MATERIALS AND METHODS FOR MONITORING INFLAMMATION

1. FIELD

Provided herein, in some embodiments, are methods of using certain biomarkers in assessing and monitoring a pharmacodynamics or pharmacokinetic effect of an inhibitor of Interleukin 1 alpha (IL1α), and in predicting and monitoring clinical sensitivity and therapeutic response to an inhibitor of IL1α, and kits for performing same. Also provided herein, in certain embodiments, are methods of screening or identifying an inhibitor of IL1α.

2. BACKGROUND

Inflammation is a common pathogenesis of many diseases, including cardiovascular and bowel diseases, diabetes, arthritis, and cancer. Inflammation is a biological response of the immune system that can be triggered by a variety of factors, including pathogens, damaged cells and toxic compounds. These factors may induce acute and/or chronic inflammatory responses in the heart, pancreas, liver, kidney, lung, brain, intestinal tract and reproductive system, potentially leading to tissue damage or disease. Chen et al., Oncotarget. 9(6): 7204-7218 (2018).

In response to tissue injury, the body initiates a chemical signaling cascade that stimulates responses aimed at healing affected tissues. These signals activate leukocyte chemotaxis from the general circulation to sites of damage. Id.

IL1α is a cytokine of the interleukin 1 family that is encoded by the IL1A gene in humans. In general, Interleukin 1 is responsible for the production of inflammation. IL1α inhibitors are being developed to interrupt those processes and treat diseases. Blocking the activity of IL1α has the potential to treat various diseases or conditions including skin diseases such as acne. See, e.g., Valente Duarte de Sousa, Expert Opinion on Investigational Drugs. 23 (10): 1389-410 (2014).

There is a need in the art to assess effects (including a pharmacodynamics or pharmacokinetic effect) of IL1α inhibitors as well as identifying additional IL1α inhibitors for therapeutic or other uses.

3. SUMMARY OF THE INVENTION

In one aspect, provided herein is a method of identifying a subject who is likely to be responsive to a treatment comprising an inhibitor of IL1a, or predicting the responsiveness of a subject to a treatment comprising an inhibitor of IL1α, comprising:

- i. providing a sample from the subject;
- ii. administering the inhibitor of IL1α to the sample;
- iii. measuring levels of one or more biomarkers in the sample;
- iv. identifying the subject as being likely to be responsive to the treatment comprising the inhibitor of IL1α, or predicting the responsiveness of the subject to the treatment comprising the inhibitor of IL1α, based on the levels of the one or more biomarkers as measured in step (iii).

In another aspect, provided herein is a method of selectively treating a subject with a treatment comprising an inhibitor of IL1α, comprising administering a therapeutically effective amount of the treatment comprising the inhibitor of IL1α to the subject identified as being likely to be responsive to the treatment comprising the inhibitor of IL1α according to the method provided herein.

In some embodiments, the sample comprises a skin cell. In some embodiments, the sample comprises an injured skin cell.

In some embodiments, the sample is obtained by a skin biopsy procedure. In some embodiments, the size of the sample is about 3.5 mm to 4.5 mm.

In some embodiments, the method further comprises culturing the sample ex vivo prior to administering the inhibitor of IL1α to the sample. In other embodiments, the method further comprises culturing the sample ex vivo after administering the inhibitor of IL1α to the sample. In yet other embodiments, the inhibitor of IL1α is administered to the sample while culturing the sample ex vivo.

In some embodiments, the levels of the one or more biomarkers are measured at least 4 hours, at least 5 hours, at least 10 hours, at least 15 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, or at least 24 hours post the administration of the inhibitor of IL1α and/or post the sample is obtained from the subject. In some embodiments, the level of the one or more biomarkers are measured at about 24 hours post the administration of the inhibitor of IL1α and/or post the sample is obtained from the subject.

In some embodiments, the method further comprises comparing the levels of the one or more biomarkers with reference levels of the one or more biomarkers. In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject prior to administration of the inhibitor of IL1α. In other embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject without administration of the inhibitor of IL1α. In yet other embodiments, the reference levels of the one or more biomarkers are pre-determined levels of the one or more biomarkers. In other embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample administered with a control agent. In some embodiments, the control agent is a positive control agent that inhibits IL1α. In other embodiments, the control agent is a negative control agent that does not inhibit IL1α.

In some embodiments, the lower levels of the one or more biomarkers as compared with reference levels of the one or more biomarkers indicates the subject is likely to be responsive to the treatment comprising the inhibitor of IL1α, or wherein the subject is identified likely to be responsive to the treatment comprising the inhibitor of IL1α if the levels of the one or more biomarkers in the sample are at least 30%, at least 35%, at least 40%, or at least 50% less than the reference levels.

In some embodiments, the levels of the one or more biomarkers are the expression levels of one or more biomarkers selected from a group consisting of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In some embodiments, the levels of the one or more biomarkers are the expression levels of one or more biomarkers selected from a group consisting of GCSF (CSF3), CXCL1, IL6, GMCSF (CSF2), CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In some embodiments, the levels of the one or more biomarkers are the expression levels of one or more biomarkers selected from a group consisting of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2. In other embodiments, the levels of the one or more biomarkers are the expression levels of one or more biomarkers selected from a group consisting of CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3, and PTGS2.

In some embodiments, the one or more biomarkers comprises CCL20. In some embodiments, the one or more biomarkers comprises CCL22. In some embodiments, the one or more biomarkers comprises CSF3. In other embodiments, the one or more biomarkers comprises CXCL1. In other embodiments, the one or more biomarkers comprises CXCL2. In other embodiments, the one or more biomarkers comprises CXCL3. In other embodiments, the one or more biomarkers comprises CXCL5. In other embodiments, the one or more biomarkers comprises CXCL6. In yet other embodiments, the one or more biomarkers comprises IL6. In yet other embodiments, the one or more biomarkers comprises IL8. In yet other embodiments, the one or more biomarkers comprises PTGS2. In yet other embodiments, the one or more biomarkers comprises CCL3. In yet other embodiments, the one or more biomarkers comprises CCL4. In yet other embodiments, the one or more biomarkers comprises CCL8. In yet other embodiments, the one or more biomarkers comprises CFB. In yet other embodiments, the one or more biomarkers comprises IL1B. In yet other embodiments, the one or more biomarkers comprises MMP3.

In one embodiment, the one or more biomarkers comprise CSF3 (GCSF). In another embodiment, the one ore more biomarkers comprise CXCL1. In another embodiment, the one or more biomarkers comprise IL6.

In some embodiments, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1. In some embodiments, a composite score is calculated based on the levels of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, GMCSF (CSF2), CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1. In some embodiments, a composite score is calculated based on the levels of GCSF (CSF3), CXCL1, IL6, GMCSF (CSF2), CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1, and wherein the method further comprises comparing the composite score to a reference score. In some embodiments, the one or more biomarkers are CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3. In some embodiments, a composite score is calculated based on the levels of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2. In some embodiments, a composite score is calculated based on the levels of CCL20, CCL22, CSF3, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2, and wherein the method further comprises comparing the composite score to a reference score.

In other embodiments, the one or more biomarkers are CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3, and PTGS2. In some embodiments, a composite score is calculated based on the levels of CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3, and PTGS2, and wherein the method further comprises comparing the composite score to a reference score.

In other embodiments, the one or more biomarkers are CCL20, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL6, IL8, and PTGS2. In some embodiments, a composite score is calculated based on the levels of CCL20, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL6, IL8, and PTGS2, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the levels of the one or more biomarkers are determined by measuring the nucleic acid levels (for example, mRNA) of the one or more biomarkers. In other embodiments, the levels of the one or more biomarkers are determined by measuring the protein levels of the one or more biomarkers.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL6 and IL8.

In some embodiments, the one or more biomarkers comprises GCSF. In some embodiments, the one or more biomarkers comprises CXCL1. In other embodiments, the one or more biomarkers comprises IL4. In other embodiments, the one or more biomarkers comprises IL6. In other embodiments, the one or more biomarkers comprises IL8. In other embodiments, the one or more biomarkers comprises MDC. In other embodiments, the one or more biomarkers comprises IP10. In other embodiments, the one or more biomarkers comprises GMCSF. In other embodiments, the one or more biomarkers comprises MIP1a. In other embodiments, the one or more biomarkers comprises TGFa.

In one embodiment, the one or more biomarkers comprise CSF3 (GCSF). In another embodiment, the one or more biomarkers comprise CXCL1. In another embodiment, the one or more biomarkers comprise IL6.

In some embodiments, the one or more biomarkers are GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa. In some embodiments, a composite score is calculated based on the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10. In some embodiments, a composite score is calculated based on the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa. In some embodiments, a composite score is calculated based on the levels of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are GCSF, CXCL1, IL6 and IL8. In some embodiments, a composite score is calculated based on the levels of GCSF, CXCL1, IL6 and IL8, and wherein the method further comprises comparing the composite score to a reference score.

In one embodiment, the one or more biomarkers are CSF3 (GCSF), CXCL1 and IL6. In one embodiment, a composite score is calculated based on the levels of CSF3 (GCSF), CXCL1 and IL6, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the levels of the one or more biomarkers are determined by measuring the protein levels of the one or more biomarkers.

In another aspect, provided herein is a method of identifying a subject having a IL1α mediated disease who is likely to be responsive to a treatment comprising an inhibitor of IL1α or predicting the responsiveness of a subject having a IL1α mediated disease to a treatment comprising an inhibitor of IL1α, comprising:

- i. providing a sample from the subject;
- ii. measuring levels of one or more biomarkers in the sample;
- iii. identifying the subject with the IL1α mediated disease as responsive to the treatment with the inhibitor of IL1α when the levels of one or more biomarkers in the sample are higher than the reference levels of one or more biomarkers; and/or identifying the subject with the IL1α mediated disease as non-responsive to the treatment with the inhibitor of IL1α when the levels of one or more biomarkers in the sample are not higher than the reference levels of one or more biomarkers.

In yet another aspect, provided herein is a method of selectively treating a subject having a IL1α mediated disease with a treatment comprising an inhibitor of IL1α, comprising administering a therapeutically effective amount of the treatment comprising the inhibitor of IL1α to the subject identified responsive to the treatment comprising the inhibitor of IL1αaccording to the method provided herein.

In some embodiments, the sample comprises a skin cell, and wherein optionally the skin cell is an injured skin cell.

In some embodiments, the sample is obtained by a skin biopsy procedure.

In some embodiments, the sample is a blood sample.

In some embodiments, the reference levels of the one or more biomarkers are pre-determined levels of the one or more biomarkers. In other embodiments, the reference level of the one or more biomarkers are the levels of the one or more biomarkers in a sample comprising an uninjured skin cell.

In one embodiment, the one or more biomarkers comprise CSF3 (GCSF). In another embodiment, the one ore more biomarkers comprise CXCL1. In another embodiment, the one or more biomarkers comprise IL6.

In some embodiments, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1. In some embodiments, a composite score is calculated based on the levels of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the one or more biomarkers are CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3. In some embodiments, a composite score is calculated based on the levels of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3, and wherein the method further comprises comparing the composite score to a reference score.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL6 and IL8.

In some embodiments, the levels of the one or more biomarkers are determined by measuring the protein levels of the one or more biomarkers.

In yet another aspect, provided herein is a method of predicting the responsiveness of a subject having an IL1α mediated disease to an IL1α inhibitor or determining an effective dose of an IL1α inhibitor to be administered to a subject having an IL1α mediated disease comprising:

- i. providing a first sample from a subject;
- ii. administering an IL1α inhibitor to the subject at a predefined dose;
- iii. providing a second sample from the subject;
- iv. measuring levels of one or more biomarkers in the first sample and the second sample; and
- v. determining that the subject is likely to be responsive to the IL1α inhibitor or the predefined dose is effective when the levels of the one or more biomarkers in the second sample are lower than the levels of one or more biomarkers in the first sample.

In some embodiments, the second sample is obtained at 1.5 to 2 hours post the administration of the IL1α inhibitor.

In one embodiment, the one or more biomarkers comprise CSF3 (GCSF). In another embodiment, the one ore more biomarkers comprise CXCL1. In another embodiment, the one or more biomarkers comprise IL6.

In some embodiments, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1. In some embodiments, a composite score is calculated based on the levels of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1, and wherein the method further comprises comparing the composite score to a reference score.

In one embodiment, the one or more biomarkers are CSF3 (GCSF), CXCL1 and IL6. In some embodiments a composite score is calculated based on the levels of CSF3 (GCSF), CXCL1 and IL6, and wherein the method further comprises comparing the composite score to a reference score. In some embodiments, the levels of the one or more biomarkers are determined by measuring the nucleic acid levels (for example, mRNA) of the one or more biomarkers. In other embodiments, the levels of the one or more biomarkers are determined by measuring the protein levels of the one or more biomarkers.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL6 and IL8.

In some embodiments, the levels of the one or more biomarkers are determined by measuring the protein levels of the one or more biomarkers.

In yet another aspect, provided herein is a kit for identifying a subject who is likely to be responsive to a treatment comprising an inhibitor of IL1a, predicting the responsiveness of a subject to a treatment comprising an inhibitor of IL1a, or monitoring the response of a subject to a treatment comprising an inhibitor of IL1α, comprising: (a) an agent for measuring levels of one or more biomarkers in a sample, wherein the one or more biomarkers are selected from (i) a group consisting of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3, or (ii) a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa, or (iii) a group consisting of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1 and (b) a positive control agent that inhibits IL1α and/or a negative control agent that does not inhibit IL1α.

In some embodiments of the kit, the one or more biomarkers comprise CSF3 (GCSF), CXCL1 and IL6.

In some embodiments of the kit, the one or more biomarkers are GCSF, CXCL1, IL6 and IL8.

In some embodiments of the kit, the one or more biomarkers are CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa.

In some embodiments of the kit, the one or more biomarkers are GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10.

In some embodiments of the kit, the one or more biomarkers are GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa.

In other embodiments of the kit, the one or more biomarkers are CCL20, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL6, IL8, and PTGS2.

In other embodiments of the kit, the one or more biomarkers are CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3, and PTGS2.

In some embodiments of the kit, the one or more biomarkers are CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2.

In some embodiments of the kit, the one or more biomarkers are CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3.

In some embodiments of the kit, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, GMCSF (CSF2), CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, G0S2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In one embodiment of the kit, the one or more biomarkers are GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In some embodiments, the kit further comprises a tool for obtaining the sample from a subject. In other embodiments, the tool is suitable for obtaining a skin sample from the subject. In other embodiments, the kit further comprises a tool for administering the inhibitor of IL1α to the sample.

In some embodiments, the positive control agent is an antibody that binds to IL1α.

In some embodiments of the various methods and kits provided herein, the subject has atopic dermatitis, hidradenitis suppurativa, psoriasis, cutaneous lupus erythematosus, or autoimmune bullous disease. In one embodiment, the subject has atopic dermatitis. In another embodiment, the subject has hidradenitis suppurativa.

4. BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the level of IL1α is elevated in ex vivo biopsy explant culture model at both 4-hour and 24-hour time point. FIG. 1A illustrates the ex vivo biopsy explant culture experimental setup. FIG. 1B shows that secreted IL1α level was measured using Luminex and was shown to be upregulated both at 4 hr and 24 hr time point compared to control (n=4 mm biopsy, 5-8 donors).

FIGS. 2A and 2B show the effects of IL1α blockade on secreted cytokine levels in supernatant of skin biopsy explant culture at 4 hr (3 mm, 4 mm or 6 mm). FIG. 2A illustrates the ex vivo biopsy explant culture experimental setup to evaluate PD effects of IL1α blockade. FIG. 2B shows the level of secreted cytokine was measured using Luminex (Millipore, HCYTMAG-60K-PX41) after 4 hours of ex vivo biopsy explant culture using 3 mm, 4 mm or 6 mm skin biopsies (1 donor, 3 replicates per donor). Treatment using anti-IL1α(open round dot) significantly reduced cytokine level compared to samples cultured with no treatment (black round dot) for all skin biopsy size evaluated. However, dynamic range (difference between 4 hr and 4 hr+anti-IL1α) was much smaller in 3 mm than 4 mm or 6 mm biopsy size.

FIG. 3 shows lidocaine injection affects ability of anti-IL1α to reduce level of GCSF, CXCL1 and IL8 in culture supernatant at 4-hour, but not at 24-hour time point. The level of secreted cytokine was measured using Luminex after 4 hours or 24 hours of ex vivo biopsy explant culture using biopsies (4 mm) that were obtained from skin that were injected with PBS (black circle) or 1% lidocaine (black square), (n=5 donors for 4-hour, n=3 donors for 24-hour, each dot or square represent the average of 3-5 replicates from each donor).

FIGS. 4A and 4B show that anti-IL1α treatment can reduce elevated level of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10 cytokines in supernatant of ex vivo biopsy explant culture model (with lidocaine injection) at 24-hour time point. The level of secreted cytokine was measured using Luminex after 24 hours of ex vivo biopsy explant culture using biopsies (4 mm) that were obtained from skin that were injected with 1% lidocaine (3 donors, 1-5 replicates per donor).

FIGS. 4C to 4F show the evaluation of the ex vivo biopsy explant culture model in 10 healthy volunteers. FIG. 4C shows that luminex analysis of the culture supernatant showed the induction of various cytokines following 24 hour of culture. FIG. 4D shows that treatment with anti-IL1α can reduce the level of a subset of cytokines, i.e., CXCL1, GCSF, GMCSF, IL8, IL8, MIP1a and TGFa. The level of these cytokines were reduced by anti-IL1α treatment (>50% inhibition) and display low variance (<100%) in their inhibition response. FIG. 4E shows the induction fold change of these cytokines in the 10 healthy volunteers. FIG. 4F shows the concentrations of these cytokines in the 10 healthy volunteers.

FIGS. 5A and 5B show that anti-IL1α treatment reduces elevated level in gene expression of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2 in ex vivo biopsy explant culture model (with lidocaine injection) at 24-hour time point. Gene expression in ex vivo biopsy explant culture model (with lidocaine injection) was measured using Nanostring platform (Nanostring, nCounter Human Inflammation v2 panel). 40 of the 249 tested genes were induced at 24-hour time point, with geometric mean of fold change>2 among skin samples derived from three donors (FIG. 5A). Elevated gene expression in 11 of the 40 induced genes was inhibited by anti-IL1α treatment, with an average of inhibition>30% (FIG. 5B). D1-3: three donors.

FIGS. 5C, 5D, and 5E show anti-IL1α treatment reduces elevated level in gene expression of CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GMCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3, and PTGS2 in ex vivo biopsy explant culture model (with lidocaine injection) at 24-hour time point as measured using Nanostring in 30 skin biopsies from ten donors, with three skin biopsies per donor. FIG. 5C shows that 25 of the 249 tested genes were induced at 24-hour time point, with fold change>2 in skin samples derived from all ten donors. Elevated gene expression in 15 of the 25 cut-induced genes was inhibited by anti-IL1α treatment, with an average of inhibition>30%. FIG. 5D shows induction of the 15 genes (upper panel) and inhibition by anti-IL1α(lower pannel) among ten donors. FIG. 5E shows that elevated gene expression in 15 of the 29 induced genes was inhibited by anti-IL1α treatment, with an average of inhibition>30%. These 15 genes were defined as IL1α signature. Geomean of induced fold change of the 15 genes (upper panel) and the average of % inhibition on induction by anti-IL1α(lower panel) among ten donors are shown.

FIG. 6 shows anti IL-1a treatment can reduce elevated level in gene expression of a subset of injury-induced genes in ex vivo biopsy explant culture model at 24-hour time point. Gene expression in ex vivo biopsy explant culture model was measured using RNAseq. 1287 genes were induced after 24-hour culture (vs control) with geometric average of fold change>2 among the 10 donors, and false discovery rate (FDR)<0.05; all these 1287 genes were also induced (fold change>1.5) in each of the 10 donors. Elevated gene expression in 139 of these 1287 injury-induced genes was inhibited by anti-IL1α treatment, with an average of inhibition>20%.

FIG. 7 shows bermekimab can reduce elevated level of a subset of induced cytokines in ex vivo biopsy explant culture model at 24-hour time point. FIG. 7A indicates that measurement of cytokine levels in the supernatant showed that bermekimab can reduce the level of GCSF, CXCL1, IL6 and IL8 in a dose responsive manner. FIG. 7B indicates that measurement of cytokine levels in the skin tissue lysate showed that bermekimab can reduce the level of CXCL1, IL6 and IL8 in a dose responsive manner.

FIG. 8 is a schematic summarizing the Phase 1 study of Example 7.

5. DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides in part an effective ex vivo skin biopsy-based assay and novel biomarkers that allow for the measurement of various effects of an anti IL1α treatment.

5.1. Definitions

As used herein, and unless otherwise specified, the terms “treat,” “treating,” and “treatment” refer to an action that occurs while a patient is suffering from a disease or disorder (e.g., such as a disease or disorder related to inflammation), which reduces the severity of the disease or disorder or retards or slows the progression of the disease or disorder.

The term “responsiveness” or “responsive” when used in reference to a treatment refers to the degree of effectiveness of the treatment in lessening or decreasing the symptoms of a disease or disorder being treated. An improvement in a disease or disorder can be characterized as a complete or partial response. “Complete response” refers to an absence of clinically detectable disease with normalization of any previously abnormal measurements. “Partial response” refers to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% decrease in any measurable parameters of a disease or disorder.

As used herein, and unless otherwise specified, the term “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the presence of the disease or disorder. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or disorder. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent. The term also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which is being sought by a researcher, veterinarian, medical doctor, or clinician.

The term “likelihood” generally refers to an increase in the probability of an event. The term “likelihood” when used in reference to the effectiveness of a patient response can mean the increase of indicators, such as mRNA or protein expression, that may evidence an increase in the progress in treating a disease or disorder.

The term “predict” generally means to determine or tell in advance. When used to “predict” the effectiveness or responsiveness of a treatment, for example, the term “predict” can mean that the likelihood of the outcome of the treatment can be determined at the outset, before the treatment has begun, or before the treatment period has progressed substantially.

The term “monitor,” as used herein, generally refers to the overseeing, supervision, regulation, watching, tracking, or surveillance of an activity. For example, the term “monitoring the response of a subject to a treatment” refers to tracking the effectiveness or responsiveness in treating a disease or disorder in a patient. The monitoring can be performed, for example, by following the expression of mRNA or protein biomarkers.

A “biological marker” or “biomarker” is a substance whose detection indicates a particular biological state, such as, for example, the responsiveness to a treatment. In some embodiments, biomarkers can be determined individually. In other embodiments, several biomarkers can be measured simultaneously. In some embodiments, a “biomarker” indicates a change in the level of mRNA expression that may correlate with the progression of a disease, or with the susceptibility of the disease to a given treatment. In some embodiments, the biomarker is a nucleic acid, such as mRNA or cDNA. In additional embodiments, a “biomarker” indicates a change in the level of polypeptide or protein expression that may correlate with the risk or progression of a disease, or patient's susceptibility to treatment. In some embodiments, the biomarker can be a polypeptide or protein, or a fragment thereof. The relative level of specific proteins can be determined by methods known in the art. For example, antibody based methods, such as an immunoblot, enzyme-linked immunosorbent assay (ELISA), or other methods can be used.

The terms “polypeptide” and “protein,” as used interchangeably herein, refer to a polymer of three or more amino acids in a serial array, linked through peptide bonds. The term “polypeptide” includes proteins, protein fragments, protein analogues, oligopeptides, and the like. The term “polypeptide” as used herein can also refer to a peptide. The amino acids making up the polypeptide may be naturally derived, or may be synthetic. The polypeptide can be purified from a biological sample. The polypeptide, protein, or peptide also encompasses modified polypeptides, proteins, and peptides, e.g., glycopolypeptides, glycoproteins, or glycopeptides; or lipopolypeptides, lipoproteins, or lipopeptides.

The term “expressed” or “expression” as used herein refers to the transcription from a gene to give an RNA nucleic acid molecule at least complementary in part to a region of one of the two nucleic acid strands of the gene. The term “expressed” or “expression” as used herein also refers to the translation from the RNA molecule to give a protein, a polypeptide, or a portion thereof.

The term “expression level” refers to the amount, accumulation, or rate of a biomarker molecule or a gene set. An expression level can be represented, for example, by the amount or the rate of synthesis of a messenger RNA (mRNA) encoded by a gene, the amount or the rate of synthesis of a polypeptide or protein encoded by a gene, or the amount or the rate of synthesis of a biological molecule accumulated in a cell or biological fluid. The term “expression level” refers to an absolute amount of a molecule in a sample or a relative amount of the molecule, determined under steady-state or non-steady-state conditions.

An mRNA that is “upregulated” is generally increased upon a given treatment or condition, or in certain patient groups. An mRNA that is “downregulated” generally refers to a decrease in the level of expression of the mRNA in response to a given treatment or condition, or in certain patient groups. In some situations, the mRNA level can remain unchanged upon a given treatment or condition. An mRNA from a patient sample can be “upregulated” when treated with a drug, as compared to a non-treated control. This upregulation can be, for example, an increase of about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1,000%, about 5,000%, or more of the comparative control mRNA level. Alternatively, an mRNA can be “downregulated”, or expressed at a lower level, in response to administration of certain compounds or other agents. A downregulated mRNA can be, for example, present at a level of about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1%, or less of the comparative control mRNA level.

Similarly, the level of a polypeptide or protein biomarker from a patient sample can be increased when treated with a drug, as compared to a non-treated control. This increase can be about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 200%, about 300%, about 500%, about 1,000%, about 5,000%, or more of the comparative control protein level. Alternatively, the level of a protein biomarker can be decreased in response to administration of certain compounds or other agents. This decrease can be, for example, present at a level of about 99%, about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 1%, or less of the comparative control protein level.

The terms “determining,” “measuring,” “evaluating,” “assessing,” and “assaying” as used herein generally refer to any form of measurement, and include determining whether an element is present or not. These terms include quantitative and/or qualitative determinations. Assessing may be relative or absolute. “Assessing the presence of” can include determining the amount of something present, as well as determining whether it is present or absent.

The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, or compounds produced synthetically, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. As used herein in the context of a polynucleotide sequence, the term “bases” (or “base”) is synonymous with “nucleotides” (or “nucleotide”), i.e., the monomer subunit of a polynucleotide. The terms “nucleoside” and “nucleotide” are intended to include those moieties that contain not only the known purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the terms “nucleoside” and “nucleotide” include those moieties that contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. “Analogues” refer to molecules having structural features that are recognized in the literature as being mimetics, derivatives, having analogous structures, or other like terms, and include, for example, polynucleotides incorporating non-natural nucleotides, nucleotide mimetics such as 2′-modified nucleosides, peptide nucleic acids, oligomeric nucleoside phosphonates, and any polynucleotide that has added substituent groups, such as protecting groups or linking moieties.

The term “complementary” refers to specific binding between polynucleotides based on the sequences of the polynucleotides. As used herein, a first polynucleotide and a second polynucleotide are complementary if they bind to each other in a hybridization assay under stringent conditions, e.g., if they produce a given or detectable level of signal in a hybridization assay. Portions of polynucleotides are complementary to each other if they follow conventional base-pairing rules, e.g., A pairs with T (or U) and G pairs with C, although small regions (e.g., fewer than about 3 bases) of mismatch, insertion, or deleted sequence may be present.

The terms “isolated” and “purified” refer to isolation of a substance (such as mRNA, DNA, or protein) such that the substance comprises a substantial portion of the sample in which it resides, i.e., greater than the portion of the substance that is typically found in its natural or un-isolated state. Typically, a substantial portion of the sample comprises, e.g., greater than 1%, greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%-100% of the sample. For example, a sample of isolated mRNA can typically comprise at least about 1% total mRNA. Techniques for purifying polynucleotides are well known in the art and include, for example, gel electrophoresis, ion-exchange chromatography, affinity chromatography, flow sorting, and sedimentation according to density.

As used herein, the term “bound” indicates direct or indirect attachment. In the context of chemical structures, “bound” (or “bonded”) may refer to the existence of a chemical bond directly joining two moieties or indirectly joining two moieties (e.g., via a linking group or any other intervening portion of the molecule). The chemical bond may be a covalent bond, an ionic bond, a coordination complex, hydrogen bonding, van der Waals interactions, or hydrophobic stacking, or may exhibit characteristics of multiple types of chemical bonds. In certain instances, “bound” includes embodiments where the attachment is direct and embodiments where the attachment is indirect.

The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in a tissue biopsy form, containing one or more components of interest. In some embodiments, the sample is a biological sample, which refers to a sample obtained from a biological subject, including a sample of biological tissue or fluid origin, obtained, reached, or collected in vivo or in situ. A biological sample also includes samples from a region of a biological subject containing injured cells or tissues. Such samples can be, but are not limited to, organs, tissues, and cells isolated from a mammal. Exemplary biological samples include but are not limited to cell lysate, cells, tissues, organs, organelles, a biological fluid, a blood sample, a urine sample, a skin sample, and the like.

The term “inhibit” as used herein in connection with IL1α refers to reducing any biological activity of IL1α, and an “inhibitor of IL1α” refers to an agent that can reduce any biological activity of IL1α. For example, such an inhibitor may partially, substantially or completely inhibit the biological activity of IL1α.

The term “polymerase chain reaction” or “PCR” as used herein generally refers to a procedure wherein small amounts of a nucleic acid, RNA and/or DNA, are amplified as described, for example, in U.S. Pat. No. 4,683,195. Generally, sequence information from the ends or beyond of the region of interest needs to be available, such that oligonucleotide primers can be designed; these primers will be identical or similar in sequence to opposite strands of the template to be amplified. The 5′ terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage, or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. 1987, 51:263-273; PCR Technology (Stockton Press, NY, Erlich, ed., 1989).

The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.

As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

It is understood that wherever embodiments are described herein with the term “comprising” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. It is also understood that wherever embodiments are described herein with the phrase “consisting essentially of” otherwise analogous embodiments described in terms of “consisting of” are also provided.

The term “between” as used in a phrase as such “between A and B” or “between A-B” refers to a range including both A and B.

The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

It should be noted that if there is a discrepancy between a depicted structure and a name given to that structure, the depicted structure is to be accorded more weight. In addition, if the stereochemistry of a structure or a portion of a structure is not indicated with, for example, bold or dashed lines, the structure or portion of the structure is to be interpreted as encompassing all stereoisomers of it.

The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology, which are within the skill of those working in the art. Such techniques are explained fully in the literature. Examples of particularly suitable texts for consultation include the following: Sambrook et al., Molecular Cloning: A Laboratory Manual (4th ed. 2014); Glover, ed., DNA Cloning, Volumes I and II (2^nded. 1995); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, N.Y., 3rd ed. 1993); and Weir & Blackwell, eds., Handbook of Experimental Immunology, Volumes I-IV (5^thed. 1996).

Bermekimab is a monoclonal anti-IL-1a antibody having heavy and light chain amino acid sequences of SEQ ID NO: 158 and SEQ ID NO: 159 respectively.

5.2. Biomarkers and Methods of Use Thereof

5.2.1. Biomarkers for Pharmacodynamics or Pharmacokinetic Effects

As shown in Section 6 below, elevated levels of gene expression of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3 or a subset thereof in injured skin biopsy (e.g., following 24 hours of culture) are reduced by treatment with an IL1α inhibitor (in this case, an anti-IL1α antibody), and thus expression levels of these biomarkers or a subset thereof can be used to evaluate effects of IL1α blockade (in particular, IL1α blockade with an anti-IL1α antibody). Similarly, elevated levels of gene expression of GCSF (CSF3), CXCL1, IL6, GMCSF (CSF2), CCL20, CCL22, CXCL2, CXCL3, CXCL5, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL5, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, G0S2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1 are reduced by treatment with an IL1α inhibitor (in this case, an anti-IL1α antibody), and thus expression of these biomarkers or a subset thereof can be used to evaluate the effects of IL1α blockade (in particular, IL1α blockade with an anti-IL1α antibody). Similarly, secretion of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa or a subset thereof in injured skin biopsy are reduced by treatment with an IL1α inhibitor (for example, an anti-IL1α antibody, such as bermekimab), and thus these cytokines can also be used to evaluate effects of IL1α blockade (in particular, IL1α blockade with an anti-IL1α antibody, such as bermekimab).

In one aspect, provided herein is a method of determining a pharmacodynamics (PD) or pharmacokinetic (PK) effect of an inhibitor of IL1α comprising providing a sample from a subject; administering the inhibitor of IL1α to the sample; measuring levels of one or more biomarkers in the sample; and determining the pharmacodynamic or the pharmacokinetic effect of the inhibitor of IL1α based on the levels of the one or more biomarkers. In some embodiments, the method is used for determining the PD effect of the inhibitor of IL1α.

In some embodiments, the sample is obtained by biopsy procedure and contain injured skin cells. Typically the injury arises as a result of the biopsy procedure.

In one embodiment, the sample is a skin sample.

The size of a skin sample may affect the measurement of a pharmacodynamic (PD) or pharmacokinetic (PK) effect of an inhibitor of IL1α. For example, as shown in Section 6 below, evaluation of the ex vivo skin explant-based assay at 4-hour time point indicate that 4 mm biopsy size is effective for measurement of PD response. For clinical implementation, it may be advantageous for a skin biopsy to be as small as possible as this minimizes disruption to a subject. The inventors have found that the measurements obtained in a 4 mm skin biopsy were comparable to a larger 6 mm biopsy when assessed 4 hours post-biopsy. Thus, in some embodiments, the size of the skin biopsy sample is about 3.5 mm to 4.5 mm. In some embodiments, the size of the skin biopsy sample is at least 4 mm. In some embodiments, the size of the skin biopsy sample is about 3.5 mm. In some embodiments, the size of the skin biopsy sample is about 3.6 mm. In some embodiments, the size of the skin biopsy sample is about 3.7 mm. In some embodiments, the size of the skin biopsy sample is about 3.8 mm. In some embodiments, the size of the skin biopsy sample is about 3.9 mm. In some embodiments, the size of the skin biopsy sample is about 4 mm. In some embodiments, the size of the skin biopsy sample is about 4.1 mm. In some embodiments, the size of the skin biopsy sample is about 4.2 mm. In some embodiments, the size of the skin biopsy sample is about 4.3 mm. In some embodiments, the size of the skin biopsy sample is about 4.4 mm. In some embodiments, the size of the skin biopsy sample is about 4.5 mm. In some embodiments, the size of the skin biopsy sample is about 4.6 mm. In some embodiments, the size of the skin biopsy sample is about 4.7 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.9 mm. In some embodiments, the size of the skin biopsy sample is about 5.0 mm.

In one embodiment, the size of the skin sample is at least 4.0 mm. In another embodiment, the size of the skin sample is about 4.0 mm. In some embodiments, the biopsy sample (for example, a skin sample) obtained from the subject is cultured ex vivo. An inhibitor of IL1α can be administered to the sample (for example, a skin sample) prior to the ex vivo culturing. In some embodiments, an inhibitor of IL1α can be administered to the sample (for example, a skin sample) during the ex vivo culturing. In other embodiments, the sample (for example, a skin sample) is cultured ex vivo for a period of time prior to administering to the sample the inhibitor of IL1α.

In one embodiment the levels of the one or more biomarkers provided herein are measured in a skin sample at 4 hours post the biopsy procedure. In certain such embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of at least 4.0 mm at 4 hours post the biopsy procedure. In other embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of about 4.0 mm at 4 hours post the biopsy procedure.

In another embodiment, the levels of the one or more biomarkers provided herein are measured in a skin sample at 24 hours post the biopsy procedure. In certain such embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of at least 4.0 mm at 24 hours post the biopsy procedure. In other embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of about 4.0 mm at 24 hours post the biopsy procedure.

In addition, a local anaesthetic that is used during a biopsy procedure may affect the ability of an IL1α inhibitor to reduce the level of one or more biomarkers. For example, as shown in Section 6 below, lidocaine injection affects ability of anti-IL1α to reduce level of GCSF, CXCL1 and IL8 in culture supernatant at 4-hour, but not at 24-hour time point. Therefore, the levels of the one or more biomarkers can be measured at least 4 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 5 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 6 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 7 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 8 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 9 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 10 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 11 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 12 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 13 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 14 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 15 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 16 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 17 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 18 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 19 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 20 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 21 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 22 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 23 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 25 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 30 hours post administration of the inhibitor of IL1α.

In one embodiment, the levels of the one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In certain embodiments, the levels of one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab) and the one or more biomarkers are selected from the group consisting of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In one embodiment, the level of CSF3 (GCSF) is measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab). In another embodiment, the level of CXCL1 is measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab). In another embodiment, the level of IL6 is measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CSF3 (GCSF), CXCL1 and IL6 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of GCSF, CXCL1, IL6 and IL8 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of CCL20, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL6, IL8, and PTGS2 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

The time point at which the biomarkers are measured may also be defined according to the time elapsed since the biopsy procedure was performed. As discussed above, a local anaesthetic that is used in the biopsy procedure may affect the ability of an IL1α inhibitor to reduce the level of one or more biomarkers. Thus, in some embodiments, the levels of the one or more biomarkers are measured at least 4 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 5 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 6 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 7 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 8 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 9 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 10 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 11 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 12 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 13 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 14 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 15 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 16 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 17 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 18 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 19 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 20 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 21 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 22 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 23 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 24 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 25 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 30 hours post the biopsy procedure.

In one embodiment, the levels of the one or more biomarkers are measured at least 24 hours post the biopsy procedure.

In certain embodiments, the levels of one or more biomarkers are measured at least 24 hours post the biopsy procedure and the one or more biomarkers are selected from the group consisting of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAMD1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In one embodiment, the level of CSF3 (GCSF) is measured at least 24 hours post the biopsy procedure. In another embodiment, the level of CXCL1 is measured at least 24 hours post the biopsy procedure. In another embodiment, the level of IL6 is measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of CSF3 (GCSF), CXCL1 and IL6 are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of GCSF, CXCL1, IL6 and IL8 are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10 are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, and PTGS2 are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of CCL20, CCL3, CCL4, CCL8, CFB, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL1B, IL6, IL8, MMP3 are measured at least 24 hours post the biopsy procedure.

In one embodiment, the levels of CCL20, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, IL6, IL8, and PTGS2 are measured at least 24 hours post the biopsy procedure).

The effects of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab) can be determined by comparing the levels of the one or more biomarkers provided herein with reference levels of these biomarkers.

The reference levels of these biomarkers can be the levels of these biomarkers in a reference injured skin sample (for example, a skin biopsy sample) without any influence by an inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab), and the levels of the biomarkers in the sample and the levels of the biomarkers in the reference sample are measured at the same time point (for example, at least 4 hours or at least 24 hours post the biopsy procedure).

In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject prior to administration of the inhibitor of IL1α. In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject without administration of the inhibitor of IL1α. In some embodiments, the reference levels of the one or more biomarkers are pre-determined levels of the one or more biomarkers. In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample administered with a control agent (e.g., a positive control agent that inhibits IL1α, such as the anti-IL1α antibody R&D, clone 4414, or a negative control agent that does not inhibit IL1α).

When the levels of the biomarkers are compared to the references levels that represent the elevated levels of these biomarkers post injury (e.g. biopsy induced injury) but without the influence of an inhibitor of IL1α, the lower levels of the one or more biomarkers as compared with the reference levels indicate that the inhibitor is effective in inhibiting IL1α.

In some embodiments, the inhibitor is identified as effective in inhibiting IL1α if the levels of the one or more biomarkers in the sample are at least 20%, are at least 30%, at least 35%, at least 40%, or at least 50% less than the reference levels.

In one embodiment, the inhibitor is identified as effective in inhibiting IL1α if the levels of the one or more cytokine biomarkers secreted by the sample (for example, as measured by a Luminex assay) are at least 50% less than the reference levels. In another embodiment, the inhibitor is identified as effective in inhibiting IL1α if the mRNA levels (for example, as measured by a Nanostring assay) of the one or more biomarkers in the sample are at least 30% less than the reference levels. In another embodiment, the inhibitor is identified as effective in inhibiting IL1α if the mRNA levels (for example, as measured by an RNAseq assay) of the one or more biomarkers in the sample are at least 20% less than the reference levels.

In another aspect, a pharmacodynamics or pharmacokinetic effect of an inhibitor of IL1α is determined by administering the inhibitor directly to the subject. The subjects who received an effective IL1α inhibitor may be protected from injury-mediated upregulation (e.g. by biopsy procedure) of IL1α and subsequent downstream effects.

Thus, in some more embodiments, provided herein is a method for determining a pharmacodynamics or pharmacokinetic effect of an inhibitor of IL1α, comprises: i. obtaining a first sample from a subject; ii. administering an IL1α inhibitor to the subject; iii. obtaining a second sample from the subject; iv. measuring levels of one or more biomarkers in the first sample and the second sample; and v. determining that the treatment is effective when the levels of the one or more biomarkers in the second sample are lower than the levels of one or more biomarkers in the first sample. In some embodiments, the first sample and the second sample are from the same source.

In some embodiments, the second sample is obtained from the subject at 1.5 to 2.5 hours post the administration of the IL1α inhibitor to the subject. In some embodiments, the second sample is obtained from the subject at about 1.5 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 1.6 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 1.7 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 1.8 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 1.9 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.0 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.1 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.2 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.3 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.4 hours post the administration. In some embodiments, the second sample is obtained from the subject at about 2.5 hours post the administration.

The first and second samples may contain skin cells. In some embodiments, the sample is obtained by biopsy procedure and contain injured skin cells. In some embodiments, the size of the skin biopsy sample is about 3.5 mm to 4.5 mm. In some embodiments, the size of the skin biopsy sample is at least 4 mm. In some embodiments, the size of the skin biopsy sample is about 3.5 mm. In some embodiments, the size of the skin biopsy sample is about 3.6 mm. In some embodiments, the size of the skin biopsy sample is about 3.7 mm. In some embodiments, the size of the skin biopsy sample is about 3.8 mm. In some embodiments, the size of the skin biopsy sample is about 3.9 mm. In some embodiments, the size of the skin biopsy sample is about 4 mm. In some embodiments, the size of the skin biopsy sample is about 4.1 mm. In some embodiments, the size of the skin biopsy sample is about 4.2 mm. In some embodiments, the size of the skin biopsy sample is about 4.3 mm. In some embodiments, the size of the skin biopsy sample is about 4.4 mm. In some embodiments, the size of the skin biopsy sample is about 4.5 mm. In some embodiments, the size of the skin biopsy sample is about 4.6 mm. In some embodiments, the size of the skin biopsy sample is about 4.7 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.9 mm. In some embodiments, the size of the skin biopsy sample is about 5.0 mm.

In one embodiment, the size of the skin sample is at least 4.0 mm. In another embodiment, the size of the skin sample is about 4.0 mm.

In some embodiments, the biopsy sample obtained from the subject is cultured ex vivo.

In other embodiments, the sample used in the methods provided herein may comprise body fluids from a subject. In some embodiments, the sample is a blood sample. More detailed description of a sample is provided in Section 5.3 below.

In some embodiments, the levels of the one or more biomarkers provided herein can be measured at a time when IL1α would have been elevated should no inhibitor of IL1α be administrated (e.g., 4 to 24 hours post the biopsy procedure). In some embodiments, the levels of the one or more biomarkers are measured at least 4 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 5 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 6 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 7 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 8 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 9 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 10 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 11 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 12 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 13 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 14 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 15 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 16 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 17 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 18 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 19 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 20 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 21 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 22 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 23 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 24 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 25 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 30 hours post the biopsy procedure.

In one embodiment, the levels of the one or more biomarkers are measured at least 4 hours post the biopsy procedure. In another embodiment, the levels of the one or more biomarkers are measured at least 24 hours post the biopsy procedure.

In one embodiment, the one or more biomarkers comprise CSF3 (GCSF). In another embodiment, the one ore more biomarkers comprise CXCL1. In another embodiment, the one or more biomarkers comprise IL6.

In some embodiments, the method provided herein comprises measuring all of CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3. Totality of the expression levels of these biomarkers is used to determine the effects of the inhibitor. Any classification methods or algorithms useful for comparing the totality of the expressions of these biomarkers with a reference are included herein. For example, a composite score based on the expression levels of these biomarkers can be used. In some instances a composite score may be calculated based on the geometric mean of the expression levels of these biomarkers. A composite score based on comparing the expression levels of these biomarkers with and without treatment may be used. In some instances, a composite score may be calculated using the average of percentage of reduction on the expression levels (following treatment) of these biomarkers. This approach may allow variations of individual gene expression in different subjects. Similarly, this method also applies to a subset including 2 or more biomarkers selected from CCL20, CCL22, CSF3(GCSF), CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, IL6, IL8, PTGS2, CCL3, CCL4, CCL8, CFB, IL1B, and MMP3.

The levels of the biomarkers provided herein can be measured by determining protein levels or nucleic acid levels (e.g., mRNA, DNA or cDNA levels) of these biomarkers. In one embodiment, the levels of the biomarkes provided herein are measured by determining protein levels of these biomarkers. In another embodiment, the levels of the biomarkers provided herein are measured by determining mRNA levels of these biomarkers. Detailed description of these measurement methods are provided in the following sections.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of GCSF, CXCL1, IL4, IL6, IL8, MDC and IP10.

In some embodiments, the levels of the one or more biomarkers are the levels of cytokines secreted by the sample selected from a group consisting of CXCL1, GCSF, GMCSF, IL6, IL8, MIP1a and TGFa.

In some embodiments, the method provided herein comprises measuring all of GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa. Totality of the expression levels of these biomarkers is used to determine the effects of the inhibitor. Any classification methods or algorithms useful for comparing the totality of the expressions of these biomarkers with a reference are included herein. For example, a composite score based on the expression levels of these biomarkers can be used. In some instances a composite score may be calculated based on the geometric mean of the expression levels of these biomarkers. A composite score based on comparing the expression levels of these biomarkers with and without treatment may be used. In some instances, a composite score may be calculated using the average of percentage of reduction on the expression levels (following treatment) of these biomarkers. Similarly, this method also applies to a subset including 2 or more biomarkers selected from GCSF, CXCL1, IL4, IL6, IL8, MDC, IP10, GMCSF, MIP1a and TGFa.

In some embodiments, the levels of the biomarkers provided herein can be measured by determining protein levels of these biomarkers. Detailed description of these measurement methods are provided in the following sections.

In some embodiments of the various methods provided herein, the level(s) of the one or more biomarker(s) post treatment with the IL1α inhibitor is compared with the reference level(s) of the same biomarker(s) from a sample cultured for the same amount of time without the treatment, and compared with another reference level(s) of the same biomarker(s) in a control sample. In some embodiments, the control sample is a positive control using another IL1α inhibitor (for example, the anti-IL1α antibody R&D, clone 4414). In other embodiments, the control sample is a negative control using a compound that is not an IL1α inhibitor. In yet other embodiments, another reference level(s) of the same biomarker(s) in a control sample is the level(s) of the biomarker(s) in a control sample just obtained from the subject, e.g., within 30 mins after obtaining the sample from the subject or within the period before IL1α level is elevated.

In some embodiments, the various methods provided herein can be used to measure PD and/or PK effects of an anti-IL1α treatment in healthy subjects. In other embodiments, the various methods provided herein can be used to measure PD and/or PK effects of an anti-IL1α treatment in a subject having atopic dermatitis, hidradenitis suppurativa, psoriasis, cutaneous lupus erythematosus, or autoimmune bullous disease. In one embodiment, the various methods provided herein are used to measure PD and/or PK effects of an anti-IL1α treatment in a subject having atopic dermatitis. In another embodiment, the various methods provided herein are used to measure PD and/or PK effects of an anti-IL1α treatment in a subject having hidradenitis suppurativa.

5.2.2. Biomarkers for Predicting Responses to Treatments and Selective Treatments Using Biomarkers

The methods and biomarkers provided herein can be used to predict and evaluate the responsiveness to a treatment comprising an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In some embodiments, provided herein is a method of identifying a subject who is likely to be responsive to a treatment comprising an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In other embodiments, provided herein is a method of predicting the responsiveness of a subject to a treatment comprising an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In other embodiments, provided herein is a method of monitoring the response of a subject to a treatment comprising an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In yet other embodiments, provided herein is a method for determining dosing regimens of an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab).

In some embodiments, the method provided herein comprises contacting an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab) with a sample ex vivo. In other embodiments, the method provided herein comprises administering an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab) directly to a subject for predicting or monitoring responsiveness of the subject to the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab).

In one aspect, provided herein is a method for monitoring the response of a subject to a treatment comprising an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab) comprising providing a sample from the subject; administering the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab) to the sample; measuring levels of one or more biomarkers in the sample; and identifying the subject as being likely to be responsive to the treatment comprising the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab), predicting the responsiveness of the subject to the treatment comprising the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab), or monitoring the response of the subject to a treatment comprising the inhibitor of IL1α (for example an anti-IL1α antibody, such as bermekimab), based on the levels of the one or more biomarkers.

The effects of the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab) can be determined by comparing the levels of the one or more biomarkers provided herein with reference levels of these biomarkers.

The reference levels of these biomarkers can be the levels of these biomarkers in a reference injured skin sample (for example a skin biopsy sample) without any influence by an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab), and the levels of the biomarkers in the sample and the levels of the biomarkers in the reference sample are measured at the same time point (for example, at least 4 hours or at least 24 hours post the biopsy procedure).

In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject prior to administration of the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample from the subject without administration of the inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab). In some embodiments, the reference levels of the one or more biomarkers are pre-determined levels of the one or more biomarkers. In some embodiments, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference sample administered with a control agent (e.g., a positive control agent that inhibits IL1α, such as the anti-IL1α antibody R&D, clone 4414, or a negative control agent that does not inhibit IL1α).

In one embodiment, the reference levels of the one or more biomarkers are the levels of the one or more biomarkers in a reference skin biopsy sample from the subject without administration of the inhibitor of IL1α, and the levels of the biomarkers in the sample and the levels of the biomarkers in the reference sample are measured at the same time point.

When the levels of the biomarkers are compared to the references level that represent the elevated levels of these biomarkers post injury (e.g. biopsy induced injury) but without the influence of an inhibitor of IL1α(for example an anti-IL1α antibody, such as bermekimab), the lower levels of the one or more biomarkers as compared with the references indicate that subject is likely to be responsive to the treatment comprising the inhibitor of IL1α (for example an anti-IL1α antibody, such as bermekimab).

In some embodiments, the subject is identified likely to be responsive to the treatment comprising the inhibitor of IL1α if the levels of the one or more biomarkers in the sample are at least 20%, at least 30%, at least 35%, at least 40%, or at least 50% less than the reference levels.

In one embodiment, the subject is identified likely to be responsive to the treatment comprising the inhibitor of IL1α if the levels of the one or more cytokine biomarkers secreted by the sample (for example, as measured by a Luminex assay) are at least 50% less than the reference levels. In another embodiment, the subject is identified likely to be responsive to the treatment comprising the inhibitor of IL1α if the mRNA levels (for example, as measured by a Nanostring assay) of the one or more biomarkers in the sample are at least 30% less than the reference levels. In another embodiment, the subject is identified likely to be responsive to the treatment comprising the inhibitor of IL1α if the mRNA levels (for example, as measured by an RNAseq assay) of the one or more biomarkers in the sample are at least 20% less than the reference levels.

In some embodiments, the sample is obtained by biopsy procedure and contain injured skin cells. In some embodiments, the size of the skin biopsy sample is about 3.5 mm to 4.5 mm. In some embodiments, the size of the skin biopsy sample is at least 4 mm. In some embodiments, the size of the skin biopsy sample is about 3.5 mm. In some embodiments, the size of the skin biopsy sample is about 3.6 mm. In some embodiments, the size of the skin biopsy sample is about 3.7 mm. In some embodiments, the size of the skin biopsy sample is about 3.8 mm. In some embodiments, the size of the skin biopsy sample is about 3.9 mm. In some embodiments, the size of the skin biopsy sample is about 4 mm. In some embodiments, the size of the skin biopsy sample is about 4.1 mm. In some embodiments, the size of the skin biopsy sample is about 4.2 mm. In some embodiments, the size of the skin biopsy sample is about 4.3 mm. In some embodiments, the size of the skin biopsy sample is about 4.4 mm. In some embodiments, the size of the skin biopsy sample is about 4.5 mm. In some embodiments, the size of the skin biopsy sample is about 4.6 mm. In some embodiments, the size of the skin biopsy sample is about 4.7 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.8 mm. In some embodiments, the size of the skin biopsy sample is about 4.9 mm. In some embodiments, the size of the skin biopsy sample is about 5.0 mm.

In one embodiment, the size of the skin sample is at least 4.0 mm. In another embodiment, the size of the skin sample is about 4.0 mm.

In other embodiments, the sample used in the methods provided herein may comprises body fluids from a subject. In some embodiments, the sample is a blood sample. More detailed description of a sample is provided in Section 5.3 below.

In some embodiments, the biopsy sample (for example, a skin sample) obtained from the subject is cultured ex vivo. An inhibitor of IL1α can be administered to the sample (for example, a skin sample) prior to the ex vivo culturing. In some embodiments, an inhibitor of IL1α can be administered to the sample (for example, a skin sample) during the ex vivo culturing. In other embodiments, the sample (for example, a skin sample) is cultured ex vivo for a period of time prior to administering to the sample the inhibitor of IL1α.

In some embodiments the levels of the one or more biomarkers provided herein are measured in a skin sample at 4 hours post the biopsy procedure. In certain such embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of at least 4.0 mm at 4 hours post the biopsy procedure. In other embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of about 4.0 mm at 4 hours post the biopsy procedure.

In another emboimdent, the levels of the one or more biomarkers provided herein are measuered in a skin sample at 24 hours post the biopsy procedure. In certain such embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of at least 4.0 mm at 24 hours post the biopsy procedure. In other embodiments, the levels of one or more biomarkers provided herein are measured in a skin sample that has a size of about 4.0 mm at 24 hours post the biopsy procedure.

The levels of the one or more biomarkers can be measured at least 4 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 5 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 6 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 7 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 8 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 9 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 10 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 11 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 12 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 13 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 14 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 15 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 16 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 17 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 18 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 19 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 20 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 21 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 22 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 23 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 25 hours post administration of the inhibitor of IL1α. In some embodiments, the levels of the one or more biomarkers are measured at least 30 hours post administration of the inhibitor of IL1α.

In one embodiment, the levels of the one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In certain embodiments, the levels of one or more biomarkers are measured at least 24 hours post administration of the inhibitor of IL1α and the one or more biomarkers are selected from the group consisting of GCSF (CSF3), CXCL1, IL6, IL8, IL4, MDC, IP10, GMCSF (CSF2), MIP1a, TGFa, CCL20, CCL22, CXCL2, CXCL3, CXCL5, CXCL6, IL1B, PTGS2, CCL3, CCL4, CCL8, CFB, MMP3, FDCSP, PTX3, CCL3L3, CXCL8, FGF2, NEFM, TNIP3, MMP10, ADORA2A, DEFB4A, GPR84, AC243829.4, COLQ, TFPI2, SPRR2A, OXTR, CCL4L2, MMP1, TNFAIP6, HMX3, LIF, CD274, AC073862.2, TNFRSF11B, AC003092.1, SALL1, AC084871.4, CD38, NFKBIZ, ZC3H12A, SLC4A4, AL139393.3, MSC, SLC1A2, CCL2, GOS2, SLC34A2, AC073611.1, AC106865.1, AL078604.2, INHBA, LCN2, AC009303.4, TDO2, NGF, IER3, BCL2A1, SOD2, CEMIP, SAA1, TRAF1, GCH1, AC007780.1, AC007998.3, TMC1, GRM1, SPINK6, CD1D, IL17C, AC004264.1, EDNRB, DRAM1, CH25H, FOXD4L1, PTGES, SAA2, SLC39A14, RIPK2, PANX2, HS3ST3B1, IL23A, ROR1-AS1, AP002784.1, TMEM132A, ADAMTS5, OSM, RGS3, BMP6, STC1, SIRPA, RFX8, TMEM158, PAX1, IL24, ODAM, BCAT1, AC099494.2, ZNF697, C2CD4A, CCRL2, LAMA1, LSS, TGFB3, TM4SF1, CCL7, FFAR3, QPCTL, GRAIVID1A, DNAJB5, ARSG, NRP2, INSM1, ICAM1, REP15, ACSL4, AC073862.5, LYN, AKR1C1, SDK1, THBS2, IL4I1, PILRA, SLC39A8, NEU4, MT1A, NRIP3, C2CD4B, ASPHD1, SLC11A1, SPRR2B, NFKB2, TNFRSF9, and NKX3-1.

In one embodiment, the levels of CSF3 (GCSF), CXCL1 and IL6 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In one embodiment, the levels of GCSF, CXCL1, IL6 and IL8 are measured at least 24 hours post administration of the inhibitor of IL1α(for example, an anti-IL1α antibody, such as bermekimab).

In some embodiments, the levels of the one or more biomarkers are measured at least 4 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 5 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 6 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 7 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 8 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 9 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 10 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 11 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 12 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 13 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 14 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 15 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 16 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 17 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 18 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 19 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 20 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 21 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 22 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 23 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 24 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 25 hours post the biopsy procedure. In some embodiments, the levels of the one or more biomarkers are measured at least 30 hours post the biopsy procedure.