavage at selected sites. Furthermore, when hybridizing with nucleic acids that also serve as templates for the synthesis of new nucleic acid chains, the use of 2'-amino-modified sugars often favors substitution rather than digestion of oligonucleotides.

Genotypes of individuals (e.g., those with IL-33-mediated diseases such as asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis) or at risk of them) can be determined using a number of assays well known in the art. Most assays involve one of several general protocols: hybridization using allele-specific oligonucleotides, primer extension, allele-specific ligation, sequencing, or electrophoretic separation techniques, such as single-strand conformation polymorphism (SSCP) and heteroduplex analysis. Exemplary assays include 5’-nuclease assays, template-guided dye-terminator incorporation, molecular beacon allele-specific oligonucleotide assays, single-base extension assays, and SNP scoring based on real-time pyrosequencing sequences. Analysis of amplified sequences can be performed using a variety of techniques such as microchips, fluorescence polarization assays, and MALDI-TOF (matrix-assisted laser desorption/ionization-time-of-flight) mass spectrometry. Two other methods are also available: assays based on invasive cleavage by Flap nuclease and methodologies using padlock probes.

The presence or absence of a specific allele is typically determined by analyzing nucleic acid samples obtained from the individual being analyzed. Nucleic acid samples often contain genomic DNA. Genomic DNA is generally obtained from blood samples, but can also be obtained from other cells or tissues.

It is also possible to analyze RNA samples for the presence of polymorphic alleles. For example, mRNA can be used to determine an individual's genotype at one or more polymorphic sites. In this case, nucleic acid samples are obtained from cells expressing the target nucleic acid (e.g., helper T-2 (Th2) cells and mast cells). This analysis can be performed by first reverse transcribing the target RNA using, for example, viral reverse transcriptase, and then amplifying the resulting cDNA; or by using a combined high-temperature reverse transcription-polymerase chain reaction (RT-PCR), as described in U.S. Patent Nos. 5,310,652; 5,322,770; 5,561,058; 5,641,864; and 5,693,517.

Samples can be taken from patients suspected of having or diagnosed with IL-33-mediated disease and therefore potentially requiring treatment, or from healthy individuals not suspected of having any disease. To determine genotype, patient samples, such as those containing cells or nucleic acids produced by those cells, can be used in the methods of this invention. Bodily fluids or secretions that can be used as samples in this invention include, for example, hematuria, saliva, feces, pleural fluid, lymph, sputum, ascites, prostatic fluid, cerebrospinal fluid (CSF), or any other bodily secretion or derivatives thereof. The term "blood" is intended to include whole blood, plasma, serum, or any blood derivative. Sample nucleic acids used in the methods described herein can be obtained from any cell type or tissue of the subject. For example, bodily fluids (e.g., blood) of the subject can be obtained using known techniques. Alternatively, dried samples (e.g., hair or skin) can be tested for nucleic acids.

Samples can be frozen, fresh, fixed (e.g., formalin fixed), centrifuged, and/or embedded (e.g., paraffin embedded). Of course, cell samples can undergo a variety of well-known post-collection preparation and storage techniques (e.g., nucleic acid and/or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) before genotyping. Similarly, biopsy samples can also undergo post-collection preparation and storage techniques, such as fixation.

The following is a brief description of the frequently used methodologies for analyzing nucleic acid samples to detect SNPs that can be used in this invention. However, any method known in the art can be used in this invention to detect the presence of single nucleotide substitutions.

a. Allel-specific hybridization

This technique, also commonly known as allele-specific oligonucleotide hybridization (ASO) (e.g., Stoneking et al., Am. J. Hum. Genet. 48:70-382, 1991; Saiki et al., Nature 324, 163-166, 1986; EP 235, 726; and WO 1989/11548), relies on hybridizing an oligonucleotide probe specific to one of the variants with the amplification product obtained from an amplified nucleic acid sample to distinguish two DNA molecules that differ by only one base. This method typically uses short oligonucleotides, for example, 15-20 bases in length. The probe is designed to hybridize differentially with one variant relative to the other. The principles and guidelines for designing such probes are available in the art, for example, in the references cited herein. Hybridization conditions should be sufficiently stringent to ensure a significant difference in hybridization strength between alleles and produce a substantially binary response, so that the probe hybridizes only with one of the alleles. Some probes are designed to hybridize with segments of target DNA in such a way that the polymorphic site aligns with the central position of the probe (e.g., at position 7 in a 15-base oligonucleotide; at position 8 or 9 in a 16-base oligonucleotide), but this design is not required.

The amount and/or presence of alleles can be determined by measuring the amount of allele-specific oligonucleotides that hybridize with a sample. Generally, the oligonucleotides are labeled with markers such as fluorescent markers. For example, allele-specific oligonucleotides are applied to immobilized oligonucleotides representing SNP sequences. After rigorous hybridization and washing conditions, the fluorescence intensity of each SNP oligonucleotide is measured.

In one embodiment, the nucleotide present at the polymorphic site is identified by hybridization under sequence-specific hybridization conditions with an oligonucleotide probe or primer that is exactly complementary to one of the polymorphic alleles in a region covering the polymorphic site. The probe or primer hybridization sequence and sequence-specific hybridization conditions are selected such that a single mismatch at the polymorphic site sufficiently destabilizes the hybrid duplex, thereby effectively preventing its formation. Therefore, under sequence-specific hybridization conditions, a stable duplex is formed only between the probe or primer and the exactly complementary allele. Thus, oligonucleotides of about 10 to about 35 nucleotides in length, typically about 15 to about 35 nucleotides in length, that are exactly complementary to the allele sequence in the region covering the polymorphic site are within the scope of this invention.

In one alternative embodiment, the presence of nucleotides at polymorphic sites is identified by hybridization under sufficiently stringent hybridization conditions with oligonucleotides substantially complementary to one of the SNP alleles in a region covering the polymorphic site and exactly complementary to the allele at the polymorphic site. Because mismatches occurring at non-polymorphic sites are mismatches with both allele sequences, the difference in the number of mismatches in duplexes formed with the target allele sequence and duplexes formed with the corresponding non-target allele sequence is the same when using oligonucleotides exactly complementary to the target allele sequence. In this embodiment, the hybridization conditions are sufficiently relaxed to allow the formation of stable duplexes with the target sequence while maintaining sufficient stringency to exclude the formation of stable duplexes with non-target sequences. Under such sufficiently stringent hybridization conditions, stable duplexes are formed only between the probe or primer and the target allele. Therefore, oligonucleotides of about 10 to about 35 nucleotides in length, typically about 15 to about 35 nucleotides in length, substantially complementary to the allele sequence in a region covering the polymorphic site and exactly complementary to the allele sequence at the polymorphic site, are within the scope of this invention.

Using oligonucleotides that are substantially complementary rather than exactly complementary may be desirable in assay modalities where hybridization conditions are limited. For example, in common multi-target immobilized oligonucleotide assay modalities, the probe or primer for each target is immobilized on a single solid support. Hybridization is performed simultaneously by contacting the solid support with a solution containing the target DNA. Because all hybridizations are performed under identical conditions, the hybridization conditions cannot be optimized separately for each probe or primer. When the assay modality precludes adjusting hybridization conditions, incorporating mismatches into the probe or primer can be used to modulate duplex stability. The effects of introducing specific mismatches on duplex stability are well known, and as described above, duplex stability can be routinely estimated and empirically determined using the guidance provided herein and known in the art, empirically selecting appropriate hybridization conditions that depend on the precise size and sequence of the probe or primer. For example, Conner et al., Proc. Natl. Acad. Sci. USA 80:278-282, 1983 and U.S. Patent Nos. 5,468,613 and 5,604,099 describe the use of oligonucleotide probes or primers to detect single base pair differences in sequences.

The ratio of stability between perfectly matched hybrid duplexes and single-base mismatched hybrid duplexes depends on the length of the hybridized oligonucleotide. Duplexes formed with shorter probe sequences are destabilized more proportionally due to the presence of mismatches. Oligonucleotides between approximately 15 and 35 nucleotides in length are frequently used for sequence-specific detection. Furthermore, because the ends of hybridized oligonucleotides undergo continuous random dissociation and refolding due to thermal energy, mismatches at either end cause less destabilization of the hybrid duplex than mismatches occurring internally. To distinguish single-base pair variations in the target sequence, probe sequences are selected such that the polymorphic sites appear in the internal regions of the probe.

The above criteria for selecting probe sequences that hybridize with specific alleles apply to the probe hybridization region, i.e., the portion of the probe involved in hybridization with the target sequence. The probe may bind to additional nucleic acid sequences, such as poly-T tails used to immobilize the probe, without significantly altering the probe's hybridization characteristics. Those skilled in the art will recognize that, for use in the methods of this invention, a probe bound to an additional nucleic acid sequence that is not complementary to the target sequence and therefore does not involve hybridization is substantially equivalent to an unbound probe.

Suitable assay modes for detecting hybrid molecules formed between probe and target nucleic acid sequences in a sample are known in the art and include immobilized target (dot blot) mode and immobilized probe (reverse dot blot or line blot) assay modes. Dot blot and reverse dot blot assay modes are described in U.S. Patent Nos. 5,310,893; 5,451,512; 5,468,613; and 5,604,099.

In dot blot mode, the amplified target DNA is immobilized on a solid support (such as a nylon membrane). The membrane-target complex is incubated with labeled probes under appropriate hybridization conditions. Unhybridized probes are removed by washing under appropriately stringent conditions, and the presence of bound probes is monitored on the membrane.

In reverse dot blot (or line blot) mode, the probe is immobilized on a solid support (such as a nylon membrane or microtiter plate). The target DNA is labeled, typically during amplification by incorporating labeled primers. One or two primers may be used. The membrane-probe complex is incubated with the labeled amplified target DNA under suitable hybridization conditions. Unhybridized target DNA is removed by washing under appropriately stringent conditions, and the presence of bound target DNA is monitored on the membrane. Reverse line blot detection analysis is described in the examples.

Allele-specific probes specific to one polymorphic variant are often used in combination with allele-specific probes targeting another polymorphic variant. In some embodiments, the probes are immobilized on a solid support and both probes are used simultaneously to analyze the target sequence in an individual. An example of a nucleic acid array is described in WO 95/11995. The same array or different arrays can be used to analyze characterized polymorphisms. WO 95/11995 also describes subarrays optimized for detecting variant forms of pre-characterized polymorphisms. Such subarrays can be used to detect the presence of the polymorphisms described herein.

b. Allele-specific primers

Allele-specific amplification or primer extension methods are also commonly used to detect polymorphisms. These reactions generally involve using primers designed to specifically target polymorphisms via mismatches at the 3' ends of the primers. The presence of mismatches affects the polymerase's ability to extend primers when the polymerase lacks error-correcting activity. For example, to detect allele sequences using allele-specific amplification or extension methods, primers complementary to an allele polymorphism are designed so that the 3' ends of the nucleotides hybridize at the polymorphic position. The presence of a specific allele can be determined by the primer's ability to initiate the extension process. If there is a 3' end mismatch, extension is inhibited.

In some embodiments, the primer is used in conjunction with a second primer in the amplification reaction. The second primer hybridizes at a site unrelated to the polymorphic location. Amplification proceeds from both primers, resulting in a detectable product indicating the presence of a specific allelic form. Methods based on allelic-specific amplification or elongation are described, for example, in WO 93/22456; U.S. Patent Nos. 5,137,806; 5,595,890; 5,639,611; and U.S. Patent No. 4,851,331.

Genotyping based on allele-specific amplification only requires detecting the presence or absence of the amplified target sequence. Methods for detecting the amplified target sequence are well-known in the art. For example, the described gel electrophoresis and probe hybridization analyses are frequently used to detect the presence of nucleic acids.

In alternative methods with fewer probes, the amplified nucleic acid is detected by monitoring an increase in the total amount of double-stranded DNA in the reaction mixture, as described, for example, in U.S. Patent No. 5,994,056; and European Patent Publications Nos. 487,218 and 512,334. Detection of the double-stranded target DNA relies on an increase in fluorescence observed when various DNA-binding dyes (e.g., SYBR Green) bind to the double-stranded DNA.

As those skilled in the art will appreciate, allele-specific amplification methods can be performed in reactions that utilize multiple allele-specific primers to target specific alleles. Primers used for such multiple applications are typically labeled with distinguishable markers or selected such that the amplification products generated from the alleles are distinguishable by size. Thus, for example, single amplification can be used to identify two alleles in a single sample by gel analysis of the amplification products.

In the case of allele-specific probes, allele-specific oligonucleotide primers may be exactly complementary to a polymorphic allele in the hybridization region or may have some mismatch at a position outside the 3' end of the oligonucleotide, where the mismatch occurs at non-polymorphic sites in both allele sequences.

c. Detectable probes

i) 5’-Nuclease analysis probe

Genotyping can also be performed using methods such as the “5’-nuclease assay” described in U.S. Patent Nos. 5,210,015; 5,487,972; and 5,804,375; and Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1988. In this assay, a labeled detection probe that hybridizes within the amplification region is added during the amplification reaction. These probes are modified to prevent them from functioning as primers for DNA synthesis. Amplification is performed using a DNA polymerase with 5’ to 3’ exonuclease activity. During each synthetic step of amplification, any probe that hybridizes with the target nucleic acid downstream of the primer being elongated is degraded by the 5’ to 3’ exonuclease activity of the DNA polymerase. Thus, the synthesis of a new target strand also leads to probe degradation, and the accumulation of degradation products provides a measurement of target sequence synthesis.

Hybridization probes can be allele-specific probes that distinguish SNP alleles. Alternatively, this method can be performed using allele-specific primers and labeled probes that bind to the amplification products.

Any method suitable for detecting degradation products can be used in 5'-nuclease assays. Often, the detection probe is labeled with two fluorescent dyes, one of which quenches the fluorescence of the other. These dyes are attached to the probe, typically one dye to the 5' end and the other to an internal site, so that quenching occurs when the probe is in an unhybridized state, and the probe is cleaved between the two dyes by the 5' to 3'-exonuclease activity of a DNA polymerase. Amplification results in the cleavage of the probe between the dyes, accompanied by the simultaneous elimination of quenching and an observable increase in fluorescence from the initially quenched dye. The accumulation of degradation products is measured by monitoring the increase in reaction fluorescence. U.S. Patent Nos. 5,491,063 and 5,571,673 describe alternative methods for detecting probe degradation accompanying amplification.

ii) Secondary structure probe

Probes that detect changes in secondary structure are also suitable for detecting polymorphisms, including SNPs. Examples of secondary structure or stem-loop structure probes include molecular beacons or primers/probes. Molecular beacon probes are single-stranded oligonucleotide probes that can form hairpin structures, in which the fluorophore and quencher are typically located at opposite ends of the oligonucleotide. Either end of the short complementary sequence of the probe allows the formation of an intramolecular stem, which makes it possible for the fluorophore and quencher to approach each other. The loop portion of the molecular beacon is complementary to the target nucleic acid. The binding of this probe to its target nucleic acid forms a hybrid molecule that forces the stem to separate. This results in a conformational change that moves the fluorophore and quencher away from each other, leading to a stronger fluorescence signal. However, molecular beacon probes are highly sensitive to small sequence variations in the probe target (see, for example, Tyagi et al., Nature Biotech. 14:303-308, 1996; Tyagi et al., Nature Biotech. 16:49-53, 1998; Piatek et al., Nature Biotech. 16:359-363, 1998; Marras et al., Genetic Analysis: Biomolecular Engineering 14:151-156, 1999; Tapp et al., BioTechniques 28:732-738, 2000). Primers/probes contain stem-loop probes covalently linked to the primers.

d. DNA sequencing and single base elongation

SNPs can also be detected by direct sequencing. Methods include dideoxy sequencing-based methods and others such as Maxam and Gilbert sequencing (see, for example, Sambrook and Russell, above).

Other detection methods include PYROSEQUENCING ^™ for oligonucleotide length products. These methods often utilize amplification techniques such as PCR. For example, in pyrosequencing, sequencing primers hybridize to a PCR-amplified single-stranded DNA template and are incubated with DNA polymerase, ATP phosphorylsulfonase, luciferase, and adenosine triphosphate diphosphate enzyme, along with the substrate adenosine 5'-phosphate sulfate (APS) and luciferin. The first of four deoxynucleotide triphosphates (dNTPs) is added to the reaction. If complementary to a base in the template strand, the DNA polymerase catalyzes the incorporation of the deoxynucleotide triphosphate into the DNA strand. Each incorporation event is accompanied by the release of pyrophosphate (PPi) in an amount equimolar to the amount of nucleotide incorporated. In the presence of APS, ATP sulfate enzyme quantitatively converts PPi into ATP. This ATP drives the luciferase-mediated conversion of luciferin to oxoluciferin, which produces visible light in an amount proportional to the amount of ATP. The light generated during the luciferase reaction is detected by a charge-coupled device (CCD) camera and displayed as a peak in PYROGRAM ^™ . Each light signal is proportional to the number of nucleotides incorporated. Adenosine triphosphate diphosphate enzyme (a nucleotide-degrading enzyme) continuously degrades unincorporated dNTPs and excess ATP. When degradation is complete, another dNTP is added.

Another similar method for characterizing SNPs does not require full PCR, but generally only utilizes primers extended by a single dideoxyribonucleic acid molecule (ddNTP) complementary to the nucleotide being studied and labeled with fluorescent markers. The nucleotide at the polymorphic site can be identified by detecting primers that have been extended by one base and are fluorescently labeled (e.g., Kobayashi et al., Mol. Cell. Probes, 9:175-182, 1995).

e. Electrophoresis

The amplification products generated by polymerase chain reaction can be analyzed using denaturing gradient gel electrophoresis. Different alleles can be identified based on different sequence-dependent melting properties and electrophoretic migration in DNA solution (see, for example, Erlich, ed., PCR Technology, Principles and Applications for DNA Amplification, W.H. Freeman and Co., 1992).

Capillary electrophoresis can be used to distinguish microsatellite polymorphisms. Capillary electrophoresis conveniently allows for the identification of the number of repetitive sequences in specific microsatellite alleles. The application of capillary electrophoresis to analyze DNA polymorphisms is well known to those skilled in the art (see, for example, Szantai et al., J Chromatogr A. 1079(1-2):41-9, 2005; Bjorheim et al., Electrophoresis 26(13):2520-30, 2005; and Mitchelson, Mol. Biotechnol. 24(1):41-68, 2003).

Allelic identity can also be obtained by analyzing the movement of nucleic acids containing polymorphic regions in a polyacrylamide gel containing a denaturing agent gradient, the nucleic acids being analyzed using denaturing gradient gel electrophoresis (DGGE) (see, for example, Myers et al., Nature 313:495-498, 1985). When using DGGE as the analytical method, the DNA is modified to ensure it is not completely denatured, for example, by GC clamp modification with approximately 40 bp of GC-rich, high-denaturation-temperature DNA added via PCR. In yet another embodiment, a temperature gradient is used instead of a denaturing agent gradient to identify differences in migration rates between control and sample DNA (see, for example, Rosenbaum et al., Biophys. Chem. 265:1275, 1987).

f. Single-strand conformational polymorphism analysis

Single-strand conformational polymorphism (SCM) analysis can be used to distinguish allele target sequences. This method identifies base differences based on changes in the electrophoretic migration of single-stranded PCR products, as described, for example, in Orit et al., Proc. Nat. Acad. Sci. 86, 2766-2770, 1989; Cotton Mutat. Res. 285:125-144, 1993; and Hayashi Genet. Anal. Tech. Appl. 9:73-79, 1992. The amplified PCR product can be generated and heated or denatured as described above to form a single-stranded amplification product. The single-stranded nucleic acid can refold or form secondary structures that are partially dependent on the base sequence. Different electrophoretic mobilities of the single-stranded amplification product may be correlated with base sequence differences between target alleles, and the resulting changes in electrophoretic mobility can enable the detection of even single-base changes. DNA fragments can be labeled or detected using labeled probes. The sensitivity of the assay can be enhanced by using RNA (instead of DNA), where secondary structure is more sensitive to changes in the sequence. In another preferred embodiment, the subject method utilizes heteroduplex analysis based on changes in electrophoretic mobility to separate double-stranded heteroduplex molecules (see, for example, Keen et al., Trends Genet. 7:5-10, 1991).

SNP detection methods frequently utilize labeled oligonucleotides. Oligonucleotides can be labeled by incorporating markers detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. Useful markers include fluorescent dyes, radiolabeled substances such as ^32P , electron-dense reagents, enzymes such as peroxidase or alkaline phosphatase, biotin, or haptens and proteins from which antiserum or monoclonal antibodies are obtained. Labeling techniques are well-known in the art (see, for example, Current Protocols in Molecular Biology, above; Sambrook et al., above).

g. Additional methods for determining an individual's genotype at polymorphisms

DNA microarray technology can be used, such as DNA chip devices, high-density microarrays for high-throughput screening applications, and low-density microarrays. Methods for fabricating microarrays are known in the art and include a variety of inkjet and microjet deposition or spotting techniques and methods, in-situ or on-chip photolithographic oligonucleotide synthesis methods, and DNA probe electronic addressing methods. DNA microarray hybridization applications have been successfully applied to gene expression analysis and genotyping of point mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs). Additional methods include interfering RNA microarrays and combinations of microarrays with other methods such as laser capture microdissection (LCM), comparative genomic hybridization (CGH), array CGH, and chromatin immunoprecipitation (ChIP). See, for example, He et al., Adv. Exp. Med. Biol. 593:117-133, 2007 and Heller Annu. Rev. Biomed. Eng. 4:129-153, 2002.

In some implementations, protection against cleavage agents (such as nucleases, hydroxylamine, or osmium tetroxide and piperidine) can be used to detect mismatched bases in RNA/RNA, DNA/DNA, or RNA/DNA heteroduplexes (see, for example, Myers et al., Science 230:1242, 1985). Typically, the “mismatch cleavage” technique begins by providing a heteroduplex formed by hybridizing a control nucleic acid (optionally labeled) (e.g., RNA or DNA) containing the nucleotide sequence of an allelic gene with a sample nucleic acid (e.g., RNA or DNA) obtained from a tissue sample. The double-stranded duplex is then treated with a reagent that cleaves the single-stranded regions of the duplex (e.g., a duplex formed based on base pair mismatches between the control and sample strands). For example, RNA/DNA duplexes can be treated with RNases and DNA/DNA hybrid molecules can be treated with S1 nucleases to enzymatically digest the mismatched regions. Alternatively, DNA/DNA duplexes or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and then with piperidine to digest the mismatched regions. After digesting the mismatched regions, the resulting material is then separated by size on a denaturing polyacrylamide gel to determine whether the control nucleic acid and the sample nucleic acid have the same nucleotide sequence or on which nucleotides they differ. See, for example, U.S. Patent No. 6,455,249; Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401, 1988; Saleeba et al., Meth. Enzymol. 217:286-295, 1992.

In some cases, the presence of a specific allele in a subject's DNA can be revealed by restriction enzyme analysis. For example, a particular nucleotide polymorphism can produce a nucleotide sequence containing a restriction site that is not present in the nucleotide sequence of another allele.

In another embodiment, allelic identification is performed using oligonucleotide ligation assays (OLA), for example, as described in U.S. Patent No. 4,998,617 and Laridegren et al., Science 241:1077-1080, 1988. The OLA protocol uses two oligonucleotides designed to hybridize with adjacent sequences on a single strand of the target. One oligonucleotide is ligated to a separating marker, for example, by biotinylation, and the other is labeled in a detectable manner. If precisely complementary sequences are present in the target molecule, the oligonucleotides hybridize so that their ends are adjacent and a ligation substrate is generated. Using an avidin or another biotin ligand, the ligation then allows for the recovery of the labeled oligonucleotide. Nucleic acid detection assays combining the properties of PCR and OLA are also known in the art (see, for example, Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923-8927, 1990). In this method, PCR is used to achieve exponential amplification of the target DNA, which is then detected using OLA.

Single base polymorphisms (SNPs) can be detected using specialized nucleotides resistant to exonucleases, for example, as described in U.S. Patent No. 4,656,127. According to this method, a primer complementary to the allele immediately adjacent to the 3' of the polymorphic site is allowed to hybridize with a target molecule obtained from a specific animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to a specific nucleotide derivative present that is resistant to the exonuclease, the derivative is incorporated into the end of the hybridization primer. This incorporation makes the primer resistant to the exonuclease and thus allows for its detection. Since the identity of the exonuclease-resistant derivative of the sample is known, the observation that the primer has become resistant to the exonuclease reveals that the nucleotide present at the polymorphic site of the target molecule is complementary to the nucleotide derivative used in the reaction. This method has the advantage that it does not require the determination of large amounts of external sequence data.

A solution-based method can also be used to determine the nucleotide identity of a polymorphic site (see, for example, WO1991/02087). As described above, a primer complementary to the allelic sequence immediately adjacent to the 3' of the polymorphic site is used. This method uses a labeled dideoxynucleotide derivative to determine the nucleotide identity of the site, wherein the dideoxynucleotide derivative is incorporated into the end of the primer if it is complementary to the nucleotide of the polymorphic site.

Alternative methods that can be used are described in WO 92/15712. This method uses a mixture of a labeled terminator and a primer complementary to the sequence immediately adjacent to the 3' of the polymorphic site. The incorporated labeled terminator is thus identified by and complementary to the nucleotide present in the polymorphic site of the target molecule being evaluated. This method is typically a heterogeneous assay, in which the primer or target molecule is immobilized on a solid phase.

Many other primer-guided nucleotide incorporation methods for analyzing polymorphic sites in DNA have been described (Komher et al., Nucl. Acids. Res. 17:7779-7784, 1989; Sokolov, Nucl. Acids. Res. 18:3671, 1990; Syvanen et al., Genomics 8:684-692, 1990; Kuppuswamy et al., Proc. Natl. Acad. Sci. USA 88:1143-1147, 1991; Prezant et al., Hum. Mutat. 1:159-164, 1992; Ugozzoli et al., GATA 9:107-112, 1992; Nyren et al., Anal. Biochem. 208:171-175, 1993). All of these methods rely on incorporating labeled deoxynucleotides to distinguish bases at polymorphic sites.

V. Biomarkers

The treatment and diagnostic methods of the present invention may involve determining the levels of one or more biomarkers (e.g., periosteal protein and/or sST2). Determining the levels of biomarkers can be carried out by any method known in the art or described below.

A. Detecting gene expression

The genetic biomarkers described herein (e.g., periosteal proteins) can be detected using any method known in the art. For example, mRNA or DNA from mammalian tissue or cell samples can be conveniently analyzed using RNA blotting, dot blotting, PCR analysis, array hybridization, RNase protection assays, or commercially available DNA SNP microarrays (including DNA microarray snapshots). Real-time PCR (RT-PCR) assays, such as quantitative PCR assays, are well known in the art. In one illustrative embodiment of the invention, a method for detecting mRNA from a target genetic biomarker (e.g., periosteal proteins and/or sST2) in a biological sample includes generating cDNA from the sample by reverse transcription using at least one primer; amplifying the thus generated cDNA; and detecting the presence of the amplified cDNA. Furthermore, such methods may include one or more steps that allow determination of the mRNA level in the biological sample (e.g., by simultaneously examining the level of a control mRNA sequence of a "housekeeping gene" such as a member of the actin family). Optionally, the sequence of the amplified cDNA may be determined.

i. Nucleic acid testing

In one specific implementation, the expression of biomarkers (e.g., periosteal protein and/or sST2) can be detected using RT-PCR technology. Probes used for PCR can be labeled with detectable markers (e.g., radioisotopes, fluorescent compounds, bioluminescent compounds, chemiluminescent compounds, metal chelators, or enzymes). Such probes and primers can be used to detect the presence of expressed biomarkers (e.g., periosteal protein) in a sample. As those skilled in the art will understand, a variety of different primers and probes can be prepared based on the sequences provided herein and efficiently used to amplify, clone, and/or determine the presence and/or level of biomarkers (e.g., periosteal protein).

Other methods include protocols for examining or detecting mRNAs derived from biomarkers (e.g., periosteal protein mRNA and/or sST2 mRNA) in tissue or cell samples using microarray technology. Using nucleic acid microarrays, test mRNA samples and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to a nucleic acid array immobilized on a solid support. The array is configured such that the sequence and location of each array member are known. For example, selected genes with the potential for expression in certain disease states can be analyzed on a solid support. Hybridization of a labeled probe with a specific array member indicates that the sample from which the probe was derived expresses that gene. Differential gene expression analysis in diseased tissues can provide valuable information. Microarray technology utilizes nucleic acid hybridization and computational techniques to evaluate the mRNA expression profiles of thousands of genes in a single experiment (see, for example, WO 2001/75166). For a discussion of array fabrication, see, for example, U.S. Patent Nos. 5,700,637, 5,445,934 and 5,807,522, Lockart, Nat. Biotech. 14:1675-1680, 1996; and Cheung et al., Nat. Genet. 21(Suppl):15-19, 1999.

Furthermore, the DNA profiling and detection method utilizing microarrays described in European Patent EP 1753878 can be used. This method rapidly identifies and distinguishes different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. In one embodiment, a labeled STR target sequence hybridizes with a DNA microarray carrying complementary probes. These probes vary in length to cover a range of possible STRs. Using post-hybridization enzymatic digestion, labeled single-stranded regions of the hybridized DNA molecules are selectively removed from the surface of the microarray. Based on the pattern of the target DNA still hybridized with the microarray, the number of repeat sequences in unknown targets is inferred.

An example of a microarray processor is the Affymetrix system, which is commercially available and comprises an array manufactured by directly synthesizing oligonucleotides on a glass surface. Other systems can be used, as is known to those skilled in the art.

The specialized microarrays described herein, such as oligonucleotide microarrays or cDNA microarrays, may contain one or more biomarkers having expression profiles associated with sensitivity or resistance to one or more IL-33 axis-binding antagonists (e.g., ST2-binding antagonists, such as ST2-Fc protein). Other methods used in this invention for detecting nucleic acids involve high-throughput RNA sequence expression analysis, including RNA-based genomic analyses such as RNASeq.

Numerous references are available to provide guidance on the use of the above techniques (Kohler et al., Hybridoma Techniques, Cold Spring Harbor Laboratory, 1980; Tijssen, Practice and Theory of Enzyme Inimonoassays, Elsevier, 1985; Campbell, Monoclonal Antibody Technology, Elsevier, 1984; Hurrell, Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, 1982; and Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc., 1987). RNA blot analysis is a well-known and routine technique in the field and is described, for example, in Sambrook et al. above. Common protocols for evaluating the state of genes and gene products exist, for example, in Ausubel et al. above.

ii. Protein detection

For the detection of protein biomarkers such as periosteal protein and/or sST2, various protein assays are available, including antibody-based methods as well as mass spectrometry and other similar techniques known in the art. In the case of antibody-based methods, for example, the sample can be contacted with an antibody specific to the biomarker (e.g., periosteal protein or sST2 protein) under conditions sufficient to form an antibody-biomarker complex, and the complex can then be detected. The presence of protein biomarkers can be detected in many ways, such as by analyzing various types of tissues and samples (including plasma or serum) using Western blotting (with or without immunoprecipitation), two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoprecipitation, fluorescence-activated cell sorting (FACS ^™ ), flow cytometry, and enzyme-linked immunosorbent assay (ELISA). A wide range of immunoassay techniques using this assay modality are available, see, for example, U.S. Patent Nos. 4,016,043; 4,424,279; and 4,018,653. These methods include non-competitive types as well as single-site and two-site or "sandwich" assays in routine competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

Sandwich assays are among the most useful and commonly used assays. Various variations of sandwich assay techniques exist, and all are intended to be covered by this invention. In short, in a common forward assay, an unlabeled antibody is immobilized on a solid substrate, and the sample to be tested is brought into contact with the bound molecule. After incubation for an appropriate time (sufficient to allow for antibody-antigen complex formation), an antigen-specific secondary antibody labeled with a reporter molecule capable of generating a detectable signal is subsequently added, and incubation is continued for a time sufficient to allow for the formation of another complex: antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observing the signal generated by the reporter molecule. The result can be qualitative by simply observing a visible signal, or quantitative by comparison with a control sample containing a known amount of the biomarker.

Variations of forward assays include simultaneous assays, in which the sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art and are readily apparent, including any subtle variations. In common forward sandwich assays, a first antibody specific to the biomarker is covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, most commonly cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene. The solid support can be in the form of a tube, bead, disc of microplates, or any other surface suitable for performing the immunoassay. The binding process is well known in the art and typically consists of cross-linking, covalent binding, or physisorption, with the polymer-antibody complex washed during preparation for use with the test sample. The test sample, in aliquots, is then added to the solid-phase complex and incubated for a time sufficient to allow binding of any subunits present in the antibody (e.g., 2–40 minutes or, if more convenient, overnight) under suitable conditions allowing binding of any subunits present in the antibody (e.g., from room temperature to 40°C, such as between 25°C and 32°C, inclusive). After the incubation period, the antibody subunits are washed, dried, and incubated with a second antibody specific to a portion of the biomarker. The second antibody is linked to a reporter molecule used to demonstrate the binding of the second antibody to the molecular marker.

Alternative methods involve immobilizing a target biomarker in a sample and subsequently exposing the immobilized target to a specific antibody, which may or may not be labeled with a reporter molecule. Depending on the amount of target and the intensity of the reporter molecule signal, the bound target may be detectable by direct labeling with the antibody. Alternatively, a second labeled antibody specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody ternary complex. This complex is detected by the signal emitted by the reporter molecule. As used in this specification, a reporter molecule means a molecule that, by virtue of its chemical properties, provides an analytically recognizable signal that allows the detection of an antigen-binding antibody. The most commonly used reporter molecules in this type of assay are enzymes, fluorophores or molecules containing radionuclides (i.e., radioisotopes), and chemiluminescent molecules.

In the case of enzyme immunoassay (EIA), the enzyme is conjugated to a second antibody, typically using glutaraldehyde or periodate. However, it will be readily apparent that various types of different conjugation techniques exist, which are readily available to those skilled in the art. Examples of commonly used enzymes suitable for the methods of this invention include horseradish peroxidase, glucose oxidase, β-galactosidase, and alkaline phosphatase. A substrate is typically chosen to be used with the specific enzyme to produce a detectable color change upon hydrolysis by the corresponding enzyme. Fluorescent substrates, which produce fluorescent products instead of the chromogenic substrates described above, may also be used. In all cases, an enzyme-labeled antibody is added to a first antibody-molecular marker complex, allowing binding and subsequently washing away excess reagent. A solution containing a suitable substrate is then added to the antibody-antigen-antibody complex. The substrate reacts with the enzyme linked to the second antibody, producing a qualitative visual signal, which can be further quantified, typically spectrophotometrically, to indicate the amount of biomarkers (e.g., periosteal proteins and/or sST2) present in the sample. Alternatively, fluorescent compounds, such as fluorescein and rhodamine, can be chemically conjugated to antibodies without altering their binding affinity. When activated by light of a specific wavelength, the fluorescently labeled antibody absorbs the light energy, is induced to an excited state within the molecule, and subsequently emits light in a characteristic color detectable by optical microscopy. In EIA, for example, the fluorescently labeled antibody is allowed to bind to a first antibody-molecule label complex. After washing away unbound reagents, the remaining ternary complex is then exposed to light of an appropriate wavelength, and the observed fluorescence indicates the presence of the target molecular label. Immunofluorescence and EIA techniques are well-established in the art. However, other reporter molecules, such as radioisotopes, chemiluminescent molecules, or bioluminescent molecules, can also be used.

In some embodiments of the present invention, the total periosteal protein assay, as described in WO 2012/083132, is used to determine the level of periosteal proteins in samples derived from patients.

For example, the following describes a highly sensitive (sensitivity of approximately 1.88 ng/ml) periosteal protein capture ELISA assay called the E4 assay. Antibodies recognize periosteal protein isoforms 1-4 (SEQ ID NO: 5-8 of WO 2012/083132) with nanomolar affinity.

The procedure is as follows: Dilute 80 μL of purified monoclonal antibody 25D4 (coated antibody, SEQ ID NO: 1 and 2 of WO 2012/083132, expressed from hybridoma or CHO cell lines) with phosphate-buffered saline (PBS) to a final concentration of 2 μg/mL. Coat microtiter plates overnight at 2–8 °C, covering with 100 μL of coated antibody per well. Wash the plates three times with 400 μL of washing buffer (PBS/0.05% Tween (polysorbate 20)) per well at room temperature. Add 200 μL of blocking buffer to each well of the plate. Incubate the covered plates at room temperature with shaking for 1.5 hours.

A standard curve for recombinant human periosteal protein (rhuPeriostin) was prepared in analytical diluent (PBS/0.5% bovine serum albumin (BSA)/0.05% polysorbate 20/0.05% ProClin 300, pH 7.4). The standard curve diluent was PBS/0.5% BSA/0.05% polysorbate 20, 0.05% ProClin 300, pH 7.4.

Prepare controls and samples. Three types of controls: spiked source control (full-length rhuPeriostin, isoform 1, R&D Systems #3548-F2), normal matrix control (normal human serum aggregate, Bioreclamation, Inc.), and high matrix control (normal human serum aggregate, with 100 ng/ml rhuPeriostin spike).

For example:

10 μL of control (or sample) serum + 1.99 mL of sample/control diluent = 1:200

300 μL 1:200 diluent + 300 μL sample/control diluent = 1:400

300 μL 1:400 diluent + 300 μL sample/control diluent = 1:800

300 μL 1:800 diluent + 300 μL sample/control diluent = 1:1600

Each dilution was tested in single-sample tests.

A matrix control was established using a pooled collection of normal human serum. Unspecified pooled human serum served as the normal control. A high-concentration control was generated by spiked 100 ng/mL rhuPeriostin into the pooled serum as described above. For each control on each plate, the mean, standard deviation (SD), and % coefficient of variation (CV, expressed as a percentage) were calculated for four dilutions. CV quantifies the magnitude of variability relative to the mean of replicates in repeated measurements (e.g., %CV = 100 * (SD/mean)). These mean concentrations were evaluated across all plates to determine inter-plate accuracy. This control table was then used to define pass/fail criteria for the normal and high-concentration controls, setting an allowable variability of ±20% of the mean concentration for each control.

Wash the plate three times with 400 μL of wash buffer (PBS/0.05% polysorbate 20) for each wash buffer cycle in each well. Add 100 μL of diluted standard (replicas), control (all four dilutions), and sample (all four dilutions) to each well. Incubate the covered plates at room temperature with shaking for 2 hours. Dilute 80 μL of the detection MAb stock solution I (biotinylated mouse anti-human periosteal protein, MAb23B9, 7.5 μg/mL in analytical dilution) to 12 mL with analytical dilution buffer = 50 ng/mL. Wash the plate four times with 400 μL of wash buffer for each wash buffer cycle in each well. Add 100 μL of diluted detection MAb to each well. Incubate the covered plates at room temperature with shaking for one hour. Dilute 80 μL of streptavidin-HRP stock solution I (AMDEX streptavidin-HRP, GE Healthcare #RPN4401, approximately 1 mg/ml) diluted 1:12k in analytical dilution buffer to 12 mL. Wash the plate four times with 400 μL of wash buffer per well per wash buffer cycle. Add 100 μL of streptavidin-HRP to each well. Incubate the covered plates at room temperature with shaking for 45 minutes. Allow the Kirkegaard and Perry (KPL) two-step TMB reagent to reach room temperature; do not mix. Wash the plate four times with 400 μL of wash buffer per well per wash buffer cycle. Mix an equal volume of the KPL TMB substrate component and add 100 μL to each well. Incubate the plates at room temperature with shaking for 20 minutes. Add 1M phosphate to each well. The plate was read using a 450nm read wavelength and a 650nm reference wavelength.

In other embodiments, the periosteal protein assay described in WO 2012/083132 is used to determine the level of periosteal proteins in samples derived from patients, as described below.

The quantitative detection of periosteal proteins was evaluated in an automated Roche cobas e601 analyzer (Roche Diagnostics GmbH). The assay was performed in a sandwich configuration, where the analyte periosteal protein was sandwiched between two monoclonal antibodies that bind to two different epitopes on the protein. One antibody was biotinylated and capable of capturing immune complexes to streptavidin-coated magnetic beads. The second antibody carried a complexed ruthenium cation as a signal transduction moiety, which allowed for voltage-dependent electrochemiluminescence detection of the bound immune complexes.

In detail, the reagents used are as follows:

- Beads (M): 0.72 mg/mL of streptavidin-coated magnetic microparticles; preservative.

-Reagent 1 (R1): Anti-periostein antibody ~ Biotin:

This purified mouse monoclonal antibody corresponds to the coated antibody 25D4 described above relative to the E4 assay and is used in biotinylated form >1.0 mg/L; TRIS buffer >100 mmol/L, pH 7.0; preservatives.

-Reagent 2 (R2): Anti-periosteal protein-antibody ~Ru(bpy):

This purified mouse monoclonal anti-periosteal protein antibody corresponds to the detection antibody 23B9 described above relative to the E4 assay and is used in labeled form (labeled with (tris(2,2’-bipyridine)ruthenium(II)-complex (Ru(bpy)) complex) >1.0 mg/L; TRIS buffer >100 mmol/L, pH 7.0; preservatives.

This immunoassay was performed using a two-stage incubation process. During the first incubation of approximately 9 minutes, periosteal protein in a 20 μL sample and biotinylated monoclonal anti-periosteal protein antibody (R1) formed a complex. In the second incubation step, an additional 9 minutes, ruthenium-modified monoclonal anti-periosteal protein antibody (R2) and streptavidin-coated microparticles (M) were added to the vial from the first incubation, resulting in the formation of a ternary sandwich complex that became bound to the solid phase (microparticles) through the interaction of biotin and streptavidin.

The reaction mixture is drawn into the measuring chamber, where microparticles are magnetically trapped onto the surface of a platinum electrode. Unbound material is washed away, and the measuring chamber is rinsed with ProCell (a reagent containing tripropylamine). Applying a voltage to the electrode induces chemiluminescence emission, which is measured by a photomultiplier.

Results were determined using an instrument-specific calibration curve, which was generated through two-point correction and a master curve provided via reagent barcodes. Calibrator 1 contained no analyte, while calibrator 2 contained 50 ng/mL rhuPeriostin in a buffered matrix. To validate the calibration, two controls were used with approximately 30 ng/mL and 80 ng/mL periostrin.

In some implementations, for example, when using the E4 assay described above, an exemplary reference level for periosteal protein levels is 23 ng/ml. For example, when using the E4 assay, if a patient's periosteal protein level in serum or plasma is 23 ng/ml or higher, 24 ng/ml or higher, 25 ng/ml or higher, 26 ng/ml or higher, 27 ng/ml or higher, 28 ng/ml or higher, 29 ng/ml or higher, 30 ng/ml or higher, 31 ng/ml or higher, 32 ng/ml or higher, 33 ng/ml or higher, 34 ng/ml or higher, 35 ng/ml or higher, 36 ng/ml or higher, 37 ng/ml or higher, 38 ng/ml or higher, 39 ng/ml or higher, 40 ng/ml or higher, 41 ng/ml or higher, 42 ng/ml or higher, 43 ng/ml or higher, 44 ng/ml or higher, 45 ng/ml or higher, or 46 ng/ml... Patients with periosteal protein levels of 47 ng/ml or higher, 48 ng/ml or higher, 49 ng/ml or higher, 50 ng/ml or higher, 51 ng/ml or higher, 52 ng/ml or higher, 53 ng/ml or higher, 54 ng/ml or higher, 55 ng/ml or higher, 56 ng/ml or higher, 57 ng/ml or higher, 58 ng/ml or higher, 59 ng/ml or higher, 60 ng/ml or higher, 61 ng/ml or higher, 62 ng/ml or higher, 63 ng/ml or higher, 64 ng/ml or higher, 65 ng/ml or higher, 66 ng/ml or higher, 67 ng/ml or higher, 68 ng/ml or higher, 69 ng/ml or higher, or 70 ng/ml or higher may have periosteal protein levels at or above the reference level.

When using the E4 assay, a patient may have a periosteal protein level at or below the reference level if the patient's periosteal protein level is 23 ng/ml or lower, 22 ng/ml or lower, 21 ng/ml or lower, 20 ng/ml or lower, 19 ng/ml or lower, 18 ng/ml or lower, 17 ng/ml or lower, 16 ng/ml or lower, 15 ng/ml or lower, 14 ng/ml or lower, 13 ng/ml or lower, 12 ng/ml or lower, 11 ng/ml or lower, 10 ng/ml or lower, 9 ng/ml or lower, 8 ng/ml or lower, 7 ng/ml or lower, 6 ng/ml or lower, 5 ng/ml or lower, 4 ng/ml or lower, 3 ng/ml or lower, 2 ng/ml or lower, or 1 ng/ml or lower.

In other embodiments, for example, when using the periosteal protein assay described above, an exemplary reference level for periosteal protein levels is 50 ng/ml. For example, when using the periosteal protein assay, if a patient's periosteal protein level is 50 ng/ml or higher, 51 ng/ml or higher, 52 ng/ml or higher, 53 ng/ml or higher, 54 ng/ml or higher, 55 ng/ml or higher, 56 ng/ml or higher, 57 ng/ml or higher, 58 ng/ml or higher, 59 ng/ml or higher, 60 ng/ml or higher, 61 ng/ml or higher, 62 ng/ml or higher, 63 ng/ml or higher, 64 ng/ml or higher, 65 ng/ml or higher, 66 ng/ml or higher, 67 ng/ml or higher, 68 ng/ml or higher, 69 ng/ml or higher, 70 ng/ml or higher, 71 ng/ml or higher, 72 ng/ml or higher, 73 ng/ml or higher, 74 ng/ml or higher, or higher, the following values are considered: Patients may have periosteal protein levels at or above the reference level, including high levels of 75 ng/ml or higher, 76 ng/ml or higher, 77 ng/ml or higher, 78 ng/ml or higher, 79 ng/ml or higher, 80 ng/ml or higher, 81 ng/ml or higher, 82 ng/ml or higher, 83 ng/ml or higher, 84 ng/ml or higher, 85 ng/ml or higher, 86 ng/ml or higher, 87 ng/ml or higher, 88 ng/ml or higher, 89 ng/ml or higher, 90 ng/ml or higher, 91 ng/ml or higher, 92 ng/ml or higher, 93 ng/ml or higher, 94 ng/ml or higher, 95 ng/ml or higher, 96 ng/ml or higher, 97 ng/ml or higher, 98 ng/ml or higher, or 99 ng/ml or higher.

When using the periosteal protein assay, if a patient's periosteal protein level is 50 ng/ml or lower, 49 ng/ml or lower, 48 ng/ml or lower, 47 ng/ml or lower, 46 ng/ml or lower, 45 ng/ml or lower, 44 ng/ml or lower, 43 ng/ml or lower, 42 ng/ml or lower, 41 ng/ml or lower, 40 ng/ml or lower, 39 ng/ml or lower, 38 ng/ml or lower, 37 ng/ml or lower, 36 ng/ml or lower, 35 ng/ml or lower, 34 ng/ml or lower, 33 ng/ml or lower, 32 ng/ml or lower, 31 ng/ml or lower, 30 ng/ml or lower, 29 ng/ml or lower, 28 ng/ml or lower, 27 ng/ml or lower, or 26 ng/ml, the following conditions should be considered: Patients with periosteal protein levels of 1 or lower, 25 ng/ml or lower, 24 ng/ml or lower, 23 ng/ml or lower, 22 ng/ml or lower, 21 ng/ml or lower, 20 ng/ml or lower, 19 ng/ml or lower, 18 ng/ml or lower, 17 ng/ml or lower, 16 ng/ml or lower, 15 ng/ml or lower, 14 ng/ml or lower, 13 ng/ml or lower, 12 ng/ml or lower, 11 ng/ml or lower, 10 ng/ml or lower, 9 ng/ml or lower, 8 ng/ml or lower, 7 ng/ml or lower, 6 ng/ml or lower, 5 ng/ml or lower, 4 ng/ml or lower, 3 ng/ml or lower, 2 ng/ml or lower, or 1 ng/ml or lower may have periosteal protein levels at or below the reference level.

In some implementations, the level of sST2 in a patient sample can be determined using any suitable method known in the art and/or described herein (e.g., in Example 3). In some implementations, if a patient's sST2 level is 0.1 ng/ml or higher, 0.5 ng/ml or higher, 1 ng/ml or higher, 2 ng/ml or higher, 3 ng/ml or higher, 4 ng/ml or higher, 5 ng/ml or higher, 6 ng/ml or higher, 7 ng/ml or higher, 8 ng/ml or higher, 9 ng/ml or higher, 10 ng/ml or higher, 11 ng/ml or higher, 12 ng/ml or higher, 13 ng/ml or higher, 14 ng/ml or higher, 15 ng/ml or higher, 16 ng/ml or higher, 17 ng/ml or higher, 18 ng/ml or higher, 19 ng/ml or higher, 20 ng/ml or higher, 21 ng/ml or higher, 22 ng/ml or higher, 23 ng/ml or higher, 24 ng/ml or higher, or 25 ng/ml or higher... Patients with sST2 levels of 26 ng/ml or higher, 27 ng/ml or higher, 28 ng/ml or higher, 29 ng/ml or higher, 30 ng/ml or higher, 31 ng/ml or higher, 32 ng/ml or higher, 33 ng/ml or higher, 34 ng/ml or higher, 35 ng/ml or higher, 36 ng/ml or higher, 37 ng/ml or higher, 38 ng/ml or higher, 39 ng/ml or higher, 40 ng/ml or higher, 41 ng/ml or higher, 42 ng/ml or higher, 43 ng/ml or higher, 44 ng/ml or higher, 45 ng/ml or higher, 46 ng/ml or higher, 47 ng/ml or higher, 48 ng/ml or higher, 49 ng/ml or higher, 50 ng/ml or higher, or higher than 50 ng/ml, may have sST2 levels at or above the reference level.

VI. Reagent Kit

In some embodiments, the present invention provides kits for performing the methods of the present invention (e.g., determining a patient's genotype at a polymorphism as described herein). In some embodiments, the present invention provides kits for determining whether a patient is at risk of IL-33-mediated disease (e.g., asthma or pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis)). Such kits generally contain one or more compositions described above and instructions for use. As an example only, the present invention also provides a kit for determining whether a patient is at risk of IL-33-mediated disease (e.g., asthma), the kit containing first and second oligonucleotides specific to polymorphic regions of IL1RL1 (e.g., specific to polymorphisms rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); or rs10206753 (SEQ ID NO:5)). In another example, the present invention also provides a kit for determining whether a patient is at risk of IL-33-mediated disease (e.g., asthma), the kit containing first and second oligonucleotides specific to polymorphic regions of IL33 (e.g., polymorphism rs4742165 (SEQ ID NO:6)). In yet another example, the present invention provides a kit for determining whether a patient is at risk of IL-33-mediated disease (e.g., asthma), the kit containing first and second oligonucleotides specific to linkage disequilibrium polymorphisms (e.g., any polymorphisms listed in Table 3 or Table 4) selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6). As an example only, the present invention also provides a kit for determining whether a patient is likely to respond to a treatment comprising an IL-33 axis binding antagonist (e.g., an ST2 binding antagonist), said kit containing first and second oligonucleotides specific to polymorphic regions of IL1RL1 (e.g., specific to polymorphisms rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157 (SEQ ID NO:4); or rs10206753 (SEQ ID NO:5)). As yet another example, the present invention also provides a kit for determining whether a patient is likely to respond to treatment comprising an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist), said kit containing first and second oligonucleotides specific to polymorphic regions of IL33 (e.g., polymorphism rs4742165 (SEQ ID NO:6)). In yet another example, the present invention provides a kit for determining whether a patient is likely to respond to a treatment comprising an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist), the kit containing first and second oligonucleotides specific to linkage-disequilibrium polymorphisms (e.g., any polymorphisms listed in Table 3 or Table 4) selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6).

"Locus-specific" oligonucleotides bind to the polymorphic region of the locus or to a polymorphic region adjacent to the locus. For oligonucleotides to be used as primers for amplification, they are adjacent if the primers are close enough to generate a polynucleotide containing the polymorphic region. In one embodiment, oligonucleotides are adjacent if they bind within approximately 1-2 kb (e.g., less than 1 kb) of the polymorphism. Specific oligonucleotides are capable of hybridizing to sequences and, under suitable conditions, will bind to sequences that are not mononucleotide different.

The oligonucleotides contained in the kit, whether or not used as probes or primers, can be labeled in a detectable manner. The labels can be detected directly (e.g., for fluorescent labels) or indirectly. Indirect detection can include any detection method known to those skilled in the art, including biotin-avidin interactions, antibody binding, etc. Fluorescently labeled oligonucleotides may also contain quenching molecules. The oligonucleotides can bind to a surface. In some embodiments, the surface is silica or glass. In some embodiments, the surface is a metal electrode.

Other kits of the present invention contain at least one reagent necessary for performing the assay. For example, the kit may contain an enzyme. Alternatively, the kit may contain a buffer or any other necessary reagent.

The kit may include all or some of the following: positive controls, negative controls, reagents, primers, sequencing markers, probes, and antibodies described herein, to determine the genotype of a subject at one or more polymorphisms in the IL1RL1 gene (e.g., polymorphism rs4988956 (SEQ ID NO:1); polymorphism rs10204137 (SEQ ID NO:2); polymorphism rs10192036 (SEQ ID NO:3); polymorphism rs10192157 (SEQ ID NO:4); or polymorphism rs10206753 (SEQ ID NO:5), and one or more polymorphisms in the IL33 gene (e.g., polymorphism rs4988956 (SEQ ID NO:1); polymorphism rs10204137 (SEQ ID NO:2); polymorphism rs10192036 (SEQ ID NO:3); polymorphism rs10192157 (SEQ ID NO:4); or polymorphism rs10206753 (SEQ ID NO:5), and one or more polymorphisms in the IL33 gene (e.g., polymorphism rs10206753 (SEQ ID NO:5)). rs4742165 (SEQ ID NO:6) or one or more polymorphisms that are linked out of balance with polymorphisms in the IL1RL1 or IL33 gene (e.g., polymorphisms that are linked out of balance with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5) and rs4742165 (SEQ ID NO:6), e.g., any polymorphisms listed in Table 3 or Table 4).

This invention also provides kits or manufactures for detecting biomarkers (e.g., periosteal proteins). Such kits can be used to determine whether a patient with IL-33-mediated disease is likely to respond to treatment containing an IL-33 axis-binding antagonist (e.g., an ST2-binding antagonist). These kits may contain carrier means that are compartmentalized to restrict the reception of one or more container devices, such as vials, tubes, etc., each containing a separate element to be used in the method. For example, a container device may contain a detectable label or a probe that may be a detectable label. Such probes may be antibodies or polynucleotides, respectively, specific to proteins or messengers. In cases where the kit utilizes nucleic acid hybridization to detect target nucleic acids, the kit may also have containers containing nucleotides for amplifying the target nucleic acid sequence, and/or containers containing reporter molecule devices, such as biotin-binding proteins (e.g., avidin or streptavidin) that bind to reporter molecules (e.g., enzyme markers, fluorescent markers, or radioisotope markers).

These kits will typically contain the containers described above and one or more other containers containing materials deemed necessary from a commercial and user perspective, including buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. Labels may be present on the containers to indicate that the composition is intended for a specific application and may also display instructions for in vivo or in vitro use, as described above.

The kit of the present invention has many embodiments. A common embodiment is a kit comprising a container, a label on the container, and a composition contained within the container, wherein the composition comprises a first antibody that binds to a protein or autoantibody biomarker (e.g., periosteal protein), and the label on the container indicates that the composition can be used to evaluate the presence of such a protein or antibody in a sample, and wherein the kit includes instructions for evaluating the presence of a biomarker protein in a specific sample type using the antibody. The kit may also include a set of instructions and materials for preparing a sample and applying the antibody to the sample. The kit may also include first and second antibodies, wherein the second antibody is conjugated to a label (e.g., an enzyme label).

Another embodiment is a kit comprising a container, a label on the container, and a composition contained within the container, wherein the composition contains one or more polynucleotides that hybridize under stringent conditions to the complementary strand of a biomarker (e.g., periosteal protein), and the label on the container indicates that the composition can be used to evaluate the presence of a biomarker (e.g., periosteal protein) in a sample, and wherein the kit includes instructions for evaluating the presence of a biomarker RNA or DNA in a specific sample type using the polynucleotide.

Other optional components of the kit include one or more buffers (e.g., blocking buffer, washing buffer, substrate buffer, etc.), other reagents such as substrates chemically modified by enzyme labeling (e.g., chromogens), epitope retrieval solutions, control samples (positive and/or negative controls), control slides, etc. The kit may also include instructions for interpreting results obtained using the kit.

In other specific embodiments, for antibody-based kits, the kit may, for example, comprise: (1) a first antibody (e.g., bound to a solid support) that binds to a biomarker protein (e.g., periosteal protein); and optionally, (2) a different second antibody that binds to said protein or the first antibody and is conjugated to a detectable marker.

For oligonucleotide-based kits, the kit may, for example, comprise: (1) oligonucleotides, such as detectable labeled oligonucleotides, said oligonucleotides being associated with polymorphic regions of the IL1RL1 gene as described above (e.g., polymorphisms rs4988956 (SEQ ID NO:1); rs10204137 (SEQ ID NO:2); rs10192036 (SEQ ID NO:3); rs10192157). (SEQ ID NO:4); or polymorphism rs10206753 (SEQ ID NO:5); polymorphic regions of the IL33 gene (e.g., polymorphism rs4742165 (SEQ ID NO:6)) or polymorphisms linked out of balance with polymorphisms in the IL1RL1 gene or IL33 gene (e.g., with polymorphisms selected from rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), r The kit may contain linkage-disequilibrium polymorphisms of s10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and rs4742165 (SEQ ID NO:6), such as any polymorphisms listed in Table 3 or Table 4, and/or hybridization of nucleic acid sequences encoding biomarker proteins (e.g., periostein or sST2), or (2) a pair of primers for amplifying the biomarker nucleic acid molecule. The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may also contain components (e.g., enzymes or substrates) necessary for detecting the detectable marker. The kit may also contain a control sample or a series of control samples that can be analyzed and compared with the test sample. Each component of the kit may be sealed in an individual container and multiple containers may be contained in a single package, along with instructions for interpreting the results of assays performed using the kit.

VII. Pharmaceutical preparations

Therapeutic formulations of the antagonists used in this invention (e.g., IL-33 axis binding antagonists (e.g., ST2 binding antagonists, such as ST2-Fc protein), trypsin-β binding antagonists, CRTH2 antagonists, IL-13 binding antagonists, IL-17 binding antagonists, JAK1 antagonists, and/or IL-5 binding antagonists) are prepared for storage by mixing the antagonist, having the desired purity, with an optional pharmaceutically acceptable carrier, excipient, or stabilizer in the form of a lyophilized formulation or an aqueous solution. For general information about the formulation, see, for example, Gilm et al. (eds.), The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990; A. Gennaro (ed.), Remington’s Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Pennsylvania, 1990; Avis et al. (eds.), Pharmaceutical Dosage Forms: Parental Medications Dekker, New York, 1993; Lieb Erman et al. (eds.), Pharmaceutical Dosage Forms: Tablets, Dekker, New York, 1990; Lieberman et al. (eds.), Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York, 1990; and Walters (ed.), Dermatological and Transdermal Formulations (Drugs and the Pharmaceutical Sciences), Vol. 119, Marcel Dekker, 2002.

Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphates, citric acid, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (such as octadecylbenzyldimethylammonium chloride; hexamethylammonium chloride; benzalkonium chloride, benzalkonium bromide; phenol, butanol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); and low molecular weight (less than about 10 residues). Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannose, trehalose, or sorbitol; counterions that form salts such as sodium; metal complexes (e.g., Zn-protein complexes) and/or nonionic surfactants such as TWEEN ^™ , PLURONICS ^™ , or polyethylene glycol (PEG).

The formulations described herein may also contain more than one active compound, preferably those with complementary activities that do not adversely affect each other. The type and effective amount of such drugs depend, for example, on the amount and type of antagonist present in the formulation and the clinical parameters of the subject.

The active ingredient can also be encapsulated in microcapsules (e.g., hydroxymethyl cellulose microcapsules or gelatin microcapsules and poly(methyl methacrylate) microcapsules), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or emulsions, prepared by coagulation techniques or interfacial polymerization, respectively. Such techniques are disclosed in Remington’s Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

Sustained-release articles can be prepared. Suitable examples of sustained-release articles include a solid hydrophobic polymer semi-permeable matrix containing an antagonist, said matrix being in the form of a molded article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol), polylactic acid (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-glutamic acid esters, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ^™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

Preparations intended for internal administration must be sterile. This can be easily achieved by filtration through a sterile filter membrane.

Example

The following embodiments are provided to illustrate, but do not limit, the invention currently claimed.

Example 1. Identification of causative SNPs that provide protection against asthma risk

In this analysis, we identified variants of IL1RL1 with amino acid alterations associated with asthma risk (see Tables 1 and 2), which are closely linked to intron SNP rs3771166 ( ^r² = 1.0). These amino acid altered SNPs were identified in 1000 genomic samples from Europe and their association with asthma risk was tested in our case-control dataset. The asthma cases included 522 samples from patients of European descent in the Genentech clinical trials BOBCAT, EXTRA, MILLY, and MOLLY, which were compared with control samples from 4,465 individuals of European descent in the genome-wide association study (GWAS) of genetic markers of cancer susceptibility (CGEMS) (Jia et al., J. Allergy Clin. Immunol. 130:647-654, 2012; Hanania et al., Am. J. Respir. Crit. Care Med. 187:804-811, 2013; Corren et al., N. Engl. J. Med. 365:1088-1098, 2011; Noonen et al., J. Allergy Clin. Immunol. 132:567-574, 2013). These same polymorphisms have also been reported to be associated with cardiovascular disease risk and elevated levels of soluble ST2 (sST2) and IL-33 (Ho et al., J. Clin. Invest. 123:4208-4218, 2013).

This analysis revealed that each of these SNPs protected against asthma risk in the study population (see Table 1). Two SNPs, rs10192036 and rs10204137, located in the same codon, resulted only in Q501R due to tight linkage disequilibrium (LD) between the two SNPs ( ^r² = 1.0).

Table 1: SNPs with altered amino acids in IL1RL1 protect against asthma risk

Chr, chromosome; MAF, minor allele frequency; OR, odds ratio.

The IL1RL1 locus on chromosome 2 is complex, containing multiple linkage disequilibrium (LD) regions, each containing SNPs that contribute to asthma susceptibility. Furthermore, SNPs in IL1RL1 are in LD with SNPs in IL18R1, making it difficult to assign causal relationships to either gene. To address this issue, conditional analysis of the SNPs at this locus was performed on rs3771166 within 500 kb in either direction. Conditionalization of the rs3771166 genotype eliminated most of the signal in this region, with only one SNP retaining its unconditional p-value (rs17766515; p = 0.01). From this same window, we selected SNPs that were not in LD with rs3771166 (D’ < 0.6) and performed conditional analysis on these SNPs. For these analyses, rs3771166 retained its statistical significance (maximum p = 0.01). The conditional and functional analyses presented below show that the amino acid-altering SNP in IL1RL1 captured by tag SNP rs3771166 is the causal SNP in this region. Based on these results, individuals with genotypes containing common IL1RL1 variants face an increased risk of asthma compared to individuals with genotypes containing protective IL1RL1 variants. Table 2 shows the genotypes of each causal SNP in patients facing an increased risk of asthma.

Table 2: SNP genotypes associated with increased asthma risk

Investigating the in vivo functional significance of these amino acid mutations to determine how these protective variants affect the IL-33 response. These variants result in alterations encoding the intracellular ST2 region of the receptor-containing signal-regulating Toll/IL-1R (TIR) domain. The TIR domain is crucial for downstream signal transduction of the IL-1 cytokine family and Toll-like receptors (TLRs), and mutations or deletions in this domain may lead to attenuated or absent responses to ligands. IL-33-induced ST2 and IL-1RAcP dimerization is thought to promote TIR-TIR domain interactions, subsequently promoting the combined recruitment of the adaptor molecules MyD88 and Myddosome (see Figure 1A). Two variants in IL1RL1, A443T and Q501R, are located within the TIR domain, while the T549I and L551S variants are localized to poorly characterized C-terminal regions not involved in signal propagation (see Figure 1A). To further determine how polymorphisms within the TIR domains might affect IL-33 signaling, each variant was localized to the known structure of the TLR10TIR dimer (Fig. 1B). The Q501R variant was localized to an αD helix within the TIR domain, which is partially disordered in the TLR10TIR dimer, while the A433T variant was localized to an αB helix immediately adjacent to the B-B ring (Fig. 1B). The conserved B-B rings of the TIR are considered to mediate the dimerization of the TIR domains linked to TLR10 (Nyman et al., J. Biol. Chem. 283: 11861-11865, 2008).

To test the effects of these missense variants on IL-33-mediated signaling, cell lines expressing multiple protective IL1RL1 variants were generated. Single TIR mutants, C-terminal double mutants, or mutants containing all four polymorphisms were generated and incorporated into expression vectors. To circumvent endogenous IL-33 activity, HEK-BLUE ^™ (Invivogen) IL-1β cells, responsive to IL-1β but lacking IL-33 activity, were used. Stimulation of HEK-BLUE ^™ IL-1β cells with IL-1β resulted in robust activation of NF-κB and AP-1, which could be measured by the activity of the NF-κB/AP-1-driven secretory alkaline phosphatase (SEAP) reporter molecule. Stable transfection of the ST2 expression vector into HEK-BLUE ^™ IL-1β cells resulted in IL-33-dependent reporter molecule activity, thus enabling the evaluation of IL-1β and IL-33 responses using the same reporter molecule system. Although IL-1β activation produced similar reporter gene induction across different cell lines, the response to IL-33 was attenuated in cells expressing only mutations in the TIR domain or expressing all four missense variants (see Figures 1C, 1D, 2A, and 2B). As a control, measurements of receptor expression revealed equivalent surface levels in all ST2 mutants (see Figures 3A–3C).

To further elucidate how protective ST2 variants can affect IL-33 activity in the context of asthma, we compared IL-33 activity and ST2 expression between human donors carrying protective IL1RL1 variants or common IL1RL1 variants. Consistent with the reported molecular cell lines, we observed reduced secretion of IL-33-mediated interleukin-8 (IL-8) from purified blood eosinophils derived from individuals carrying protective IL1RL1 variants compared to individuals carrying common IL1RL1 variants (Fig. 4). Additionally, we observed greater expression of soluble ST2 in these individuals (Figs. 5A and 5B).

These results provide a link between asthma genetic predisposition and IL-33-mediated responses. Given the pro-inflammatory role of IL-33 in Th2-mediated immunity, perturbing this pathway to weaken the IL-33 response may promote protection against asthma risk. Positional alterations of variants within the TIR domain of ST2 predict MyD88-mediated signaling. The reduced sensitive IL-33 response induced by these variants is consistent with the nature of the amino acid substitutions, the moderately protective OR of variants in IL1RL1 in asthma genetics studies, and the chemical properties of their side chains. The fact that individuals carrying common IL1RL1 variants face an increased risk of asthma compared to those carrying protective IL1RL1 variants suggests that genotyping at the SNPs with these amino acid alterations could be used in diagnostic methods to determine whether a patient faces an increased risk of asthma. Additionally, these patients may respond to therapies containing IL-33 axis-binding antagonists (e.g., anti-IL-33 antibodies) or ST2-binding antagonists (e.g., ST2-Fc protein).

method

Stable expression of ST2L and protective variants

ST2L cDNA was cloned into the pCMV Neo expression vector and a protective variant was generated by site-directed mutagenesis based on PCR. The linearized plasmid was stably transfected into HEK-BLUE ^™ IL-1β reporter cells (Invivogen) using Life Technologies. HEK-BLUE ^™ IL-1β cells were maintained in DMEM, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum (FBS), NORMOCIN ^™ (100 μg/ml), hygromycin B (200 μg/ml), ZEOCIN ^™ (100 μg/ml), 50 U/ml penicillin, and 50 μg/ml streptomycin. After 48 hours, transfected cells were selected for 2 weeks in HEK-BLUE ^™ IL-1β growth medium supplemented with 2 mg/ml G418. Stable expression of ST2L was confirmed by flow cytometry and mRNA analysis. Single clonal cultures were generated using a limiting dilution method of batch cultures.

Cell culture and stimulation

IL-33 pathway activity in stably transfected HEK-BLUE ^™ IL-1β reporter cells was measured using a colorimetric assay performed according to the manufacturer's instructions. Briefly, stably transfected HEK-BLUE ^™ IL-1β reporter cells (50,000 cells/well in 96-well plates) were stimulated with increasing concentrations of IL-33 or IL-1β at 37°C for 20 h at 5% _CO₂ . SEAP reporter molecule activity was detected from the supernatant using a spectrophotometer at 620 nm using the QUANTI-BLUE ^™ assay (Invivogen).

RNA isolation and quantitative RT-PCR

RNA was isolated using a micro-quantity kit (Qiagen). An ABI 7500 real-time PCR system (Applied Biosystems) and a one-step RT-PCR master mix (Applied Biosystems) were used for real-time RT-PCR (primers and probe sets from Applied Biosystems). Results were normalized for RPL19 and relative expression was calculated by changes in thresholds (ΔΔCT method).

Recombinant protein

Recombinant human IL-33 (IL-33 _112-270 ) was prepared in-house. Recombinant IL-1β was purchased from R&D Systems.

Flow cytometry analysis

ST2L surface expression was detected using a biotinylated polyclonal antibody (BAF523, R&D Systems). IL-1RAcP surface expression was detected using an allophycocyanin (APC)-conjugated monoclonal antibody (FAB676A, R&D Systems). Mean fluorescence intensity (MFI) was calculated using FLOWJO ^™ software.

Isolation of human eosinophils and basophils

Primary human eosinophils and basophils were enriched from whole blood using a Miltenyi Biotec kit with negative selection. Purity (>92%) was confirmed by flow cytometry. Eosinophils were seeded at 1 x ^10⁶ cells/ml in DMEM supplemented with 10% FBS, GLUTAMAX ^™ , penicillin/streptomycin, and containing 10 ng/ml recombinant human IL-3 (R&D Systems). Cell culture supernatant was collected after 24 hours.

ELISA analysis

The levels of IL-8 secreted from culture supernatant and plasma sST2 were measured using an ELISA kit obtained from R&D Systems.

Genotyping

Asthma cases were genotyped on a 2.5M Omni array and variants were retrieved using Illumina's GENOMESTUDIO ^™ software. Population controls were derived from the Cancer Susceptibility Genetic Markers Study (CGEMS) (cgems.cancer.gov). Population controls consisted of a control group from the Cancer Susceptibility Genetic Markers Study (CGEMS) and were accessed and downloaded using the authorized genotype and phenotype (dbGAP) database.

Sample quality control

Various quality control measures were performed on asthma cases and controls. Samples with more than 10% genotypic loss were removed (n=29). Samples with heterozygosity ± 3 standard deviations (SD) from the mean were removed (n=47). Ancestry identity (IBD) analysis was performed to identify and remove relevant samples with a certain proportion of allele-shared IBD > 0.4 (n=11). We assessed population substructure by filtering GWAS data by minor allele frequency (MAF) and linkage disequilibrium. This subset of SNPs was combined with HapMap data and subsequently analyzed in EIGENSTRAT (Price et al., Nat. Genet. 38:904-909, 2006) to remove ancestral outliers not clustered in Caucasian samples using principal component analysis (n=242). After applying quality control filtering procedures, we analyzed 4,987 Nordic Caucasian samples, including 522 asthma cases and 4,465 controls.

SNP Quality Control

Quality control was performed to identify and remove low-quality SNPs. SNPs with a genotyping call rate <95% were excluded from the analysis. SNPs showing signs of deviation from Hardy Weinberg Equilibrium (HWE) were also removed (Purcell et al., Am. J. Hum. Genet. 81(3):559-575, 2007). In addition, any SNPs that failed to be converted into human genome assembly hg19 or had a 1000 genome project (kgp) identifier that was not localized to the reference SNP ID (rsid) were removed from the dataset. After these quality control measures, 297,157 SNPs remained for attribution.

Genotype attribution

Genotypic attribution of those samples was performed using the following workflow, which included pre-selecting a phase using Shapeit (Delaneau et al., Nat. Methods 9:179-181, 2012), followed by attribution using IMPUTE2 (Marchini et al., Nat. Genet. 39:906-913, 2007) and reference haplotypes from the 1000 Genomes Project (Durbin et al., Nature 467:1061-1073, 2010).

Example 2. Periosteal protein levels predict asthma risk in individuals carrying protective ST2 variants.

To investigate whether asthma biomarkers could be used to refine diagnostic and prognostic methods for determining whether asthma patients are likely to respond to IL-33 axis binding antagonists, we examined whether periosteal protein levels predicted asthma susceptibility in individuals carrying the protective SNPs shown in Figure 6. Individuals were divided into those with high or low periosteal protein levels, and the association between each group and asthma was determined. Individuals with low periosteal protein levels were less likely to develop asthma compared to a reference level carrying the protective SNP (i.e., lower odds ratio) (Figure 6) compared to those with high periosteal protein levels (Figure 6). However, both groups were less likely to develop asthma compared to individuals carrying common variants.

Example 3. Association between serum sST2 levels and genetic susceptibility factors of the IL-33 axis

To investigate whether peripheral blood sST2 levels are associated with IL-33 pathway activity by leveraging the association of genetic susceptibility factors to the IL-33 axis in asthma, we extended previous findings on the association between serum sST2 levels and IL1RL1 genetic variants in healthy donors (described in Example 1) and measured baseline serum sST2 levels in 760 well-characterized individuals with moderate to severe asthma from the clinical studies BOBCAT (Jia et al., J. Allergy Clin. Immunol. 130:647-654, 2012), MILLY (Corren et al., N. Engl. J. Med. 365:1088-1098, 2011), and COSTA (Jeffrey et al., C101. Allergic airway inflammation and hyper-responsiveness: novel mechanisms and therapy American Thoracic Society; 2015. p. A5168-A).

Using the aforementioned asthma discovery set (Ramirez-Carrozzi et al., J. Allergy Clin. Immunol. 135:1080-1083, 2015), we scanned the IL33 locus previously identified as an asthma risk locus (see, e.g., Moffatt et al., N. Engl. J. Med. 363:1211-1221, 2010) and identified rs4742165 (SEQ ID NO: 6) as the top SNP at this locus based on the p-value (OR = 1.71; P = 5.26 x ^10⁻⁴ ). The SNP (rs1342326; SEQ ID NO:7) identified by Moffatt et al. above was not present in our dataset, and its strongest indicator was not associated with disease risk. However, the ^r² ( ^r² = 0.66) of this SNP with rs1342326 was below the threshold ( ^r² > 0.8) commonly used to identify strongly linked SNPs (Moffatt et al., above). Therefore, we performed quantitative trait linkage (eQTL) analysis of rs3771166 (SEQ ID NO:8) and rs4742165 (SEQ ID NO:6) expression in asthma serum sST2 to assess their combined genetic effects. IL1RL1 and IL33 are located on chromosomes 2 and 9, respectively, and are therefore completely independent of each other.

Figures 7A and 7B show the observed distribution and summary statistics of serum sST2 based on genotypes rs3771166 and rs4742165, and sex, respectively. As previously reported, the median level of serum sST2 is higher in males (Ramirez-Carrozzi et al., J. Allergy Clin. Immunol. 135:1080-1083, 2015 and Ho et al., J. Clin. Invest. 123:4208-4218, 2013). Furthermore, the median level of serum sST2 increases with increasing minor allele counts in the genotype. Multiple regression analysis of serum sST2 levels _log₂ transformed with rs3771166 and rs4742165 genotypes, adjusted for sex, was used to assess these SNPs to simultaneously predict the strength and significance of serum sST2 levels. All results were statistically significant (p < 0.05, ANOVA F-test), indicating that rs4742165 predicts serum sST2 levels, even after considering rs3771166. The magnitude and variability of genotype predictive power are represented by curves of serum sST2 least-squares mean (lsmean) and standard error (Figure 7C). The magnitude of the genetic effect of rs4742165 is approximately 1.6 times greater than that of rs3771166. For each minor allele count of rs4742165 and rs3771166, soluble ST2 levels increased by 23% and 14%, respectively. Serum sST2 levels were 43% higher in males compared to females.

IL-33 is a potent stimulant for the production of type II cytokines (e.g., IL-13) in mast cells and group 2 innate lymphoid cells (ILC2) (Nagarkar et al., J. Allergy Clin. Immunol. 136:202-205, 2015). Therefore, we assessed the paired relationships between serum periosteal protein, exhaled nitric oxide fraction (FeNO), and blood eosinophil count (each a type II biomarker predicting response to IL-13 blockade in asthmatic patients (Arron et al., Ann. Am. Thorac. Soc. 10(Suppl): S206-213, 2013)) and serum sST2 levels (Figure 8). Interestingly, no correlation was observed between serum sST2 and each type II biomarker (Spearman p-evaluation values ranging from -0.15 to -0.078). These data suggest that sST2-associated biology may play a role in both type II hyperasthma and type II hypoasthma.

These data demonstrate a positive association between serum sST2 levels and the risk of IL33-related asthma, suggesting that sST2 may be a biomarker of IL-33 axis activity in asthma and could be useful in predicting clinical response to IL-33 axis-targeted therapies containing IL-33 axis binding antagonists (such as anti-IL-33 antibodies) and in measuring their pharmacokinetic effects.

method

biomarkers

sST2 was measured by ELISA (#DST200, R&D Systems, Quantikine). Serum periosteal protein was measured by immunoassay using a Cobas e601 analyzer (Roche Professional Diagnostics, Penzberg, Germany), as previously reported (Jia et al., J. Allergy Clin. Immunol. 130:647-654, 2012). FeNO was measured using a NIOX device (Aerocrine, Solna, Sweden). Blood eosinophil counts were assessed as part of a complete blood count (CBC) on an automated hematology analyzer in the central laboratory.

statistics

R software (RCoreteam.R: A Language and Environment for Statistical Computing) was used for plotting and analysis. Spearman rank correlation was used to assess the correlation of baseline biomarker levels. Expression quantitative trait linkage (eQTL) was performed as described above (Stranger et al., Nat. Genet. 39:1217-1224, 2007). Multiple linear regression was used to model _log2 transformed serum sST2 levels relative to genotypes rs4742165 (SEQ ID NO:6) and rs3771166, with sex adjustments. The significance of each term in the model was determined by the F-test.

Genotyping

As described in Example 1 above, genotyping was performed on asthma cases.

Example 4. SNPs highly linked to IL-33 axis genetic susceptibility factors in equilibrium

HapMap (International HapMap Consortium, Nature 437(7063):1299-1320, 2005) linkage disequilibrium (LD) data and the 1000 Genomes dataset (McVean et al., Nature.491:56-65, 2012) were used to identify SNPs with high LDs for the IL-33 axis genetic susceptibility factors (selected SNPs) described in Examples 1 and 3. SNPs with high LD, such as those listed in Tables 3 and 4, including the selected SNPs rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), rs10206753 (SEQ ID NO:5), and/or rs4742165 (SEQ ID NO:6), serve as alternative SNPs that can be used as biomarkers for IL-33-mediated diseases (e.g., asthma). Genotyping at these selected or alternative SNPs can be used in diagnostic methods to determine whether a patient faces an increased risk of IL-33-mediated diseases (e.g., asthma). Additionally, patients with equivalent alleles of alternative SNPs may respond to therapies containing an IL-33 axis-binding antagonist (e.g., an anti-IL-33 antibody or an ST2-binding antagonist (e.g., ST2-Fc protein)). Minor alleles in a population are generally equivalent alleles relative to the SNPs described in Tables 3 and 4, although in some cases the major allele may be equivalent. Conventional methods in the art can be used to confirm whether a given allele of a SNP listed in Tables 3 and 4 is an equivalent allele.

HapMap samples were based on ancestral segregation to identify ancestral-specific LD SNPs. These groups were divided into several categories: (1) ASW (African ancestry of the southwestern United States), LWK (Luhy of Webuye, Kenya), MKK (Maasai of Kinyawa, Kenya), and YRI (Yoruba of Ibadan, Nigeria; West Africa); (2) CEU (Utah residents of Nordic and Western European ancestry from the CEPH center) and TSI (Tuscans of Italy); (3) CHB (Han Chinese of Beijing, China), CHD (Chinese of Metropolitan Denver, Colorado), and JPT (Japanese of Tokyo, Japan); and (4) GIH (Gujarati Indians of Houston, Texas) and MEX (Mexican ancestry of Los Angeles, California). Within each ancestral group, LD was assessed for SNPs in the region surrounding rs4988956 (SEQ ID NO:1) or rs4742165 (SEQ ID NO:6), and those with a D’ value greater than or equal to 0.6 were included. There exists a subset of SNPs where LD information is available in HapMap data, but allele frequency data is not. For these SNPs, information from the 1000 Genomes Project (1000GP) is used. In Tables 3 and 4, these SNPs are indicated in the "Freq Source" column with the label "1000GP".

For SNPs in Tables 3 and 4 that lack HapMap allele frequencies, the following labels are used:

For CEU ancestry, this is shown as “EUR_MAF” and the minor alleles and frequencies of SNPs from the Phase I 1000 Genomes cohort combined with the European population. For Asian populations, this is shown as “ASN_MAF” and the minor alleles and frequencies of SNPs from the Phase I 1000 Genomes cohort combined with the Asian population. For YRI ancestry, this is shown as “AFR_MAF” and the minor alleles and frequencies of SNPs from the Phase I 1000 Genomes cohort combined with the African population.

Table 3 shows the SNPs that are not linked to rs4988956 (SEQ ID NO:1). Since rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), and rs10206753 (SEQ ID NO:5) are all 100% linked, the SNPs in Table 3 are also not linked to rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4), and rs10206753 (SEQ ID NO:5). Table 4 shows the SNPs that are not linked to rs4742165 (SEQ ID NO:6).

The terms used in Tables 3 and 4 are defined as follows: (1) ancestry or “ANC” refers to the ancestry of the population used to determine ^r² and D’ values; (2) “LD_SNP” refers to an SNP that is LD-positive with IL-33 axis genetic susceptibility SNPs rs4988956 (SEQ ID NO:1) (relative to Table 3) and rs4742165 (SEQ ID NO:6) (relative to Table 4) (rsID names are from NCBI dbSNP version 137 (June 6, 2012)); (3) CHR refers to the chromosomal location of the LD_SNP (genome version hg19; UCSC HG19 genome assembly; February 2009); (4) BP refers to the DNA base pair location of the LD_SNP (genome version hg19; UCSC HG19 genome assembly; February 2009); (5) “RSQ” refers to the r-squared ( ^r²) of the IL-33 axis genetic susceptibility of the SNP and LD_SNP. (6) "LD SOURCE" refers to the database from which LD data for a given LD_SNP is obtained; (7) "FREQ SOURCE" refers to the database from which allele frequency data for a given LD_SNP is obtained (i.e., HapMap or 1000gp); (8) "A1" refers to allele 1 of LD_SNP; (9) "A1_FREQ" refers to the allele frequency of allele 1 of LD_SNP; (10) "A2" refers to allele 2 of LD_SNP; (11) "A2FREQ" refers to the allele frequency of allele 2 of LD_SNP; and (12) "ALLELES" refers to alleles of LD_SNP.

Other implementation plans

While the invention has been described in detail to some extent by way of illustration and example for the purpose of clarity, such description and example should not be construed as limiting the scope of the invention. All disclosures of patents and scientific literature cited herein are expressly and entirely incorporated herein by reference.

Claims (6)

1. Use of an effective amount of an IL-33 axis-binding antagonist in the manufacture of a medicament for treating patients with IL-33-mediated disease, wherein the patient has been identified to contain the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); or the C allele at polymorphism rs10206753 (SEQ ID NO:4). The presence of the T allele at 5), wherein each G allele at polymorphism rs4988956 (SEQ ID NO:1); each A allele at polymorphism rs10204137 (SEQ ID NO:2); each C allele at polymorphism rs10192036 (SEQ ID NO:3); each C allele at polymorphism rs10192157 (SEQ ID NO:4); or each T allele at polymorphism rs10206753 (SEQ ID NO:5) indicates an increased likelihood of a patient responding to treatment containing an IL-33 axis binding antagonist. The IL-33 axial binding antagonist is an IL-33 binding antagonist, an ST2 binding antagonist, or an IL-1RAcP binding antagonist, and Among them, the IL-33-mediated disease is asthma. 2. The use according to claim 1, wherein the identified patient further comprises one or more of the following alleles: a T allele at polymorphism rs4742165 (SEQ ID NO:6); and/or an equivalent allele at a polymorphism selected from polymorphisms of rs4988956 (SEQ ID NO:1), rs10204137 (SEQ ID NO:2), rs10192036 (SEQ ID NO:3), rs10192157 (SEQ ID NO:4) and rs4742165 (SEQ ID NO:6). 3. The use according to claim 1 or 2, wherein the pharmaceutical formulation is for administration in combination with a trypsin-β binding antagonist, a CRTH2 binding antagonist, an IL-13 binding antagonist, an IL-17 binding antagonist, a JAK1 antagonist, and/or an IL-5 binding antagonist. 4. The use according to claim 1, wherein: (i) IL-33 binding antagonists are anti-IL33 antibodies or their antigen-binding fragments; (ii) The ST2 binding antagonist is the ST2-Fc protein, an anti-ST2 antibody, or its antigen-binding fragment; or (iii) IL-1RAcP binding antagonists are anti-IL-1RAcP antibodies; and Among them, IL-33-mediated diseases include asthma. 5. The use according to claim 4, wherein the IL-33 axis-binding antagonist is an anti-ST2 antibody or its antigen-binding fragment. 6. Use of an effective amount of anti-ST2 antibody or its antigen-binding fragment in the manufacture of a medicament for treating patients with IL-33-mediated disease, wherein the patient has been identified to include the G allele at polymorphism rs4988956 (SEQ ID NO:1); the A allele at polymorphism rs10204137 (SEQ ID NO:2); the C allele at polymorphism rs10192036 (SEQ ID NO:3); the C allele at polymorphism rs10192157 (SEQ ID NO:4); or the T allele at polymorphism rs10206753 (SEQ ID NO:5). The presence of each G allele at polymorphism rs4988956 (SEQ ID NO:1); each A allele at polymorphism rs10204137 (SEQ ID NO:2); each C allele at polymorphism rs10192036 (SEQ ID NO:3); each C allele at polymorphism rs10192157 (SEQ ID NO:4); or each T allele at polymorphism rs10206753 (SEQ ID NO:5) indicates an increased likelihood of a patient responding to treatment containing an anti-ST2 antibody or its antigen-binding fragment, wherein the IL-33-mediated disease is asthma.