HK1215711B - Diagnosis and treatments relating to th2 inhibition - Google Patents

Description

Diagnosis and treatment related to TH2 suppression

This application is a divisional application of Chinese patent application 201180067674.8 (PCT/US2011/065410), filed on December 16, 2011, and entitled “Diagnosis and Treatment Related to TH2 Inhibition”.

Cross-reference to related applications

This application claims the benefit of priority to Provisional U.S. Application No. 61/459,760, filed December 16, 2010, Provisional U.S. Application No. 61/465,425, filed March 18, 2011, Provisional U.S. Application No. 61/484,650, filed May 10, 2011, Provisional U.S. Application No. 61/574,485, filed August 2, 2011, and Provisional U.S. Application No. 61/557,295, filed November 8, 2011, all of which are incorporated herein by reference in their entirety.

Sequence Listing

This application includes a sequence listing submitted in ASCII format via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy was created on November 17, 2011, is named P4569R1WO_PCTSequenceListing.txt, and is 73,118 bytes in size.

Technical Field

The present invention provides methods for diagnosing and treating conditions associated with TH2 inhibition, including but not limited to asthma. The present invention also provides methods for selecting or identifying patients for treatment with a particular therapeutic agent, wherein the therapeutic agent is a TH2 pathway inhibitor.

Background Art

Asthma is a complex disease with an increasing incidence worldwide. Among other things, eosinophilic inflammation has been reported in the airways of asthmatics. The pathophysiology of this disease is characterized by variable airflow obstruction, airway inflammation, mucus hypersecretion, and subepithelial fibrosis. Clinically, patients may present with cough, wheezing, and shortness of breath. Although many patients are adequately treated with currently available therapies, some asthmatics still have persistent disease despite using current therapies.

A wide variety of drugs for the treatment of asthma are on sale or in development. One of the many targets for the treatment of asthma is IL-13. IL-13 is a pleiotropic TH2 cytokine produced by activated T cells, NKT cells, basophils, eosinophils and mast cells, and it has been strongly suggested to be involved in the pathogenesis of asthma in preclinical models. Although there are many connections between IL-13, IL-4 and asthma in the literature, many IL13 and/or IL4 antagonist therapies have disappointing results in the clinic. Currently, no IL-13 or IL-4 antagonist therapy is approved for use in asthma. In addition, moderate to severe asthma patients continue to lack good alternative treatment options. Therefore, it is necessary to identify better therapies for the treatment of asthma and improved methods for understanding how to treat asthma patients.

All references cited herein, including patent applications and publications, are incorporated by reference in their entirety for any purpose.

SUMMARY OF THE INVENTION

The present application provides therapeutic agents for inhibiting the TH2 pathway and better methods of using the same. The present application also provides better disease diagnosis methods for use in the treatment of diseases (optionally with TH2 pathway inhibitors).

The therapeutic and diagnostic methods as provided herein can be applied to patients suffering from asthma, eosinophilic disorders, respiratory disorders, IL-13 mediated disorders and/or IgE mediated disorders, or symptoms associated with these disorders. Patients suffering from asthma-like symptoms, including patients who have not yet been diagnosed as having asthma, can be treated according to the methods provided herein.

According to one embodiment, the patient treated according to the methods provided herein has asthma, an eosinophilic disorder, a respiratory disorder, an IL-13 mediated disorder and/or an IgE mediated disorder, or symptoms associated with these disorders, and does not have cancer or a tumor. According to another embodiment, the patient treated according to the methods provided herein has asthma, an eosinophilic disorder, a respiratory disorder, an IL-13 mediated disorder and/or an IgE mediated disorder, or symptoms associated with these disorders, and is 12 years of age or older, 18 years of age or older, or 19 years of age or older, or between 12-17 years of age, or between 18-75 years of age.

In one embodiment, a patient treated with a TH2 pathway inhibitor according to the present invention is also treated with one, two, three or more therapeutic agents. In one embodiment, the patient is an asthma patient. According to one embodiment, the patient is treated with a TH2 pathway inhibitor and one, two, three or more therapeutic agents, wherein at least one therapeutic agent other than the TH2 inhibitor is a corticosteroid, a leukotriene antagonist, a LABA, a corticosteroid/LABA combination, theophylline, cromolyn sodium, nedocromil sodium, omalizumab, a LAMA, a MABA, a 5-lipoxygenase activating protein (FLAP) inhibitor, or a PDE-4 inhibitor. According to one aspect of the present invention, a TH2 pathway inhibitor is administered to an asthma patient diagnosed with an EIP state, wherein the diagnosis includes the use of an EID assay (alone or in combination with other assays) to determine the EIP state. In a further embodiment, the asthma patient was not controlled with corticosteroids prior to treatment. In another embodiment, the asthma patient is also being treated with a second controller. In one embodiment, the second controller is a corticosteroid, a LABA, or a leukotriene antagonist. In a further embodiment, the asthma patient has moderate to severe asthma. Thus, in one embodiment, the patient to be treated with a TH2 pathway inhibitor is a patient with moderate to severe asthma who was uncontrolled with corticosteroids prior to treatment with the TH2 pathway inhibitor, and the patient is subsequently treated with a TH2 pathway inhibitor and one, two, three, or more controllers. In one embodiment, at least one controller is a corticosteroid. In a further embodiment, such a patient is treated with a TH2 pathway inhibitor, a corticosteroid, and another controller. In another embodiment, the patient has mild asthma but is not currently being treated with a corticosteroid. It should be understood that the therapeutic agent may have a different treatment cycle than the TH2 inhibitor and, therefore, may be administered at a different time as part of the patient's treatment. Thus, according to one embodiment, a treatment method according to the present invention comprises administering to the patient a TH2 pathway inhibitor and, optionally, administering at least one, two, or three additional therapeutic agents. In one embodiment, the TH2 pathway inhibitor is present in a composition with the additional therapeutic agents. In another embodiment, the TH2 pathway inhibitor is not present in the composition with the additional therapeutic agent.

According to another embodiment, the present invention comprises a method for treating asthma comprising administering an anti-IL-13 antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10, or comprising HVRH1, HVRH2, HVRH3, HVRL1, HVRL2, and HVRL3 having the amino acid sequences of SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16, respectively, at a flat dose. In one embodiment, the anti-IL-13 antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10 is administered at a flat dose of 125-1000 mg (i.e., independent of body weight) by subcutaneous injection or by intravenous injection at a frequency selected from the group consisting of: every 2 weeks, every 3 weeks, and every 4 weeks. In one embodiment, an anti-IL-13 antibody comprising HVRH1, HVRH2, HVRH3, HVRL1, HVRL2, and HVRL3 having the amino acid sequences of SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16, respectively, is administered at a fixed dose of 125-1000 mg (i.e., independent of body weight) by subcutaneous injection or by intravenous injection at a frequency selected from: every 2 weeks, every 3 weeks, and every 4 weeks. In one embodiment, the anti-IL-13 antibody is lebrikizumab, which is administered at a fixed dose of 125-1000 mg (i.e., independent of body weight) by subcutaneous injection or by intravenous injection at a frequency selected from: every 2 weeks, every 3 weeks, and every 4 weeks. In another embodiment, a patient is diagnosed with EIP status by determining EIP status using a total periostin assay.

According to another embodiment, an antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10 is administered in a therapeutically effective amount to treat asthma, the therapeutically effective amount being sufficient to reduce the exacerbation rate over time or improve FEV1 in the patient. In yet another embodiment, the invention includes a method for treating asthma comprising administering an anti-IL-13 antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10, or comprising HVRH1, HVRH2, HVRH3, HVRL1, HVRL2, and HVRL3 having the amino acid sequences of SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16, respectively, at a fixed dose of 37.5 mg (i.e., independent of weight), or a fixed dose of 125 mg, or a fixed dose of 250 mg. In a specific embodiment, the dose is administered by subcutaneous injection once every 4 weeks for a period of time. In certain embodiments, the time period is 6 months, 1 year, 2 years, 5 years, 10 years, 15 years, 20 years, or the lifetime of the patient. In certain embodiments, the asthma is severe asthma, and the patient is inadequately controlled or uncontrolled on inhaled corticosteroids plus a second controller medication. In another embodiment, the patient is diagnosed with EIP status using a total periostin assay, and the patient is selected for treatment with an anti-IL-13 antibody as described above. In another embodiment, the method of the present invention comprises treating an asthma patient with an anti-IL-13 antibody as described above, wherein the patient has previously been diagnosed with EIP status using a total periostin assay. In one embodiment, the asthma patient is 18 years of age or older. In one embodiment, the asthma patient is 12-17 years of age, and the anti-IL-13 is administered at a fixed dose of 250 mg or a fixed dose of 125 mg. In one embodiment, the asthma patient is 6-11 years of age, and the anti-IL-13 antibody is administered at a fixed dose of 125 mg or a fixed dose of 62.5 mg.

The present invention provides periostin assays. In one embodiment, the periostin assay is a total periostin assay. In another embodiment, the total periostin assay comprises using one or more anti-periostin antibodies of the present invention to bind total periostin in a biological sample obtained from a patient. In another embodiment of the present invention, the biological sample is serum obtained from whole blood. In one embodiment, the biological sample is obtained from an asthma patient. In a further embodiment, the asthma patient is a moderate to severe asthma patient. In yet a further embodiment, the moderate to severe asthma patient is not controlled with corticosteroids and is optionally being treated with one, two, three or more controllers.

The anti-periostin assays and antibody assays disclosed herein can be used in other diseases in which periostin is elevated, such as idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), and cancer.

The present invention provides a TH2 pathway inhibitor as a therapeutic agent for treating asthma or respiratory disorders in a patient, wherein the patient expresses elevated levels of total periostin. In one embodiment, the target of inhibition in the TH2 pathway is selected from the group consisting of IL-9, IL-5, IL-13, IL-4, OX40L, TSLP, IL-25, IL-33, and IgE; and receptors such as IL-9 receptor, IL-5 receptor, IL-4 receptor α, IL-13 receptor α1 and IL-13 receptor α2, OX40, TSLP-R, IL-7Rα (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcεRI, and FcεRII/CD23 (receptor for IgE). In one embodiment, the patient to be treated according to the methods of the present invention has mild to severe asthma, optionally moderate to severe asthma, and their asthma is not controlled with corticosteroids. In a further embodiment, in patients with moderate to severe asthma not controlled on corticosteroids, the serum level of total periostin is greater than 20 ng/ml, 21 ng/ml, 22 ng/ml, 23 ng/ml, 24 ng/ml or 25 ng/ml in the E4 assay. In yet further embodiments, the patient to be treated also has elevated expression levels of any one, combination, or all of the following: CEA, TARC (CCL17), and MCP-4 (CCL13) mRNAs or proteins. In yet further embodiments, the patient to be treated, in addition to having elevated expression levels of periostin as described herein, also has FE _NO levels greater than 21 ppb or greater than 35 ppb.

The present invention provides the use of a therapeutic agent that binds to a target of a TH2-induced asthma pathway in the preparation of a medicament for treating a patient with asthma or a respiratory condition, wherein the patient expresses elevated levels of total periostin, and wherein the target is IL-9, IL-5, IL-13, IL-4, OX40L, TSLP, IL-25, IL-33, and IgE; and receptors such as IL-9 receptor, IL-5 receptor, IL-4 receptor α, IL-13 receptor α1 and IL-13 receptor α2, OX40, TSLP-R, IL-7Rα (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2, FcεRI, and FcεRII/CD23 (receptor for IgE). In one embodiment, the patient has EIP. According to one embodiment, the patient has been identified as having EIP using an assay according to the present invention. In another embodiment, the assay is a total periostin assay. In another embodiment, the assay measures the level of total periostin in a biological sample obtained from a patient. In one embodiment, the assay measures the level of total periostin protein in a serum sample obtained from a patient.

The present invention includes a kit or article of manufacture for diagnosing asthma subtype in a patient, comprising:

(1) determining the total periostin level and, optionally, the protein expression level of one or more proteins selected from TARC and MCP-4 in a serum sample obtained from a patient; and

(2) Instructions for measuring the expression levels of total periostin and optionally TARC and/or MCP-4 proteins in a serum sample, wherein elevated expression levels of any one, combination, or all of said proteins are indicative of an asthma subtype.

In another embodiment, a method for identifying an asthma patient or a respiratory disorder patient who may respond to treatment with a TH2 pathway inhibitor is provided. In a specific embodiment, the method comprises determining whether the patient is eosinophilic inflammation positive (EIP) using an eosinophilic inflammation diagnostic assay (EIDA), wherein an EIP status indicates that the patient may respond to treatment with a TH2 pathway inhibitor.

In another embodiment, a method of identifying an asthma patient or a respiratory disorder patient who is likely to suffer a severe exacerbation is provided. In a specific embodiment, the method comprises using EIDA to determine whether the patient is an EIP, wherein the EIP status indicates an increase in the patient's likelihood of suffering a severe exacerbation.

In another embodiment, a method of identifying an asthma patient or a respiratory disorder patient who is less likely to respond to treatment with a TH2 pathway inhibitor is provided. In a specific embodiment, the method comprises using EIDA to determine whether the patient is eosinophilic inflammation negative (EIN), wherein an EIN status indicates that the patient is less likely to respond to treatment with a TH2 pathway inhibitor.

In another embodiment, a method for monitoring an asthma patient being treated with a TH2 pathway inhibitor is provided. In a specific embodiment, the method comprises using EIDA to determine whether the patient has EIP or EIN. In one embodiment, the method comprises determining a treatment regimen for the TH2 pathway inhibitor. In one embodiment, a determination of EIP indicates continuation of treatment with the TH2 pathway inhibitor, while a determination of EIN indicates discontinuation of treatment with the TH2 pathway inhibitor.

In a specific embodiment, the EIDA used in the method described above comprises the steps of: (a) determining the total periostin amount in a sample obtained from an asthma patient; (b) comparing the total periostin amount determined in step (a) with a reference amount; and (c) stratifying the patient into a responder type or a non-responder type based on the comparison obtained in step (b). In a specific embodiment, the total periostin is serum periostin, and the periostin is measured using an immunoassay. In a specific embodiment, the immunoassay is a sandwich immunoassay. In a specific embodiment, the sandwich immunoassay is performed by an analyzer (Roche Diagnostics GmbH). In a specific embodiment, the sandwich immunoassay is an E4 assay. In one embodiment, when the E4 assay is used in step (a), the reference amount of EIP is 23 ng/ml or greater. In one embodiment, when the analyzer is used in step (a), the reference amount of EIP is 50 ng/ml or greater. In one embodiment, when the E4 assay is used in step (a), the reference amount of EIN is 21 ng/ml or less. In one embodiment, when an analyzer is used in step (a), the reference amount of EIN is 48 ng/ml or less.

In a specific embodiment, the patient according to the method described above suffers from moderate to severe asthma. In a specific embodiment, the asthma or respiratory condition is not controlled when using corticosteroids. In a specific embodiment, the corticosteroid is an inhaled corticosteroid. In a specific embodiment, the inhaled corticosteroid is or in one embodiment, the patient is also being treated with a second controller. In a specific embodiment, the second controller is a long-acting bronchodilator (LABD). In a specific embodiment, LABD is a long-acting beta-2 agonist (LABA), a leukotriene receptor antagonist (LTRA), a long-acting muscarinic antagonist (LAMA), theophylline, or an oral corticosteroid (OCS). In a specific embodiment, LABD is Perforomist ^™ or

In certain embodiments, the TH2 pathway inhibitor according to the above method inhibits targets ITK, BTK, IL-9 (e.g., MEDI-528), IL-5 (e.g., Mepolizumab, CAS No. 196078-29-2; resilizumab), IL-13 (e.g., IMA-026, IMA-638 (also known as anrukinzumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), tralokinumab (also known as CAT-354, CAS No. 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody), IL-4 (e.g., AER-001, IL4/IL13 trap), OX40L, TSLP, IL-25, IL-33, and IgE (e.g., XOLAIR, QGE-031; MEDI-4212); and receptors such as IL -9 receptor, IL-5 receptor (e.g., MEDI-563 (benralizumab, CAS No. 1044511-01-4)), IL-4 receptor alpha (e.g., AMG-317, AIR-645), IL-13 receptor alpha 1 (e.g., R-1671) and IL-13 receptor alpha 2, OX40, TSLP-R, IL-7Rα (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2 (e.g. AMG-853, AP768, AP-761, MLN6095, ACT129968), FcεRI and FcεRII/CD23 (receptors for IgE), Flap (e.g. GSK2190915), Syk kinase (R-343, PF3526299); CCR4 (AMG-761), TLR9 (QAX-935), or CCR3, IL5, IL3, GM-CSF (e.g. TPI ASM8). In certain embodiments, the TH2 pathway inhibitor is an anti-IL13/IL4 pathway inhibitor or an anti-IgE binder. In certain embodiments, the TH2 pathway inhibitor is an anti-IL-13 antibody. In certain embodiments, the anti-IL-13 antibody is an antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10, or an antibody comprising VH comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively. NO.: 16, or lebrikizumab. In certain embodiments, the anti-IL-13 antibody is a bispecific antibody that also binds to IL-4. In certain embodiments, the TH2 pathway inhibitor is an anti-IgE antibody. In certain embodiments, the anti-IgE antibody is (i) an antibody, (ii) an anti-M1 'antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain is SEQ ID NO: 24 and the variable light chain is SEQ ID NO: 25, or (iii) an anti-M1 'antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3, and the variable light chain further comprises HVR-L1, HVR-L2 and HVR-L3, and: (a) HVR-H1 is SEQ ID NO: NO:24, residues 26-35, [GFTFSDYGIA]; (b) HVR-H2 is residues 49-66 of SEQ ID NO:24, [AFISDLAYTIYYADTVTG]; (c) HVR-H3 is residues 97-106 of SEQ ID NO:24, [ARDNWDAMDY]; (d) HVR-L1 is residues 24-39 of SEQ ID NO:25, [RSSQSLVHNNANTYLH]; (e) HVR-L2 is residues 55-61 of SEQ ID NO:25, [KVSNRFS]; (f) HVR-L3 is residues 94-102 of SEQ ID NO:25, [SQNTLVPWT].

In another aspect, the present invention relates to the use of a kit for detecting total periostin in a sample obtained from an asthma patient for stratifying/classifying asthma patients into potential responders and non-responders for therapeutic treatment with a TH2 pathway inhibitor. In a specific embodiment, total periostin is detected using an EIDA comprising the steps of: (a) determining the amount of total periostin in a sample obtained from an asthma patient; (b) comparing the amount of total periostin determined in step (a) with a reference amount; and (c) stratifying the patient into a responder type or a non-responder type based on the comparison obtained in step (b). In a specific embodiment, total periostin is serum periostin, which is measured using an immunoassay. In a specific embodiment, the immunoassay is a sandwich immunoassay. In a specific embodiment, the sandwich immunoassay is performed by an analyzer (Roche Diagnostics GmbH). In a specific embodiment, the sandwich immunoassay is an E4 assay. In one embodiment, when the E4 assay is used in step (a), the reference amount of EIP is 23 ng/ml or greater. In one embodiment, when an analyzer is used in step (a), the reference amount of EIP is 50 ng/ml or greater.

In a specific embodiment, according to the uses described in the above paragraphs, the patient suffers from moderate to severe asthma. In a specific embodiment, the asthma or respiratory condition is not controlled with corticosteroids. In a specific embodiment, the corticosteroid is an inhaled corticosteroid. In a specific embodiment, the inhaled corticosteroid is or in one embodiment, the patient is also being treated with a second controller. In a specific embodiment, the second controller is a long-acting bronchodilator (LABD). In a specific embodiment, LABD is a long-acting beta-2 agonist (LABA), a leukotriene receptor antagonist (LTRA), a long-acting muscarinic antagonist (LAMA), theophylline, or an oral corticosteroid (OCS). In a specific embodiment, LABD is Perforomist ^™ or

In certain embodiments, according to the uses above, the TH2 pathway inhibitor inhibits targets ITK, BTK, IL-9 (e.g., MEDI-528), IL-5 (e.g., mepolizumab, CAS No. 196078-29-2; resilizumab), IL-13 (e.g., IMA-026, IMA-638 (also known as anrulizumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), tra lokinumab (also known as CAT-354, CAS No. 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody), IL-4 (e.g., AER-001, IL4/IL13 trap), OX40L, TSLP, IL-25, IL-33, and IgE (e.g., XOLAIR, QGE-031; MEDI-4212); and receptors such as IL-9 receptor, IL-5 receptors (e.g., MEDI-563 (benralizumab, CAS No. 1044511-01-4)), IL-4 receptor alpha (e.g., AMG-317, AIR-645), IL-13 receptor alpha 1 (e.g., R-1671) and IL-13 receptor alpha 2, OX40, TSLP-R, IL-7Rα (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, C RTH2 (e.g. AMG-853, AP768, AP-761, MLN6095, ACT129968), FcεRI and FcεRII/CD23 (IgE receptors), Flap (e.g. GSK2190915), Syk kinase (R-343, PF3526299); CCR4 (AMG-761), TLR9 (QAX-935), or CCR3, IL5, IL3, GM-CSF (e.g. TPI ASM8). In certain embodiments, the TH2 pathway inhibitor is an anti-IL13/IL4 pathway inhibitor or an anti-IgE binder. In certain embodiments, the TH2 pathway inhibitor is an anti-IL-13 antibody. In certain embodiments, the anti-IL-13 antibody is an anti-IL-13 antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10, or comprising VLs having SEQ ID NOs.: 11, 12, 13, 14, 15, and 16, respectively. NO.: 16, or lebrikizumab. In certain embodiments, the anti-IL-13 antibody is a bispecific antibody that also binds to IL-4. In certain embodiments, the TH2 pathway inhibitor is an anti-IgE antibody. In certain embodiments, the anti-IgE antibody is (i) an antibody, (ii) an anti-M1 'antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain is SEQ ID NO: 24 and the variable light chain is SEQ ID NO: 25, or (iii) an anti-M1 'antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain further comprises HVR-H1, HVR-H2 and HVR-H3, and the variable light chain further comprises HVR-L1, HVR-L2 and HVR-L3, and: (a) HVR-H1 is SEQ (a) HVR-L2 is residues 55-61 of SEQ ID NO:25, [KVSNRFS]; (b) HVR-H2 is residues 49-66 of SEQ ID NO:24, [AFISDLAYTIYYADTVTG]; (c) HVR-H3 is residues 97-106 of SEQ ID NO:24, [ARDNWDAMDY]; (d) HVR-L1 is residues 24-39 of SEQ ID NO:25, [RSSQSLVHNNANTYLH]; (e) HVR-L2 is residues 55-61 of SEQ ID NO:25, [KVSNRFS]; (f) HVR-L3 is residues 94-102 of SEQ ID NO:25, [SQNTLVPWT].

In another aspect, a kit for measuring total periostin in a biological sample obtained from an asthmatic patient or a patient with a respiratory condition is provided, wherein the kit comprises a first nucleic acid molecule hybridized to a second nucleic acid molecule, wherein the second nucleic acid molecule encodes total periostin or a portion thereof, or the kit comprises an antibody that binds to total periostin. In a specific embodiment, the kit comprises a package insert containing information describing the uses provided above.

In yet another aspect, a kit for diagnosing asthma subtype in a patient is provided, the kit comprising: (1) determining the level of total periostin and, optionally, the protein expression level of one or more proteins selected from TARC and MCP-4 in a serum sample obtained from the patient; and (2) instructions for measuring the level of total periostin and, optionally, TARC and/or MCP-4 in the serum sample, wherein elevated expression levels of any one, combination, or all of the proteins indicate an asthma subtype. In a specific embodiment, the kit further comprises a package insert for determining whether an asthma patient or a respiratory disorder patient has EIP or EIN. In a specific embodiment, the kit further comprises a package insert for determining whether an asthma patient is likely to respond to a TH2 pathway inhibitor. In a specific embodiment, the kit further comprises a package insert containing information describing any of the uses provided above. In a specific embodiment, the kit further comprises an empty container for holding a biological sample. In a specific embodiment, the kit comprises two anti-periostin antibodies for use in an immunoassay for determining total periostin levels.

In another aspect, a method of treating asthma or a respiratory condition comprises administering an anti-IL-13 antibody comprising HVRH1, HVRH2, HVRH3, HVRL1, HVRL2, and HVRL3, wherein each HVR has the amino acid sequence of SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15, and SEQ ID NO.: 16, to a patient suffering from asthma or a respiratory condition at a fixed dose of 125-500 mg every 2-8 weeks. In certain embodiments, the patient has moderate to severe asthma. In certain embodiments, the asthma or respiratory condition is uncontrolled with corticosteroids. In certain embodiments, the asthma or respiratory condition is uncontrolled with inhaled corticosteroids. In certain embodiments, the asthma or respiratory condition is uncontrolled with a total daily dose of at least 500 mcg of fluticasone propionate (FP). In certain embodiments, the corticosteroid is an inhaled corticosteroid, and in certain embodiments, the patient is being treated with a secondary controller. In certain embodiments, the patient continues to be treated with corticosteroids, optionally inhaled corticosteroids, during treatment with the anti-IL-13 antibody. In certain embodiments, the patient continues to be treated with a second control agent during treatment with the anti-IL-13 antibody. In certain embodiments, the second control agent is a long-acting bronchodilator. In certain embodiments, the long-acting bronchodilator is a LABA, LTRA, LAMA, theophylline, or OCS. In certain embodiments, the patient has been identified as EIP. In certain embodiments, the patient has been identified as EIP using the kit described above. In certain embodiments, the patient has been identified as EIP using the methods described above. In certain embodiments, the patient is administered a fixed dose of 125 mg or 250 mg every four weeks. In certain embodiments, the patient is 18 years of age or older, or the patient is 12-17 years of age or 12 years of age and older, or the patient is 6-11 years of age or 6 years of age and older.

In certain embodiments, the anti-IL-13 antibody is administered subcutaneously. In certain embodiments, the anti-IL-13 antibody is administered using a prefilled syringe or an automatic injection device. In certain embodiments, the asthma patient to be treated according to the above methods is 18 years of age or older and has a serum periostin of ≥50 ng/mL and is uncontrolled with inhaled corticosteroids and a second controller medication. In certain embodiments, serum periostin is measured using an immunoassay selected from a periostin assay and an E4 assay. In certain embodiments, serum periostin is measured using a kit as described above. In certain embodiments, the anti-IL-13 antibody is an antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10. In certain embodiments, the anti-IL-13 antibody is lebrikizumab. In certain embodiments, the asthma patient to be treated according to the above methods is 12 years of age or older and is uncontrolled with inhaled corticosteroids and a second controller medication.

In another aspect, methods of treating asthma or respiratory disease are provided, comprising administering a therapeutically effective amount of lebrikizumab to a patient. In certain embodiments, treatment results in a relative improvement in FEV1 of greater than 5% compared to before treatment with lebrikizumab. In certain embodiments, the relative improvement in FEV1 is greater than 8% compared to before treatment with lebrikizumab. In certain embodiments, treatment results in a reduction in severe exacerbations.

In another aspect, a method of treating a patient suffering from asthma or a respiratory disorder is provided, comprising administering a TH2 pathway inhibitor to a patient diagnosed with EIP. In certain embodiments, the method comprises the step of diagnosing the patient with EIP using a total periostin assay. In certain embodiments, the method further comprises the step of re-treating the patient with a TH2 pathway inhibitor if the patient is determined to have EIP. In certain embodiments, serum from the patient is used to determine whether the patient has EIP.

In a specific embodiment, according to the above method, the determination of EIP status uses an EIDA comprising the following steps: (a) determining the total periostin amount in a sample obtained from the patient; (b) comparing the total periostin amount determined in step (a) with a reference amount; and (c) stratifying the patient into a responder type or a non-responder type based on the comparison obtained in step (b). In a specific embodiment, the total periostin is serum periostin, and the periostin is measured using an immunoassay. In a specific embodiment, the immunoassay is a sandwich immunoassay. In a specific embodiment, the sandwich immunoassay is performed by an analyzer (Roche Diagnostics GmbH). In a specific embodiment, the sandwich immunoassay is an E4 assay. In one embodiment, when the E4 assay is used in step (a), the reference amount of EIP is 23 ng/ml or greater. In one embodiment, when the analyzer is used in step (a), the reference amount of EIP is 50 ng/ml or greater.

In certain embodiments, according to the methods described above, the patient suffers from moderate to severe asthma. In certain embodiments, the asthma or respiratory condition is not controlled with corticosteroids. In certain embodiments, the corticosteroid is an inhaled corticosteroid. In certain embodiments, the inhaled corticosteroid is or in one embodiment, the patient is also being treated with a second controller. In certain embodiments, the second controller is a long-acting bronchodilator (LABD). In certain embodiments, the LABD is a long-acting beta-2 agonist (LABA), a leukotriene receptor antagonist (LTRA), a long-acting muscarinic antagonist (LAMA), theophylline, or an oral corticosteroid (OCS). In certain embodiments, the LABD is Perforomist ^™ or

In certain embodiments, according to the above methods, the TH2 pathway inhibitor inhibits targets ITK, BTK, IL-9 (e.g., MEDI-528), IL-5 (e.g., mepolizumab, CAS No. 196078-29-2; resilizumab), IL-13 (e.g., IMA-026, IMA-638 (also known as anrulizumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), tr alokinumab (also known as CAT-354, CAS No. 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody), IL-4 (e.g., AER-001, IL4/IL13 trap), OX40L, TSLP, IL-25, IL-33, and IgE (e.g., QGE-031; MEDI-4212); and receptors such as IL-9 receptor, IL-5 receptor (e.g., Such as MEDI-563 (benralizumab, CAS No. 1044511-01-4)), IL-4 receptor α (such as AMG-317, AIR-645), IL-13 receptor α1 (such as R-1671) and IL-13 receptor α2, OX40, TSLP-R, IL-7Rα (TSLP co-receptor), IL17RB (IL-25 receptor), ST2 (IL-33 receptor), CCR3, CCR4, CRT H2 (e.g. AMG-853, AP768, AP-761, MLN6095, ACT129968), FcεRI and FcεRII/CD23 (IgE receptors), Flap (e.g. GSK2190915), Syk kinase (R-343, PF3526299); CCR4 (AMG-761), TLR9 (QAX-935), or CCR3, IL5, IL3, GM-CSF (e.g. TPI In certain embodiments, the TH2 pathway inhibitor is an anti-IL13/IL4 pathway inhibitor or an anti-IgE binder. In certain embodiments, the TH2 pathway inhibitor is an anti-IL-13 antibody. In certain embodiments, the anti-IL-13 antibody is an antibody comprising a VH comprising SEQ ID NO: 9 and a VL comprising SEQ ID NO: 10, or an anti-IL-13 antibody comprising HVRH1, HVRH2, HVRH3, HVRL1, HVRL2, and HVRL3 having the amino acid sequences of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively, or lebrikizumab. In certain embodiments, the anti-IL-13 antibody is a bispecific antibody that also binds to IL-4.

In certain embodiments, the TH2 pathway inhibitor is an anti-IgE antibody. In certain embodiments, the anti-IgE antibody is (i) an antibody, (ii) an anti-M1′ antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain is SEQ ID NO: 24 and the variable light chain is SEQ ID NO: 25, or (iii) an anti-M1′ antibody comprising a variable heavy chain and a variable light chain, wherein the variable heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3, and the variable light chain further comprises HVR-L1, HVR-L2, and HVR-L3, and: (a) HVR-H1 is residues 26-35 of SEQ ID NO: 24, [GFTFSDYGIA]; (b) HVR-H2 is residues 49-66 of SEQ ID NO: 24, [AFISDLAYTIYYADTVTG]; (c) HVR-H3 is residues 97-106 of SEQ ID NO: 24, [ARDNWDAMDY]; (d) HVR-L1 is residues 107-111 of SEQ ID NO: 24, [ARDNWDAMDY]; NO:25, residues 24-39, [RSSQSLVHNNANTYLH]; (e) HVR-L2 is residues 55-61 of SEQ ID NO:25, [KVSNRFS]; (f) HVR-L3 is residues 94-102 of SEQ ID NO:25, [SQNTLVPWT].

In another aspect, a method for assessing adverse events associated with treating asthma with lebrikizumab in a patient is provided. In a specific embodiment, the method includes the step of monitoring the number and/or severity of events, wherein the event is an exacerbation, community acquired pneumonia, allergic reaction, musculoskeletal pain, musculoskeletal disorders, connective tissue pain, or a connective tissue disorder. In a specific embodiment, the musculoskeletal or connective tissue disorder is arthralgia, back pain, pain in the limbs, myalgia, neck pain, arthritis, bone dysplasia, bursitis, costochondritis, exostoses, flank pain, musculoskeletal chest pain, musculoskeletal pain, jaw pain, or tendinitis.

In another aspect, an anti-periostin antibody is provided. In a specific embodiment, the anti-periostin antibody comprises the HVR sequence of SEQ ID NO: 1 and the HVR sequence of SEQ ID NO: 2. In a specific embodiment, the anti-periostin antibody comprises the sequence of SEQ ID NO: 1 and SEQ ID NO: 2. In a specific embodiment, the anti-periostin antibody comprises the HVR sequence of SEQ ID NO: 3 and the HVR sequence of SEQ ID NO: 4. In a specific embodiment, the anti-periostin antibody comprises the sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In a specific embodiment, a total periostin assay comprising the use of the above-mentioned anti-periostin antibody is provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 provides a schematic diagram of the allergen challenge assay described in Example 1.

Figure 2 shows allergen-induced changes in FEV1 at screening (A) and after challenge at week 13 (B). After allergen challenge, FEV1 was measured every 10 minutes for the first 90 minutes and then every hour from 2 to 8 hours. Error bars represent standard errors of the mean.

Figure 3 shows serum levels of IgE, CCL13 (MCP-4), and CCL17 (TARC). Serum levels are expressed as percentages (%) of pre-dose levels over time (A) in placebo-treated patients and (B) in lebrikizumab-treated patients. Lines represent individual patients; group medians are not shown. Arrows indicate dosing at weeks 0, 4, 8, and 12 (days 0, 28, 56, and 84). (C) Reduction of IgE, CCL13, and CCL17 at week 13 in individual patients (relative to those marked baseline levels).

Figure 4 shows lebrikizumab-induced suppression of late asthmatic response (LAR) in biomarker-high and biomarker-low patient subgroups. Data are presented as placebo-corrected mean reduction in AUC (area under the curve) of LAR at Week 13 (n=5-8 active patients/group).

FIG5 provides a schematic diagram of the asthma assay described in Example 2.

FIG6 provides baseline characteristics of patients participating in the asthma trial of Example 2.

FIG7 provides the results of the asthma trial from Example 2.

Figure 8 provides FEV1 results from the asthma trial of Example 2 for all efficacy evaluable subjects (A) and for only periostin high subjects (B).

FIG9 provides the reduction rate of exacerbations from the asthma trial of Example 2.

FIG10 provides the percent change in FE _NO from the asthma trial of Example 2.

FIG11 provides safety results from the asthma trial of Example 2.

FIG12 provides a schematic diagram of the asthma assay described in Example 3.

FIG13 provides a schematic diagram of the asthma observational study as described in Example 5.

Figure 14 shows the correlation between serum periostin levels across multiple visits within subjects in the BOBCAT cohort, as described in Example 5. (A) Correlation between Visit 1 and Visit 2; (B) Correlation between Visit 1 and Visit 3; (C) Correlation between Visit 2 and Visit 3; (D) Correlation between Visit 1 and the average serum periostin across all visits; (E) Correlation between Visit 2 and the average serum periostin across all visits; and (F) Correlation between Visit 3 and the average serum periostin across all visits.

Figure 15 shows that FE _NO can differentiate moderate to severe asthma uncontrolled on high-dose ICS based on airway eosinophilic inflammation (BOBCAT cohort), as described in Example 5. (A) Asthma with >3% sputum eosinophils had significantly elevated FE _NO compared to asthma with <3% sputum eosinophils (p<0.001 by Wilcoxon rank sum test). (B) Asthma with >22 eosinophils/mm2 of total bronchial tissue had a trend toward elevated FE _NO compared to asthma with <22 eosinophils/mm2 of total bronchial tissue (p=0.07 by Wilcoxon rank sum test). (C) A strong positive trend (p=0.001 by logistic regression) of increasing FE NO levels with increasing composite airway eosinophil scores was demonstrated by composite airway eosinophil score, where 0=sputum eosinophils <3% and tissue ("Bx") eosinophils <22/mm2; 1=sputum eosinophils >3% or tissue eosinophils >22/mm2 (mutually exclusive); 2=sputum eosinophils >3% and tissue eosinophils >22 _{/mm2. Serum periostin status is shown in the legend. (D) Serum periostin and FE NO were} elevated in most subjects with elevated sputum or tissue eosinophils, but a subset of subjects had elevations in only periostin or FE _NO . Most subjects who lacked elevated sputum and tissue eosinophils showed low serum periostin and low FE _NO . (E) Receiver operating characteristic (ROC) curve analysis of sensitivity and specificity of serum periostin, FE _NO , blood eosinophils, and serum IgE for composite airway eosinophil status. AUC = area under the curve.

FIG16 provides the percentage of patients without treatment failure in the asthma trial described in Example 3.

Figure 17 shows a comparison between serum periostin levels for samples from the clinical trials described in Examples 3 and 5, as measured on an assay similar to the Example 4 assay (E4 assay) or the periostin assay (Example 7).

FIG18 shows serum periostin pharmacodynamics in periostin-low patients (A) and periostin-high patients (B), as described in Example 2.

19 shows the percent change in median periostin levels over time in placebo- and lebrikizumab-treated patients, as described in Example 2.

20 shows the correlation between % change in FEV1 and body weight at Week 12 for subjects in the Phase II study described in Example 2 (A) and the Phase II study described in Example 3 (B), as described in Example 6.

FIG21 shows the predicted proportions of patients having steady-state trough concentrations above various levels, as described in Example 6.

FIG22 shows the effect of lebrikizumab on serum CCL17 (TARC) levels over time for Study 2 (A) and Study 3 (B), as described in Example 6.

Figure 23 shows simulated population PK curves as described in Example 6 at the following Phase III doses: 250 mg every 4 weeks, 125 mg every 4 weeks, and 37.5 mg every 4 weeks.

Detailed Description of the Invention

Unless defined otherwise, technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry: Reactions, Mechanisms and Structure, 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide those skilled in the art with a general guidance as to many of the terms used herein.

Some definitions

For the purpose of interpreting this specification, the following definitions will apply and, where appropriate, terms used in the singular will also encompass the plural, and vice versa. In the event of a conflict between any definition given below and any document incorporated herein by reference, the definition given below will control.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include reference to the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" or "an antibody" includes a plurality of proteins or antibodies, respectively; reference to "a cell" includes a mixture of cells, etc.

As used herein, the term "total periostin" refers to at least isoforms 1, 2, 3, and 4 of periostin. According to the NCBI database, human periostin isoforms 1, 2, 3, and 4 are known in the art and comprise the following amino acid sequences, respectively: NP_006466.2 (SEQ ID NO: 19); NP_001129406.1 (SEQ ID NO: 20), NP_001129407.1 (SEQ ID NO: 21), and NP_001129408.1 (SEQ ID NO: 22). In addition, the applicant has detected an additional form of periostin. This new isoform is referred to herein as "isoform 5" and has been partially sequenced. Isoform 5 comprises the amino acid sequence of SEQ ID NO: 23. In one embodiment, the periostin isoform is human periostin. In a further embodiment, the term total periostin includes isoform 5 of human periostin plus isoforms 1-4. In another embodiment, the total periostin is total serum periostin or total plasma periostin (ie, total periostin in a serum sample obtained from whole blood or in a plasma sample obtained from whole blood, the whole blood being obtained from a patient).

The term "total periostin assay" refers to an assay that measures the total periostin level in a biological sample. In one embodiment, the total periostin level is measured using an anti-periostin antibody. In another embodiment, the anti-periostin antibody is an anti-periostin antibody described herein. In another example, the total periostin level is measured using one or more nucleic acid sequences that are antisense to the mRNA encoding periostin isoforms 1-4. In another example, the total periostin assay is the assay described in Example 4 ("Example 4 assay" or "E4 assay"). In one embodiment, a total periostin assay comprises: (1) using an antibody comprising the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 ("25D4" antibody) and/or an antibody comprising the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 ("23B9" antibody) to bind periostin in a biological sample, (2) using an antibody comprising the variable region sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and/or an antibody comprising the variable region sequences of SEQ ID NO: 3 and SEQ ID NO: 4 to bind periostin in a biological sample, (3) using an antibody comprising the HVR sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and/or an antibody comprising the HVR sequences of SEQ ID NO: 3 and SEQ ID NO: 4 to bind periostin in a biological sample, (4) using an antibody comprising an HVR sequence that is 95% or more identical to the HVR sequences of SEQ ID NO: 1 and SEQ ID NO: 2 and/or an antibody comprising an HVR sequence that is 95% or more identical to the HVR sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

As used herein, an "Eosinophilic Inflammation Diagnostic Assay," abbreviated as "EIDA," is an assay in which a patient with eosinophilic inflammation in the body or TH2 pathway inflammation in the body is diagnosed by measuring the level of a marker of eosinophilic inflammation in a biological sample from the patient, wherein the marker is selected from periostin mRNA levels or periostin protein levels, iNOS mRNA levels or iNOS protein levels, or FE _NO levels or CCL26 mRNA or CCL26 protein levels, serpinB2 mRNA levels or serpinB2 protein levels, serpinB4 mRNA levels or serpinB4 protein levels, CST1 mRNA levels or CST1 protein levels, CST2 mRNA levels or CST2 protein levels, CST4 mRNA levels or CST4 protein levels. In one embodiment, total periostin serum or plasma levels are measured. Examples of highly validated assays include, but are not limited to, those described in Example 4 below (also referred to as the E4 assay), or other periostin assays that measure total periostin serum or plasma levels in a biological sample. Two or more assays can be performed to make a diagnosis of eosinophilic inflammation in a patient. In one embodiment, the EID assay includes a total periostin assay combined with a FE _NO assay. In another embodiment, the EID assay includes a total periostin assay +/- FE _NO level assay, and a combination of assays that measure the level of any one or combination of the following markers: CST1, CST2, CCL26, CLCA1, PRR4, PRB4, SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, and SERPINB10.

The term "periostin antibody" or "anti-periostin antibody" refers to an antibody that binds to an isoform of periostin. In one embodiment, the periostin is human periostin. In one embodiment, the antibody comprises the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 ("25D4" antibody), or comprises the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 ("23B9" sequence). In another embodiment, the antibody comprises the variable region sequence of SEQ ID NO: 1 and SEQ ID NO: 2, or comprises the variable region sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the antibody comprises the HVR sequence of SEQ ID NO: 1 and SEQ ID NO: 2 or the HVR sequence of SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the antibody comprises an HVR sequence that is 95% or more identical to the HVR sequence of SEQ ID NO: 1 and SEQ ID NO: 2, and/or the antibody comprises an HVR sequence that is 95% or more identical to the HVR sequence of SEQ ID NO: 3 and SEQ ID NO: 4.

Eosinophilic inflammation positive (EIP) patient or condition: refers to a patient who, if a serum or plasma sample from the patient had been tested for serum or plasma periostin levels using the E4 assay (Example 4), would have a total serum periostin level of 20 ng/ml or greater (eosinophil positive). According to one embodiment, the total periostin level in a patient with EIP may be selected from the group consisting of: 21 ng/ml or more, 22 ng/ml or more, 23 ng/ml or more, 24 ng/ml or more, 25 ng/ml or more, 26 ng/ml or more, 27 ng/ml or more, 28 ng/ml or more, 29 ng/ml or more, 30 ng/ml or more, 31 ng/ml or more, 32 ng/ml or more, 33 ng/ml or more, 34 ng/ml or more, 35 ng/ml or more, 36 ng/ml or more, 37 ng/ml or more, 38 ng/ml or more, 39 ng/ml or more, 40 ng/ml or more, 41 ng/ml or more, 42 ng/ml or more, 43 ng/ml or more, 44 ng/ml or more, ng/ml or more, 45 ng/ml or more, 46 ng/ml or more, 47 ng/ml or more, 48 ng/ml or more, 49 ng/ml or more, 50 ng/ml or more, 51 ng/ml or more, 52 ng/ml or more, 53 ng/ml or more, 54 ng/ml or more, 55 ng/ml or more, 56 ng/ml or more, 57 ng/ml or more, 58 ng/ml or more, 59 ng/ml or more, 60 ng/ml or more, 61 ng/ml or more, 62 ng/ml or more, 63 ng/ml or more, 64 ng/ml or more, 65 ng/ml or more, 66 ng/ml or more, 67 ng/ml or more, 68 ng/ml or more, 69 ng/ml or more, and 70 ng/ml or more. It will be understood that EIP status represents the patient's status and is independent of the type of assay used to determine that status. Therefore, other eosinophilic inflammation diagnostic assays, including other periostin assays such as the periostin assay shown in Example 7, can be used or developed to test for eosinophilic inflammation-positive status.

Eosinophilic inflammation negative (EIN) patient or state refers to such patient, if serum or plasma sample from this patient has been tested serum or plasma periostin level using E4 assay method, then this patient will have the total serum periostin level of less than 20ng/ml.It should be understood that EIN state represents the state of patient, and does not rely on the assay method type for measuring this state.Therefore, other eosinophilic inflammation diagnostic assays, including other periostin assays such as the periostin assay method shown in Example 7, can be used for or developed for testing eosinophilic inflammation negative state.

As used herein, the term "biological sample" includes, but is not limited to, blood, serum, plasma, sputum, tissue biopsy (eg, lung sample), and nasal samples, including nasal swabs or nasal polyps.

The FE _NO assay refers to an assay that measures the level of FE _NO (fractional exhaled nitric oxide). This level can be assessed using, for example, the handheld portable device NIOX (Aerocrine, Solna, Sweden) according to the guidelines published by the American Thoracic Society (ATS) in 2005. FE _NO may be referred to as other similar methods such as FeNO or FENO, and it should be understood that all such similar variations have the same meaning.

The age of the patient to be tested or treated according to the methods provided herein includes all ages. In one embodiment, the age is 18+ years old. In another embodiment, the age is 12+ years old. In yet another embodiment, the age is 2+ years old. In one embodiment, the age is 18-75 years old, 12-75 years old, or 2-75 years old.

Asthma is a complex disorder characterized by changing and recurring symptoms, reversible airflow obstruction (e.g., with bronchodilators), and bronchial hyperresponsiveness, which may or may not be associated with underlying inflammation. Examples of asthma include aspirin-sensitive/exacerbated asthma, atopic asthma, severe asthma, mild asthma, moderate to severe asthma, corticosteroid-naive asthma, chronic asthma, corticosteroid-resistant asthma, corticosteroid-refractory asthma, newly diagnosed and untreated asthma, smoking-induced asthma, asthma that is uncontrolled with corticosteroids, and other asthma as described in J Allergy Clin Immunol (2010) 126(5):926-938.

Eosinophilic disorders refer to disorders associated with excess eosinophil numbers, which may present with atypical symptoms due to the level or activity of eosinophils locally or systemically. Disorders associated with excess eosinophil numbers or activity include, but are not limited to, asthma (including aspirin-sensitive asthma), atopic asthma, atopic dermatitis, allergic rhinitis (including seasonal allergic rhinitis), non-allergic rhinitis, asthma, severe asthma, chronic eosinophilic pneumonia, allergic bronchopulmonary aspergillosis, celiac disease, Churg-Strauss syndrome (atopic periarteritis nodularis), eosinophilic myalgia syndrome, hypereosinophilic syndrome, edema reactions, including episodic angioedema, helminthic infections in which eosinophils may have a protective role, onchocercal dermatitis, and eosinophilic myalgia syndrome. Eosinophil-dermatitis), and eosinophil-associated gastrointestinal disorders, including but not limited to eosinophilic esophagitis, eosinophilic gastritis, eosinophilic gastroenteritis, eosinophilic enteritis and eosinophilic colitis, nasal micropolyposis and polyposis, aspirin intolerance, asthma and obstructive sleep apnea. Eosinophil-derived secretory products have also been associated with the promotion of angiogenesis and connective tissue formation in tumors, conditions such as chronic asthma, Crohn's disease, scleroderma, and the fibrotic response seen in endomyocardial fibrosis (Munitz A, Levi-Schaffer F. Allergy 2004; 59: 268-75, Adamko et al. Allergy 2005; 60: 13-22, Oldhoff, et al. Allergy 2005; 60: 693-6). Other examples include cancer (e.g., glioblastoma (e.g., glioblastoma multiforme), non-Hodgkin's lymphoma (NHL)), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease, pulmonary fibrosis (including idiopathic pulmonary fibrosis (IPF) and pulmonary fibrosis secondary to cirrhosis), COPD, liver fibrosis.

IL-13 mediated disorders refers to disorders associated with excessive IL-13 levels or activity, which may manifest as atypical symptoms due to local and/or systemic IL-13 levels or activity. Examples of IL-13 mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease (e.g., Crohn's disease), pulmonary inflammatory disorders (e.g., pulmonary fibrosis such as IPF), COPD, and liver fibrosis.

An IL-4-mediated disorder is a disorder associated with excessive IL-4 levels or activity, which may manifest as atypical symptoms due to local and/or systemic IL-4 levels or activity. Examples of IL-4-mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease (e.g., Crohn's disease), pulmonary inflammatory disorders (e.g., pulmonary fibrosis such as IPF), COPD, and liver fibrosis.

An IL-5-mediated disorder is a disorder associated with excessive IL5 levels or activity, wherein atypical symptoms may manifest due to local and/or systemic IL5 levels or activity. Examples of IL5-mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease (e.g., Crohn's disease), pulmonary inflammatory disorders (e.g., pulmonary fibrosis such as IPF), COPD, and liver fibrosis.

IL-9 mediated disorders refers to disorders associated with excessive IL-9 levels or activity, wherein atypical symptoms may be manifested due to local and/or systemic IL-9 levels or activity. Examples of IL-9 mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease (e.g., Crohn's disease), pulmonary inflammatory disorders (e.g., pulmonary fibrosis such as IPF), COPD, and liver fibrosis.

TSLP-mediated disorders refer to disorders associated with excessive TSLP levels or activity, wherein atypical symptoms may manifest due to local and/or systemic TSLP levels or activity. Examples of TSLP-mediated disorders include: cancer (e.g., non-Hodgkin's lymphoma, glioblastoma), atopic dermatitis, allergic rhinitis, asthma, fibrosis, inflammatory bowel disease (e.g., Crohn's disease), pulmonary inflammatory disorders (e.g., pulmonary fibrosis such as IPF), COPD, and liver fibrosis.

IgE-mediated disorders are disorders associated with excess IgE levels or activity, in which atypical symptoms may manifest due to local and/or systemic IgE levels in the body. Such disorders include asthma, atopic dermatitis, allergic rhinitis, and fibrosis (e.g., pulmonary fibrosis, such as IPF).

Asthma-like symptoms include symptoms selected from the group consisting of shortness of breath, cough (changes in sputum production and/or sputum quality and/or cough frequency), wheezing, chest tightness, bronchioconstriction, and nighttime awakenings attributable to one or a combination of the above symptoms (Juniper et al. (2000) Am. J. Respir. Crit. Care Med., 162(4), 1330-1334.).

The term "respiratory disorder" includes, but is not limited to, asthma (e.g., allergic and non-allergic asthma (e.g., due to infection, such as respiratory syncytial virus (RSV) infection, e.g., in young children)); bronchitis (e.g., chronic bronchitis); chronic obstructive pulmonary disease (COPD) (e.g., emphysema (e.g., cigarette-induced emphysema); conditions involving airway inflammation, eosinophilia, fibrosis, and excess mucus production, such as cystic fibrosis, pulmonary fibrosis, and allergic rhinitis. Examples of diseases that can be characterized by airway inflammation, excess airway secretions, and airway obstruction include asthma, chronic bronchitis, bronchiectasis, and cystic fibrosis.

Exacerbation (commonly referred to as asthma attack or acute asthma) is a new onset or progressive increase in the following conditions: shortness of breath, cough (sputum production and/or sputum quality and/or cough frequency), wheezing, chest tightness, nocturnal awakenings attributed to one of the above symptoms or a combination of these symptoms. Exacerbation is usually characterized by a decrease in exhaled airflow (PEF or FEV1). However, PEF variability does not usually increase during an exacerbation, but it can increase to cause recovery from an exacerbation, or it can increase in the process of recovering from an exacerbation. The severity of an exacerbation can range from mild to life-threatening and can be assessed based on symptoms and lung function. Severe asthma exacerbations as described herein include causing any one or combination of the following exacerbations: subsequent hospitalization for asthma treatment, high corticosteroid use (e.g., quadrupling the total daily dose of corticosteroids, or being greater than or equal to the total daily dose of 500 micrograms of FP or equivalent, for three consecutive days or more), or oral/parenteral corticosteroid use.

TH2 pathway inhibitors are agents that inhibit the TH2 pathway.

Examples of TH2 pathway inhibitors include inhibitors of the activity of any one of the following targets: ITK, BTK, IL-9 (e.g., MEDI-528), IL-5 (e.g., mepolizumab, CAS No. 196078-29-2; resilizumab), IL-13 (e.g., IMA-026, IMA-638 (also known as anrulizumab, INN No. 910649-32-0; QAX-576; IL4/IL13 trap), tralokinumab (also known as CAT-354, CAS No. 1044515-88-9); AER-001, ABT-308 (also known as humanized 13C5.5 antibody), IL-4 (e.g., AER-001, IL4/IL13 trap), OX40L, TSLP, IL-25, IL-33, and IgE (e.g., XOLAIR, QGE-031; MEDI-4212); and receptors such as: IL-9 receptor, IL-5 receptor (e.g., MEDI-563 (benralizumab, CAS No. 10 44511-01-4)), IL-4 receptor alpha (e.g., AMG-317, AIR-645), IL-13 receptor alpha 1 (e.g., R-1671) and IL-13 receptor alpha 2, OX40, TSLP-R, IL-7Rα (co-receptor for TSLP), IL17RB (receptor for IL-25), ST2 (receptor for IL-33), CCR3, CCR4, CRTH2 (e.g., AMG-853, AP768, AP-761, MLN6095, ACT12996 8), FcεRI, FcεRII/CD23 (receptor for IgE), Flap (e.g., GSK2190915), Syk kinase (R-343, PF3526299); CCR4 (AMG-761), TLR9 (QAX-935), and multi-cytokine inhibitors of CCR3, IL5, IL3, GM-CSF (e.g., TPIASM8). Examples of inhibitors of the above targets are disclosed in, for example, WO2008/086395; WO2006/085938; US 7,615,213; US 7,501,121; WO2006/085938; WO 2007/080174; US 7,807,788; WO2005007699; WO2007036745; WO2009/009775; WO2007/082068; WO201 0/073119; WO2007/045477; WO2008/134724; US2009/0047277; and WO2008/127,271).

The therapeutic agents provided herein include active agents that can bind to the targets identified above, such as polypeptide(s) (e.g., antibodies, immunoadhesins, or peptibodies), aptamers or small molecules that can bind to proteins, or nucleic acid molecules that can bind to nucleic acid molecules encoding the targets identified herein (i.e., siRNA).

"Anti-IL-13/IL4 pathway inhibitors" refer to therapeutic agents that inhibit IL-13 and/or IL-4 signaling. Examples of anti-IL-13/IL4 pathway inhibitors include inhibitors of the interaction between IL13 and/or IL4 and their receptors, such inhibitors including but not limited to anti-IL13 binders, anti-IL4 binders, anti-IL3/IL4 bispecific binders, anti-IL4 receptor α binders, anti-IL13 receptor α1 binders, and anti-IL13 receptor α2 binders. Inhibitors specifically include single domain antibodies that can bind to IL13, IL4 (including bispecific antibodies having a single domain that binds to IL13 and a single domain that binds to IL4), IL-13Rα1, IL-13Rα2, or IL-4Rα. It should be understood that molecules that can bind to more than one target are included.

An "anti-IL4 binder" refers to an agent that binds to human IL-4. Such binders may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the binder binds to human IL-4 sequences with an affinity of 1 uM–1 pM. Specific examples of anti-IL4 binders may include soluble IL4 receptor α (e.g., the extracellular domain of the IL4 receptor fused to a human Fc region), anti-IL4 antibodies, and soluble IL13 receptor α1 (e.g., the extracellular domain of the IL13 receptor α1 fused to a human Fc region).

An "anti-IL4 receptor alpha binding agent" refers to an agent that binds to human IL4 receptor alpha. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the binding agent binds to the human IL-4 receptor alpha sequence with an affinity of 1 uM–1 pM. Specific examples of anti-IL4 receptor alpha binding agents may include anti-IL4 receptor alpha antibodies.

An "anti-IL-13 binding agent" refers to an agent that binds to human IL-13. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the binding agent binds to the human IL-13 sequence with an affinity of 1 uM–1 pM. Specific examples of anti-IL-13 binding agents may include anti-IL-13 antibodies, soluble IL-13 receptor α2 fused to a human Fc, soluble IL4 receptor α fused to a human Fc, and soluble IL-13 receptor α fused to a human Fc. According to one embodiment, the anti-IL-13 antibody comprises (1) HVRH1 comprising the amino acid sequence of SEQ ID NO: 11, (2) HVRH2 comprising the amino acid sequence of SEQ ID NO: 12, (3) HVRH3 comprising the amino acid sequence of SEQ ID NO: 13, (4) HVRL1 comprising the amino acid sequence of SEQ ID NO: 14, (5) HVRL2 comprising the amino acid sequence of SEQ ID NO: 15, and (6) HVRL3 comprising the amino acid sequence of SEQ ID NO: 16. In another embodiment, the anti-IL-13 antibody comprises a VH domain comprising the amino acid sequence of SEQ ID NO: 9 and a VL domain comprising the amino acid sequence of SEQ ID NO: 10. According to one embodiment, the antibody is an IgG1 antibody. According to another embodiment, the antibody is an IgG4 antibody. According to one embodiment, the IgG4 antibody comprises an S228P mutation in its constant domain.

An "anti-IL13 receptor α1 binding agent" refers to an agent that specifically binds to human IL13 receptor α1. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the binding agent binds to the human IL-13 receptor α1 sequence with an affinity of 1 uM–1 pM. Specific examples of anti-IL13 receptor α1 binding agents may include anti-IL13 receptor α1 antibodies.

An "anti-IL13 receptor α2 binding agent" refers to an agent that specifically binds to human IL13 receptor α2. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the binding agent binds to the human IL-13 receptor α2 sequence with an affinity of 1 uM–1 pM. Specific examples of anti-IL13 receptor α2 binding agents may include anti-IL13 receptor α2 antibodies.

"Anti-IgE binding agent" refers to an agent that specifically binds to human IgE. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the anti-IgE antibody comprises a VL sequence comprising the amino acid sequence of SEQ ID NO: 17 and a VH sequence comprising the amino acid sequence of SEQ ID NO: 18.

An "anti-M1' binding agent" refers to an active agent that specifically binds to the membrane-proximal M1' region of IgE expressed on the surface of B cells. Such binding agents may include small molecules, aptamers, or polypeptides. Such polypeptides may include, but are not limited to, polypeptides selected from immunoadhesins, antibodies, peptibodies, and peptides. According to one embodiment, the anti-IgE antibody comprises an antibody described in WO2008/116149 or a variant thereof. According to another embodiment, the anti-M1' antibody comprises a variable heavy chain and a variable light chain, wherein the variable heavy chain is SEQ ID NO: 24 and the variable light chain is SEQ ID NO: 25. According to another embodiment, the anti-IgE/M1′ antibody comprises a variable heavy chain and a variable light chain, wherein the variable heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3, and the variable light chain further comprises HVR-L1, HVR-L2, and HVR-L3, and: (a) HVR-H1 is residues 26-35 of SEQ ID NO: 24, [GFTFSDYGIA]; (b) HVR-H2 is residues 49-66 of SEQ ID NO: 24, [AFISDLAYTIYYADTVTG]; (c) HVR-H3 is residues 97-106 of SEQ ID NO: 24, [ARDNWDAMDY]; (d) HVR-L1 is residues 24-39 of SEQ ID NO: 25, [RSSQSLVHNNANTYLH]; (e) HVR-L2 is residues 107-111 of SEQ ID NO: 1 Residues 55-61 of SEQ ID NO: 25, [KVSNRFS]; (f) HVR-L3 is residues 94-102 of SEQ ID NO: 25, [SQNTLVPWT].

The term "small molecule" refers to an organic molecule having a molecular weight of 50 Daltons - 2500 Daltons.

The term "antibody" is used in the broadest sense and specifically encompasses, for example, monoclonal antibodies, polyclonal antibodies, antibodies with polyepitopic specificity, single-chain antibodies, multispecific antibodies, and antibody fragments. Such antibodies can be chimeric, humanized, human, and synthetic. Such antibodies and methods of generating them are described in more detail below.

The terms "uncontrolled" or "uncontrolled" refer to a treatment regimen that is insufficient to minimize disease symptoms. As used herein, the terms "uncontrolled" and "inadequately controlled" are used interchangeably and refer to the same state. A patient's control state can be determined by the treating physician based on a number of factors, including the patient's clinical history, responsiveness to treatment, and the current level of treatment prescribed by the physician. For example, the physician may consider factors such as FEV1 <75% of predicted or personal best, frequency of SABA need in the past 2-4 weeks (e.g., greater than or equal to two doses/week), nighttime awakenings/symptoms in the past 2-4 weeks (e.g., less than or equal to two nights/week), limitation of activities in the past 2-4 weeks, and daytime symptoms in the past 2-4 weeks.

The term "therapeutic agent" refers to any active agent used to treat a disease.

The term "controller" or "preventer" refers to any therapeutic agent used to control asthma inflammation. Examples of controllers include corticosteroids, leukotriene receptor antagonists (e.g., inhibiting the synthesis or activity of leukotrienes, such as montelukast, zileuton, pranlukast, zafirlukast), LABAs, corticosteroid/LABA combination compositions, theophylline (including aminophylline), sodium cromoglycate, nedocromil sodium, omalizumab, LAMAs, MABAs (e.g., dual-function muscarinic antagonist-β2 agonist), 5-lipoxygenase activating protein (FLAP) inhibitors, and enzyme PDE-4 inhibitors (e.g., roflumilast). A "second controller" typically refers to a controller that is different from the first controller.

The term "corticosteroid-sparing" or "CS" means that in a patient taking corticosteroids for a condition, the frequency and/or amount of corticosteroids used to treat the condition is reduced, or the use of corticosteroids is eliminated, due to the administration of another therapeutic agent. A "CS agent" refers to a therapeutic agent that can cause CS in a patient taking corticosteroids.

The term "corticosteroid" includes, but is not limited to, fluticasone (including fluticasone propionate (FP)), beclometasone, budesonide, ciclesonide, mometasone, flunisolide, betamethasone, and triamcinolone. "Inhalable corticosteroid" means a corticosteroid suitable for delivery by inhalation. Exemplary inhalable corticosteroids are fluticasone, beclometasone dipropionate, budesonide, mometasone furoate, ciclesonide, flunisolide, triamcinolone acetonide, and any other corticosteroid currently available or available in the future. Examples of corticosteroids that can be inhaled and combined with a long-acting beta2-agonist include, but are not limited to, budesonide/formoterol and fluticasone/salmeterol.

Examples of corticosteroid/LABA combination drugs include fluticasone furoate/vilanterol trifenatate and indacaterol/mometasone.

The term "LABA" means a long-acting beta2-agonist, which includes, for example, salmeterol, formoterol, bambuterol, albuterol, indacaterol, arformoterol, and clenbuterol.

The term "LAMA" means long-acting muscarinic antagonist, which antagonists include: tiotropium bromide.

Examples of LABA/LAMA combinations include, but are not limited to, olodaterol tiotropium (Boehringer Ingelheim's) and indacaterol glycopyrronium (Novartis).

The term "SABA" means a short-acting beta-2 agonist including, but not limited to, salbutamol, levosalbutamol, fenoterol, terbutaline, pirbuterol, procaterol, bitolterol, rimiterol, carbuterol, tulobuterol, and reproterol.

Leukotriene receptor antagonists (sometimes called leukast) (LTRAs) are drugs that inhibit leukotrienes. Examples of leukotriene inhibitors include montelukast, zileuton, pranlukast, and zafirlukast.

The term "FEV1" refers to the volume of air exhaled in the first second of a forced exhalation. It is a measure of airway obstruction. The provocative concentration of methacholine required to induce a 20% decrease in FEV1 (PC20) is a measure of airway hyperresponsiveness. FEV1 may be expressed in other similar terms, such as _FEV1 , and it should be understood that all such variations have the same meaning.

The term "relative change in FEV1" = (FEV1 at treatment week 12 - FEV1 before the start of treatment) divided by FEV1.

The term "mild asthma" refers to patients who generally experience fewer than two symptoms or exacerbations per week, fewer than two nocturnal symptoms per month, and are asymptomatic between exacerbations. Mild, intermittent asthma is often treated as needed with: inhaled bronchodilators (short-acting inhaled beta2-agonists); avoidance of known triggers; annual influenza vaccination; pneumococcal vaccination every 6–10 years, and in some cases, inhaled beta2-agonists, cromolyn, or nedocromil before exposure to identified triggers. If a patient has an increased need for short-acting beta2-agonists (e.g., more than three to four times a day for acute exacerbations or more than one canister per month for symptoms), the patient may require a step-up in treatment.

The term "moderate asthma" generally refers to asthma in which the patient experiences more than two exacerbations per week that affect sleep and activity; the patient has more than two nighttime awakenings due to asthma per month; the patient has chronic asthma symptoms that require a short-acting inhaled beta2-agonist every day or every other day; and the patient's pre-treatment baseline PEF or FEV1 is 60%-80% predicted and the PEF variability is 20-30%.

The term "severe asthma" generally refers to asthma in which the patient has nearly continuous symptoms, frequent exacerbations, frequent nighttime awakenings due to asthma, limited activity, a baseline PEF or FEV1 less than 60% predicted, and a PEF variability of 20-30%.

Examples of rescue medications include albuterol, ventolin, and others.

"Resistant" refers to a disease that shows little or no clinically significant improvement after treatment with a therapeutic agent. For example, asthma that requires treatment with high-dose ICS (e.g., quadrupling the total daily dose of corticosteroids or greater than or equal to 500 micrograms of FP (or equivalent) for at least three consecutive days or more, or a two-week trial of systemic corticosteroids to establish whether the asthma remains uncontrolled or FEV1 does not improve) is generally considered severe refractory asthma.

As provided herein, therapeutic agents can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In one embodiment, therapeutic agent is an inhalant. According to another embodiment, administration is by injection such as intravenous or subcutaneous injection. In another embodiment, therapeutic agent is administered using a syringe (such as preloaded or non-preloaded) or an automatic syringe.

For the prevention or treatment of disease, the appropriate dose of therapeutic agent can depend on the type of disease to be treated, the severity and process of the disease, the therapeutic agent to be used for prevention or treatment purposes, previous treatment, the patient's clinical history and the response to the therapeutic agent and the judgment of the attending physician. Therapeutic agent can be applied to the patient aptly at one time or through a series of treatments. Can prepare, dose and administer therapeutic agent compositions in a manner consistent with good medical practice. The factor considered in this case comprises the specific disease to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the disease, the delivery site of the activating agent, the method of administration, the time of administration and other factors known to medical practitioners.

For TH2 therapy for eosinophilic diseases (including asthma) and for the treatment of other diseases, the dosage of lebrikizumab is as follows: lebrikizumab can be administered at 0.1 mg/kg to 100 mg/kg of patient body weight. In one embodiment, the dosage administered to the patient is 0.1 mg/kg to 20 mg/kg of patient body weight. In another embodiment, the dosage is 1 mg/kg to 10 mg/kg of patient body weight.

In alternative embodiments, lebrikizumab can be administered as a fixed dose. In one embodiment, lebrikizumab is administered as a 125-1000 mg fixed dose (i.e., not weight dependent) by subcutaneous injection or by intravenous injection at a time frequency selected from the following: every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 1 month, 2 months, 3 months, or 4 months. In another embodiment, if the patient is overweight, lebrikizumab can be administered, for example, 125-250 mg 3 times a month. In one embodiment, lebrikizumab is administered every 4 weeks as a fixed dose of 125 mg, 250 mg, or 500 mg. In another embodiment, lebrikizumab is administered every 4 weeks as a fixed dose of 37.5 mg, 125 mg, 250 mg, or 500 mg in patients >40 kg.

In one embodiment, the patient is 18 years of age or older. In one embodiment, the asthma patient is 12-17 years of age, and lebrikizumab is administered as a fixed dose of 250 mg or a fixed dose of 125 mg. In one embodiment, the asthma patient is 6-11 years of age, and lebrikizumab is administered as a fixed dose of 125 mg.

"Patient response" or "response" (and grammatical variations thereof) may be evaluated using any endpoint indicative of benefit to the patient, including but not limited to (1) some inhibition of disease progression, including slowing and complete arrest; (2) a reduction in the number of disease attacks and/or symptoms; (3) a reduction in the size of lesions; (4) inhibition (i.e., reduction, slowing, or complete cessation) of disease cell infiltration into adjacent peripheral organs and/or tissues; (5) inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; (6) a reduction in the autoimmune response, which may, but need not, result in regression or elimination of disease lesions; (7) some relief of one or more symptoms associated with the disorder; (8) an increase in the length of disease-free presentation after treatment; and/or (9) a reduction in mortality at a given time point after treatment.

"Affinity" refers to the total strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen-binding arm). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

An "affinity matured" antibody is one with one or more alterations in one or more hypervariable regions (HVRs) compared to a parent antibody which does not possess such alterations, such alterations resulting in improvements in the affinity of the antibody for antigen.

The terms "anti-target antibody" and "antibody that binds to a target" refer to an antibody that is capable of binding to a target with sufficient affinity, such that the antibody can be used as a diagnostic and/or therapeutic agent targeting the target. In one embodiment, the degree of binding of the anti-target antibody to an unrelated, non-target protein, as measured by radioimmunoassay (RIA) or biacore assay, is less than about 10% of the binding of the antibody to the target. In a specific embodiment, the antibody that binds to the target has a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM or ≤0.001 nM (e.g., 10 ^-8 M or less, e.g., 10 ^-8 M to 10 ^-13 M, e.g., 10 ^-9 M to 10 ^-13 M). In a specific embodiment, the anti-target antibody binds to an epitope of the target that is conserved across species.

The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

"Antibody fragments" refer to molecules other than intact antibodies that contain a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

An "antibody that binds to the same epitope as a reference antibody" means that the antibody blocks the binding of the reference antibody to its antigen by 50% or more in a competition assay, and conversely, the reference antibody blocks the binding of the antibody to its antigen by 50% or more in a competition assay. Exemplary competition assays are provided herein.

For the purposes of this paper, an "acceptor human framework" is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or a heavy chain variable (VH) framework derived from a human immunoglobulin framework or a human consensus framework (defined below). An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework can comprise the same amino acid sequence, or it can contain amino acid sequence changes. In some embodiments, the number of amino acid changes is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the VL acceptor human framework is identical in sequence to a VL human immunoglobulin framework sequence or a human consensus framework sequence.

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

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

As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cell function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C); C), chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and various anti-tumor or anti-cancer agents disclosed below.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

An "effective amount" of an active agent, such as a pharmaceutical agent, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. Unless otherwise indicated herein, the amino acid residues in the Fc region or constant region are numbered according to the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

"Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the HVR and FR sequences generally appear in the following order in VH (or VL): FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody that has a structure substantially similar to a native antibody structure or has heavy chains that contain an Fc region as defined herein.

The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical to the parent cell in nucleic acid content but may contain mutations. Mutant progeny that possess the function or biological activity screened or selected for the original transformed cell are encompassed herein.

A "human antibody" is an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source that utilizes human antibody repertoires or other human antibody coding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.

A "human consensus framework" is a framework that represents the most commonly occurring amino acid residues in a selected human immunoglobulin VL or VH framework sequence. Typically, the selected human immunoglobulin VL or VH sequence is from a subgroup of variable domain sequences. Typically, the subgroup of sequences is as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Volumes 1-3. In one embodiment, for VL, the subgroup is subgroup κI as described in Kabat et al., supra. In one embodiment, for VH, the subgroup is subgroup III as described in Kabat et al., supra.

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

As used herein, the term "hypervariable region" or "HVR" refers to each region of an antibody variable domain that is highly variable in sequence and/or forms structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). HVRs generally comprise amino acid residues from hypervariable loops and/or from "complementarity determining regions" (CDRs), the latter of which generally have the highest sequence variability and/or are involved in antigen recognition. As used herein, the HVR region comprises any number of residues located within positions 24-36 (for HVRL1), 46-56 (for HVRL2), 89-97 (for HVRL3), 26-35B (for HVRH1), 47-65 (for HVRH2), and 93-102 (for HVRH3).

An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to a cytotoxic agent.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

An "isolated" antibody is one that has been separated from the components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for evaluating antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

"Isolated nucleic acid encoding an anti-target antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including such (one or more) nucleic acid molecules in a single vector or in separate vectors, and such (one or more) nucleic acid molecules present at one or more locations in a host cell.

The term "monoclonal antibody" as used herein refers to an antibody derived from a substantially homogeneous antibody population, and the so-called substantially homogeneous antibody population refers to that, except for possible variant antibodies (e.g., containing natural mutations or occurring during the production of monoclonal antibody products) generally present in trace amounts, each antibody in the population is identical and/or binds to the same epitope. In contrast to polyclonal antibody preparations generally comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a determinant on the antigen. Therefore, the modifier "monoclonal" refers to the characteristic that an antibody is derived from a substantially homogeneous antibody population and is not to be construed as requiring the antibody to be produced by any particular method. For example, the monoclonal antibody to be used according to the method provided herein can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of human immunoglobulin loci, and these methods and other exemplary methods for preparing monoclonal antibodies are described herein.

A "naked antibody" is an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical formulations.

"Natural antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light chains and two identical heavy chains connected by disulfide bonds. From N to C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from N to C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chains of antibodies can be divided into one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information concerning the use of such therapeutic products, such as indications, usage, dosage, administration, combination therapy, contraindications, and/or warnings. The term "package insert" is also used to refer to instructions customarily included in commercial packages of diagnostic products, that contain information regarding the intended use, test principles, preparation and handling of reagents, sample collection and preparation, calibration of assays and assay protocols, and performance and precision data, such as the sensitivity and specificity of the assay.

"Percent (%) amino acid sequence identity" relative to a reference polypeptide sequence is defined as the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the reference and candidate sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways in the art, such as using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. A skilled person can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the sequences to be compared. However, for the purposes of this article, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington, D.C., 20559, along with user documentation, where it is registered as U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and do not change.

Where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A with or relative to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a particular % amino acid sequence identity with or relative to a given amino acid sequence B) is calculated as follows:

100 is multiplied by the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the ALIGN-2 program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B is not equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained by the ALIGN-2 computer program, as described in the immediately preceding paragraph.

The term "pharmaceutical formulation" refers to a preparation that is in such form that the biological activity of the active ingredient contained therein is effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

"Pharmaceutically acceptable carrier" refers to a component in a pharmaceutical formulation other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

Unless otherwise indicated, as used herein, the term "target" refers to any naturally occurring molecule from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed targets, as well as any form of the target that results from processing in the cell. The term also encompasses naturally occurring variants of the target, such as splice variants or allelic variants.

As used herein, "treatment" (and grammatical variations thereof) refers to clinical intervention that attempts to alter the natural course of the individual being treated, which can be performed for prevention or in the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, antibodies are used to delay the development of the disease or slow the progression of the disease.

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. In addition, antibodies that bind to a particular antigen can be isolated using a VH or VL domain from an antibody that binds to that antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are autonomously replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

Compositions and methods

In one aspect, the present invention is based, in part, on new diagnostic assays and better treatments. In a specific embodiment, antibodies that bind to periostin are provided. The antibodies of the present invention can be used, for example, to diagnose or treat asthma and other diseases.

Exemplary antibodies

Anti-periostin antibodies

In one aspect, the present invention provides isolated antibodies that bind to periostin. In certain embodiments, the anti-periostin antibodies can bind to human periostin isoforms 1-4 with good affinity.

In one embodiment, the antibody comprises the sequences of SEQ ID NO: 1 and SEQ ID NO: 2 ("25D4" antibody), or comprises the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 ("23B9" antibody). In another embodiment, the antibody comprises the variable region sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or comprises the variable region sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the antibody comprises the HVR sequences of SEQ ID NO: 1 and SEQ ID NO: 2, or the HVR sequences of SEQ ID NO: 3 and SEQ ID NO: 4. In another embodiment, the antibody comprises HVR sequences that are 95% or more identical to the HVR sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and/or the antibody comprises HVR sequences that are 95% or more identical to the HVR sequences of SEQ ID NO: 3 and SEQ ID NO: 4.

In any of the above embodiments, the anti-periostin antibody may be humanized.In one embodiment, the anti-periostin antibody may comprise HVRs as in any of the above embodiments, and further comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In another aspect, the anti-periostin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1. In a specific embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-periostin antibody comprising the sequence retains the ability to bind to periostin. In a specific embodiment, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 1. In a specific embodiment, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-periostin antibody comprises the VH sequence in SEQ ID NO: 1, including post-translational modifications of the sequence.

In another aspect, an anti-periostin antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-periostin antibody comprising the sequence retains the ability to bind to periostin. In a specific embodiment, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 2. In a specific embodiment, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-periostin antibody comprises the VL sequence in SEQ ID NO: 2, including post-translational modifications of the sequence.

In another aspect, the anti-periostin antibody comprises a heavy chain variable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3. In a specific embodiment, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-periostin antibody comprising the sequence retains the ability to bind to periostin. In a specific embodiment, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 3. In a specific embodiment, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-periostin antibody comprises the VH sequence in SEQ ID NO: 3, including post-translational modifications of the sequence.

In another aspect, an anti-periostin antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2. In a specific embodiment, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions (e.g., conservative substitutions), insertions or deletions relative to the reference sequence, but the anti-periostin antibody comprising the sequence retains the ability to bind to periostin. In a specific embodiment, a total of 1-10 amino acids have been substituted, inserted and/or deleted in SEQ ID NO: 4. In a specific embodiment, the substitution, insertion or deletion occurs in a region outside the HVRs (i.e., in the FRs). Optionally, the anti-periostin antibody comprises the VL sequence in SEQ ID NO: 4, including post-translational modifications of the sequence.

In another aspect, an anti-periostin antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-periostin antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as the anti-periostin antibodies are provided, wherein the anti-periostin antibodies comprise a VH sequence of SEQ ID NO: 1 and a VL sequence of SEQ ID NO: 2. For example, in certain embodiments, antibodies that bind to the same epitope as the anti-periostin antibodies are provided, wherein the anti-periostin antibodies comprise a VH sequence of SEQ ID NO: 3 and a VL sequence of SEQ ID NO: 4.

In a further aspect of the invention, the anti-periostin antibody according to any of the above embodiments is a monoclonal antibody, including chimeric, humanized or human antibodies. In one embodiment, the anti-periostin antibody is an antibody fragment, such as Fv, Fab, Fab', scFv, diabody or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 or IgG4 antibody or other antibody class or isotype as defined herein. In another embodiment, the antibody is a bispecific antibody.

In a further aspect, an anti-periostin antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-7 below, alone or in combination.

Anti-IL13 antibodies

In one aspect, the invention provides isolated antibodies that bind to human IL-13.

In one embodiment, the anti-IL-13 antibody comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO: 14; HVR-L2 comprising the amino acid sequence of SEQ ID NO: 15; HVR-L3 comprising the amino acid sequence of SEQ ID NO: 16; HVR-H1 comprising the amino acid sequence of SEQ ID NO: 11; HVR-H2 comprising the amino acid sequence of SEQ ID NO: 12; and HVR-H3 comprising the amino acid sequence of SEQ ID NO: 13.

In another embodiment, the antibody comprises the variable region sequences of SEQ ID NO:9 and SEQ ID NO:10.

In any of the above embodiments, the anti-IL-13 antibody may be humanized. In one embodiment, the anti-IL-13 antibody comprises HVRs as in any of the above embodiments, and further comprises an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

In another aspect, the anti-IL-13 antibody comprises a heavy chain variable domain (VH) sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In specific embodiments, the VH sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-IL-13 antibody comprising such sequence retains the ability to bind to human IL-13. In specific embodiments, a total of 1-10 amino acids have been substituted, altered, inserted, and/or deleted in SEQ ID NO: 9. In specific embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., within the FRs). Optionally, the anti-IL-13 antibody comprises the VH sequence in SEQ ID NO: 9, including post-translational modifications of that sequence.

In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a light chain variable domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g., conservative substitutions), insertions, or deletions relative to the reference sequence, but the anti-IL-13 antibody comprising the sequence retains the ability to bind to IL-13. In certain embodiments, a total of 1-10 amino acids have been substituted, inserted, and/or deleted in SEQ ID NO: 10. In certain embodiments, the substitutions, insertions, or deletions occur in regions outside the HVRs (i.e., in the FRs). Optionally, the anti-IL-13 antibody comprises the VL sequence in SEQ ID NO: 10, including post-translational modifications of that sequence.

In yet another embodiment, the anti-IL-13 antibody comprises a VL region that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10, and a VH region that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9. In yet a further embodiment, the anti-IL-13 antibody comprises: HVR-L1 comprising the amino acid sequence of SEQ ID NO:14; HVR-L2 comprising the amino acid sequence of SEQ ID NO:15; HVR-L3 comprising the amino acid sequence of SEQ ID NO:16; HVR-H1 comprising the amino acid sequence of SEQ ID NO:11; HVR-H2 comprising the amino acid sequence of SEQ ID NO:12; and HVR-H3 comprising the amino acid sequence of SEQ ID NO:13.

In another aspect, an anti-IL-13 antibody is provided, wherein the antibody comprises a VH as in any of the embodiments provided above and a VL as in any of the embodiments provided above.

In a further aspect, the present invention provides antibodies that bind to the same epitope as the anti-IL-13 antibodies provided herein. For example, in certain embodiments, antibodies that bind to the same epitope as or can be competitively inhibited by anti-IL-13 antibodies are provided, wherein the anti-IL-13 antibody comprises a VH sequence of SEQ ID NO: 9 and a VL sequence of SEQ ID NO: 10.

In a further aspect of the invention, the anti-IL-13 antibody according to any of the above embodiments may be a monoclonal antibody, including chimeric, humanized, or human antibodies. In one embodiment, the anti-IL-13 antibody is an antibody fragment, such as an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another embodiment, the antibody is a full-length antibody, such as a complete IgG1 or IgG4 antibody, or other antibody class or isotype as defined herein. According to another embodiment, the antibody is a bispecific antibody. In one embodiment, the bispecific antibody comprises the HVRs described above or comprises the VH and VL regions described above.

In a further aspect, an anti-IL-13 antibody according to any of the above embodiments may incorporate any of the features described in Sections 1-7 below, alone or in combination.

1. Antibody affinity

In certain embodiments, the antibodies provided herein have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 ⁻⁸ M or less, e.g., 10 ⁻⁸ M to 10 ⁻¹³ M, e.g., 10 ⁻⁹ M to 10 ⁻¹³ M).

In one embodiment, Kd is measured by performing a radiolabeled antigen binding assay (RIA) using a Fab form of the antibody of interest and its antigen, as described by the following assay. The solution binding affinity of Fabs for antigen is measured by equilibrating the Fab with the lowest concentration of ( ¹²⁵ I)-labeled antigen in the presence of a titration series of unlabeled antigen, followed by capturing the bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999)). To determine the conditions for this assay, multiwell plates (ThermoScientific) were coated overnight with 5 μg/ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6) and then blocked with 2% (w/v) bovine serum albumin in PBS at room temperature (about 23° C.) for two to five hours. In a non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen is mixed with a serial dilution of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody Fab-12 in Presta et al., Cancer Res. 57:4593-4599 (1997)). The Fab of interest is then incubated overnight; however, the incubation can be continued for longer periods (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixture is transferred to a capture plate for incubation at room temperature (e.g., one hour). The solution is then removed and the plate is washed eight times with 0.1% polysorbate 20 in PBS. When the plate is dry, 150 μl/well of scintillant (MICROSCINT-20™; Packard) is added and the plate is counted for tens of minutes in a TOPCOUNT™ gamma counter (Packard). Concentrations of each Fab that give less than or equal to 20% maximal binding are selected for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmon resonance assay, using -2000 or -3000 (BIAcore, Inc., Piscataway, NJ) at 25°C, using an immobilized antigen CM5 chip with ～10 response units (RU). Briefly, according to the supplier's instructions, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg/ml (～0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl/minute to achieve approximately 10 response units (RU) of coupled protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM - 500 nM) in PBS (PBST) with 0.05% polysorbate 20 (TWEEN-20™) surfactant were injected at 25° C. at a flow rate of approximately 25 μl/min. Association rates (k on ) and dissociation rates (k off ) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model ( ELISA® Evaluation Software version 3.2). The equilibrium dissociation constant was calculated as the ratio of k off / k on . See, e.g., Chen et al., J. Mol. Biol. 293: 865-881 (1999). If the on-rate exceeds 10 ⁶ M ^-1 s ^-1 by the surface plasmon resonance assay above, the on-rate can be determined by using a fluorescence quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM antigen-antibody (Fab form) in PBS, pH 7.2 at 25° C. in the presence of increasing concentrations of antigen, wherein the measurement can be made using, for example, a spectrophotometer, such as a spectrophotometer equipped with stopped flow (Aviv Instruments) or a 8000 series SLM-AMINCO™ spectrophotometer with a stirred cuvette (ThermoSpectronic).

2. Antibody fragments

In certain embodiments, the antibodies provided herein are antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv and scFv fragments and other fragments described below. For a review of some antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthün, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, eds. (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93/16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. For a discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Patent No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that can be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments that contain all or part of the heavy chain variable domain or all or part of the light chain variable domain of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516B1).

Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

3. Chimeric and humanized antibodies

In certain embodiments, the antibodies provided herein are chimeric antibodies. Specific chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed relative to the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

In certain embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies comprise one or more variable domains, wherein HVRs such as CDRs (or parts thereof) are derived from non-human antibodies, and FRs (or parts thereof) are derived from human antibody sequences. Humanized antibodies optionally can also comprise at least a portion of a human constant region. In some embodiments, some FR residues in humanized antibodies are replaced with corresponding residues from non-human antibodies (such as the antibody from which HVR residues are derived), such as to restore or improve antibody specificity or affinity.

Humanized antibodies and methods of making the same are reviewed, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" approach to FR shuffling).

Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)). )); human mature (somatically mature) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions obtained from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271: 22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, the antibodies provided herein are human antibodies. Human antibodies can be produced using a variety of techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic attack. Such animals generally contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or exists outside the chromosome or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™ technology; 5,770,429 describing technology; 7,041,870 describing K-M technology, and U.S. Patent Application Publication No. US 2007/0061900 describing technology). The human variable regions of the complete antibodies generated from such animals can be further modified, for example, by combining with different human constant regions.

Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cell lines for producing human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147:86 (1991).) Human antibodies generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include, for example, those described in U.S. Pat. No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

Human antibodies can also be generated by isolating the Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. The technology for selecting human antibodies from antibody libraries is described below.

5. Library-derived Antibodies

The antibodies of the present invention can be isolated by screening for antibodies with one or more desired activities in a combinatorial library. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding characteristics. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348: 552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132 (2004).

In a specific phage display method, the VH and VL gene repertoires are cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phage generally display antibody fragments, either in the form of single-chain Fv (scFv) fragments or in the form of Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, a naive repertoire (e.g., from humans) can be cloned without any immunization to provide a single source of antibodies directed against a wide range of non-self and self antigens, as described by Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the hypervariable CDR3 regions to achieve rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373 and U.S. Patent Application Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, the antibodies provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies having binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for IL-13, and the other is for any other antigen. In certain embodiments, bispecific antibodies can bind to two different epitopes of IL-13. Bispecific antibodies can also be used to localize cytotoxic agents to cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments.

Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), WO 93/08829 and Traunecker et al., EMBO J. 10:3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (WO 93/08829 and Traunecker et al., EMBO J. 10:3655 (1991)). 2009/089004A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229:81 (1985)); using leucine zippers to generate bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology for preparing bispecific Antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and preparation of trispecific antibodies as described, e.g., in Tutt et al., J. Immunol. 147:60 (1991).

Engineered antibodies with three or more functional antigen-binding sites, including "Octopus antibodies," are also contemplated herein (see, e.g., US 2006/0025576A1).

The antibodies or fragments herein also include "dual-acting FAbs" or "DAFs" comprising an antigen binding site that binds to IL-13 and another, different antigen (see, eg, US 2008/0069820).

7. Antibody variants

In a specific embodiment, it is contemplated that the amino acid sequence variants of the antibodies provided herein. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. The amino acid sequence variants of the antibody can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and/or insertions and/or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be prepared to reach the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

Substitution, insertion, and deletion variants

In a specific embodiment, antibody variants with one or more amino acid substitutions are provided. The target sites for substitution mutagenesis include HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Conservative Substitutions". More substantial changes are provided under the heading "Exemplary Substitutions" in Table 1 and are further described below with reference to amino acid side chain types. Amino acid substitutions can be introduced into the target antibody and the product can be screened for desired activity, such as retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

Table 1

Original residue Exemplary substitutions Conservative substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn；Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu

Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

Amino acids can be grouped according to common side chain properties:

(1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

(2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect chain direction: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions result in exchanging a member of one of these classes for a member of another class.

A class of substitutional variants involves replacing one or more hypervariable region residues of a parent antibody (e.g., humanized or human antibody). Generally, the resulting variants selected for further study will have modifications (e.g., improvements) relative to the parent antibody in specific biological properties (e.g., increased affinity, reduced immunogenicity), and/or substantially retain the specific biological properties of the parent antibody. Exemplary substitutional variants are affinity matured antibodies, which can be conveniently generated, for example, using affinity maturation techniques based on phage display, such as described herein. In short, one or more HVR residues are mutated, and the variant antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity).

Changes (e.g., substitutions) can be made in HVRs, for example to improve antibody affinity. Such changes can be made in HVR "hot spots" (i.e., residues encoded by codons that undergo mutations at high frequency during somatic maturation) (see, e.g., Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)) and/or SDRs (a-CDRs), and the resulting variant VH or VL binding affinity is tested. Affinity maturation by constructing a secondary library and reselecting therefrom has been described, for example, in Hoogenboom et al., Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).). In some embodiments of affinity maturation, diversity can be introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then prepared. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves an HVR-guided approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often specifically targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more HVRs, as long as such changes do not substantially reduce the ability of the antibody to bind to antigen. For example, conservative changes that do not substantially reduce binding affinity (e.g., conservative substitutions as provided herein) may be made in HVRs. Such changes may be outside HVR "hot spots" or SDRs. In certain embodiments of the variant VH and VL sequences provided above, each HVR is either unchanged or contains no more than one, two, or three amino acid substitutions.

A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, residues or target residue groups (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that were shown to be functionally sensitive to the initial substitution. Alternatively or additionally, the crystal structure of the antigen-antibody complex can be used to identify contact points between the antibody and the antigen. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Variants can be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino and/or carboxyl terminal fusions (ranging in length from one residue to polypeptides containing one hundred or more residues), as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody.

Glycosylation variants

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation.Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence to create or remove one or more glycosylation sites.

When the antibody comprises the Fc district, the carbohydrate attached thereto can be changed. The natural antibody produced by mammalian cells generally comprises branched, diantennary oligosaccharides, which are generally attached to the Asn297 of the CH2 domain in the Fc district by N connection. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose on the GlcNAc in the " stem " of the diantennary oligosaccharide structure. In some embodiments, modification of oligosaccharides can be carried out in the antibody of the present invention to produce antibody variants with specific improved properties.

In one embodiment, antibody variants are provided that lack carbohydrate structures that are attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain of Asn297 relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297, as measured by MALDI-TOF mass spectrometry, as described in, for example, WO 2008/077546. Asn297 refers to the asparagine residue located at approximately position 297 of the Fc region (Eu numbering of Fc region residues); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells with a protein fucosylation defect (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al., particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003/085107).

Further provided are antibody variants having bisected oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

Fc region variants

In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region variant can include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

In certain embodiments, the present invention contemplates antibody variants with some but not all effector functions, which makes it desirable candidates for applications where the half-life of the antibody in vivo is important, while some effector functions (e.g., complement and ADCC) are unwanted or harmful. In vitro and/or in vivo cytotoxicity assays can be performed to confirm the reduction/depletion of CDC and/or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (thereby potentially lacking ADCC activity), but retains FcRn binding capacity. NK cells, the main cells for mediating ADCC, express only RIII, while monocytes express RI, RII, and RIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, e.g., Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods can be employed (see, e.g., the ACTI ^™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and the CytoTox non-radioactive cytotoxicity assay (Promega, Madison, WI). Effector cells that can be used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, e.g., WO 2006/029879 and WO 2005/100402. C1q and C3c binding ELISA. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996); Cragg, MS et al., Blood 101: 1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103: 2738-2743 (2004)). FcRn binding and in vivo clearance/half-life determination can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18 (12): 1759-1769 (2006)).

Antibodies with reduced effector function include those with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 ( U.S. Patent No. 6,737,056 ). Such Fc mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitutions of residues 265 and 297 to alanine ( U.S. Patent No. 7,332,581 ).

Specific antibody variants with improved or decreased binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO 2004/056312 and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, the antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues).

In some embodiments, alterations are made in the Fc region to result in altered (e.g., improved or reduced) C1q binding and/or complement-dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half-life and improved binding to the neonatal Fc receptor (FcRn) are described in US 2005/0014934A1 (Hinton et al.), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)). These antibodies comprise an Fc region having one or more substitutions therein that improve binding of the Fc region to FcRn. Such Fc variants include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434, e.g., substitution of Fc region residue 434 ( U.S. Pat. No. 7,371,826 ).

For other examples of Fc region variants, see also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94/29351.

Cysteine-engineered antibody variants

In certain embodiments, it may be desirable to prepare cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced by cysteine residues. In certain embodiments, the replaced residues are located on accessible sites of the antibody. By replacing these residues with cysteine, reactive sulfhydryl groups are placed on accessible sites of the antibody, which can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to produce immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be replaced by cysteine residues: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) in the heavy chain Fc region. Cysteine engineered antibodies can be generated as described in, for example, U.S. Patent No. 7,521,541.

Antibody derivatives

In certain embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous parts known in the art and readily available. Suitable parts for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers) and dextran or poly-(n-vinyl pyrrolidone) polyethylene glycol, propropylene glycol homopolymers, polyoxypropylene/polyoxyethylene copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Polyethylene glycol propionaldehyde, due to its stability in water, can have advantages in manufacturing. Polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can change, and if more than one polymer is attached, they can be identical or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the specific property or function of the antibody to be improved, whether the antibody derivative is to be used in therapy under defined conditions, etc.

In another embodiment, a conjugate of an antibody and a nonproteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength, including, but not limited to, a wavelength that does not harm normal cells but heats the nonproteinaceous moiety to a temperature that kills cells in the vicinity of the antibody-nonproteinaceous moiety.

Recombinant methods and compositions

Antibodies can be produced using recombinant methods and compositions, for example as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an anti-periostin antibody described herein is provided. This nucleic acid can encode an amino acid sequence comprising the VL of the antibody and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chain of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising this nucleic acid are provided. In a further embodiment, a host cell comprising this nucleic acid is provided. In one embodiment, the host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, such as a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., a Y0, NS0, Sp20 cell). In one embodiment, a method of preparing an anti-periostin antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody as provided above under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of anti-periostin antibodies, nucleic acids encoding the antibodies, such as those described above, are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibodies).

Suitable host cells for cloning or expressing antibody encoding vectors include prokaryotic or eukaryotic cells described herein. For example, particularly when glycosylation and Fc effector functions are not required, antibodies can be produced in bacteria. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C.Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing expression of antibody fragments in E. coli). After expression, the antibody can be separated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in production of antibodies with partially or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004) and Li et al., Nat. Biotech. 24: 210-215 (2006).

Suitable host cells for expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES ^™ technology for producing antibodies in transgenic plants).

Vertebrate cells can also be used as hosts. For example, suspension-grown adapted mammalian cell lines can be useful. Other examples of useful mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (such as 293 or 293 cells as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; Buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT060562); TRI cells as described, for example, in Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR ^- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2/0. For a review of some mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

Assay

The anti-periostin antibodies provided herein can be identified, screened, or characterized for their physical/chemical properties and/or biological activity by various assays known in the art.

Binding and other assays

In one aspect, the antibodies of the invention can be tested for their antigen binding activity, for example, by known methods such as ELISA, Western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with IL-13 or periostin for binding to IL-13 or periostin, respectively. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitope) as lebrikizumab or another anti-IL-13 antibody described in detail herein or an anti-periostin antibody described in detail herein. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology, Vol. 66 (Humana Press, Totowa, NJ).

In an exemplary competitive assay, immobilized periostin is incubated in a solution comprising a first labeled antibody (e.g., 25D4) that binds to periostin and a second unlabeled antibody to be tested for its ability to compete with the first antibody for binding to periostin. The second antibody can be present in the hybridoma supernatant. As a control, immobilized periostin is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to periostin, excess unbound antibody is removed and the amount of label bound to the immobilized periostin is measured. If the amount of label bound to the immobilized periostin is substantially reduced in the test sample relative to the control sample, this indicates that the second antibody competes with the first antibody for binding to periostin. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

Activity assay

In one aspect, assays are provided for identifying anti-IL-13 antibodies having biological activity. The biological activity can include, for example, activity in asthma. Antibodies having such biological activity in vivo and/or in vitro are also provided.

In certain embodiments, the antibodies of the invention are tested for such biological activities.

Immunoconjugates

The invention also provides immunoconjugates comprising an anti-periostin antibody herein conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, the immunoconjugate is an antibody-drug conjugate (ADC) in which the antibody is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235). B1); auristatin such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. Res. 58:2925-2928 (1998)); anthracyclines such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; trichothecenes; and CC1065.

In another embodiment, the immunoconjugate comprises an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

In another embodiment, the immunoconjugate comprises an antibody as described herein that is conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes can be used for the production of radioconjugates. Examples include At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioisotopes of Lu. When the radioconjugate is used for detection, it can include radioactive atoms for scintillation studies, such as tc 99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, mri), such as iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl 3-(2-pyridyldithiol) propionate (SPDP), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclides to antibodies. See WO94/11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug in the cell. For example, an acid-sensitive linker, a peptidase-sensitive linker, a photosensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al. Cancer Research 52: 127-131 (1992)).

Immunoconjugates or ADCs are specifically contemplated herein, but are not limited to, such conjugates prepared with cross-linker reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which is commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., U.S.A.).

Methods and compositions for diagnosis and detection

In certain embodiments, any of the anti-periostin antibodies provided herein can be used to detect the presence of periostin in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissues, such as serum, plasma, nasal swabs, and sputum.

In one embodiment, anti-periostin antibodies are provided for use in a diagnostic or detection method. In a further aspect, a method for detecting the presence of periostin in a biological sample is provided. In a specific embodiment, the method comprises contacting the biological sample with an anti-periostin antibody as described herein under conditions that allow the anti-periostin antibody to bind to periostin, and detecting whether a complex is formed between the anti-periostin antibody and periostin. This method can be an in vitro or in vivo method. In one embodiment, the anti-periostin antibody is used to select eligible subjects for a therapy that uses an anti-IL-13 antibody or any other TH2 pathway inhibitor, for example, when periostin is a biomarker for patient selection.

Exemplary disorders that can be diagnosed using the antibodies of the invention are provided herein.

In certain embodiments, labeled anti-periostin antibodies are provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent, chromogenic, electron-dense, chemiluminescent, and radioactive labels), as well as indirectly detectable moieties such as enzymes or ligands (e.g., via enzymatic reactions or molecular interactions). Exemplary labels include, but are not limited to, radioisotopes ³² P, ¹⁴ C, ¹²⁵ I, ³ H, and ¹³¹ I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled to enzymes that utilize hydrogen peroxide to oxidize dye precursors such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, phage labels, stable free radicals, and the like.

pharmaceutical preparations

Pharmaceutical formulations of anti-IL-13 antibodies or other TH2 pathway inhibitors as described herein can be prepared by mixing an antibody or molecule having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Mark (1980)) in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) Polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersants such as soluble neutral-active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (Baxter International, Inc.). Some exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinase.

Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.

The formulations herein may also contain more than one active ingredient as needed for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a control agent along with a TH2 pathway inhibitor. Such active ingredients are suitably present in combination in amounts effective for their intended purpose.

The active ingredient can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980).

Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices can be in the form of shaped articles, eg, films, or microcapsules.

Formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

Methods of treatment and compositions

Eosinophilic inflammation is associated with a variety of allergic and non-allergic lesions (Gonlugur (2006) Immunol. Invest. 35 (1): 29-45). Inflammation is a restorative response of living tissue to injury. The inflammatory response is characterized by the accumulation of leukocytes in damaged tissues due to some chemicals produced in the damaged tissue itself. Eosinophils accumulate in a wide variety of conditions, such as allergic conditions, helminth infections, and neoplastic diseases (Kudlacz et al., (2002) Inflammation 26: 111–119). As a component of the immune system, eosinophils are defensive elements of mucosal surfaces. They respond not only to antigens, but also to parasites, chemicals, and trauma.

Tissue eosinophilia occurs in skin diseases such as eczema, pemphigus, acute urticaria and toxic epidermal necrolysis and atopic dermatitis ([Rzany et al., 1996]). In the allergic skin reaction of IgE mediation, eosinophils gather in tissue and empty granule protein ([Nielsen et al., 2001]). Eosinophils and mast cell combinations may cause joint inflammation (Miossec et al., 1997). Eosinophilic inflammation is sometimes associated with joint trauma. Synovial fluid eosinophilia may be relevant to the following disease, such as rheumatoid arthritis, parasitic diseases, high eosinophil syndrome, Lyme disease and allergic process and hemarthrosis and arthrography ([Atanes et al., 1996]). Eosinophilic inflammation can also affect bone ([Yetiser et al., 2002]). The example of eosinophilic myopathy includes eosinophilic perimyositis, eosinophilic polymyositis and focal eosinophilic myositis ([Lakhanpal et al., 1988]). The eosinophilic inflammation affecting skeletal muscle can be associated with the features of some systemic conditions (such as idiopathic hypereosinophilic syndrome and eosinophilic myalgia syndrome) of parasitic infection or medicine or excessive eosinophilia. Eosinophils participate in the inflammatory reaction ([Engineer et al., 2001]) of the epi-position identified for autoimmune antibodies. Connective tissue disease can cause neutrophil, eosinophil or lymphocytic vasculitis ([Chen et al., 1996]). Tissue and peripheral blood eosinophilia can occur in active rheumatic diseases. Elevated serum ECP levels in ankylosing spondylitis, a connective tissue disease, suggest that eosinophils are also involved in this underlying process (Feltelius et al., 1987). Wegener's granulomatosis can rarely be associated with pulmonary nodules, pleural effusions, and peripheral blood eosinophilia ([Krupsky et al., 1993]).

Peripheral blood eosinophilia of at least 400 cells/ ^mm3 can occur in 7% of systemic sclerosis cases, 31% of localized scleroderma cases, and 61% of eosinophilic fasciitis cases ([Falanga and Medsger, 1987]). Scleroderma produces an inflammatory process that closely resembles the submucosal and myenteric plexuses, composed of mast cells and eosinophils in the gastrointestinal system. Eosinophil-derived neurotoxins can contribute to gastrointestinal motility disorders, such as those that occur in scleroderma ([de Schryver Kecskemeti and Clouse, 1989]).

Eosinophils can be accompanied by localized ([Varga and Kahari, 1997]) or systemic ([Bouros et al., 2002]) connective tissue hyperplasia. They can cause fibrosis ([Hernnas et al., 1992]) by inhibiting the degradation of proteoglycans in fibroblasts, and fibroblasts mediate eosinophil survival ([Vancheri et al., 1989]) by secreting GM-CSF. Eosinophils can be found in the nose ([Bacherct et al., 2001]), bronchus ([Arguelles and Blanco, 1983]) and gastrointestinal polyp tissue ([Assarian and Sundareson, 1985]). Similarly, eosinophils can be located in inflammatory pseudotumors (myofibroblastoma). Eosinophils are often accompanied by inflammatory pseudotumors in the orbital region, and in the case of the condition, the condition can be similar to angioedema or allergic rhinoconjunctivitis ([Li et al., 1992]).

Eosinophilic inflammation can be found in tissue trauma (e.g., due to surgery or injury). Eosinophilic inflammation can also be associated with cardiovascular disease (e.g., eosinophilic myocarditis, eosinophilic coronary arteritis, ischemic heart disease, acute myocardial infarction, heart rupture). Necrotizing inflammatory processes can also involve eosinophilic inflammation (necrotic damage, dementia, cerebral infarction in polymyositis, coronary artery dissection, neuro-Behcet's disease).

Provided herein are methods for identifying eosinophilic inflammation positive (EIP) patients predicted to respond (or will respond) to treatment with a TH2 pathway inhibitor by measuring total serum periostin levels in the patients.

Also provided herein are methods of treating asthma, an eosinophilic disorder, an IL-13-mediated disorder, an IL4-mediated disorder, an IL9-mediated disorder, an IL5-mediated disorder, an IL33-mediated disorder, an IL25-mediated disorder, a TSLP-mediated disorder, an IgE-mediated disorder, or asthma-like symptoms comprising administering a TH2 pathway inhibitor to a patient positive for eosinophilic inflammation, wherein the patient has been diagnosed with EIP by measuring total serum periostin levels in the patient.

In certain embodiments, methods of treating asthma, eosinophilic disorders, IL-13-mediated disorders, IL-4-mediated disorders, or IgE-mediated disorders are provided, comprising administering lebrikizumab to a patient positive for eosinophilic inflammation.

In certain embodiments, methods of treating asthma, eosinophilic disorders, IL-13-mediated disorders, IL-4-mediated disorders, or IgE-mediated disorders are provided, comprising administering a fixed dose of 125-500 mg of lebrikizumab every 4 weeks to a patient suffering from the disorder.

Also provided are methods of treating asthma (or respiratory disease) comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in a relative change in FEV1 greater than 5%. In another embodiment, the FEV1 is greater than 6%, 7%, 8%, 9% or 10% FEV1. In another embodiment, the patient has been diagnosed with EIP using a total periostin assay. In another embodiment, the asthma patient has been diagnosed using a total serum periostin assay.

In certain embodiments, methods of treating asthma (or respiratory disease) are provided, comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in a greater than 35% reduction in exacerbation rate (other embodiments, greater than 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, up to 85%; in another embodiment, wherein the patient has been diagnosed with EIP).

In certain embodiments, methods of treating asthma (or respiratory disease) are provided, comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in a reduction in nighttime awakenings. In one embodiment, the patient is diagnosed by measuring the total serum periostin level in the patient. In another embodiment, the patient's asthma is uncontrolled with corticosteroids. In another embodiment, the patient is diagnosed with EIP.

Also provided are methods of treating asthma (or respiratory disease) comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in improved asthma control. In one embodiment, the patient is diagnosed by measuring total serum periostin levels in the patient. In another embodiment, the asthma is uncontrolled with corticosteroid treatment. In another embodiment, the patient is diagnosed with EIP.

Provided are methods for treating asthma (or respiratory disease) comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in a reduction in inflammation in the lungs. In one embodiment, the patient is diagnosed by measuring total serum periostin levels in the patient. In another embodiment, the asthma is not controlled with corticosteroid therapy. In another embodiment, the patient is diagnosed with EIP.

In certain embodiments, methods for treating eosinophilic disorders in patients who are suffering from eosinophilic disorders and are being treated with corticosteroids are provided, the methods comprising administering a therapeutically effective amount of Lebrikizumab to an asthma patient, wherein the treatment results in a reduction or elimination of the amount or frequency of corticosteroid therapy used to treat the disease. In one embodiment, the patient is diagnosed by measuring the total serum periostin level in the patient. In another embodiment, the patient's asthma is not controlled with corticosteroids. In another embodiment, the patient is diagnosed with EIP prior to treatment.

Also provided are methods for treating a patient suffering from asthma (or a respiratory disease) comprising: diagnosing the patient as EIP using a total periostin assay, administering a therapeutically effective amount of a TH2 pathway inhibitor to the asthma patient, diagnosing the patient as EIP, and if the status is EIP, re-treating the patient with the TH2 pathway inhibitor. The diagnosis is performed using a total periostin assay alone or in combination with FE _NO levels, and optionally in combination with other biomarkers selected from the group consisting of CST1, CST2, CCL26, CLCA1, PRR4, PRB4, SERPINB2, CEACAM5, iNOS, SERPINB4, CST4, and SERPINB10. In yet another embodiment, the patient to be treated has a FE _NO level greater than 21 ppb in addition to elevated expression levels of periostin as described herein. In yet another embodiment, the patient to be treated has a FE _NO level greater than 35 ppb in addition to elevated expression levels of periostin as described herein.

In certain embodiments, a method of identifying an eosinophilic inflammation negative (EIN) patient is provided, comprising the steps of measuring total periostin levels in the patient and determining that the patient is EIN.

Any TH2 pathway inhibitor provided herein can be used in the methods of treatment described herein, particularly in asthma. In one embodiment, the asthma patient is being treated with corticosteroids and has been diagnosed as responsive to a TH2 pathway inhibitor using the periostin assay described herein. In a further embodiment, the asthma patient is suffering from moderate to severe asthma. In another embodiment, the patient is suffering from mild asthma but is not being treated with corticosteroids.

The antibodies of the present invention (and any additional therapeutic agents) can be administered by any suitable method, including parenteral, intrapulmonary and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection (depending in part on whether administration is temporary or long-term). Various dosing regimens are contemplated herein, including, but not limited to, single or multiple administrations over multiple time points, bolus administration, and pulse infusions.

The antibodies of the present invention will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered include the specific condition to be treated, the specific mammal to be treated, the clinical condition of the individual patient, the cause of the condition, the active agent delivery site, the method of administration, the administration schedule, and other factors known to medical practitioners. The antibodies need not but may optionally be formulated with one or more active agents currently used to prevent or treat the condition in question. The effective amount of such other active agents depends on the amount of antibody present in the formulation, the type of condition or treatment, and the other factors discussed above. These are generally used in the same dosages and routes of administration as described herein, or in about 1-99% of the dosages described herein, or in any dosage and any route determined empirically/clinically to be suitable.

For disease prevention or treatment, the appropriate dosage of the antibodies of the present invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prevention or treatment, previous therapy, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibodies of the present invention are suitable for administration to the patient once or over a series of treatments. Depending on the type of disease and severity, about 1 μg/kg-15 mg/kg (e.g., 0.1 mg/kg-10 mg/kg) of antibody can be the starting candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose can be about 1 μg/kg-100 mg/kg or more. For repeated administration over several days or longer periods, depending on the situation, treatment generally continues until desired disease symptoms are suppressed. An exemplary antibody dosage is about 0.05 mg/kg-about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) can be administered to the patient. This dosage can be administered intermittently, for example, weekly or once every three weeks (e.g., so that the patient receives about two to about twenty, e.g., about six doses of the antibody). However, other dosage regimens are also available. The progress of this therapy can be easily monitored by conventional techniques and assays.

In a specific embodiment, the antibody of the present invention is administered at a fixed dose of 37.5 mg (i.e., not weight-dependent), or a fixed dose of 125 mg, or a fixed dose of 250 mg. In a specific embodiment, the dose is administered by subcutaneous injection once every 4 weeks for a period of time. In a specific embodiment, the period of time is 6 months, 1 year, 2 years, 5 years, 10 years, 15 years, 20 years, or the patient's lifetime. In a specific embodiment, the asthma is severe asthma, and the patient is not adequately controlled or uncontrolled when treated with inhaled corticosteroids plus a second controller medication. In another embodiment, the EIP status is determined using a total periostin assay, the patient is diagnosed with EIP status, and the patient is selected for treatment with an anti-IL-13 antibody as described above. In another embodiment, the method comprises treating an asthma patient with an anti-IL-13 antibody as described above, wherein the patient has previously been diagnosed with EIP status by determining EIP status using a total periostin assay. In one embodiment, the asthma patient is 18 years of age or older. In one embodiment, the asthma patient is 12-17 years old and the anti-IL-13 is administered as a fixed dose of 250 mg or a fixed dose of 125 mg. In one embodiment, the asthma patient is 6-11 years old and the anti-IL-13 antibody is administered as a fixed dose of 125 mg.

It will be understood that any of the above-described formulations or treatment methods may be practiced using the immunoconjugates of the invention in place of or in addition to anti-target antibodies or anti-periostin antibodies.

manufactured products

In another aspect of the present invention, an article of manufacture containing materials for treating, preventing, and/or diagnosing the conditions described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition, alone or in combination with another composition effective for treating, preventing, and/or diagnosing the condition, and can have a sterile access port (e.g., the container can be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody of the present invention. The label or package insert indicates that the composition is used to treat the selected condition. Additionally, the article of manufacture can comprise (a) a first container containing a composition comprising an antibody of the present invention; and (b) a second container containing a composition comprising another cytotoxic agent or additional therapeutic agent. In this embodiment of the invention, the article of manufacture can further comprise a package insert indicating that the composition can be used to treat a specific condition. Alternatively or additionally, the article of manufacture may further include a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

It will be understood that any of the above articles of manufacture may include an immunoconjugate of the invention in place of or in addition to an anti-target antibody or an anti-periostin antibody.

Example

Example 1 - Allergen challenge clinical study

Phase II randomized, double-blind, placebo-controlled study; 5 mg/kg lebrikizumab:placebo (1:1 ratio) subcutaneously every 4 weeks for 12 weeks. Table 2 and Figure 1 provide a high-level overview of the trial design. The primary outcome measure was allergen-induced late asthmatic reaction (LAR) at week 13. Patients received an allergen challenge at screening and at week 13, followed by a methacholine challenge 18-24 hours later. Serum biomarkers were also evaluated to confirm IL13 pathway inhibition and identify patients who derive increased benefit from lebrikizumab.

Table 2

Patients included in the study were mild asthmatics, 18-55 years of age, with: (a) a positive skin test (> or = 3 mm over negative control) at screening for house dust mites, cat dander, or ragweed; (b) a forced expiratory volume in 1 second (FEV1) > or = 70% predicted; and (c) an early asthmatic reaction of > or = 20% reduction in FEV1 within 5-30 minutes after allergen challenge, and a late asthmatic reaction (LAR) of > or = 15% reduction in FEV1 within 2-8 hours after challenge.

Twenty-eight patients were included in the analysis of the primary endpoint (n=16 placebo, 12 lebrikizumab). At week 13, lebrikizumab suppressed the LAR. The mean AUC (area under the curve) of FEV1 2-8 hours after allergen challenge (LAR) was reduced by 48% in lebrikizumab-treated patients compared with placebo (26.3% vs. 50.5%; 95% CI: -19 to 90%), with no effect on the early response (EAR) at week 13. The mean AUC of FEV1 0-2 hours after allergen challenge and the maximum reduction in FEV1 were similar in the lebrikizumab and placebo groups (27.5% vs. 26.4% for both parameters; Table 3). See also Figure 2. No significant differences were observed between the lebrikizumab and placebo groups in airway hyperresponsiveness to methacholine. The arithmetic mean of methacholine doublings in the lebrikizumab group was 0.33 doublings higher than in the placebo group (1.58 versus 1.25, 95% CI -0.64 to 1.3), which is not considered clinically meaningful inhibition.

Table 3

Lebrikizumab treatment significantly produced a systemic effect on Th2 inflammatory markers, wherein serum IgE, MCP-4/CCL13 and TARC/CCL17 had an average placebo-adjusted reduction of 24%, 25% and 26% respectively (p < 0.01). Referring to Figures 3A and 3B. Serum periostin levels slightly decreased (5-10%) after treatment. Serum IL-13 levels were mostly below the detection level (<150pg/ml) after treatment. Figure 3C shows the reduction of IgE, CCL13 and CCL17 (relative to the baseline levels of these markers) in individual patients at week 13. Generally, periostin, YKL-40, CEA and blood eosinophil levels did not change significantly after lebrikizumab treatment (data not shown).

Subjects with baseline levels above the median for peripheral blood eosinophils, serum IgE, or serum periostin showed greater placebo-adjusted lebrikizumab-dependent reductions in LAR than subjects with baseline levels below the median for these biomarkers. See Figure 4.

Patients were classified as "biomarker high" or "biomarker low" based on whether the following inflammatory biomarkers were above or below the median level at baseline: serum IgE, CLL13, CCL17, CEA, periostin, YKL-40, and peripheral blood eosinophils. As shown in Figure 4, the results for serum IgE, eosinophils, and periostin indicate that patients with elevated (compared to the median) baseline serum IgE, periostin, or increased peripheral blood eosinophils are more likely to respond to IL13 blockade (e.g., by lebrikizumab).

In summary, the study met its primary endpoint: a 48% reduction in mean late AUC (90% CI [-19%, 90%]). Lebrikiuzumab significantly reduced LAR compared to placebo. No safety signals were observed in the safety data. Therapeutic blockade of IL-13 may be an effective treatment for allergic asthma, particularly in patients with elevated airway TH2 inflammatory markers.

Example 2 - Clinical Study I in Asthma Patients

A randomized, double-blind, placebo-controlled study was conducted to assess the effect of lebrikizumab in patients with asthma who were unable to obtain adequate control or control during long-term treatment with ICS. Patients continued their standard-of-care therapy, which includes inhaled corticosteroids (ICS) and may also include LABA. In this two-arm study, patients were randomly assigned to receive lebrikizumab or placebo for a total of 6 months. During the 14-20 day run-in period (visit 1 to visit 3), patients must show compliance with ICS and be able to use the equipment required for daily monitoring throughout the study. The eligibility of the patients for the study was subsequently assessed and randomly assigned (1: 1) to the study drug (lebrikizumab or placebo), wherein stratification was performed based on IL-13 label surrogate status (further described below), LABA use, and research site. The first SC dose occurred within 24 hours of random assignment (study day 1, i.e., the day of random assignment, regardless of the first study drug administration date). Administration of study drug was repeated every 4 weeks for the next 20 weeks (a total of six study drug doses, providing a 24-week treatment period).Measures of the efficacy of lebrikizumab were assessed during the treatment period.

Study drug was administered to selected patients via subcutaneous (SC) injection at the following time points: Day 1 and Weeks 4, 8, 12, 16, and 20. Each dose of lebrikizumab was 250 mg. Each placebo dose was 2 mL of the same solution without lebrikizumab. SC injections were administered in the arm, thigh, or abdomen. A schematic diagram of the trial design can be seen in Figure 5.

The primary analysis was performed after all (approximately 200) patients had been treated and followed for 24 weeks after randomization (Day 1). Safety was evaluated throughout the study. After the last dose of study drug (Week 20), patients were monitored for an additional 12 weeks; the first 4 weeks after the last dose were considered part of the (24-week) treatment period, while the last 8 weeks constituted the follow-up period. The 8-week follow-up period, together with the last 4 weeks of the treatment period, allowed patients to be monitored for 3-4 half-lives after the last dose. Therefore, patients were generally included in the study for a total of approximately 34 weeks. See Figure 5. Efficacy-evaluable patients (n=180) were defined for decision-making purposes to exclude subjects based on two reasons: (1) they were not part of the target population on key characteristics, and (2) the baseline FEV1 on which the treatment effect was measured was unreliable. See Figure 6 for baseline characteristics of patients participating in the trial.

Table 4

Key inclusion criteria included the following: ability to perform spirometry according to the study-defined pulmonary function test (PFT) manual at Visits 1-3; no evidence of clinically significant abnormalities on chest radiograph within 12 months of Visit 1; ability to complete study materials as measured by compliance with daily completion and PEF measurements between Visits 1-3; and uncontrolled asthma selected based on all of the following criteria:

->12-month asthma diagnosis

- Bronchodilator response at visit 1 or 2

- Pre-bronchodilator FEV1 ≥ 40% predicted and ≤ 80% predicted at Visits 1 and 3

- Use of ICS ≥200 mcg and ≤1000 mcg total daily dose of fluticasone propionate (FP) or equivalent for at least 6 consecutive months prior to Visit 1

- Visit 3 Asthma Control Questionnaire (ACQ) score ≥1.5 despite ICS compliance.

Key exclusion criteria include the following:

Medical conditions:

- Asthma exacerbation, significant airflow obstruction, or respiratory tract infection starting from Visits 1-3

- Known malignancy or current evaluation for potential malignancy

- Known immunodeficiency, including but not limited to HIV infection

- Pre-existing non-asthmatic lung disease, including acute infections

- Uncontrolled clinically significant medical illness

Exposure:

-Current smokers or former smokers with a lifetime smoking history >10 pack-years

- Concomitant medications prohibited (non-ICS steroids, non-SABA short-acting bronchodilators, immunomodulators)

- Pregnancy or unwillingness to use highly effective contraception.

Given the practical difficulties of measuring IL-13 in the lung itself, eosinophil and IgE levels in peripheral blood were used as surrogate measures, denoted as "IL-13 signature surrogate." IL-13 signature surrogate-positive patients were defined as those with total IgE>100 IU/mL and blood eosinophils x 10xe9 cells/L, while IL-13 signature surrogate-negative patients had total IgE≤100 IU/mL or blood eosinophils <0.14 x 10xe9 cells/L. The criteria for IgE and eosinophil levels used to determine a patient's IL-13 signature surrogate status were established by patients with asthma in whom IL-13-induced gene expression (IL-13 signature) in the bronchial epithelium correlated with peripheral blood IgE and eosinophils (Corren et al., N Engl J Med 365:1088-1098 [2011]).

The efficacy of lebrikizumab was evaluated using multiple measures of asthma activity, including lung function (i.e., FEV1, peak flow, including variability in peak flow, response to methacholine at selected centers, fractional exhaled nitric oxide [ _FENO ]) and measures of disease activity or control (i.e., patient-reported outcomes [PROs], use of rescue medication, exacerbation rate). The change in FEV1 was the primary outcome. The following markers were used for pharmacodynamic analysis after treatment with lebrikizumab: TARC (CCL17), MCP-4 (CCL13), and IgE.

Primary efficacy outcome measure

The primary efficacy outcome measure was the relative change from baseline to Week 12 in pre-bronchodilator FEV1 (volume).

Secondary efficacy outcome measures

Secondary efficacy outcome measures were as follows: relative change in pre-bronchodilator FEV1 (volume) from baseline to Week 24; relative change in pre-bronchodilator FEV1 (volume) from baseline to Week 12 for patients with positive IL-13 signature surrogate status; change in Asthma Control Questionnaire (ACQ) score from baseline to Week 12; change in asthma symptom score measured by the Asthma Control Daily Diary (ACDD) from baseline to Week 12; change in morning pre-bronchodilator peak flow value from baseline to Week 12; rate of asthma exacerbations during the 24-week treatment period; rate of severe asthma exacerbations during the 24-week treatment period; and change in rescue medication use (measured by the number of times per day that rescue medication was inhaled or nebulized).

Exploratory metrics:

The following exploratory outcome measures were assessed: change in the number of days per week with well-controlled asthma as measured by ACDD from baseline to week 12; change in the weekly frequency of nighttime awakenings due to asthma from baseline to week 12; and change in FE _NO levels from baseline to week 12.

Initial observation

In this study of patients with poorly controlled asthma, lebrikizumab treatment was associated with a statistically significant improvement in pre-bronchodilator FEV1 (the primary outcome variable). The improvement in FEV1 occurred shortly after treatment began, indicating that IL-13 inhibition affects airflow measurements relatively quickly. Although lebrikizumab treatment did not result in a statistically significant reduction in protocol-defined and severe exacerbations using the periostin assay (described below, as values could not be obtained to facilitate this analysis), a trend toward a decrease in the rate of severe exacerbations was observed (especially for patients with high periostin levels). However, using the E4 assay described below, lebrikizumab treatment did result in a statistically significant reduction in protocol-defined and severe exacerbations (84% (95% CI 14%, 97%, p=0.03) in the periostin-high subgroup. See also below. In addition, the FE _NO subgroup analysis did achieve a statistically significant reduction in severe exacerbations (p=0.04). Lebrikizumab treatment did not improve asthma symptoms as measured by the symptom-only ACQ5 (excluding rescue SABA use and FEV1) or daily diary measures.

The reduction in serum Th2 chemokines CCL13 and CCL17 and IgE supports the biological effects mediated by lebrikizumab, which underlie the clinical effects measured in the airways. The slight increase in blood eosinophil counts is consistent with the overall reduced trafficking of eosinophils from the blood to the lung compartment after inhibition of chemokines that attract eosinophils. The finding that lebrikizumab reduced FE _NO is consistent with this suggestion. However, lebrikizumab may have led to the reduction in FE _NO by indirectly inhibiting nitric oxide synthase expression through IL-13 blockade, rather than by modifying eosinophil inflammation (which is also thought to affect FE _NO ).

For this study, patient eligibility criteria required at least 12% reversibility for 400 μg of albuterol (no SABA use for at least 4 hours before the visit and no LABA use for at least 12 hours). This ensured that patients had "wiggle room" when looking for an overall relative change of at least 10% in lung function. This simple clinical trial may be a limitation on the ability to generalize these data to an unselected, more general population of asthma patients. In fact, the most common reason for patient ineligibility was failure to pass the trial (92 of the 263 patients who failed screening).

In this study, we first hypothesized that the combination of high serum IgE and high blood eosinophil counts was a surrogate for identifying patients with increased expression of IL-13-related genes in the lungs (IL-13 signature surrogate, or Th2 high). Although the data were still masked with respect to treatment assignment, we wrote a statistical analysis plan to use a distinction based on serum periostin levels. As described in more detail below, this subgroup analysis showed that the effectiveness of lebrikizumab treatment was enhanced in patients with high periostin relative to those with low periostin, as observed by a stronger increase in FEV1 and a greater decrease in FE _NO , as well as a significant interaction test. These findings suggest that a pre-specified marker, serum periostin, can potentially be used to select asthma patients who may be more responsive to lebrikizumab treatment.

The baseline characteristics of the patients participating in this study are shown in Figure 6. Unless otherwise indicated, the numbers refer to the mean (SD). Periostin high refers to patients with a baseline periostin level of more than 23 ng/ml according to the E4 assay described below. Periostin low refers to patients with a baseline periostin level of less than 23 ng/ml according to the E4 assay described below.

The relative changes (95% confidence intervals) in FEV1 (liters) at Weeks 12 and 24 for treated patients are shown in Figure 7. Periostin-positive patients experienced a greater relative change in FEV1 compared to IL-13 signature-positive patients.

Figure 7 shows that the placebo-corrected relative change in FEV1 from baseline to week 12 was 5.9% (95% CI 0.9%, 10.9%, p = 0.2) for all participants and 10% (95% CI 2.5%, 17.5%, p = 0.01) for the periostin-high subgroup. The placebo-corrected relative change in FEV1 from baseline to week 24 was 4.8% (95% CI 0.3%, 9.3%, p = 0.04) for all participants and 6.1% (95% CI -1.4%, 13.6%, p = 0.11) for the periostin-high subgroup. Figure 8 shows the relative change in FEV1 over the entire treatment period (32 weeks) for all efficacy-evaluable subjects (A) and only periostin-high subjects (B). The efficacy of lebrikizumab did not diminish at week 32 of treatment, suggesting that the frequency of lebrikizumab administration may be reduced.

We also examined the effect of lebrikizumab treatment on post-bronchodilator FEV1 as an exploratory outcome and an indirect surrogate for airway remodeling. Changes in post-bronchodilator FEV1 at 20 weeks were measured after four 100mcg inhalations of salbutamol. At baseline before study drug administration, mean post-bronchodilator FEV1 was 2.50 liters (0.71) and 2.54 liters (0.73) in the placebo and lebrikizumab groups, respectively. This corresponded to 77.9% of predicted in both groups. The overall placebo-corrected change in post-bronchodilator FEV1 was 4.9% (95% CI 0.2% to 9.6%; p = 0.04). In the periostin-high group, lebrikizumab treatment increased post-bronchodilator FEV1 at 20 weeks (8.5%; 95% CI 1.1% to 16%; p = 0.03). There was no evidence of improvement in postbronchodilator FEV1 in the low periostin group (1.8%; 95% CI -4.1 to 7.7; p = 0.55). In addition, lebrikizumab increased absolute postbronchodilator FEV1 in the high periostin group (170 mls; 95% CI 10 to 320 ml). We conclude that lebrikizumab improved postbronchodilator FEV1 in patients with uncontrolled asthma who had evidence of increased Th2 airway inflammation (periostin high). Based on these data, lebrikizumab may have the potential to reduce airway remodeling in asthma.

Figure 9 shows the exacerbation rate and severe exacerbation rate observed at 24 weeks of treatment. Compared with placebo, the exacerbation rate was reduced by 37% (95% CI -22%, 67%, p = 0.17) in all patients treated with lebrikizumab, and by 61% (95% CI -6%, 86%, p = 0.07) in the periostin high subgroup. The severe exacerbation rate was reduced by 51% (95% CI -33%, 82%, p = 0.16) for all patients, and by 84% (95% CI 14%, 97%, p = 0.03) in the periostin high subgroup. We also examined the severe exacerbation rate in the ITT population at 32 weeks (end of follow-up, 12 weeks after the last dose of lebrikizumab). As shown in Table 10, the severe exacerbation rate was significantly reduced by 50% (p = 0.03) in all patients treated with lebrikizumab at 32 weeks compared to placebo. The rate of severe exacerbations was lowest in patients with high periostin levels treated with lebrikizumab (0.13); however, the reduction in this rate relative to placebo did not reach statistical significance in this subanalysis. We conclude that lebrikizumab has a significant benefit in reducing severe exacerbations in patients who are inadequately controlled on ICS. The greatest reduction in severe exacerbations was seen in patients with high periostin levels before treatment. The duration of observation to capture events is important for running studies on this endpoint; to detect at least a 50% reduction in approximately 200 subjects, 32 weeks of observation may be required compared with 24 weeks.

Table 10. Rate of severe exacerbations at Week 32 (12 weeks after final dose).

Lebrikizumab met its primary endpoint and was very effective in reducing severe exacerbations. The data suggest that the patient's periostin status can prognose exacerbation events and, to a lesser extent, prognose FEV1. The data support the predictive value of periostin levels for determining the response to Lebrikizumab treatment. For example, using the E4 assay, those patients with serum or plasma periostin levels below 20 ng/ml are less likely to benefit from lebrikizumab, while those patients with serum or plasma periostin levels exceeding 20 ng/ml are more likely to benefit from lebrikizumab. In addition, those patients with serum or plasma periostin levels exceeding 23 ng/ml (as determined by the E4 assay) have an even greater likelihood of benefiting from lebrikizumab.

Patient-Reported Outcomes: The ACQ and Mini-AQLQ did not show consistent differences between treatment groups. After several visits, the ACDD data showed some differences in mean scores between treatment groups for well-controlled days, asthma symptoms, nighttime awakenings, and rescue medication use, all in favor of lebrikizumab treatment. For all endpoints in the ACDD data (well-controlled days, asthma symptoms, nighttime awakenings, and rescue medication use), there was a greater placebo-adjusted treatment effect in periostin-high subjects compared to all subjects; however, none of the differences between treatment groups were statistically significant at 12 weeks.

Pulmonary function: Improvements in pulmonary function (PEF) relative to baseline were observed in all lebrikizumab-treated subjects at most time points and in periostin-high lebrikizumab-treated subjects at all time points. Placebo-treated subjects experienced a decrease relative to baseline at all time points.

Pulmonary Inflammation: For lebrikizumab-treated subjects and subjects in the periostin-high subgroup, FE _NO decreased (improved) throughout the study, while placebo-treated subjects had an FE _NO increase (worsening). The percent change in FE _NO at 12 weeks was -20.1% for lebrikizumab-treated subjects (n=83) and 15% for placebo-treated subjects (n=86) [p<0.01]. The percent change in FE _NO at 12 weeks was -24.2% for lebrikizumab-treated subjects and 17.5% for placebo-treated subjects (n=44) [p<0.01]. The differences between treatment groups at 12 weeks were statistically significant. See Figure 10. In post hoc analyses, high baseline FE _NO was also associated with efficacy in improving FEV1 and a lower rate of severe exacerbations compared to placebo. However, we noted that baseline FE _NO showed greater intra-patient variability during the run-in period compared to periostin (19.8% vs. 5.0%).

We also examined the data to determine whether patients with low periostin and high FE _NO benefited from lebrikizumab treatment to the same extent as patients with high periostin. At baseline, we observed a moderate correlation between FE _NO and serum periostin (rs = 0.31, P <0.001; N = 210 with matched data). Using the median cutoff, periostin and FE _NO status generally but not completely overlapped (Table 11). For the data shown in Table 11, the periostin cutoff was 25 ng/mL using the E4 assay described below; the FENO cutoff was 21 ppb measured using standard methods known in the art. FEV1 assessment based on composite periostin and FE _NO status revealed that the placebo-adjusted lebrikizumab effect at 12 weeks was greatest in the group with high levels of periostin and FE _NO (Table 12). In contrast, the treatment benefit observed in the periostin low/FE _NO high group was small (2.3%, P = 0.73). Therefore, FE _NO does not appear to identify a subset of periostin-low patients who may benefit from lebrikizumab. The relative predictive value of periostin and FE _NO for lebrikizumab treatment response warrants further investigation.

Table 11. Distribution of patients according to baseline periostin and FE _NO status.

p = 0.0004, Fisher's exact test (1-sided)

Table 12. Mean relative change from baseline in FEV ₁ at week 12 in ITT patients. For all patients meeting protocol-defined inclusion criteria, the median baseline level of FE _NO or periostin was used to define the subsets described in the table (high = median or higher; low = less than median).

The pharmacokinetic profile of lebrikizumab was similar to that seen in previous studies. Subject weight had an effect on the pharmacokinetics of lebrikizumab.

Several markers were evaluated for their ability to provide predictive value, in addition to or in place of serum periostin levels, with respect to the above treatment benefit. These markers included CEA, IgE, TARC (CCL17), and MCP-4 (CCL13).

Additionally, we hypothesized that the excess serum periostin in patients with high periostin levels was due to the effects of IL-13. Therefore, we sought to evaluate the relative contribution of IL-13 to total systemic periostin levels in patients with uncontrolled asthma treated with placebo and lebrikizumab. For these experiments, we measured serum periostin using the periostin assay described in Example 7. As shown in Figures 18 and 19, we found that the majority (>90%) of patients with low periostin at baseline remained low in both arms of the study (Figure 18A), while 72% of patients with high periostin treated with placebo and 40% of patients with high periostin treated with lebrikizumab remained high at week 12 (Figure 18B). We conclude that in adults with uncontrolled asthma despite ICS use, patients with high periostin levels showed a significant reduction in serum periostin levels after lebrikizumab treatment compared to placebo, but patients with low periostin levels did not show a significant reduction in response to lebrikizumab. These findings suggest that in patients with uncontrolled asthma, excess serum periostin is due to IL-13 activity and that inhibition of IL-13 with lebrikizumab can reduce serum periostin levels.

Four lebrikizumab-treated patients experienced serious adverse events (SAEs) (asthma exacerbation [n=2], community-acquired pneumonia, and traumatic pneumothorax related to an automobile accident). Six placebo-treated patients experienced seven SAEs (asthma exacerbation [n=2], headache, cerebrospinal fluid leak after epidural surgery, shingles, herpes zoster, acute purulent meningitis, and pain medication addiction). See Figure 11 for safety results from this trial.

The overall frequency of AEs was similar in both treatment arms (lebrikizumab, 74.5%; placebo, 78.6%), as was the frequency of serious AEs (lebrikizumab, 3.8%; placebo, 5.4%) (Table 9). Musculoskeletal events were more common with lebrikizumab (lebrikizumab, 13.2%; placebo, 5.4%; P = 0.045) (Table 9). Twenty-five patients (11.5%) discontinued the study early, including 12 placebo-treated and 13 lebrikizumab-treated patients.

Table 9

Example 3 - Asthma Patient Study II (Dose Ranging Study)

A randomized, double-blind, placebo-controlled, four-arm, dose-ranging study was conducted to further evaluate the relationship between lebrikizumab dose and response in terms of efficacy, safety, and tolerability in asthma patients not using inhaled corticosteroids. A schematic diagram of the trial design is shown in Figure 12. Selected patients had at least a 15% bronchodilator response and a pre-bronchodilator FEV1 of 60% and 85% predicted, and demonstrated disease stability during the run-in period. Selected patients for the study who had previously been managed with ICS could not have received ICS for a minimum of 30 days prior to the first study visit (Visit 1). There was no withdrawal period; patients were not withdrawn from corticosteroids for the sole purpose of study eligibility.

The first study-related procedure begins an approximately 2-week lead-in period at visit 1. During the lead-in period (visits 1-3), asthma control (measured by the Asthma Control Questionnaire (ACQ) score) and lung function were evaluated. The patient's asthma was further characterized using a skin prick test and related biomarkers (including IgE and peripheral blood eosinophils). Patients were classified based on the patient's IL-13 signature surrogate status using IgE and eosinophil levels. At the end of the lead-in period, eligible patients were randomly assigned (1:1:1:1) to receive one of three lebrikizumab doses (500 mg, 250 mg, or 125 mg) or placebo via SC administration. The study drug was administered four times during the 12-week treatment period. After the last dose of the study drug, the patient was monitored for another 8-week follow-up period. Therefore, after visit 1, most patients participated in the study for a total of approximately 22 weeks, during which intermittent PK samples were obtained. In addition to obtaining additional PK samples during the study and follow-up periods, and 16 weeks after the last study drug administration, intensive PK sampling was performed until 70 subjects had completed intensive PK sampling. Therefore, for patients in the intensive PK sampling group, study participation lasted approximately 26 weeks after Visit 1. Safety was evaluated throughout the study, and a predetermined treatment failure threshold was established so that if patients had clinically significant decline, they could be weaned from study drug and restarted standard of care.

Initial observation

Efficacy: For all lebrikizumab-treated subjects, the placebo-corrected relative change in FEV1 from baseline to week 12 was a 4.1% improvement (95% CI -0.4, 8.6%; p = 0.075). Throughout the 12-week treatment period, the estimated risk of treatment failure in all lebrikizumab-treated subjects was 75% lower than that in placebo-treated patients (HR 0.25, 95% CI: 0.11, 0.78; p = 0.001). These results were considered clinically and statistically significant. There was no evidence of a dose response (see Figure 16).

Safety: With the exception of injection site reactions (23% vs. 6% for lebrikizumab and placebo, respectively), there were no clinically significant imbalances in the incidence of lebrikizumab- and placebo-treated patients.

Example 4 - Periostin Assay (E4 Assay)

A very sensitive periostin capture ELISA assay (sensitivity 1.88 ng/ml) is described below. The antibodies recognize periostin isoforms 1-4 (SEQ ID NOs: 5-8) with nM affinities.

80uL of purified monoclonal antibody 25D4 (coating antibody, expressed by hybridoma or CHO cell line, SEQ ID NOs: 1 and 2) was diluted to a final concentration of 2ug/mL with phosphate buffered saline. 100μL/well of coating antibody was coated on a microtiter plate at 2-8°C overnight and the plate was covered. The plate was washed three times at room temperature with 400μL wash buffer (PBS/0.05% Tween (polysorbate 20)/well/cycle wash buffer. 200μL/well blocking buffer was added to the plate. The covered plate was incubated at room temperature for 1.5 hours under shaking.

Prepare a rhuPeriostin standard curve (rhuPeriostin standard stock = rhuPeriostin isoform 1, R&D Systems #3548-F2, 5.25 ng/ml in assay diluent (PBS/0.5% bovine serum albumin (BSA)/0.05% polysorbate 20/0.05% ProClin 300, pH 7.4). Standard curve diluent = PBS/0.5% BSA/0.05% polysorbate 20, 0.05% ProClin 300, pH 7.4). For example:

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

For example:

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

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

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

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

Each dilution was run in duplicate.

Normal human serum was used to construct matrix controls. Unspiked combined human serum was used as a normal control. As described above, high control was generated by incorporating 100 ng/mL rhuPOSTN into the serum combined. For the four dilutions of each control on each plate, mean values, standard deviations (SD), and % coefficient of variation (CV, expressed as a percentage) were calculated. CV was quantitatively analyzed for the magnitude of variation in the measured values of the replicates with respect to the mean values of the replicates. %CV=100*(SD/ mean values). Across all plates, these mean concentrations were assessed to determine inter-plate accuracy. This table of comparisons was subsequently used to define normal and high control pass/fail criteria, with allowable variation being set to ± 20% of the mean concentration of each control.

With 400 μ L/ hole/cycle wash buffer (PBS/0.05% polysorbate 20), the plate is washed three times. The diluted standard (wells in duplicate), control (all four dilutions) and sample (all four dilutions) are added to the plate, 100 μ L/ hole. The plate on the cover is incubated at room temperature for 2 hours under shaking. With determination diluent, 80 uL detection MAb stock solution I (biotinylated mouse anti-human periostin, MAb 23B9, 7.5 ug/ml in determination diluent) is diluted to 12 mL, = 50 ng/mL. With 400 μ L/ hole/cycle wash buffer, the plate is washed four times. The diluted detection MAb is added to the plate, 100 μ L/ hole. The plate on the cover is incubated at room temperature for one hour under shaking. 80 μL of Streptavidin-HRP stock solution I (AMDEX Streptavidin-HRP, GE Healthcare #RPN4401, approximately 1 mg/ml) diluted 1:80 in assay diluent was diluted to 12 mL with assay diluent (1:12k). The plate was washed four times with 400 μL/well/cycle wash buffer. The diluted Streptavidin-HRP was added to the plate at 100 μL/well. The covered plate was incubated at room temperature for 45 minutes with shaking. The Kirkegaard and Perry (KPL) two-step TMB reagent was allowed to reach room temperature; no combination was performed. The plate was washed four times with 400 μL/well/cycle wash buffer. Equal volumes of KPL TMB substrate components were mixed and added to the plate at 100 μL/well. The plate was incubated at room temperature for 20 minutes with shaking. 1 M phosphoric acid was added to the plate at 100 μL/well. The plate was read using a 450 nm reading wavelength and a 650 nm reference wavelength. This assay, or an assay similar to the one above, was used in the clinical trial described in Example 3.

Using a similar antibody capture format, a periostin assay was performed on different asthma patient samples using an antibody against isoform 1 (not total periostin).Preliminary studies indicated that periostin isoform 1 is not as strong a marker of TH2 inflammation as total periostin.

Example 5 - Observational Study of Asthma Patients (BOBCAT)

We previously reported biomarker findings based on studies of biological samples stored in an airway tissue bank at the University of California, San Francisco (UCSF) that were collected during bronchoscopy in healthy and asthmatic volunteers for research purposes. See, for example, International Publication No. WO2009/124090. To validate these findings in a large cohort of moderate to severe asthma and generalize them across multiple clinical sites, we conducted a multicenter, three-visit observational study ("BOBCAT") for moderate to severe asthma (ACQ>1.50 and FEV1 between 40-80%) that was uncontrolled on high-dose ICS (>1000 μg/day fluticasone DPI or equivalent), collecting induced sputum, endobronchial biopsies, and peripheral blood. We obtained matched blood and airway data from 59 subjects (see study overview in Figure 13). Details of the study are provided below.

BOBCAT (Bronchoscopy in Corticosteroid-Refractory Asthma: An Exploratory Investigation of Biomarkers) is a multicenter study conducted in the United States, Canada, and the United Kingdom to collect matched airway and blood samples from approximately 60 patients with moderate to severe asthma. Inclusion criteria required a diagnosis of moderate to severe asthma (confirmed by an FEV1 between 40-80% predicted and evidence of airway obstruction >12% reversibility with short-acting bronchodilators or methacholine sensitivity (PC20) <8 mg/ml within the past 5 years) that was uncontrolled (defined as at least 2 exacerbations in the previous year or an Asthma Control Questionnaire (ACQ) score >1.50 (Juniper, E.F., et al., Respir Med 100, 616-621 (2006)) despite a stable dosing regimen (>6 weeks) of high-dose ICS (>1000 μg fluticasone or equivalent/day) with or without a long-acting beta-agonist. Concurrent medications allowed also included leukotriene receptor antagonists and oral corticosteroids. The BOBCAT study protocol is depicted in Figure 13.

FE _NO , bronchoscopy, BAL, induced sputum, and immunohistochemistry for eosinophil counts were performed as previously described (Woodruff, PG, et al., Am J Respir Crit Care Med 180, 388-395 (2009); Boushey, HA, et al., N Engl J Med 352, 1519-1528 (2005); Brightling, CE, et al., Thorax 58, 528-532 (2003); Lemiere, et al., J Allergy Clin Immunol 118, 1033-1039 (2006)). All study protocols were approved by the relevant institutional review board, and informed consent was obtained from all subjects before enrollment. Patient demographics and lung function data are summarized in Table 5 below.

Table 5. BOBCAT demographic and clinical data.

N.D., not determined

Values are presented as mean ± SD or median (range)

*FPI, fluticasone dipropionate. Fluticasone:budesonide equivalent ratio = 1:1.6

**Of these, 6 subjects were also on systemic corticosteroids, receiving 4-20 mg prednisolone equivalents/day

***Of these, 7 subjects were also on systemic corticosteroids, receiving 5-40 mg prednisolone equivalents/day

We used predetermined cutoffs consistent with previous studies: 3% for sputum eosinophils (Green, RH, et al., Lancet 360, 1715-1721 (2002); Haldar, P., et al., N Engl J Med 360, 973-984 (2009)), and 22 eosinophils/mm ² of total biopsy area (Miranda, C., A. et al., J Allergy Clin Immunol 113, 101-108 (2004); Silkoff, PE, et al., J Allergy Clin Immunol 116, 1249-1255 (2005)). Serum periostin levels were very stable in individual subjects between three visits over a period of up to 5 weeks (data not shown). As defined by sputum or tissue eosinophil measurement, mean periostin levels were significantly higher in "eosinophil-high" subjects compared to "eosinophil-low" subjects (data not shown). Subjects stratified by a composite score (0 representing eosinophilia in neither sputum nor tissue, 1 representing eosinophilia in one of sputum and tissue, or 2 representing eosinophilia in both sputum and tissue) showed a highly significant trend toward increased serum periostin as eosinophil scores increased (data not shown). In addition, using the E4 assay described above, non-eosinophilic asthma in all cohorts consistently had serum periostin levels below 25 ng/ml. Using 25 ng/ml serum periostin as a cutoff, "eosinophil-low" and "eosinophil-high" subjects were effectively distinguished in BOBCAT with a positive predictive value of 93% (Table 6). Tissue neutrophil counts were positively correlated with tissue eosinophils and serum periostin levels (data not shown). In conclusion, these data show that serum periostin is a systemic biomarker of persistent airway eosinophilia despite steroid therapy in patients with moderate to severe asthma.

Table 6. Contingency table of cutoff values for serum periostin (N=57) and FE _NO (N=56) versus composite airway eosinophil status in BOBCAT.

Low eosinophilia: sputum eosinophils < 3% and biopsy tissue eosinophils < 22/mm2

Eosinophilia: sputum eosinophils > 3% or biopsy tissue eosinophils > 22/mm2

PPV, positive predictive value

NPV, negative predictive value

p-value is Fisher's exact test (2-tailed)

Comparison of serum periostin with fractional exhaled nitric oxide (FE _NO ), peripheral blood eosinophils, serum IgE, and serum YKL-40 as biomarkers of asthma

In recent years, other non-invasive biomarkers of asthma severity and airway inflammation have been described. Four particularly interesting markers are: fractional exhaled nitric oxide (FE _NO )—exhaled air produced by the action of the enzyme iNOS (inducible nitric oxide synthase) in the inflamed bronchial mucosa (Pavord, ID et al., J Asthma 45, 523-531 (2008)); peripheral blood eosinophils; serum IgE; and YKL-40—a chitinase-like protein detectable in peripheral blood (Chupp, GL, et al., N Engl J Med 357, 2016-2027 (2007)). In our asthma cohort, we measured these biomarkers and compared the values with airway eosinophilia and other biomarkers.

Periostin, FE _NO , IgE, and serum eosinophils were not significantly correlated with ACQ, FEV1, age, sex, or body mass index (BMI) in BOBCAT. In BOBCAT, FE _NO levels, like serum periostin levels, generally remained consistent across multiple visits, but FE _NO varied more at higher levels (data not shown), while the variability of blood eosinophils was much greater (r2=0.65 for serum periostin between visits 1 and 3, and r2=0.18 for blood eosinophils between visits 1 and 3, not shown). As shown in Figures 14A-F, in BOBCAT, serum periostin levels were highly correlated with each other and with the average periostin level across all visits at visits 1, 2, and 3.

As shown in Table 6, stratification based on sputum and biopsy eosinophil status showed significantly higher FE _NO levels in eosinophilic asthma compared to non-eosinophilic asthma. However, although both FE _NO and periostin were highly specific, showing consistently lower values for "low eosinophil" subjects, FE _NO detected fewer subjects with tissue eosinophilia and showed greater overlap between "low eosinophil" and "high eosinophil" subjects according to each metric used (Figures 15A-D). We fitted a logistic regression model incorporating age, sex, BMI, blood eosinophils, serum IgE, FE _NO , and serum periostin (Table 7); we found that periostin was the most significant single predictor of composite airway eosinophil status (p = 0.007).

Table 7. Logistic regression model of biomarkers versus eosinophil status in the BOBCAT study (N=59).

Estimated value Standard error z-score p-value age -0.0396 0.039 -1.015 0.31 Gender (Male) -0.2031 0.889 -0.229 0.82 body mass index -0.1004 0.066 -1.527 0.13 Blood eosinophils 1.7482 3.621 0.483 0.63 Serum IgE -0.0002 0.001 -0.100 0.92 0.0476 0.038 1.238 0.22 serum periostin 0.2491 0.092 2.719 0.007

Using a cutoff of 35 ppb as previously described (Dweik, RA, et al., Am J Respir Crit CareMed 181, 1033-1041 (2010)), FE _NO distinguished "eosinophil-low" from "eosinophil-high" asthma patients with comparable specificity but lower sensitivity to a periostin cutoff of 25 ng/ml (Table 6). Peripheral blood eosinophils tended to be higher in "eosinophil-high" asthma patients, but did not reach statistical significance (data not shown). To evaluate the relative performance of each marker on a continuous basis, we performed receiver operating characteristic (ROC) analyses of periostin, FE _NO , blood eosinophils, and serum IgE relative to composite airway eosinophil status and found that periostin performed favorably with FE NO (AUCs of 0.84 and 0.79, respectively), while blood eosinophils and serum IgE performed substantially worse (Figure 15E).

YKL-40 did not show a significant association with periostin in any cohort, nor with any measure of airway or peripheral eosinophilia (data not shown). Consistent with these findings at the levels of exhaled breath and blood biomarkers, we found that bronchial epithelial gene expression levels of periostin and NOS2 (the gene encoding iNOS) were significantly correlated with each other and with bronchial mucosal expression levels of IL-13 and IL-5, while expression of CHI3L1 (the gene encoding YKL-40) was not correlated with periostin, IL-13, or IL-5 (Table 8). In summary, these data suggest that peripheral blood periostin is a more reliable indicator of airway Th2/eosinophilic inflammation than FE _NO , blood eosinophils, serum IgE, or YKL-40 in asthma patients across a range of severity and steroid treatment.

Table 8. Correlation matrix between expression levels of genes encoding biomarkers and Th2 cytokines

POSTN_210809_s_at NOS2_210037_s_at CHI3L1_209395_at IL-13 0.42(0.014) 0.37(0.029) -0.23(0.198) IL-5 0.42(0.013) 0.34(0.048) -0.13(0.450) POSTN_210809_s_at - 0.72(<0.001) -0.24(0.136) NOS2_210037_s_at - - -0.34(0.027)

Values are given as Spearman rank correlations (p-values)

IL-13 and IL-5 expression levels were determined by qPCR from endobronchial biopsies

Periostin (POSTN_210809_s_at), NOS2 (NOS2_210037_s_at), and CHI3L1 (CHI3L1_209395_at), the gene encoding YKL-40, were determined from bronchial epithelial microarrays as described in Truyen, E., L. et al. Thorax 61, 202-208 (2006).

discuss

Although asthma has traditionally been viewed as an allergic disease mediated by Th2-driven inflammation (1), evidence of pathophysiological heterogeneity is emerging (3-8). We recently demonstrated that in patients with mild to moderate asthma who were not taking corticosteroids, only about half of the subjects had signs of Th2 inflammation in their airways. The "Th2-high" subset was distinguished by elevated allergic markers, eosinophilic airway inflammation, bronchial fibrosis, and sensitivity to ICS (13). Because antagonists of the Th2 cytokines IL-4, IL-5, and IL-13 are under active development as asthma therapies (35-37), it will be important to identify asthma patients who are most likely to benefit from these targeted therapies. Although bronchoscopy, induced sputum sampling, and measurement of exhaled breath allow direct characterization of inflammatory pathways in the airways, these modalities can be time-consuming, expensive, invasive, and/or not widely available in primary care settings. Furthermore, assay procedures have not been standardized in the relatively few centers equipped to analyze airway samples, making implementation in multicenter clinical trials a challenge. Therefore, in order to select patients with evidence of Th2-driven eosinophilic inflammation in their airways for targeted therapy, it would be advantageous to develop non-invasive biomarkers of Th2-driven eosinophilic airway inflammation that are widely usable on accessible assay platforms. To address this need, we have used gene expression profiling in asthmatic airway samples to enable the discovery and characterization of clinically useful peripheral biomarkers of Th2-driven eosinophilic airway inflammation.

Periostin is a secreted matrix cell protein associated with fibrosis, whose expression is upregulated by recombinant IL-4 and IL-13 in cultured bronchial epithelial cells (21, 38) and bronchial fibroblasts (39). It is expressed at elevated levels in vivo in bronchial epithelial cells (21) and the subepithelial layer (39) of bronchial epithelial cells in a mouse model of asthma (40), a rhesus macaque model of asthma (unpublished data), and human asthmatics. In human asthmatics, periostin expression levels correlate with reticular basement membrane thickness (an indicator of subepithelial fibrosis) (23). Periostin is also overexpressed in an IL-13-dependent manner in nasal polyps associated with aspirin-sensitive asthma (41, 42) and in the esophageal epithelium of patients with eosinophilic esophagitis (43), and may therefore play a role in the tissue infiltration of eosinophils during Th2-driven disease processes (44). Elevated periostin expression has also been observed in several types of epithelial-derived cancers (45-49), and elevated levels of soluble periostin have been reported in the serum of some cancer patients (24-26, 45, 46). It remains unclear whether the local and systemic expression of periostin in asthma or other conditions is due to a direct or indirect effect of IL-13, and this will be best addressed by comparing the evaluation of periostin expression before and after therapeutic blockade of IL-13.

Periostin is detectable in substantial systemic concentrations in the peripheral blood of non-asthmatic subjects but is elevated in the peripheral blood of a subset of asthmatic patients who are not treated with ICS. Periostin expression in the bronchial epithelium is suppressed by ICS treatment (13, 21), and systemic levels of periostin are generally lower in moderate asthmatics who are relatively well controlled on ICS compared with asthmatics who are not controlled on ICS, but with considerable heterogeneity. Considering that ICS primarily exert their effects locally in the airways and that systemic periostin levels (as we have previously demonstrated, see, e.g., International Patent Publication No. WO2009/124090) are suppressed in asthmatics who undergo ICS treatment, it can be concluded that a substantial portion of systemic periostin in asthmatics originates from the airways and that a 10-20% difference in systemic periostin levels is clinically significant with respect to airway inflammation.

FE _NO is associated with airway inflammation and predicts ICS responsiveness in asthma patients of varying severity (11, 34). However, FE _NO levels do not reliably reflect airway eosinophilia in severe, steroid-dependent asthma, and there is inconsistency between sputum and mucosal eosinophil quantification with respect to FE _NO (29, 50). Titration of ICS therapy to suppress sputum eosinophil counts can reduce the rate of severe asthma exacerbations (12), but titration of ICS therapy to FE _NO levels does not (51). Serum YKL-40 has been described as a marker of asthmatic airway inflammation, but its levels do not correlate with measures of Th2 inflammation such as IgE or eosinophils (33). Therefore, in the present study, we found that bronchial epithelial gene expression levels of periostin and NOS2, but not CHI3L1, were associated with bronchial IL-13 and IL-5 expression (Table 8). Although we observed a relatively strong positive correlation between bronchial or systemic eosinophilia and serum periostin levels, the correlation between eosinophilia and FE _NO was weak, and we did not observe a correlation between serum YKL-40 and eosinophilic inflammation in the asthma patients studied. Depending on allergen exposure, exacerbations, and steroid treatment, sputum and blood eosinophil counts and FE _NO undergo significant temporal variability (50, 52, 53). Although the half-life of circulating periostin is currently unknown, it is possible that if periostin is relatively long-lived in the blood, systemic periostin levels may reflect total airway Th2 inflammation that integrates over a long period of time. Consistent with this possibility, we observed relatively small intra-subject variability in serum periostin in 3 measurements over the course of up to 5 weeks (data not shown). Future studies should be directed to comparative evaluation of longitudinal intra-subject variability in serum periostin, airway eosinophilia, FE _NO , and other candidate biomarkers of Th2 inflammation over longer time periods.

The standard of care for bronchial asthma that is not well controlled with symptomatic treatment (e.g., beta-antagonists) is inhaled corticosteroids (ICS). ICS treatment substantially reduces the levels of IL-13 and IL-13-induced genes in affected tissues in patients with mild to moderate asthma who have elevated levels of IL-13 in the airways (19) and in patients with eosinophilic esophagitis who have elevated expression of IL-13 in esophageal tissue (43). We have demonstrated that corticosteroid treatment substantially reduces the expression of IL-13-induced Th2 signature genes in the airway epithelium and cultured bronchial epithelial cells of asthmatic patients after one week of ICS treatment (13, 21). This downregulation may be the result of ICS-mediated reductions in IL-13 levels, ICS-mediated reductions in target gene expression, or a combination of both. A similar proportion of subjects (approximately 40%) with detectable sputum IL-13 levels was found in patients with severe asthma refractory to ICS therapy compared to that seen in mild, ICS-naive asthma (19), which is comparable to the proportion of subjects with a bronchial epithelial Th2 signature that we have described (13). Similar observations have been reported for the persistence of IL-4 and IL-5 expressing cells in BAL (54) and eosinophilic inflammation in bronchial biopsies and sputum (8) in patients with steroid-refractory asthma. These observations suggest that although Th2-driven eosinophilic inflammation is suppressed by ICS treatment in patients with moderate asthma, it is reappeared in a subset of patients with severe asthma who are not fully controlled by steroid therapy. Additional complications can arise through incomplete adherence to prescribed ICS therapy, which may be the underlying cause of poor control in some patients with severe asthma. Therefore, future studies should be directed towards evaluating blood periostin levels in patients with controlled and uncontrolled asthma, in the context of ICS treatment status, ICS dose, inherent steroid sensitivity, and ICS treatment adherence.

Currently, there are many biological therapies in clinical development targeting IL-13 and related factors that drive Th2 inflammation in asthma (35-37), including those described herein. It is important that these treatments are targeted to patients with relevant molecular pathologies, otherwise the important therapeutic effects may be underestimated; studies on anti-IL5 therapy highlight this point (14,15,55). Our findings illustrate that approximately half of steroid-naive mild to moderate asthma patients can show activity of the Th2 pathway in their airways, and a similar proportion of moderate to severe, steroid-refractory asthma patients show activity of this pathway. Therefore, as described herein, identifying biomarkers of asthma patients who may have Th2-driven inflammation in their airways can help identify and select patients who are most likely to respond to these experimental targeted therapies.

References (Example 5)

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25.Sasaki, H., K.M.Lo, L.B.Chen, D.Auclair, Y.Nakashima, S.Moriyama, I.Fukai, C.Tam, M.Loda and Y.Fujii, Expression of Periostin, homologous with aninsect cell adhesion molecule, as a prognostic marker in non-small cell lungcancers.Jpn J Cancer Res 92, 869-873(2001)

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27. Gibson, P.G., Inflammatory phenotypes in adult asthma: clinical applications. Clin Respir J 3, 198-206 (2009)

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35.Barnes, P.J., The cytokine network in asthma and chronic obstructivepulmonary disease.J Clin Invest 118, 3546-3556 (2008)

36. Holgate, S. T. and R. Polosa, Treatment strategies for allergy and asthma. Nat Rev Immunol 8, 218-230 (2008)

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40. Hayashi, N., T. Yoshimoto, K. Izuhara, K. Matsui, T. Tanaka and K. Nakanishi, T helper 1 cells stimulated with ovalbumin and IL-18 induce airway hyperresponsiveness and lung fibrosis by IFN-gamma and IL-13production. Proc Natl Acad Sci U S A 104,14765-14770(2007)

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45. Baril, P., R. Gangeswaran, P. C. Mahon, K. Caulee, H. M. Kocher, T. Harada, M. Zhu, H. Kalthoff, T. Crnogorac-Jurcevic and N. R. Lemoine, Periostin promotes invasiveness and resistance of pancreatic cancer cells to hypoxia-induced cell death: role of the beta4 integrin and the PI3k pathway.Oncogene 26, 2082-2094(2007)

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51. Shaw, D.E., M.A.Berry, M.Thomas, R.H.Green, C.E.Brightling, A.J.Wardlaw and I.D.Pavord, The use of exhaled nitric oxide to guide asthma management: arandomized controlled trial. Am J Respir Crit Care Med 176, 231-237 (2007)

52. Pavord, I. D., P. K. Jeffery, Y. Qiu, J. Zhu, D. Parker, A. Carlsheimer, I. Naya and N. C. Barnes, Airway inflammation in patients with asthma with high-fixed or low-fixed plus as-needed budesonide/formoterol. J Allergy Clin Immunol 123, 1083-1089, 1089 e1081-1087(2009)

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55. Flood-Page, P., C. Swenson, I. Faiferman, J. Matthews, M. Williams, L. Brannick, D. Robinson, S. Wenzel, W. Busse, T. T. Hansel and N. C. Barnes, A study to evaluate safety and efficacy of mepolizumab in patients with moderate persistent asthma. Am J Respir Crit Care Med 176, 1062-1071(2007).

Example 6 - Asthma Patient Study III (Evaluation of Safety, Tolerability and Efficacy)

This study is a randomized, multicenter, double-blind, placebo-controlled, parallel-group study of lebrikizumab in patients with severe asthma whose asthma is uncontrolled despite daily treatment with ICS (500–2000 μg/day fluticasone propionate dry powder inhaler [DPI] or equivalent) plus a second controller medication, such as a long-acting beta-agonist (LABA), leukotriene receptor antagonist (LTRA), long-acting muscarinic antagonist (LAMA), or theophylline. The study will also evaluate the diagnostic value of a baseline level of serum periostin ≥50 ng/mL (as measured by an assay). While continuing their standard of care therapy (which must include ICS and a second controller medication), patients will be randomly assigned to one of three lebrikizumab doses or placebo for a total of 52 weeks of placebo-controlled period.

During a 2-week run-in period, also known as the screening period (visits 1 to 3), patients will be evaluated for compliance with their current asthma therapy, their ability to use the equipment required for monthly clinic visits throughout the study, and the degree of asthma control provided by their standard of care medication. Patients reporting an Asthma Control Questionnaire (ACQ-5) score ≥ 1.5 and one or more uncontrolled asthma symptoms (night awakenings ≥ 1 time/week, symptoms > 2 days/week, SABA use > 2 days/week, and/or interference with daily activities) will be considered uncontrolled. Patients whose symptoms remain uncontrolled at visits 1 or 2 and 3 despite adherence to controller medication will be eligible for inclusion. In cases where adherence data are incomplete, and for patients whose ACQ-5 is < 1.5 at visit 3, the screening period may be extended by 1 week if the investigator's experience with the patient suggests that this week is atypical for their disease. Patients may be eligible for up to two additional rescreenings based on selected reasons.

At the end of the run-in (screening) period, eligible patients will be randomized (1:1:1:1) to study drug (placebo or lebrikizumab 37.5 mg SC monthly, 125 mg SC monthly, or 250 mg SC every 4 weeks). Randomization will be stratified by baseline serum periostin measured using an assay (<42 ng/mL, ≥42 to <50 ng/mL, ≥50 to <58 ng/mL, ≥58 ng/mL), history of asthma exacerbations in the last 12 months (0, 1–2, ≥3 events), baseline asthma medication (ICS dose ≥1000 μg/day fluticasone propionate DPI or equivalent plus LABA [yes, no]), and geographic region (US/Canada, Europe, Asia, rest of the world). In addition to receiving double-blind study treatment for 52 weeks, patients will continue stable doses of their standard of care therapy, which must include ICS (500-2000 μg/day fluticasone propionate DPI or equivalent) and a second controller medication.

The first SC injection of study treatment will occur on the same day as randomization (Visit 3 [Day 1]), and dosing will be repeated every 4 weeks during the 52-week placebo-controlled period (a total of 13 study treatment doses). Safety, efficacy, and patient-reported outcome (PRO) measures will be evaluated throughout the 52-week placebo-controlled period. The primary efficacy endpoint is the rate of asthma exacerbations and will be evaluated over the 52-week placebo-controlled period.

Patients who complete the 52-week placebo-controlled period (i.e., those who do not prematurely discontinue study treatment) will continue into a 52-week active-treatment extension study.

All patients continuing into the 52-week active treatment extension study will receive double-blind SC lebrikizumab at a dose of 125 mg or 250 mg every 4 weeks. Patients assigned to receive 125 mg or 250 mg of lebrikizumab during the 52-week placebo-controlled period will maintain their assigned lebrikizumab dose. Patients assigned to receive placebo or lebrikizumab 37.5 mg SC every 4 weeks during the 52-week placebo-controlled period will be randomized in a 1:1 ratio to receive lebrikizumab 125 mg or 250 mg SC every 4 weeks for a 52-week active treatment extension, with randomization stratified by baseline periostin level and prior treatment assignment.

At week 76 of the 52-week active treatment extension, each patient will be evaluated for asthma control using data from the three most recent visits (weeks 68, 72, and 76). Patients whose asthma symptoms have been controlled for 12 consecutive weeks (ACQ-5 ≤ 0.75 at each evaluation) and have not worsened during the first half of the active treatment extension (weeks 52-76) will discontinue lebrikizumab therapy and enter the follow-up period. During follow-up, patients will be tracked for safety for 24 weeks after the last dose of study drug. Patients who maintain symptoms (ACQ-5> 0.75) at week 76 or who have experienced an exacerbation event during the first half of the active treatment extension (weeks 52-76) will continue lebrikizumab treatment for an additional 28 weeks. Safety, efficacy, and PRO measures will be evaluated throughout the 52-week active treatment extension.

During the follow-up period, all patients will be followed for safety for 24 weeks (>5 half-lives of drug) after the last dose of study treatment, regardless of when the last dose may have occurred (as planned in the protocol or in the event of early discontinuation of study treatment). For patients who complete the study to Visit 23 (Week 76), are well controlled, and discontinue study treatment, Visit 23 will replace Safety Follow-up Visit 1. For patients who complete the entire study to Visit 30 (Week 104), Visit 30 will replace Safety Follow-up Visit 1. In both cases, the next visit in the follow-up period will be Safety Follow-up Visit 2 at Week 12. For all other patients, the first follow-up visit will be Safety Follow-up Visit 1, at Week 4 of the safety follow-up period (approximately 4 weeks after the last dose of study treatment).

Total participation in the study, from randomization at Visit 3 (Day 1), including a 52-week placebo-controlled period, a 52-week active treatment extension, and a safety follow-up period, can be up to 124 weeks.

In this study, approximately 1,400 patients (175 patients per treatment group [SClebrikizumab 250 mg, 125 mg, 37.5 mg, or placebo] periostin group [periostin high ≥ 50 ng/mL, periostin low < 50 ng/mL]) will be enrolled at approximately 250 sites worldwide. A minimum of 650 patients will be enrolled in the periostin high group (≥ 50 ng/mL). A minimum of approximately 450 patients will be enrolled who are taking an ICS fluticasone ≥ 1000 μg/day DPI or equivalent plus a LABA.

Key inclusion criteria included the following: a diagnosis of asthma ≥12 months before screening; bronchodilator responsiveness/reversibility: patients must have had a bronchodilator response of ≥12% to four puffs of a short-acting beta-agonist (e.g., albuterol or salbutamol) during the screening period; a pre-bronchodilator FEV1 of 40%–80% predicted at Visits 2 and 3; use of an ICS ≥500 (e.g., 500–2000) μg fluticasone propionate DPI or equivalent (total daily dose) for ≥6 months before screening; use of a second controller medication (e.g., a LABA, LAMA, LTRA, or theophylline within the prescribed dose range) for 6 months before screening; and uncontrolled asthma confirmed during the screening period (i.e., Visit 1 [Day -14] or Visit 2 [Day -7]) and at randomization (Visit 3 [Day 1]) as defined by an ACQ-5 score ≥1.5 and a National Heart, Lung, and Blood Institute and National At least one of the following uncontrolled asthma symptoms according to the Asthma Education and Prevention Program Expert Panel Report 3 (2007) and the Global Initiative for Asthma (2010):

Symptoms > 2 days/week

Nighttime awakenings ≥1 time/week

SABA use as rescue medication > 2 days/week

Interference with normal daily activities;

The absence of other lung disease confirmed by chest X-ray or computed tomography (CT) scan obtained within the 12 months prior to Visit 1 or by chest X-ray during the Screening Period; and Confirmation of ≥70% adherence to controller medication during the Screening Period (adherence defined as the patient affirmatively responding that they have taken their asthma controller therapy for ≥70% of the days during the Screening Period (Visit 1 to Visit 3), as recorded on their peak airflow meter).

Key exclusion criteria included the following: history of severe allergic or anaphylactic reaction to a biologically active agent, or known hypersensitivity to any component of the lebrikizumab injection; maintenance oral corticosteroid therapy, defined as daily or every other day oral corticosteroid maintenance therapy within the 3 months prior to Visit 1; asthma exacerbation requiring systemic (oral, intravenous (IV), or intramuscular (IM)) corticosteroids for any reason (including acute exacerbation events) during the 4 weeks prior to screening (Visit 1) or at any time during the screening process; major infectious episode requiring any of the following:

1. Hospitalization for ≥24 hours within 4 weeks prior to screening (Visit 1)

2. Treatment with IV antibiotics within 4 weeks of screening (Visit 1)

3. Oral antibiotics within 2 weeks before screening (visit 1);

Active parasitic infection within 6 months prior to Visit 1; pulmonary tuberculosis requiring treatment within 12 months prior to screening (patients who have received pulmonary tuberculosis treatment and have not relapsed within 12 months after completing treatment are allowed); known immunodeficiency, including but not limited to HIV infection; current use of immunomodulatory therapy; known current malignancy or current evaluation for potential malignancy; active hepatitis B/C or signs of unstable liver disease; active parasitic infection within 6 months prior to screening; AST/ALT elevation ≥2.0 upper limit of normal; history of cystic fibrosis; chronic obstructive pulmonary disease, and/or other clinically significant lung disease other than asthma; current smoker or smoking history of >10 pack-years; use of biologic therapy at any time during the 6 months prior to screening; female patients of childbearing potential who are unwilling to use highly effective contraceptive methods for the duration of the study, or female patients who are pregnant or lactating; other unstable medical conditions; body mass index >38 kg/m2; weight <40 kg.

Phase III dosing basis

Population PK analysis

A preliminary population PK model was developed to describe the PK profile of lebrikizumab in adult patients. A total of 4914 serum concentration-time samples from 333 lebrikizumab-treated patients in the studies described above were used to develop the model. No significant differences were observed between studies, as demonstrated by the good agreement between the mean PK profiles observed in each study and the PK profiles predicted from historical data.

A two-compartment model with first-order absorption and elimination kinetics adequately described the serum lebrikizumab concentration-time data. Structural model parameters included clearance (CL) after SC administration, volume of distribution in the central compartment (V1), volume of the peripheral compartment (V2), intercompartmental clearance (Q), as well as first-order absorption rate (Ka) and bioavailability (F). All parameters were assumed to be lognormal except for V2 and Q, which were fixed at the population mean.

The population mean CL and V1 were estimated to be 0.18 L/day and 3.7 L, respectively. The population mean bioavailability was estimated to be 76%. The inter-individual variability of the PK parameters was moderate, ranging from 15% to 27%. Body weight was found to be a significant covariate for the CL and volume of distribution of lebrikizumab, with higher body weight associated with higher clearance and higher volume of distribution. Approximately 19% of the inter-individual variability in CL and 11% of the inter-individual variability in V1 were explained by body weight. The estimated population PK parameters are summarized in Table 13. The population PK analysis indicated that the PK characteristics of lebrikizumab were consistent with those typical of IgG monoclonal antibodies.

The effect of periostin on PK parameters was evaluated by comparing individual post hoc estimates of PK parameters between patients with baseline periostin levels above and below the median in Study 2 and Study 3. For CL (p = 0.54, 0.11), V1 (p = 0.83, 0.79), and F (p = 0.74, 0.37), the differences were not significant in either study (p > 0.05).

Table 13. Lebrikizumab population PK parameters.

BW = body weight; CL = clearance; PK = pharmacokinetics; Q = compartmental clearance; SE = standard error;

V ₁ = volume of distribution of the central compartment; V ₂ = volume of distribution of the peripheral compartment.

Note: The BW effect was modeled as a power function, TVP _i = θ1*(BW _i /BW _Ref ) ^θ2 , for which TVP is the typical value of the PK parameter; BW _i refers to the body weight of the i-th individual, BW _Ref refers to the reference body weight, which is the median body weight of all patients included in the population PK model (82 kg), θ1 refers to the population mean estimate of the PK parameter, and θ2 refers to the effect of body weight on the PK parameter.

Rationale for fixed-dose administration

Fixed-dose administration was used in the Phase II studies described above. Given the impact of body weight on the PK profile of lebrikizumab, heavier patients generally have lower drug exposure. However, no association was found between body weight and the proportional change in FEV1 from baseline to Week 12 in individual patients treated with lebrikizumab, indicating that the effect of body weight on exposure did not affect efficacy (see Figure 20). Furthermore, data from these studies indicate that there were no safety concerns associated with fixed-dose administration within the weight range tested (53–135 kg).

To assess the effect of fixed dose administration on the overall variability of lebrikizumab exposure, population PK simulations were performed to compare fixed dose administration and equivalent weight-based (i.e., mg/kg) dosing regimens, assuming that the weight distribution was similar to that seen in the Phase II study described above. The predicted proportions of patients with steady-state trough concentrations above different levels were similar between the fixed dose and weight-based dosing regimens (see Figure 21), suggesting that weight-based administration had no significant advantages. Therefore, given the advantages of fixed dose administration compared to individualized weight-based administration—reducing the risk of dosing errors and operational complexity (e.g., drug preparation), fixed dose administration was selected for Phase III studies.

Basis of target concentration

Exposure-response analyses were performed using Phase II data to derive target lebrikizumab concentrations to inform dose selection for Phase III studies. In the two Phase II studies described above, no significant correlation was found between the change from baseline in placebo-corrected FEV1 and serum trough drug concentrations at Week 12 in individual patients treated with lebrikizumab (data not shown), suggesting that the effect of lebrikizumab on FEV1 was saturated across the exposure range tested in both studies. In addition, evaluation of the correlation between changes in PD biomarkers (FeNO, IgE, CCL17, and CCL13) and serum trough drug concentrations in the Phase II studies suggested that these PD responses were saturated during the treatment period in both studies. These results were similar in the periostin-high group. On the basis of these results, a target serum steady-state trough concentration of 10 μg/mL was proposed to exclude the low end of the C _trough,wk12 range observed in both studies (5th-95th percentiles: 9.6–50 μg/mL in the Example 2 study and 9.4–73 μg/mL in the Example 3 study). In addition, considering the assumed serum-to-lung partitioning of lebrikizumab (1:500) and the levels of IL-13 in the lung based on available data in the literature, a serum concentration of 10 μg/mL is expected to maintain sufficiently high drug levels in the lung to neutralize IL 13 in asthma patients. Therefore, it is expected that the effective dose in Phase III will maintain a serum steady-state trough concentration>10 μg/mL.

To select a partially effective dose in Phase III, a lower target steady-state trough concentration of 5 μg/mL was proposed to ensure no overlap with the C _{trough, wk12} range observed in the Example 2 and Example 3 studies (in which efficacy was observed). In addition, in both studies, when the mean serum lebrikizumab concentration was >5 μg/mL, the effect of lebrikizumab on CCL17 (TARC) levels returned toward baseline during the drug washout period (see Figure 22; (A) Example 2 study, (B) Example 3 study). Although the effect of lebrikizumab on serum CCL17 (TARC) was not directly related to efficacy, the recovery of this biomarker suggested suboptimal inhibition of IL-13 activity in some biological pathways at this concentration. Therefore, a partially effective dose was proposed to maintain a serum steady-state trough concentration of <5 μg/mL.

Rationale for the proposed phase III dose

Given the lack of a clear dose response in the Example 3 study as described above (i.e., doses of 125 mg, 250 mg, and 500 mg appeared equally effective), Phase III doses were selected to elucidate the dose response of lebrikizumab by including both effective and partially effective dose levels. To achieve this goal, the doses of lebrikizumab proposed for the Phase III program included 250 mg, 125 mg, and 37.5 mg administered by SC injection every four weeks (q4wk). Figure 23 shows the simulated population PK profiles at these doses, assuming a body weight distribution similar to that seen in the Phase II study.

The highest dose of 250mg (two 1-mL SC injections) q4wk is expected to show clinical efficacy in Phase III. It is the only dosage regimen (Example 2) studied in Phase II proof-of-concept studies, and is shown in the efficacy of reducing severe asthma exacerbation rate in following patients, wherein, although the patient uses ICS therapy, its asthma is not controlled, and the patient population is intended to be used for treatment. Although treated with ICS together with or without another controller, Example 2 patients have asthma that is not controlled, and in Example 2, using ICS≥500 μg/day patients≥90% are also adopting LABA medication. With this dosage regimen, based on population PK simulations, almost all (99%) patients predict the target C _trough,ss (see Table 14) reaching 10 μg/mL, which means that maximum efficacy is expected. Therefore, this dosage will be tested to repeat the clinical efficacy seen in Phase II.

Table 14. Predicted Percentage of Patients with Steady-State Trough Concentrations Above Target at Proposed Phase III Doses

Based on the similar magnitude of FEV1 improvement observed at 125 and 250 mg q4wk in the Phase II dose ranging study (Example 3), an intermediate dose of 125 mg (one 1-mL SC injection) q4wk was proposed. It is reasonable to expect that the same dose-response relationship will still be correct for the exacerbation endpoint in a patient population with severe asthma, and therefore, the 125 mg dose is expected to show efficacy in Phase III. This expectation is further supported by population PK simulations, which suggest that most (78%) patients will reach the target C _through,ss of 10 μg/mL with this dose regimen (see Table 14).

A dose of 37.5 mg (one 0.3-mL SC injection) every 4 weeks was proposed to elucidate the lebrikizumab dose that is partially effective or clinically ineffective, which is important for establishing a dose-response relationship. This dose regimen was selected based on the following considerations:

Ability to maintain target C _trough,ss below 5 μg/mL in the majority (72%) of patients and below 10 μg/mL in nearly all (99%) patients (see Table 14)

Reasonable (3.3 times) separation from the intermediate dose

Minimal overlap with the middle dose in the simulated range of serum exposure to lebrikizumab (see Table 14) to elucidate differential clinical responses

Convenient injection volumes (multiple 0.1 mL) to reduce possible dosing errors

Various outcome measures for this Phase III trial are described below.

Outcome metrics

Each of the following endpoints will be evaluated separately in periostin-high and periostin-low patients.The trial will be considered positive if the primary endpoint is met in the periostin-high group when the 250 mg glebrikizumab group is compared to the placebo group.

Primary efficacy outcome measure

The primary efficacy outcome measure is the rate of asthma exacerbations during the 52-week placebo-controlled period. For this trial, an asthma exacerbation will be defined as new or increased asthma symptoms (including, for example, wheezing, cough, dyspnea, or chest tightness, or nighttime awakenings due to these symptoms) that result in treatment with systemic corticosteroids or hospitalization. Here, treatment with systemic corticosteroids is defined as treatment with oral (i.e., OCS) or parenteral corticosteroids for ≥3 days or an emergency department visit with one or more doses of parenteral (IV or IM) corticosteroids.

Secondary efficacy outcome measures

Secondary efficacy outcome measures at Week 52 were as follows: relative change in pre-bronchodilator FEV1 from baseline to Week 52; time to first asthma exacerbation during the 52-week treatment period; change in fraction of expired nitric oxide ( _FENO ) from baseline to Week 52; change in asthma-specific health-related quality of life as assessed by the standardized version of the Asthma Quality of Life Questionnaire global score (AQLQ(S)) from baseline to Week 52; change in rescue medication use (measured by the number of puffs per day of rescue medication or nebulized rescue medication (i.e., SABA)) from baseline to Week 52; and urgent asthma-related healthcare utilization (i.e., hospitalizations, emergency department visits, and acute care visits) during the 52-week placebo-controlled period.

Exploratory outcome measures

Exploratory outcome measures will include the following: proportion of patients who do not experience a protocol-specified asthma exacerbation during the 52-week placebo-controlled period; change in morning post-bronchodilator peak airflow (L/min) from baseline to Week 52; change in post-bronchodilator FEV1 from baseline to Week 52; time to a 150-mL improvement in pre-bronchodilator FEV1 during the 52-week placebo-controlled period; relative change from baseline in pre-bronchodilator FEV1 (liters) averaged over Weeks 4 to 52; relative change in forced vital capacity from baseline to Week 52; rate of exacerbations associated with lung function decline, defined as the number of days during the screening period (visit date). Questions 1-3) resulting in an exacerbation of PEF or FEV1 < 60% of maximal value requiring treatment with systemic corticosteroids; change from baseline to week 52 in work, school, and activity impairment as measured by the Work Productivity and Activity Impairment Questionnaire-Asthma (WPAI-Asthma); change from baseline to week 52 in the Asthma Control Questionnaire-5 (ACQ-5) score; change from baseline to week 52 in asthma symptoms as measured by the Asthma Symptom Utility Index (ASUI); change from baseline to week 52 in health utilities as measured by the EQ-5D; and change from baseline to week 52 in the Global Evaluation of Treatment Effectiveness (GETE).

These exploratory outcome measures may also be evaluated during the 52-week active treatment extension and 24-week follow-up period. Additional exploratory outcome measures may include the frequency and severity of adverse events in patients exposed to lebrikizumab for >52 weeks; changes in interleukin-13 (IL-13)/asthma-related PD biomarkers during the 52-week active treatment extension or 24-week follow-up period; and serum lebrikizumab concentrations during the 52-week active treatment extension or 24-week follow-up period.

Patient-Reported Outcomes

Asthma Quality of Life Questionnaire – Standardized (AQLQ(S))

The AQLQ(S) will be used to assess patients' asthma-specific health-related quality of life (Juniper 2005). This questionnaire contains four domains: activity limitations, symptoms, emotional function, and environmental stimuli. The AQLQ(S) has been approved for use in this study population. The AQLQ(S) has a 2-week recall requirement. The AQLQ(S) will be administered to patients prior to all other non-PRO assessments and before they receive any disease status information or study medication during this assessment.

Work, Productivity, and Activity Impairment-Asthma (WPAI–Asthma)

To assess impairment at work, school, and activities, the WPAI-Asthma Questionnaire (Reilly et al. 1993, 1996) will be administered. The questionnaire items are adapted from the WPAI-Allergy Specific (WPAI-AS) questionnaire, replacing all occurrences of the term "allergy" with "asthma." The WPAI-AS will be administered to patients before all other non-PRO assessments and before they receive any disease status information or study medication during this assessment.

Euro-QOL 5D Questionnaire (EQ-5D)

The EQ-5D is a universal, preference-based, health-related quality of life questionnaire that provides a single index value for health status (The EuroQol Group 1990). The EQ-5D is designed to be completed by patients themselves. The EQ-5D will be administered to patients before all other non-PRO assessments and before they receive any disease status information or study medication during the assessment.

Asthma Symptom Utility Index (ASUI)

The ASUI (Revicki 1998) is an asthma-specific symptom questionnaire that measures cough, wheezing, shortness of breath, and nighttime awakenings. The ASUI will be administered to patients before all other non-PRO assessments and before they receive any disease status information or study medication during this assessment.

Example 7 - Periostin Assay

The quantitative detection of periostin was evaluated in an automated Roche cobas e601 analyzer (Roche Diagnostics GmbH). The assay was performed in a sandwich format, wherein the analyte, periostin, was sandwiched between two monoclonal antibodies that bind to two different epitopes on periostin. One antibody was biotinylated, enabling the immune complex to be captured onto streptavidin-coated magnetic beads. The second antibody had a complexed ruthenium cation as a signaling moiety, which allowed voltage-dependent electrochemiluminescence detection of the bound immune complex.

In detail, the reagents used are as follows:

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

- Reagent 1 (R1): Anti-periostin antibody ~ biotin:

This purified mouse monoclonal antibody corresponds to the coating antibody 25D4 according to Example 4 and is used in biotinylated form at >1.0 mg/L; TRIS buffer >100 mmol/L, pH 7.0; preservative.

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

This purified mouse monoclonal anti-periostin antibody corresponds to the detection antibody 23B9 according to Example 4 and is used in labeled form (labeled with (tris(2,2'-bipyridyl)ruthenium(II)-complex (Ru(bpy))))>1.0 mg/L; TRIS buffer>100 mmol/L, pH 7.0; preservative.

The immunoassay was performed using two incubations. In the first incubation of approximately 9 minutes, periostin and biotinylated monoclonal anti-periostin antibody (R1) in the 20 μL sample formed a complex. In the second incubation step of another 9 minutes, ruthenium-containing monoclonal anti-periostin antibody (R2) and streptavidin-coated microparticles (M) were added to the vial of the first incubation, allowing a 3-member sandwich complex to form and bind to the solid phase (microparticles) via the interaction of biotin and streptavidin.

The reaction mixture is pumped into a measurement chamber where the particles are magnetically captured on the surface of a platinum electrode. Unbound material is washed away, and the chamber is rinsed with ProCell (a reagent containing tripropylamine). Applying a voltage to the electrodes can subsequently induce chemiluminescent emission, which can be measured by a photomultiplier tube.

Results were determined using an instrument-specific calibration curve generated by a 2-point calibration and a master curve provided via the reagent barcode. Calibrator 1 was analyte-free, while calibrator 2 contained 50 ng/mL recombinant human periostin in a buffered matrix. To validate the calibration, two controls were used, containing approximately 30 and 80 ng/mL periostin.

Example 8 - Comparison of E4 and Periostin Assays

Periostin levels were measured in serum samples from patients in each of the clinical trials described in Example 2 and Example 3 using methods similar to the E4 assay (Example 4) and the periostin assay (Example 7). The results from the samples of the two tests showed that the periostin values overlapped. Variability and diffusion were comparable between the assays. In general, at the median, the assay results were typically equal to the E4 assay results or >2 times higher than the E4 assay results. Referring to Figure 17, the median was 50-51 ng/ml for the periostin assay, while the median was 23 ng/ml for the E4 assay.

Although the present invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the descriptions and examples should not be construed as limiting the scope of the invention. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Sequence Listing Keywords

Claims (4)

1. An anti-periostrin antibody comprising the HVR-H1, HVR-H2 and HVR-H3 sequences of SEQ ID NO:1 and the HVR-L1, HVR-L2 and HVR-L3 sequences of SEQ ID NO:2. 2. The antibody according to claim 1, wherein the antibody comprises the sequences of SEQ ID NO:1 and SEQ ID NO:2. 3. An anti-periostrin antibody comprising the HVR-H1, HVR-H2 and HVR-H3 sequences of SEQ ID NO:3 and the HVR-L1, HVR-L2 and HVR-L3 sequences of SEQ ID NO:4. 4. The antibody of claim 3, wherein the antibody comprises the sequences of SEQ ID NO:3 and SEQ ID NO:4.