WO2016179469A1 - Méthodes et compositions de diagnostic et de traitement de la maladie intestinale inflammatoire - Google Patents

Méthodes et compositions de diagnostic et de traitement de la maladie intestinale inflammatoire Download PDF

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WO2016179469A1
WO2016179469A1 PCT/US2016/031145 US2016031145W WO2016179469A1 WO 2016179469 A1 WO2016179469 A1 WO 2016179469A1 US 2016031145 W US2016031145 W US 2016031145W WO 2016179469 A1 WO2016179469 A1 WO 2016179469A1
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gene
subject
antibody
gene signature
csf3r
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Li Li
Lichun WANG
Bradford L. Mcrae
Viswanath Devanarayan
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Abbvie Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/06—Gastro-intestinal diseases
    • G01N2800/065—Bowel diseases, e.g. Crohn, ulcerative colitis, IBS
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • IBD Inflammatory bowel disease
  • Anti-TNFa antibodies such as adalimumab and infliximab, have proven efficacious in treating IBD, including ulcerative colitis and Crohn's disease. For example, for more than a decade, studies have shown that antibodies against TNFa can induce and maintain remission in patients with Crohn's disease, with about 60% of patients responding. (Schreiber et al. (2010) Am J Gastroenterol 105: 1574). The percentage of patients who respond to a second anti-TNFa agent, however, is lower than with the first agent. (Hanauer et al. (2006) Gastroenterology 1309:323; and Danborn et al. (2007) Ann Intern Med 146:829).
  • diagnostic methods including molecular-based diagnostic methods, that can be used to objectively identify the presence of and/or classify an IBD in a patient, define pathophysiologic aspects of IBD, clinical activity, response to therapy, including response to treatment with various IBD therapeutic agents, prognosis, and/or risk of developing IBD.
  • diagnostic methods including molecular-based diagnostic methods, that can be used to objectively identify the presence of and/or classify an IBD in a patient, define pathophysiologic aspects of IBD, clinical activity, response to therapy, including response to treatment with various IBD therapeutic agents, prognosis, and/or risk of developing IBD.
  • Such associations would also benefit the identification of pathophysiologic aspects of IBD, clinical activity, response to therapy, or prognosis.
  • the identification of a genetic marker or genetic markers that would help to predict or assess the effectiveness of a given treatment for IBD remains a challenge.
  • the present invention provides methods of treating subjects having IBD, such as Crohn' s disease and ulcerative colitis, as well as methods for identifying subpopulations of IBD patients, who will be responsive to treatment with a TNFoc inhibitor.
  • the present invention is based, at least in part, on the identification of gene signatures that can be used to assess the responsiveness of a subject to treatment with a TNFoc inhibitor, e.g., treatment with a human anti-TNFoc antibodies, or antigen binding portions thereof.
  • the invention provides more effective ways of treating IBD, such as Crohn' s or ulcerative colitis, in subjects having the disease.
  • the invention identifies four sets of genetic markers (gene signatures) that may be used alone, or in combination with one another, to determine whether a subject having IBD will be responsive to treatment with a TNFa inhibitor.
  • the invention includes a method for determining the responsiveness of a subject having an inflammatory bowel disease (IBD) to treatment with a TNFa inhibitor, the method comprising determining a gene signature from a biological sample from a subject having an IBD, wherein the gene signature is obtained by determining an expression level(s) of a gene or gene group selected from the group consisting of (i) CSF1R, CSF3R, CCL18, and IL1B; (ii) CSF1R, CSF3R, CD36, and IL1B ; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1 ; and (iv) TNFAIP6; and comparing the gene signature with a responder gene signature comprising a corresponding gene or gene group of (i) to (iv), wherein the subject will be responsive to treatment with the TNFa inhibitor if the gene signature correlates with the respond
  • the invention includes a method for determining the
  • the method comprising determining a gene signature from a biological sample from a subject having an IBD, wherein the gene signature is obtained by determining an expression level(s) of a gene or gene group selected from the group consisting of (i) CSF1R, CSF3R, CCL18, and IL1B; (ii) CSF1R, CSF3R, CD36, and IL1B; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1; and (iv) TNFAIP6; and comparing the gene signature with a non- responder gene signature comprising a corresponding gene or gene group of (i) to (iv), wherein the subject will not be responsive to treatment with the TNFa inhibitor if the gene signature correlates with the non-responder gene signature.
  • the invention provides a method for treating a subject having an IBD, said method comprising determining a gene signature from a biological sample from a subject having an IBD, wherein the gene signature is obtained by determining an expression level(s) of a gene or gene group selected from the group consisting of (i) CSF1R, CSF3R, CCL18, and IL1B; (ii) CSF1R, CSF3R, CD36, and IL1B; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and
  • TREM1 TREM1; and (iv) TNFAIP6, and administering a TNFa inhibitor to the subject having IBD provided that the gene signature correlates with a responder gene signature comprising a corresponding gene or gene group of (i) to (iv).
  • the invention provides a method for treating a subject
  • said method comprising selecting a subject having an IBD who is
  • responder gene signature comprises a gene or gene group selected from the group consisting of (i) CSF1R,
  • the invention includes a method for determining a personalized medical treatment for a subject diagnosed with an IBD comprising the steps of: measuring a gene signature of a biological sample from a subject diagnosed with an IBD comprising gene expression analysis for a gene or gene group selected from the group consisting of (i) CSF1R, CSF3R, CCL18, and IL1B; (ii) CSF1R, CSF3R, CD36, and IL1B; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1; and (iv) TNFAIP6; accessing a computer database to compare the results of the gene expression analysis in step a) to a responder or non-responder gene signature to determine whether the gene signature correlates to the responder or non-responder gene signature, such that a personalized medical treatment is determined.
  • the invention includes a kit for predicting the responsiveness of a subject having IBD to treatment with a TNFa inhibitor, the kit comprising a means for determining the expression level(s) of a gene signature comprising a gene or gene group selected from the group consisting of (i) CSFIR, CSF3R, CCL18, and ILIB; (ii) CSFIR, CSF3R, CD36, and ILIB; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, and TREM1; and (iv) TNFAIP6, in a sample obtained from the subject having IBD; and instructions for recommended treatment of the subject having IBD based on the expression level(s) of the gene signature.
  • a gene signature comprising a gene or gene group selected from the group consisting of (i) CSFIR, CSF3R, CCL18, and ILIB; (ii) CSFIR, CSF3R, CD36
  • the invention includes a kit for identifying a responder to a TNFa inhibitor for treating IBD, the kit comprising a means for determining the expression level(s) of a gene signature comprising a gene or gene group selected from the group consisting of (i) CSFIR, CSF3R, CCL18, and ILIB; (ii) CSFIR, CSF3R, CD36, and ILIB; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, and TREM1; and (iv) TNFAIP6, in a patient sample obtained from the subject having IBD; and a control sample from a responder or a non-responder which can be used to determine the expression level(s) of the gene signature determined for the patient sample.
  • a gene signature comprising a gene or gene group selected from the group consisting of (i) CSFIR, CSF3R, CCL18, and ILIB; (ii)
  • the kit comprises a means for determining the
  • the means for determining the presence of the gene signature (i) CSFIR, CSF3R, CCL18, and ILIB comprises either nucleic acids that hybridize to the nucleic acid molecules encoding each of genes CSFIR, CSF3R, CCL18, and ILIB; or antibodies which specifically bind to the proteins corresponding to each of genes CSFIR, CSF3R, CCL18, and ILIB.
  • the kit comprises a means for determining the
  • the means for determining the presence of the gene signature (ii) CSFIR, CSF3R, CD36, and ILIB comprises either nucleic acids that hybridize to the nucleic acid molecules encoding each of genes CSFIR, CSF3R, CD36, and ILIB; or antibodies which specifically bind to the proteins corresponding to each of genes CSFIR, CSF3R, CD36, and ILIB.
  • the kit comprises a means for determining the presence of the gene signature (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, and TREMl in a sample obtained from the subject having Crohn' s disease.
  • the means for determining the presence of the gene signature (iii) is a means for determining the presence of the gene signature (iii)
  • CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, and TREMl comprises either: nucleic acids that hybridize to the nucleic acid molecules encoding each of genes CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, and TREMl ; or antibodies which specifically bind to the proteins corresponding to each of genes CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, and TREMl .
  • the kit comprises a means for determining the presence of the gene signature (iv) TNFAIP6 in a sample obtained from the subject having IBD.
  • the means for determining the presence of the gene signature (iv) TNFAIP6 comprises either a nucleic acid that hybridizes to the nucleic acid molecule encoding the gene TNFAIP6; or an antibody which specifically binds to the protein corresponding to the gene TNFAIP6.
  • the biological sample is a tissue sample.
  • tissue samples useful in the invention include a tissue sample is from an intestine of the subject.
  • the biological sample is a blood sample or a stool sample.
  • the biological sample comprises peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the gene signature comprises the gene group CSF1R, CSF3R, CCL18, and ILIB.
  • the gene signature comprises the gene group CSF1R, CSF3R, CD36, and ILIB.
  • the gene signature comprises the gene group CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, and TREMl .
  • the gene signature comprises TNFAIP6.
  • the expression level is determined using an microarray analysis and/or polymerase chain reaction (PCR) technology.
  • PCR polymerase chain reaction
  • the PCR technology is quantitative real time PCR (qRT-PCR).
  • the expression level is determined using an immunoassay, e.g., ELISA.
  • the TNFa inhibitor is an anti-TNFa antibody, or antigen- binding fragment thereof.
  • the anti-TNFa antibody, or antigen- binding portion thereof is selected from the group consisting of a human antibody, a chimeric antibody, and a humanized antibody.
  • the anti-TNFa is selected from the group consisting of a human antibody, a chimeric antibody, and a humanized antibody.
  • antibody is infliximab, or a bio similar thereof.
  • the anti-TNFa antibody, or antigen-binding portion thereof is an isolated human antibody that dissociates from human TNFa with a K d of 1 x 10 " M or less and
  • a k 0j f rate constant of 1 x 10 " s " or less both determined by surface plasmon resonance, and inhibits human TNFa- induced expression of ELAM- 1 in human umbilical vein endothelial cells.
  • the anti-TNFa antibody, or antigen-binding portion thereof comprises a light chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 27, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 29, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 31, and a heavy chain variable region comprising a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 30, and a CDR1 domain comprising the amino acid sequence of SEQ ID NO: 32.
  • the anti-TNFa antibody, or antigen-binding portion thereof comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 25 and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the light chain is a kappa light chain.
  • the anti-TNFa antibody, or antigen-binding portion thereof is an IgG isotype, e.g., an IgGl or an IgG4.
  • the anti-TNFa antibody is adalimumab, or a bio similar thereof.
  • the anti-TNFa antibody is golimumab, or a bio similar thereof.
  • the anti-TNFa antibody fragment is certolizumab pegol, or a bio similar thereof.
  • a first 160 mg dose of the anti-TNFa antibody, or antigen-binding portion thereof is administered to the subject
  • a second 80 mg dose of the anti-TNFa antibody, or antigen-binding portion thereof is administered to the subject two weeks after the first dose
  • a third 40 mg dose of the anti-TNFa antibody, or antigen-binding portion thereof is administered to the subject two weeks after the second dose.
  • a 40 mg dose of the anti-TNFa antibody, or antigen-binding portion thereof, is administered to the subject every other week after the third dose.
  • the IBD is Crohn's disease.
  • the Crohn's disease is moderately to severely active Crohn's disease.
  • the IBD is ulcerative colitis.
  • the subject is a human subject.
  • Figure 1 shows a boxplot of the log 2 normalized gene expression values for the before treatment Crohn's disease training data (GEO GSE16879, Arijs et al. (2009) PLoS One, Vol. 4(11): e7984) comparing non-responders and responders for the probesets of gene signature #1.
  • a diagonal line connects the median expression values.
  • Figure 2 shows a boxplot of the composite score for gene signature #1 for the Crohn's disease training data set, (GSE16879, Arijs 2009), comparing responders versus non- responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 3 shows the ROC plot of the composite score of gene signature #1 applied to Test Set #1, (GSE52746, Leal 2015) comparing responders versus non-responders. An ROC AUC of 1 was obtained.
  • Figure 4 shows the canonical Principal Component Analysis (PCA) plot of the normalized gene expressions of gene signature #1 applied to Test Set #1 comparing responders versus non-responders. Individual responders are shown as points and individual non- responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • PCA Principal Component Analysis
  • Figure 5 shows a boxplot of the composite score for gene signature #1 for Test Set #1, comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 6 shows the ROC plot of the composite score of gene signature #1 applied to Test Set #2, Crohn's disease dataset provided by UMASS, comparing responders versus non- responders. An ROC AUC of 0.79 was obtained
  • Figure 7 shows the canonical plot of the normalized gene expressions of gene signature #1 applied to Test Set #2 (UMASS dataset) comparing responders versus non- responders. Individual responders are shown as circles and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 8 shows a boxplot of the composite score for gene signature #1 for Test Set #2: Private Dataset (UMASS) comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • UASS Private Dataset
  • Figure 9 shows the ROC plot of the composite score of gene signature #1 applied to Test Set #3 UC disease dataset, (GSE16879, Arijs 2009) comparing responders versus non- responders. An ROC AUC of 0.86 was obtained.
  • Figure 10 shows the canonical PCA plot of the normalized gene expressions of gene signature #1 applied to Test Set #3 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 11 shows a boxplot of the composite score for gene signature #1 for Test Set #3 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 12 shows boxplots of the log 2 normalized gene expression values for the before treatment Crohn's disease training data (GEO GSE16879, Arijs 2009) comparing non- responders and responders for the probesets of gene signature #2.
  • a diagonal line connects the median expression values.
  • Figure 13 shows a boxplot of the composite score for gene signature #2 for the Crohn's disease training data set, (GSE16879, Arijs 2009), comparing responders versus non- responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups
  • Figure 14 shows the ROC plot of the composite score of gene signature #2 applied to Test Set #1 , (GSE52746, Leal et al. (2014) Gut 0:1-10) comparing responders versus non- responders. An ROC AUC of 0.93 was obtained.
  • Figure 15 shows the canonical PCA plot of the normalized gene expressions of gene signature #2 applied to Test Set #1 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 16 shows a boxplot of the composite score for gene signature #2 for Test Set #1 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 17 shows the ROC plot of the composite score of gene signature #2 applied to Test Set #2, Crohn's disease dataset provided by UMASS, comparing responders versus non- responders. An ROC AUC of 0.75 was obtained.
  • Figure 18 shows the canonical PCA plot of the normalized gene expressions of gene signature #2 applied to Test Set #2 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 19 shows a boxplot of the composite score for gene signature #2 for Test Set #2 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 20 shows the ROC plot of the composite score of gene signature #2 applied to Test Set #3 UC disease dataset, (GSE16879, Arijs 2009) comparing responders versus non- responders. An ROC AUC of 0.87 was obtained.
  • Figure 21 shows the canonical PCA plot of the normalized gene expressions of gene signature #2 applied to Test Set #3 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 22 shows a boxplot of the composite score for gene signature #2 for Test Set #3 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 23 show the log 2 normalized gene expression values for the before treatment Crohn's disease training data (GEO GSE16879, Arijs 2009) comparing non-responders and responders for the probesets of gene signature #3.
  • a diagonal line connects the median expression values.
  • Figure 24 shows a boxplot of the composite score for gene signature #3 for the Crohn's disease training data set, (GSE16879, Arijs 2009), comparing responders versus non- responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 25 shows the ROC plot of the composite score of gene signature #3 applied to Test Set #1 , (GSE52746, Leal 2014) comparing responders versus non-responders. An ROC AUC of 0.93 was obtained.
  • Figure 26 shows the canonical PCA plot of the normalized gene expressions of gene signature #3 applied to Test Set #1 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 27 shows a boxplot of the composite score for gene signature #3 for Test Set #1 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 28 shows the ROC plot of the composite score of gene signature #3 applied to Test Set #2, Crohn's disease dataset provided by UMASS, comparing responders versus non- responders. An ROC AUC of 0.84 was obtained.
  • Figure 29 shows the canonical PCA plot of the normalized gene expressions of gene signature #3 applied to Test Set #2 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 30 The above plot shows a boxplot of the composite score for gene signature #3 for Test Set #2 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 31 shows the ROC plot of the composite score of gene signature #3 applied to Test Set #3 UC disease dataset, (GSE16879, Arijs 2009)comparing responders versus non- responders. An ROC AUC of 0.90 was obtained.
  • Figure 32 shows the canonical PCA plot of the normalized gene expressions of gene signature #3 applied to Test Set #3 comparing responders versus non-responders. Individual responders are shown as points and individual non-responders are shown as stars. Open circles show the 95% confidence region for the true mean of each group.
  • Figure 33 shows a boxplot of the composite score for gene signature #3 for Test Set #3 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 34 show the log 2 normalized gene expression values for the before treatment Crohn's disease training data (GEO GSE16879, Arijs 2009) comparing non-responders and responders for the probesets of gene signature #4. A diagonal line connects the median expression values.
  • Figure 35 shows a boxplot of the composite score for gene signature #4 for the Crohn's disease training data set, (GEO GSE16879, Arijs 2009), comparing responders versus non-responders. Individual values are shown as circles for the responders and the non- responders. A diagonal line connects the median across groups.
  • Figure 36 shows the ROC plot of the composite score of gene signature #4 applied to Test Set #1 , (GSE52746, Leal 2014) comparing responders versus non-responders. We obtained a ROC AUC of 0.87.
  • Figure 37 shows a boxplot of the composite score for gene signature #4 for Test Set #1 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 38 shows the ROC plot of the composite score of gene signature #4 applied to Test Set #2, Crohn's disease dataset provided by UMASS, comparing responders versus non- responders. An ROC AUC of 0.84 was obtained.
  • Figure 39 shows a boxplot of the composite score for gene signature #4 for Test Set #2 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • Figure 40 shows the ROC plot of the composite score of gene signature #4 applied to Test Set #3 UC disease dataset, (GSE16879, Arijs 2009) comparing responders versus non- responders. An ROC AUC of 0.88 was obtained.
  • Figure 41 shows a boxplot of the composite score for gene signature #4 for Test Set #3 comparing responders versus non-responders. Individual values are shown as circles for the responders and the non-responders. A diagonal line connects the median across groups.
  • inflammatory bowel disease refers to inflammatory conditions of the large and small intestine.
  • IBD inflammatory bowel disease
  • Examples of inflammatory bowel disease include, but are not limited to, Crohn's disease (also referred to herein as "CD”) and ulcerative colitis.
  • gene signature refers to a gene or group of genes whose expression (e.g. expression level or activity) is uniquely characteristic of a biological phenotype or medical condition.
  • a gene signature refers to a gene or a group of genes whose expression indicates whether a subject who has inflammatory bowel disease (IBD), e.g., Crohn' s disease or ulcerative colitis, will be responsive to treatment with a TNFoc inhibitor, such as, but not limited to, adalimumab.
  • IBD inflammatory bowel disease
  • TNFoc inhibitor such as, but not limited to, adalimumab.
  • expression refers to transcription of a gene or to translation of a protein.
  • To quantitate expression refers to the act of determining the level of the transcript of the gene or the protein of the gene. Detecting and/or quantitating expression can include determining whether gene expression is upregulated as compared to a control level, downregulated as compared to a control level, or substantially unchanged as compared to a control level.
  • the step of quantitating and/or detecting expression does not require that expression of the gene is actually is upregulated or downregulated, but rather, can also include detecting no expression of the gene or detecting that the expression of the gene has not changed or is not different (i.e., detecting no significant expression of the gene or no significant change in expression of TNFa as compared to a control).
  • the term "gene expression” refers to transcription of a gene.
  • to detect gene expression refers to the act of actively determining whether the mRNA of the gene is expressed or not, e.g., using real time or quantitative PCR (RT PCR or Q PCR).
  • level refers to the measurable quantity of gene expression (transcript or protein).
  • the amount may be either (a) an absolute amount as measured in an appropriate unit, e.g., number of cells, fluorescence intensity, molecules, moles or weight per unit volume or cell or (b) a relative amount.
  • the level of expression of a gene can be considered “high”, “low”, “increased” or “decreased” relative to a control level of expression or relative to the level of expression of the gene in a "responder”, relative to either the level of expression of the gene in a "non-responder", or, in another embodiment, the level of expression of a subject who does not have an IBD.
  • the "level of expression” refers to the transcription level of expression of a gene (e.g., a gene from one of the gene signatures described herein) in a sample from a subject having IBD.
  • the term "responder gene signature” as used herein refers to a gene signature of a subject or group of subjects having a disease or disorder, such as IBD, (e.g., Crohn' s disease or ulcerative colitis), that correlates with treatment when a subject having the disease is administered a certain therapeutic agent.
  • a responder gene signature indicates that a subject having that gene signature and IBD will show improvement when given a specific therapeutic agent for the treatment of a certain disorder, such as IBD.
  • non-responder gene signature refers to a gene signature of a subject or group of subjects having a disease or disorder, such as IBD, (e.g., Crohn' s disease or ulcerative colitis), that correlates with no significant improvement or a deficient response when the subject having the disease is treated with a certain therapeutic agent.
  • IBD e.g., Crohn' s disease or ulcerative colitis
  • a non-responder gene signature is a gene signature which indicates that a subject having IBD, (e.g., Crohn's disease or ulcerative colitis), who is treated with a TNFoc inhibitor, such as, but not limited to, adalimumab, will not be responsive to or have a deficient response to the TNFoc inhibitor treatment for IBD.
  • a subject having IBD e.g., Crohn's disease or ulcerative colitis
  • a TNFoc inhibitor such as, but not limited to, adalimumab
  • correlate or “correlating”, as used herein, is intended to mean that two gene signatures substantially match in their gene expression patterns. For example, if a subject having IBD (e.g., Crohn's disease or ulcerative colitis) has a gene signature that correlates to a responder gene signature, then the subject would be expected to respond to TNFoc inhibitor treatment. In contrast, if a subject having IBD (e.g., Crohn' s disease or ulcerative colitis) has a gene signature that correlates to a non-responder gene signature, then the subject would not be expected to respond to TNFoc inhibitor treatment for IBD (e.g., Crohn' s disease or ulcerative colitis).
  • IBD e.g., Crohn's disease or ulcerative colitis
  • the gene signature of a subject having IBD could be compared to both a responder gene signature and a non-responder gene signature to determine which gene signature the subject's own gene signature correlates more closely with.
  • two gene signatures correlate if the two gene expression profiles are substantially similar.
  • gene expression levels of a gene signature are statistically higher in a non-responder versus a responder.
  • the term “substantially similar” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two or more numeric values (for example, one associated with gene expression levels of a gene signature and the other associated with a reference/comparator gene signature), such that one of skill in the art would consider the difference between the two values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said values (e.g., gene expression levels).
  • the difference between said two values may be, for example, less than about 50%, less than about 40%, less than about 30%, less than about 20%, and/or less than about 10% as a function of the reference/comparator value.
  • biological sample includes, but is not limited to, any quantity of a substance from a living thing or formerly living thing.
  • a biological sample may be obtained from an individual, body fluid, body tissue, cell line, tissue culture, or other source.
  • Body fluids are, e.g., lymph, sera, whole fresh blood, peripheral blood mononuclear cells, frozen whole blood, plasma (including fresh or frozen), urine, saliva, semen, synovial fluid, and spinal fluid.
  • Biological samples also include synovial tissue, skin, hair follicle, and bone marrow. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. If the term “sample” is used alone, it shall still mean that the "sample” is a
  • biological sample i.e., the terms are used interchangeably.
  • a biological sample may contain genetic material, such as nucleic acids (DNA and/or RNA), or proteins.
  • polynucleotide or “nucleic acid,” as used interchangeably herein, refers to polymers of nucleotides of any length, and include DNA and RNA.
  • the nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.
  • primer refers to a single stranded polynucleotide that is capable of hybridizing to a nucleic acid and allowing the polymerization of a complementary nucleic acid, generally by providing a free 3'-OH group.
  • array refers to an ordered arrangement of hybridizable array elements, preferably polynucleotide probes on a substrate.
  • the substrate can be a solid substrate, such as a glass slide, or a semi-solid substrate, such as nitrocellulose membrane.
  • Amplification refers to the process of producing one or more copies of a reference nucleic acid sequence or its complement. Amplification may be linear or exponential (e.g., PCR).
  • detection includes any means of detecting, including direct and indirect detection.
  • detection is understood to refer to an assay performed for identification of a specific gene or genes in a sample.
  • expression of a gene or group of genes is detected, e.g., by PCR (e.g., real time or quantitiative real time PCR (qRT- PCR) and/or microarray analysis.
  • PCR e.g., real time or quantitiative real time PCR (qRT- PCR) and/or microarray analysis.
  • molecular phenotype refers to a subpopulation of subjects having IBD, e.g., Crohn's disease or ulcerative colitis, where the subpopulation is characterized by a certain gene signature, e.g., a responder gene signature or a non-responder gene signature.
  • prediction is used herein to refer to the likelihood that a subject having a disease or disorder will respond either favorably or unfavorably to a therapeutic agent.
  • the invention provides a method for predicting whether a subject having IBD, e.g., Crohn's disease or ulcerative colitis, will respond to treatment with a TNFa inhibitor.
  • human TNFa (abbreviated herein as hTNFa, or simply hTNF or hTNFalpha), as used herein, is intended to refer to a human cytokine that exists as a 17 kD secreted form and a 26 kD membrane associated form, the biologically active form of which is composed of a trimer of noncovalently bound 17 kD molecules.
  • hTNFa The structure of hTNFa is described further in, for example, Pennica, D., et al. (1984) Nature 312:724-729; Davis, J.M., et al. (1987) Biochemistry 26: 1322-1326; and Jones, E.Y., et al. (1989) Nature 338:225-228.
  • human TNFa is intended to include, in one embodiment, recombinant human TNFa (rhTNFa), which can be prepared by standard recombinant expression methods or purchased commercially (R & D Systems, Catalog No. 210- TA, Minneapolis, MN).
  • rhTNFa recombinant human TNFa
  • TNFa inhibitor or "TNF inhibitor” includes agents which interfere with TNFa activity.
  • the TNFa inhibitor is an agent which interferes with human TNFa activity.
  • the term also includes each of the anti-TNFa human antibodies and antibody portions described herein as well as those described in U.S. Patent Nos.
  • the TNFa inhibitor used in the invention is an anti- TNFa antibody, or a fragment thereof, including infliximab (REMICADE ® , Janssen; described in U.S. Patent No. 5,656,272, incorporated by reference herein), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNFa antibody fragment; certolizumab pegol or CIMZIA ® ; UCB Group), an anti-TNF dAb
  • infliximab REMICADE ® , Janssen; described in U.S. Patent No. 5,656,272, incorporated by reference herein
  • CDP571 a humanized monoclonal anti-TNF-alpha IgG4 antibody
  • CDP 870 a humanized monoclonal anti-TNFa antibody fragment; certolizumab pegol or CIMZIA ® ; UCB Group
  • CNTO 148 golimumab; Medarex and Janssen, see WO 02/12502
  • adalimumab HUMIRA ® , AbbVie, a human anti-TNFa mAb, described in US 6,090,382 as D2E7.
  • the TNFa inhibitor is a TNF fusion protein, e.g., etanercept (ENBREL ® , Amgen; described in WO 91/03553 and WO 09/406476, incorporated by reference herein).
  • the TNFoc inhibitor is a recombinant TNF binding protein (r-TBP-I) (Serono).
  • antibody is intended to refer to immunoglobulin molecules comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the antibodies of the invention are described in further detail in U.S. Patent Nos. 6,090,382; 6,258,562; and 6,509,015, each of which is incorporated herein by reference in its entirety.
  • antigen-binding portion or "antigen-binding fragment” of an antibody (or simply “antibody portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., hTNFoc). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments include Fab, Fab', F(ab') 2 , Fabc, Fv, single chains, and single- chain antibodies.
  • binding fragments encompassed within the term "antigen- binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Set USA 85:5879- 5883).
  • scFv single chain Fv
  • single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure 2: 1121- 1123).
  • the antibody portions of the invention are described in further detail in U.S. Patent Nos. 6,090,382, 6,258,562, 6,509,015, each of which is incorporated herein by reference in its entirety.
  • an antibody or antigen-binding portion thereof may be part of a larger immunoadhesion molecules, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.
  • a “conservative amino acid substitution”, as used herein, is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains ⁇ e.g., lysine, arginine, histidine), acidic side chains ⁇ e.g., aspartic acid, glutamic acid), uncharged polar side chains ⁇ e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains ⁇ e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains ⁇ e.g., threonine, valine, isoleucine) and aromatic side chains ⁇ e.g., tyrosine, phenylalanine, tryptophan,
  • Chimeric antibodies refers to antibodies wherein one portion of each of the amino acid sequences of heavy and light chains is homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class, while the remaining segment of the chains is homologous to corresponding sequences from another species.
  • the invention features a chimeric antibody or antigen-binding fragment, in which the variable regions of both light and heavy chains mimics the variable regions of antibodies derived from one species of mammals, while the constant portions are homologous to the sequences in antibodies derived from another species.
  • chimeric antibodies are made by grafting CDRs from a mouse antibody onto the framework regions of a human antibody.
  • Humanized antibodies refer to antibodies which comprise at least one chain comprising variable region framework residues substantially from a human antibody chain (referred to as the acceptor immunoglobulin or antibody) and at least one complementarity determining region (CDR) substantially from a non-human-antibody (e.g., mouse). In addition to the grafting of the CDRs, humanized antibodies typically undergo further alterations in order to improve affinity and/or immmunogenicity.
  • CDR complementarity determining region
  • multivalent antibody refers to an antibody comprising more than one antigen recognition site.
  • a “bivalent” antibody has two antigen recognition sites, whereas a “tetravalent” antibody has four antigen recognition sites.
  • the terms “monospecific”, “bispecific”, “trispecific”, “tetraspecific”, etc. refer to the number of different antigen recognition site specificities (as opposed to the number of antigen recognition sites) present in a multivalent antibody.
  • a "monospecific” antibody's antigen recognition sites all bind the same epitope.
  • a “bispecific” or “dual specific” antibody has at least one antigen recognition site that binds a first epitope and at least one antigen recognition site that binds a second epitope that is different from the first epitope.
  • a “multivalent monospecific” antibody has multiple antigen recognition sites that all bind the same epitope.
  • a “multivalent bispecific” antibody has multiple antigen recognition sites, some number of which bind a first epitope and some number of which bind a second epitope that is different from the first epitope
  • human antibody is intended to include recombinant antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • isolated protein or "isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation is not associated with naturally associated components that accompany it in its native state; is substantially free of other proteins from the same species; is expressed by a cell from a different species; or does not occur in nature.
  • a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components.
  • a protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • a “neutralizing antibody”, as used herein (or an “antibody that neutralized hTNFa activity”), is intended to refer to an antibody whose binding to hTNFa results in inhibition of the biological activity of hTNFa.
  • This inhibition of the biological activity of hTNFa can be assessed by measuring one or more indicators of hTNFa biological activity, such as hTNFa- induced cytotoxicity (either in vitro or in vivo), hTNFa-induced cellular activation and hTNFa binding to hTNFa receptors.
  • hTNFa- induced cytotoxicity either in vitro or in vivo
  • hTNFa-induced cellular activation hTNFa binding to hTNFa receptors.
  • These indicators of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art (see U.S. Patent No. 6,090,382).
  • the ability of an antibody to neutralize hTNFa activity is assessed by inhibition of hTNFa-induced cytotoxicity of L929 cells.
  • the ability of an antibody to inhibit hTNFa-induced expression of ELAM-1 on HUVEC, as a measure of hTNFa-induced cellular activation can be assessed.
  • surface plasmon resonance refers to an optical phenomenon that allows for the analysis of real-time bio specific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ).
  • BIAcore Pharmaacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ.
  • k 0 ff is intended to refer to the off rate constant for dissociation of an antibody from the antibody/antigen complex.
  • the term as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.
  • IC 50 is intended to refer to the concentration of the inhibitor required to inhibit the biological endpoint of interest, e.g., neutralize cytotoxicity activity.
  • dose refers to an amount of TNFoc inhibitor which is administered to a subject.
  • treating refers to the administration of a substance (e.g., an anti-TNFa antibody) to achieve a therapeutic objective (e.g., treatment of rheumatoid arthritis).
  • a substance e.g., an anti-TNFa antibody
  • a therapeutic objective e.g., treatment of rheumatoid arthritis
  • a “dosing regimen” describes a treatment schedule for a TNFoc inhibitor, e.g., a treatment schedule over a prolonged period of time and/or throughout the course of treatment.
  • administration refers to the time course of administering a substance (e.g., an anti-TNFa antibody) to a subject to achieve a therapeutic objective, e.g., throughout the course of treatment.
  • a substance e.g., an anti-TNFa antibody
  • the biweekly dosing regimen is not intended to include a weekly dosing regimen.
  • the substance is administered every 9- 19 days, every 11- 17 days, every 13- 15 days, and every 14 days.
  • a first agent in combination with a second agent includes co-administration of a first agent and a second agent, which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of a first agent, followed by the second agent, or administration of the second agent, followed by the first agent.
  • the present invention includes methods of combination therapeutic treatment and combination pharmaceutical compositions.
  • concomitant as in the phrase “concomitant therapeutic treatment” includes administering an agent in the presence of a second agent.
  • a concomitant therapeutic treatment method includes methods in which the first, second, third, or additional agents are
  • a concomitant therapeutic treatment method also includes methods in which the first or additional agents are administered in the presence of a second or additional agents, wherein the second or additional agents, for example, may have been previously administered.
  • a concomitant therapeutic treatment method may be executed step-wise by different actors. For example, one actor may administer to a subject a first agent and a second actor may to administer to the subject a second agent, and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and additional agents) are after administration in the presence of the second agent (and additional agents).
  • the actor and the subject may be the same entity (e.g., human).
  • combination therapy refers to the administration of two or more therapeutic substances, e.g., an anti-TNFa antibody and another drug.
  • the other drug(s) may be administered concomitant with, prior to, or following the administration of an anti- TNFa antibody.
  • subject refers to either a human or non-human animal.
  • the subject is a human.
  • the subject is a human having IBD, e.g., Crohn's disease, who has had an inadequate response to conventional therapy.
  • the subject is a human having IBD, e.g., Crohn' s disease who has had an inadequate response to corticosteroids or immunomodulators such as azathioprine, 6-mercaptopurine, or methotrexate.
  • the subject has ulcerative colitis and has had an inadequate response to immunosuppressants such as corticosteroids, azathioprine or 6-mercaptopurine (6-MP).
  • a “kit” is any article of manufacture (e.g., a package or container) comprising at least one reagent, e.g., a medicament for treatment of IBD, e.g. , Crohn's disease or ulcerative colitis, or a probe for specifically detecting a gene or protein for use in the invention.
  • the article of manufacture is preferably promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • An unmet need in the treatment of IBD is to establish predictive biomarkers for therapeutic responders in order to avoid exposure of non-responders to anti-TNFa therapy, thus decreasing morbidity in patients with a low likelihood of response and enhancing safety and cost effective use of this treatment.
  • the invention provides gene signatures that have been identified as being useful for determining whether a subject having IBD, e.g., Crohn's disease or ulcerative colitis, will be responsive to treatment with a TNFa inhibitor, e.g., adalimumab.
  • a panel of gene signatures showing statistically significant differences between responders and non-responders of Crohn's disease and ulcerative colitis patients by performing global gene expression analyses in a training cohort (GSE16879) have been identified, and were cross-validated in two independent cohorts.
  • the sensitive methods disclosed in this application yielded several genes relevant to IBD, including Crohn's disease and ulcerative colitis, that can be used, for example, for determining an appropriate treatment for a subject having IBD.
  • the invention provides the following four sets of genetic markers (gene signatures): i) CSF1R, CSF3R, CCL18, and IL1B;
  • the gene signatures described herein represent genes or groups of genes whose expression patterns may be used for determining whether a subject having IBD (e.g., Crohn's disease or ulcerative colitis) will be responsive to treatment with a TNFoc inhibitor, including, for example, a human anti-TNFoc antibody, such as adalimumab.
  • a TNFoc inhibitor including, for example, a human anti-TNFoc antibody, such as adalimumab.
  • the gene signatures described herein may also be useful in the treatment of subjects having IBD and for compositions, such as kits, relating to the determination of a treatment for a subject having IBD.
  • the members of each gene signature are described in more detail below:
  • the first gene signature that may be used in the methods and compositions described herein comprises the genes CSF1R, CSF3R, CCL18, and IL1B.
  • Colony stimulating factor 1 receptor is a protein tyrosine-kinase
  • transmembrane receptor for CSF1 and IL34 The receptor controls the production
  • the nucleic acid sequence encoding CSFIR is described in SEQ ID NO: 1, and the amino acid sequence of the CSFIR protein is provided in SEQ ID NO: 2.
  • a nucleic acid encoding the CSFIR protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the CSFIR gene in accordance with the methods and compositions of the invention.
  • levels of the CSFIR protein may be determined in accordance with the invention.
  • Colony stimulating factor 3 receptor is a receptor for granulocyte colony- stimulating factor (CSF3), a cytokine that controls the production, differentiation, and function of granulocytes.
  • CSF3R may function in some adhesion or recognition events at the cell surface.
  • the nucleic acid sequence encoding CSF3R is described in SEQ ID NO: 3, and the amino acid sequence of the CSF3R protein is provided in SEQ ID NO: 4.
  • a nucleic acid encoding the CSF3R protein may be used in determining expression levels of the CSF3R gene in accordance with the methods and compositions of the invention.
  • levels of the CSFIR protein may be determined in accordance with the invention.
  • Chemokine (C-C motif) ligand 18 is a chemotactic factor that attracts lymphocytes but not monocytes or granulocytes. This chemokine may be involved in B-cell migration into B-cell follicles in lymph nodes, and also has chemotactic activity for naive T- cells, CD4+ and CD8+ T-cells and thus may play a role in both humoral and cell-mediated immunity responses.
  • the nucleic acid sequence encoding CCL18 is described in SEQ ID NO: 5, and the amino acid sequence of the CCL18 protein is provided in SEQ ID NO: 6.
  • a nucleic acid encoding the CCL18 protein may be used in determining expression levels of the CCL18 gene in accordance with the methods and compositions of the invention.
  • levels of the CCL18 protein may be determined in accordance with the invention.
  • Interleukin 1, beta is produced by activated macrophages and other non- lymphoid cells, such as epithelial cells.
  • IL-1 stimulates thymocyte proliferation by inducing IL- 2 release, B-cell maturation and proliferation, and fibroblast growth factor activity.
  • IL-1 proteins are involved in the inflammatory response, being identified as endogenous pyrogens, and are reported to stimulate the release of prostaglandin and collagenase from synovial cells.
  • the nucleic acid sequence encoding the IL1B protein is described in SEQ ID NO: 7, and the amino acid sequence of IL1B protein is provided in SEQ ID NO: 8.
  • a nucleic acid encoding the IL1B protein may be used in determining expression levels of the IL1B gene in accordance with the methods and compositions of the invention.
  • levels of the IL1B protein may be determined in accordance with the invention.
  • expression levels from the gene group comprising CSFIR, CSF3R, CCL18, and IL1B may be detected in a sample from a subject having IBD and used in the methods and compositions described herein for the treatment of the subject.
  • expression levels of CSFIR, CSF3R, CCL18, and IL1B in a sample from a subject having IBD may be use to determine whether that subject will be responsive to treatment with a TNFoc inhibitor.
  • the second signature that may be used in the methods and composition described herein comprises the genes CSF1R, CSF3R, CD36, and IL1B. Sequences and descriptions of CSF1R, CSF3R, and IL1B are provided above in the first gene signature.
  • CD36 is believed to have numerous potential physiological functions. It binds to collagen, thrombospondin, anionic phospholipids and oxidized LDL. CD36 may function as a cell adhesion molecule.
  • the nucleic acid sequence encoding CD36 is described in SEQ ID NO: 9, and the amino acid sequence of the CD36 protein is provided in SEQ ID NO: 10.
  • a nucleic acid encoding the CD36 protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the CD36 gene in accordance with the methods and compositions of the invention. In another embodiment, levels of the CD36 protein may be determined in accordance with the invention.
  • expression levels from the gene group comprising CSF1R, CSF3R, CCL18, and CD36 may be detected in a sample from a subject having IBD and used in the methods and compositions described herein for the treatment of the subject.
  • expression levels of CSF1R, CSF3R, CCL18, and CD36 in a sample from a subject having IBD may be use to determine whether that subject will be responsive to treatment with a TNFoc inhibitor.
  • the third gene signature that may be used in the methods and composition described herein comprises the genes CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1. Sequences and a description of IL1B are provided above in the second gene signature.
  • CLEC4A C-type lectin domain family 4, member A
  • CLEC4A may be involved in regulating immune reactivity. It may play a role in modulating dendritic cells (DC) differentiation and/or maturation.
  • CLEC4A may be involved via its ITIM motif (immunoreceptor tyro sine-based inhibitory motifs) in the inhibition of B -cell-receptor- mediated calcium mobilization and protein tyrosine phosphorylation.
  • ITIM motif immunomunoreceptor tyro sine-based inhibitory motifs
  • the nucleic acid sequence encoding CLEC4A is described in SEQ ID NO: 11, and the amino acid sequence of the CLEC4A protein is provided in SEQ ID NO: 12.
  • a nucleic acid encoding the CLEC4A protein may be used in determining expression levels of the CLEC4A gene in accordance with the methods and compositions of the invention.
  • levels of the CLEC4A protein may be determined in accordance with the invention.
  • C-type lectin domain family 7, member A is a type of lectin that functions as pattern receptor specific for beta-l,3-linked and beta-l,6-linked glucans, such as cell wall constituents from pathogenic bacteria and fungi. It is necessary for the TLR2-mediated inflammatory response and for TLR2-mediated activation of NF-kappa-B. CLEC7A enhances cytokine production in macrophages and dendritic cells, mediates production of reactive oxygen species in the cell, and binds T-cells in a way that does not involve their surface glycans and plays a role in T-cell activation.
  • nucleic acid sequence encoding CLEC7A is described in SEQ ID NO: 13, and the amino acid sequence of the CLEC7A protein is provided in SEQ ID NO: 14.
  • a nucleic acid encoding the CLEC7A protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the CLEC7A gene in accordance with the methods and compositions of the invention.
  • levels of the CLEC7A protein may be determined in accordance with the invention.
  • Cy statin A ( stefin A) ( CSTA ) is an intracellular thiol proteinase inhibitor, may play a role in controlling microorganism invasion.
  • the nucleic acid sequence encoding CSTA is described in SEQ ID NO: 15, and the amino acid sequence of the CSTA protein is provided in SEQ ID NO: 16.
  • a nucleic acid encoding the CSTA protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the CSTA gene in accordance with the methods and compositions of the invention.
  • levels of the CSTA protein may be determined in accordance with the invention.
  • Chitinase 3 -like 1 (cartilage glycoprotein-39) (CHI3L1) is carbohydrate-binding lectin with a preference for chitin. It may play a role in defense against pathogens, or in tissue remodeling.
  • the nucleic acid sequence encoding CHI3LI is described in SEQ ID NO: 17, and the amino acid sequence of the CHI3LI protein is provided in SEQ ID NO: 18.
  • a nucleic acid encoding the CHI3LI protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the CHI3LI gene in accordance with the methods and compositions of the invention.
  • levels of the CHI3LI protein may be determined in accordance with the invention.
  • Interleukin 1 receptor antagonist ILIRN
  • ILIRN Interleukin 1 receptor antagonist
  • the nucleic acid sequence encoding ILIRN is described in SEQ ID NO: 19, and the amino acid sequence of the ILIRN protein is provided in SEQ ID NO: 20.
  • a nucleic acid encoding the ILIRN protein mRNA or cDNA when reverse transcribed in a PCR reaction
  • probes or primers thereto may be used in determining expression levels of the ILIRN gene in accordance with the methods and compositions of the invention.
  • levels of the ILIRN protein may be determined in accordance with the invention.
  • TNFAIP6 Tumor necrosis factor, alpha-induced protein 6
  • TNFAIP6 is involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis.
  • TNFAIP6 is upregulated in response to proinflammaotry cytokines (e.g., IL-1 and TNFoc), and it is detected in many inflammatory disease.
  • proinflammaotry cytokines e.g., IL-1 and TNFoc
  • the nucleic acid sequence encoding TNFAIP6 is described in SEQ ID NO: 21, and the amino acid sequence of the TNFAIP6 protein is provided in SEQ ID NO: 22.
  • a nucleic acid encoding the TNFAIP6 protein mRNA or cDNA when reverse transcribed in a PCR reaction
  • probes or primers thereto may be used in
  • levels of the TNFAIP6 gene in accordance with the methods and compositions of the invention.
  • levels of the TNFAIP6 protein may be determined in accordance with the invention.
  • Triggering receptor expressed on myeloid cells 1 stimulates neutrophil and monocyte-mediated inflammatory responses through an unknown ligand. It triggers release of pro-inflammatory chemokines and cytokines, as well as increased surface expression of cell activation markers.
  • the nucleic acid sequence encoding TREM1 is described in SEQ ID NO: 23, and the amino acid sequence of the TREM1 protein is provided in SEQ ID NO: 24.
  • a nucleic acid encoding the TREM1 protein (mRNA or cDNA when reverse transcribed in a PCR reaction) (or probes or primers thereto) may be used in determining expression levels of the TREM1 gene in accordance with the methods and compositions of the invention. In another embodiment, levels of the TREM1 protein may be determined in accordance with the invention.
  • the fourth predictive gene signature for determining or predicting responsiveness of a subject to treatment with TNFa inhibitor contains one gene set: TNFAIP6.
  • the sequences of TNFAIP6 are described above. A sequence listing is being filed herewith to provide sequences of the markers provided herein.
  • the gene names, associated accession numbers, and corresponding nucleotide and amino acid SEQ ID NOs are provided in the summary table below. All of the associated accession numbers are incorporated herein by reference.
  • NP_001001548.1 thrombospondin receptor
  • cystatin A (stefin A) (NP_005204.1)
  • TSG6 TSG-6
  • TREM-1 TREM-1 The foregoing sequences are representative of the genes identified as useful in the gene signatures of the invention. Variants of the aforementioned gene sequences are also included in the invention.
  • the present invention provides methods for predicting or assessing responsiveness of a subject having IBD, e.g., Crohn's disease or ulcerative colitis, to a TNFa inhibitor.
  • the methods generally include determining the expression level(s) of at least one of the following gene signatures: CSF1R, CSF3R, CCL18, and IL1B ; CSF1R, CSF3R, CD36, and IL1B ; CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1 ; or TNFAIP6 in a biological sample obtained from the subject, wherein the expression level(s) of the marker(s) in the sample is an indication that the subject will respond to treatment with the TNFa inhibitor when compared to a responder gene signature or a non-responder gene signature.
  • the gene signatures provide a method for determining a personalized medical treatment for a subject diagnosed with an IBD.
  • a method includes measuring a gene signature of a biological sample from a subject diagnosed with an IBD comprising gene expression analysis for a gene or gene group selected from the group consisting of (i) CSF1R, CSF3R, CCL18, and IL1B; (ii) CSF1R, CSF3R, CD36, and IL1B; (iii) CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, and TREM1 ; and (iv) TNFAIP6. Further the method comprises accessing a computer database to compare the results of the gene expression analysis of the gene signature(s) to a responder or non-responder gene signature to determine whether the gene signature correlates to the responder or non- responder gene signature.
  • a sample from a subject may be tested for the expression level(s) of at least one of the above-mentioned gene signatures.
  • Methods for detecting the genes of the gene signatures described herein include protocols that examine the presence and/or expression of the gene(s) of the gene signature in a sample from a subject. Determining the presence or absence, and/or expression level(s) of the gene(s) of the gene signature in the biological sample may be accomplished using any well known techniques in the art, such microarrays or a polymerase chain reaction (PCR) amplification reaction, such as reverse-transcriptase PCR (RT-PCR) analysis.
  • PCR polymerase chain reaction
  • RT-PCR reverse-transcriptase PCR
  • RNA or protein include, but are not limited to, gene expression profiling, polymerase chain reaction (PCR) including quantitative real time PCR (qRT-PCR), microarray analysis, serial analysis of gene expression (SAGE), MassARRAY, Gene Expression Analysis by Massively Parallel Signature Sequencing (MPSS), proteomics, immunohistochemistry (IHC), etc.
  • PCR polymerase chain reaction
  • qRT-PCR quantitative real time PCR
  • SAGE serial analysis of gene expression
  • MassARRAY Gene Expression Analysis by Massively Parallel Signature Sequencing
  • MPSS Gene Expression Analysis by Massively Parallel Signature Sequencing
  • proteomics immunohistochemistry
  • mRNA is quantified.
  • mRNA analysis is preferably performed using the technique of polymerase chain reaction (PCR), or by microarray analysis.
  • PCR is quantitative real time PCR (qRT-PCR).
  • Samples, including tissue or cell samples, can be conveniently assayed for expression, e.g., mRNAs, using, for example, a Northern blot method, a Southern blot method, a dot-blot, PCR analysis, array hybridization, RNase protection assay, a FISH method, a CGH method, an RNA chip method, or a DNA chip method, such as a DNA chip microarray (e.g., Affymetrix' s microarray system or Illumina' s BeadArray Technology).
  • DNA chip microarrays are commercially available, including DNA microarray snapshots.
  • the methods of the invention are performed using a genechip or DNA microarray comprising nucleic acid probes specific for CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6.
  • an mRNA sample may be obtained from the subject (e.g., isolated from peripheral blood mononuclear cells, by standard methods) and expression of mRNA(s) encoding CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 in the mRNA sample may be detected using standard molecular biology techniques, such as PCR analysis.
  • a preferred method of PCR analysis is reverse transcriptase-polymerase chain reaction (RT-PCR) or quantitative real time PCR (qRT-PCR).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • qRT-PCR quantitative real time PCR
  • RT-PCR real-time PCR
  • a method for detecting CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 mRNA in a biological sample comprises producing cDNA from the sample by reverse transcription using at least one primer; amplifying the cDNA so produced using CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB
  • such methods can include one or more steps that allow one to determine the levels of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 mRNA in a biological sample (e.g., by simultaneously examining the levels of a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).
  • the sequence of the amplified CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 cDNA can be determined.
  • the method used for determining the expression level(s) of genes in the gene signatures described herein i.e., CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6, is serotyping of biological samples from a subject using, e.g., commercially available antibodies for CSFIR, CSF3R, CCL18, and ILIB; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 in an ELISA assay.
  • samples may be assayed for the expression of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 at the protein level, using a detection reagent that detects the protein product encoded by the mRNA of the marker.
  • an antibody reagent that binds specifically to the protein products of the CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 to be detected, and not to other proteins, then such an antibody reagent can be used to detect the expression of the CSFIR, CSF3R, CCL18, and ILIB; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 in a sample from the subject, or a preparation derived from the sample, using standard antibody-based techniques known in the art, such as FACS analysis, ELISA and the like
  • PCR can be used to compare mRNA levels in different sample populations, e.g., in responder and/or non-responder samples, to characterize patterns of gene expression of the identified gene signatures.
  • the first step is the isolation of mRNA from a target sample.
  • the starting material may be a blood sample from a subject having IBD.
  • General methods for mRNA extraction are well known in the art and are disclosed in standard textbooks of molecular biology, including Ausubel et al., Current Protocols of Molecular Biology, John Wiley and Sons (1997). Methods for RNA extraction from paraffin embedded tissues are disclosed, for example, in Rupp and Locker, Lab Invest. 56:A67 (1987), and De Andres et al., BioTechniques 18:42044 (1995).
  • RNA isolation can be performed using purification kit, buffer set and protease from commercial manufacturers, such as Qiagen, according to the manufacturer's instructions.
  • RNA from cells in culture can be isolated using Qiagen RNeasy mini-columns.
  • Other commercially available RNA isolation kits include MASTERPURETM. Complete DNA and RNA Purification Kit (EPICENTRETM, Madison, Wis.), and Paraffin Block RNA Isolation Kit (Ambion, Inc.).
  • Total RNA from tissue samples can be isolated using RNA Stat-60 (Tel-Test).
  • RNA prepared from a biological sample can be isolated, for example, by cesium chloride density gradient centrifugation.
  • qRT-PCR quantitative real time PCR
  • qRT-PCR is well known in the art and measures PCR product accumulation through a dual- labeled fluorigenic probe (i.e., TAQMANTM probe).
  • Real time PCR is compatible both with quantitative competitive PCR, where internal competitor for each target sequence is used for normalization, and with quantitative comparative PCR using a normalization gene contained within the sample, or a housekeeping gene for PCR. For further details see, e.g. Held et al., Genome Research 6:986- 994 (1996).
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 1, or a portion thereof, and can be used to detect gene expression levels of CSF1R.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 3, or a portion thereof, and can be used to detect gene expression levels of CSF3R.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 5, or a portion thereof, and can be used to detect gene expression levels of CCL18.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 7, or a portion thereof, and can be used to detect gene expression levels of IL1B.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 9, or a portion thereof, and can be used to detect gene expression levels of CD36.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 11, or a portion thereof, and can be used to detect gene expression levels of CLEC4A.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 13, or a portion thereof, and can be used to detect gene expression levels of CLEC7A. In one embodiment, a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 15, or a portion thereof, and can be used to detect gene expression levels of CSTA. In one embodiment, a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 17, or a portion thereof, and can be used to detect gene expression levels of CHI3L1.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 19, or a portion thereof, and can be used to detect gene expression levels of ILIRN.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 21, or a portion thereof, and can be used to detect gene expression levels of TNFAIP6.
  • a primer(s) used in the methods and compositions described herein is specific to SEQ ID NO: 23, or a portion thereof, and can be used to detect gene expression levels of TREM1.
  • the methods and compositions described herein may include one or any combination of the foregoing primers specific to the gene signatures identified herein.
  • a responder whose gene expression levels defines a responder gene signature, is a subject having IBD who has shown a clinical response when treated with a TNFa inhibitor.
  • a responder is a subject having IBD who achieves a reduction of 100 points or more in their Crohn' s Disease Activity Index (CDAI) score following treatment with a TNFa inhibitor.
  • a responder is a subject having IBD who achieves a reduction of 100 points or more in their Crohn' s Disease Activity Index (CDAI) score in a specific time frame following treatment with a TNFa inhibitor.
  • non- responder includes, but is not limited to, a subject with IBD who has no, or limited improvement in their clinical disease status following treatment with a TNFa inhibitor (e.g., lack of reduction in CDAI score, lack of reduction in use of corticosteroids).
  • a non-responder is a subject having IBD who fails to achieve a reduction of 100 points or more in their Crohn' s Disease Activity Index (CDAI) score following treatment with a TNFa inhibitor. In one embodiment, a non-responder is a subject having IBD who fails to achieve a reduction of 100 points or more in their Crohn's Disease Activity Index (CDAI) score in a specific time frame following treatment with a TNFa inhibitor.
  • CDAI Crohn' s Disease Activity Index
  • a responder or non-responder is a subject having IBD who has not yet received treatment with a TNFa inhibitor.
  • Levels of expression of the gene signature of a subject having IBD are compared to levels of expression of the same gene signature from a responder gene signature and/or a non- responder gene signature. Given the responder and non-responder expression profiles, one of ordinary skill would be able to determine which gene signature i.e., responder or non- responder, the subject correlates to. Measurement of gene signature expression levels, for example, may be performed by using a software program executed by a suitable processor. Suitable software and processors are well known in the art and are commercially available.
  • the program may be embodied in software stored on a tangible medium such as a CD-ROM, a floppy disk, a hard drive, a DVD, or a memory associated with the processor, but persons of ordinary skill in the art will readily appreciate that the entire program or parts thereof could alternatively be executed by a device other than a processor, and/or embodied in firmware and/or dedicated hardware in a well known manner.
  • the assay results, findings, diagnoses, predictions, and/or treatment recommendations may be recorded and communicated to technicians, physicians, and/or patients, for example.
  • computers will be used to communicate such information to interested parties, such as patients and/or the attending physicians.
  • the assays will be performed or the assay results analyzed in a country or jurisdiction that differs from the country or jurisdiction to which the results or diagnoses are communicated.
  • a diagnosis, prediction, and/or treatment recommendation based on the expression level of genes in a gene signature described herein, in a subject having IBD is communicated to the subject as soon as possible after the assay is completed and the diagnosis and/or prediction is generated.
  • the results and/or related information may be communicated to the subject by the subject's treating physician.
  • the results may be communicated directly to a test subject by any means of communication, including writing, electronic forms of communication, such as e-mail, or telephone. Communication may be facilitated by use of a computer, such as in the case of e- mail communications.
  • the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test may be generated and delivered automatically to the subject using a combination of computer hardware and software that will be familiar to artisans skilled in telecommunications.
  • a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761 ; however, the present invention is not limited to methods that utilize this particular communications system.
  • communicating of assay results or diagnoses may be carried out in diverse (e.g., foreign) jurisdictions.
  • Any sample obtained from a subject having IBD may be used to determine the expression level(s) of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, IL1RN, TNFAIP6, AND TREM1; and/or TNFAIP6.
  • the sample may be any fluid or subcomponent thereof, e.g., blood fluids, vomit, intra-articular fluid, saliva, lymph, cystic fluid, urine, feces, fluids collected by bronchial lavage, fluids collected by peritoneal rinsing, or gynecological fluids, obtained from the subject.
  • the fluid may be a blood sample, or a component thereof, obtained from the subject.
  • the sample may also be any tissue or fragment or sub-component thereof, e.g., bone, connective tissue, cartilage, lung, liver, kidney, muscle tissue, heart, pancreas, and skin, obtained from the subject.
  • obtaining samples from a subject include, for example, obtaining samples by a mouth swab or a mouth wash; drawing blood; or obtaining a biopsy. Isolating sub-components of fluid or tissue samples (e.g., cells or RNA or DNA) may be accomplished using well known techniques in the art.
  • the invention also provides methods for treating a subject having IBD.
  • inflammatory bowel diseases that may be treated by the methods described herein include, but are not limited to, Crohn's disease and ulcerative colitis.
  • Crohn's disease represents one of the major entities of inflammatory bowel diseases, and is characterized by a chronic relapsing inflammation of the intestinal mucosa (Strober et al. Clin Invest 117, 514-521 (2007) and Danese, S. New therapies for inflammatory bowel disease: from the bench to the bedside. Gut 61, 918-932 (2012)). Patients with this incurable disease suffer from chronic diarrhea, rectal bleeding, abdominal cramping and fistula formation and many patients require surgical intervention over time. It is general consensus that inappropriate activation of the mucosal immune system leading to augmented cytokine production contributes to disease pathogenesis (Neurath et al. Immunity 31, 357-361 (2009) and Atreya, et al. Nat Med 6, 583-588 (2000)). In this context, the pro-inflammatory cytokine tumor necrosis factor-oc (TNFoc) plays a pivotal role in Crohn's disease immunopathogenesis.
  • TNFoc pro-inflammatory cytokine
  • Ulcerative colitis is a chronic inflammatory disease of unknown etiology afflicting the large intestine.
  • the course of the disease may be continuous or relapsing, mild or severe.
  • the earliest lesion is an inflammatory infiltration with abscess formation at the base of the crypts of Lieberkuhn. Coalescence of these distended and ruptured crypts tends to separate the overlying mucosa from its blood supply, leading to ulceration.
  • Signs and symptoms of the disease include cramping, lower abdominal pain, rectal bleeding, and frequent, loose discharges consisting mainly of blood, pus, and mucus with scanty fecal particles.
  • a total colectomy may be required for acute, severe or chronic, unremitting ulcerative colitis.
  • the gene signatures described herein may be used in a method of selecting a patient having IBD, e.g., Crohn's disease or ulcerative colitis, who will be responsive to treatment with a TNFoc inhibitor, e.g., a human anti-TNFoc antibody, or antigen binding portion thereof, such as, but not limited to, adalimumab.
  • a TNFoc inhibitor e.g., a human anti-TNFoc antibody, or antigen binding portion thereof, such as, but not limited to, adalimumab.
  • the method includes determining the expression levels of a gene signature, i.e., CSF1R, CSF3R, CCL18, and IL1B ; CSF1R, CSF3R, CD36, and IL1B; CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6, in the subject, selecting a treatment regimen with an TNFa inhibitor based upon the expression level(s) of CSF1R, CSF3R, CCL18, and IL1B ; CSF1R, CSF3R, CD36, and IL1B; CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 in the subject, and administering the TNFa inhibitor according to the treatment regimen such that the subject is treated for the Crohn's disease.
  • the gene signatures described herein may be used in a method for treating a subject having an IBD comprising determining a gene signature from a biological sample from a subject having IBD, wherein the gene signature is obtained by determining an expression level(s) of any one of gene signatures (i) to (iv) described above.
  • a TNFa inhibitor may be administered to the subject having IBD provided that the gene signature correlates with a responder gene signature comprising the same gene or gene group of (i) to (iv) tested. Should the gene signature of the subject correlate to a non-responder gene signature, however, an alternative therapy other than a TNFa inhibitor may need to be considered.
  • the use of the gene signatures described herein may be incorporated into the treatment process, whereby a determination is first made as to whether or not a TNFa inhibitor is an appropriate therapy for a subject having IBD, e.g., Crohn's disease or ulcerative colitis.
  • the pharmaceutical composition used in the methods of the invention may include a "therapeutically effective amount” or a “prophylactically effective amount” of an antibody or antibody portion of the invention.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of the antibody, antibody portion, or other TNFa inhibitor may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody, antibody portion, other TNFa inhibitor to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, antibody portion, or other TNFoc inhibitor are outweighed by the therapeutically beneficial effects.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the prophylactically effective amount
  • the treatment regimen that is selected typically includes at least one of the following parameters and may include many or all of the following parameters: the type of agent chosen for administration, the dosage, the formulation, the route of administration and/or the frequency of administration.
  • the subject having IBD who is identified as a responder to TNFoc inhibitor therapy according to the methods described herein is treated with a human anti- TNFoc antibody, or antigen-binding portion thereof, according to a multiple variable dose regimen.
  • Multiple-variable dose regimens are described in US Publication No. 20060009385, which is incorporated by reference herein in its entirety.
  • a subject identified as a responder is subcutaneously administered a loading or induction dose (s) followed by subsequent treatment or maintenance doses.
  • the subject is subcutaneously administered a first dose of 160 mg, a second dose of 80 mg, and a dose of 40 mg.
  • a dose of 80 mg is administered subcutaneously followed by a dose of 40 mg for treatment of IBD, including Crohn's disease and ulcerative colitis in a subject identified as a responder.
  • the subject is treated with a TNFoc inhibitor in accordance with a biweekly dosing regimen.
  • Biweekly dosing regimens are further described in US Appln. No. 10/163657 (US 20030235585), incorporated by reference herein.
  • the subject is subcutaneously administered 40 mg of a human TNFoc antibody, or antigen binding portion thereof, every other week for the treatment of Crohn's disease or ulcerative colitis.
  • the methods described herein are used for reducing signs and symptoms and inducing and maintaining clinical remission in adult patients with moderately to severely active Crohn's disease. In certain embodiments of the invention, the methods described herein are used for reducing signs and symptoms and inducing clinical remission in patients having Crohn's disease if they have also lost response to or are intolerant to infliximab.
  • the methods described herein are used for reducing signs and symptoms and inducing and maintaining clinical remission in patients 6 years of age and older with moderately to severely active Crohn's disease who have had an inadequate response to corticosteroids or immuno modulators such as azathioprine, 6- mercaptopurine, or methotrexate. In certain embodiments of the invention, the methods described herein are used to for inducing and sustaining clinical remission in adult patients with moderately to severely active ulcerative colitis.
  • the invention provides a method for determining whether a subject having IBD will respond to treatment with a TNFa inhibitor based on the identified gene signatures (i) to (iv), as well as methods and kits based on the use of the gene signatures described herein.
  • the TNFa inhibitor used in the methods and compositions of the invention is an anti-TNFa antibody, or antigen-binding portion thereof, such as, but not limited to, a human antibody, a chimeric antibody, and a humanized antibody.
  • a chimeric antibody that may be used is infliximab, or a biosimilar thereof.
  • the anti-TNFa antibody fragment is certolizumab pegol, or a biosimilar thereof.
  • the invention features uses and composition for predicting or determining the responsiveness of a subject having an IBD to treatment with a TNFa inhibitor, or for treating said subject, wherein the TNFa antibody is an isolated human antibody, or antigen-binding portion thereof, that binds to human TNFa with high affinity and a low off rate, and also has a high neutralizing capacity.
  • TNFa antibody is an isolated human antibody, or antigen-binding portion thereof, that binds to human TNFa with high affinity and a low off rate, and also has a high neutralizing capacity. Examples of such antibodies include
  • the human antibodies used in the invention are recombinant, neutralizing human anti-hTNFa antibodies.
  • the most preferred recombinant, neutralizing antibody of the invention is referred to herein as adalimumab, also referred to as HUMIRA or D2E7 (the amino acid sequence of the adalimumab VL region is shown in SEQ ID NO: 25; the amino acid sequence of the adalimumab VH region is shown in SEQ ID NO: 26).
  • D2E7 adalimumab / HUMIRA
  • the TNFa inhibitor for use in the invention is a fully human TNFa antibody which is a biosimilar to adalimumab.
  • the TNFa inhibitor is highly similar to adalimumab, and may, for example, include minor differences in clinically inactive components.
  • the TNFa inhibitor is interchangeable with adalimumab, and is, for example, able to produce the same clinical result as adalimumab in any given patient.
  • the adalimumab biosimilar is Exemptia (Zydus Cadila), ABP 501 (Amgen), ONS-3010 (Oncobiologics), and BI 695501 (Boehringer Ingelheim).
  • the method of the invention includes determining the
  • the invention provides treatment with an isolated human antibody, or an antigen-binding portion thereof, that
  • the isolated human antibody, or antigen-binding portion thereof dissociates from human TNFa with a k a g of 5 x 10 "4 s "1 or less, or even more preferably, with a k a g of 1 x 10 "4 s "1 or less.
  • the isolated human antibody, or antigen-binding portion thereof neutralizes human TNFa cytotoxicity in a standard in vitro L929 assay with an IC 50 of 1 x 10 " M or less, even more preferably with an IC 50 of 1 x 10 "9 M or less and still more preferably with an IC 50 of 1 x 10 "10 M or less.
  • the invention pertains to treating an IBD, e.g., Crohn' s disease or ulcerative colitis, by administering human antibodies that have slow dissociation kinetics for association with hTNFa and that have light and heavy chain CDR3 domains that structurally are identical to or related to those of adalimumab.
  • Position 9 of the adalimumab VL CDR3 can be occupied by Ala or Thr without substantially affecting the k Q g.
  • a consensus motif for the adalimumab VL CDR3 comprises the amino acid sequence: Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 27). Additionally, position 12 of the adalimumab VH CDR3 can be occupied by Tyr or Asn, without substantially affecting the k Q g. Accordingly, a consensus motif for the adalimumab VH CDR3 comprises the amino acid sequence: V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 28). Moreover, as demonstrated in Example 2 of U.S. Patent No.
  • the CDR3 domain of the adalimumab heavy and light chains is amenable to substitution with a single alanine residue (at position 1, 4, 5, 7 or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 within the VH CDR3) without substantially affecting the k 0 g.
  • substitution of other amino acids within the CDR3 domains may be possible while still retaining the low off rate constant of the antibody, in particular substitutions with conservative amino acids.
  • no more than one to five conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. More preferably, no more than one to three conservative amino acid substitutions are made within the adalimumab VL and/or VH CDR3 domains. Additionally, conservative amino acid substitutions should not be made at amino acid positions critical for binding to hTNFoc.
  • Positions 2 and 5 of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab VH CDR3 are critical for interaction with hTNFoc and thus, conservative amino acid substitutions preferably are not made at these positions (although an alanine substitution at position 5 of the adalimumab VL CDR3 is acceptable, as described above) (see U.S. Patent No. 6,090,382).
  • the antibody or antigen-binding portion thereof preferably dissociates from human TNFoc with a £ 0j ⁇ rate constant of 1 x 10 " 3 s " 1 or less, as determined by surface plasmon resonance; has a light chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 27, or modified from SEQ ID NO: 27 by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8 and/or 9; and has a heavy chain CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, or modified from SEQ ID NO: 28 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
  • the antibody, or antigen-binding portion thereof dissociates from human TNFoc with a k 0 g of 5 x 10 "4 s "1 or less. Even more preferably, the antibody, or antigen- binding portion thereof, dissociates from human TNFoc with a k a g of 1 x 10 "4 s "1 or less.
  • the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 27, or modified from SEQ ID NO: 27 by a single alanine substitution at position 1, 4, 5, 7 or 8, and with a heavy chain variable region (HCVR) having a CDR3 domain comprising the amino acid sequence of SEQ ID NO: 28, or modified from SEQ ID NO: 28 by a single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the LCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 29 (i.e., the adalimumab VL CDR2) and the HCVR further has a CDR2 domain comprising the amino acid sequence of SEQ ID NO: 30 (i.e., the adalimumab VH CDR2).
  • the LCVR further has CDRl domain comprising the amino acid sequence of SEQ ID NO: 31 (i.e., the adalimumab VL CDRl) and the HCVR has a CDRl domain comprising the amino acid sequence of SEQ ID NO: 32 (i.e., the adalimumab VH CDRl).
  • the framework regions for VL preferably are from the V K I human germline family, more preferably from the A20 human germline Vk gene and most preferably from the adalimumab VL framework sequences shown in Figures 1A and IB of U.S. Patent No. 6,090,382.
  • the framework regions for VH preferably are from the VH3 human germline family, more preferably from the DP-31 human germline VH gene and most preferably from the
  • the antibody or antigen-binding portion thereof preferably contains a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 25 (i.e. , the adalimumab VL) and a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 26 (i.e., the adalimumab VH).
  • the antibody comprises a heavy chain constant region, such as an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.
  • the heavy chain constant region is an IgGl heavy chain constant region or an IgG4 heavy chain constant region.
  • the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region.
  • the antibody comprises a kappa light chain constant region.
  • the antibody portion can be, for example, a Fab fragment or a single chain Fv fragment.
  • the antibody or antigen-binding portion thereof comprises a light chain variable region (LCVR) comprising CDRs as described by the amino acid sequence of SEQ ID NO: 25 (i.e. , the adalimumab VL) and a heavy chain variable region (HCVR) comprising CDRs as described in the amino acid sequence of SEQ ID NO: 26 (i.e., the adalimumab VH).
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the TNFa antibody used in the methods and compositions of the invention may be modified for improved treatment of an IBD, e.g., Crohn's disease or ulcerative colitis.
  • the TNFa antibody or antigen binding fragments thereof is chemically modified to provide a desired effect.
  • pegylation of antibodies and antibody fragments of the invention may be carried out by any of the pegylation reactions known in the art, as described, for example, in the following references: Focus on Growth Factors 3:4- 10 (1992); EP 0 154 316; and EP 0 401 384 (each of which is incorporated by reference herein in its entirety).
  • the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
  • a preferred water-soluble polymer for pegylation of the antibodies and antibody fragments of the invention is polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (C1-C10) alkoxy- or aryloxy-poly ethylene glycol.
  • Methods for preparing pegylated antibodies and antibody fragments of the invention will generally comprise the steps of (a) reacting the antibody or antibody fragment with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby the antibody or antibody fragment becomes attached to one or more PEG groups, and (b) obtaining the reaction products.
  • polyethylene glycol such as a reactive ester or aldehyde derivative of PEG
  • Pegylated antibodies and antibody fragments may generally be used to treat IBD by administration of the TNFa antibodies and antibody fragments described herein. Generally the pegylated antibodies and antibody fragments have increased half-life, as compared to the nonpegylated antibodies and antibody fragments. The pegylated antibodies and antibody fragments may be employed alone, together, or in combination with other pharmaceutical compositions.
  • TNFa antibodies or fragments thereof can be altered wherein the constant region of the antibody is modified to reduce at least one constant region-mediated biological effector function relative to an unmodified antibody.
  • the immunoglobulin constant region segment of the antibody can be mutated at particular regions necessary for Fc receptor (FcR) interactions (see e.g., Canfield, S.M. and S.L. Morrison (1991) J. Exp. Med. 173: 1483-1491; and Lund, J. et al. (1991) J. of Immunol. 147:2657-2662).
  • Reduction in FcR binding ability of the antibody may also reduce other effector functions which rely on FcR interactions, such as opsonization and phagocytosis and antigen-dependent cellular cytotoxicity.
  • an antibody or antibody portion used in the methods of the invention can be derivatized or linked to another functional molecule ⁇ e.g., another peptide or protein). Accordingly, the antibodies and antibody portions of the invention are intended to include derivatized and otherwise modified forms of the human anti-hTNFoc antibodies described herein, including immunoadhesion molecules.
  • an antibody or antibody portion of the invention can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody e.g., a bispecific antibody or a diabody
  • a detectable agent e.g., a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate associate of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by cross-linking two or more antibodies (of the same type or of different types, e.g., to create bispecific antibodies).
  • Suitable cross- linkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • Such linkers are available from Pierce Chemical Company, Rockford, IL.
  • An antibody, or antibody portion, used in the methods and compositions of the invention can be prepared by recombinant expression of immunoglobulin light and heavy chain genes in a host cell.
  • a host cell is transfected with one or more recombinant expression vectors carrying DNA fragments encoding the
  • immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered.
  • Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F.M. et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent No. 4,816,397 by Boss et al.
  • DNA fragments encoding the light and heavy chain variable regions are first obtained. These DNAs can be obtained by amplification and modification of germline light and heavy chain variable sequences using the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Vf j Segments with Different Hypervariable Loops J. Mol. Biol. 227:776-798; and Cox, J.P.L. et al. (1994) "A Directory of Human Germ-line V 78 Segments Reveals a Strong Bias in their Usage” Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference).
  • a member of the VJJ3 family of human germline VH genes is amplified by standard PCR. Most preferably, the DP-31 VH germline sequence is amplified.
  • a member of the V K I family of human germline VL genes is amplified by standard PCR.
  • the A20 VL germline sequence is amplified.
  • PCR primers suitable for use in amplifying the DP-31 germline VH and A20 germline VL sequences can be designed based on the nucleotide sequences disclosed in the references cited supra, using standard methods.
  • germline VH and VL fragments can be mutated to encode the adalimumab, or an adalimumab-related amino acid sequences disclosed herein.
  • the amino acid sequences encoded by the germline VH and VL DNA sequences are first compared to the adalimumab, or an adalimumab-related VH and VL amino acid sequences to identify amino acid residues in the adalimumab, or an adalimumab-related sequence that differ from germline.
  • the appropriate nucleotides of the germline DNA sequences are mutated such that the mutated germline sequence encodes the adalimumab, or an adalimumab-related amino acid sequence, using the genetic code to determine which nucleotide changes should be made.
  • Mutagenesis of the germline sequences is carried out by standard methods, such as PCR-mediated mutagenesis (in which the mutated nucleotides are incorporated into the PCR primers such that the PCR product contains the mutations) or site-directed mutagenesis.
  • the "germline" sequences obtained by PCR amplification encode amino acid differences in the framework regions from the true germline configuration ⁇ i.e., differences in the amplified sequence as compared to the true germline sequence, for example as a result of somatic mutation), it may be desirable to change these amino acid differences back to the true germline sequences ⁇ i.e., "backmutation" of framework residues to the germline configuration).
  • DNA fragments encoding the anti-TNFa antibody e.g., adalimumab, VH and VL segments are obtained (by amplification and mutagenesis of germline VH and VL genes, as described above)
  • these DNA fragments can be further manipulated by standard recombinant DNA techniques, for example to convert the variable region genes to full-length antibody chain genes, to Fab fragment genes or to a scFv gene.
  • a VL- or VH-encoding DNA fragment is operatively linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker.
  • the term "operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.
  • the isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding heavy chain constant regions (CHI, CH2 and CH3).
  • CHI, CH2 and CH3 DNA molecule encoding heavy chain constant regions
  • the sequences of human heavy chain constant region genes are known in the art (see e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the heavy chain constant region can be an IgGl, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably is an IgGl or IgG4 constant region.
  • the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain CHI constant region.
  • the isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL.
  • the sequences of human light chain constant region genes are known in the art (see e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242) and DNA fragments encompassing these regions can be obtained by standard PCR amplification.
  • the light chain constant region can be a kappa or lambda constant region, but most preferably is a kappa constant region.
  • the VH- and VL-encoding DNA fragments are operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 33) such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al, Nature (1990) 348:552-554).
  • a flexible linker e.g., encoding the amino acid sequence (Gly4-Ser)3 (SEQ ID NO: 33) such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see
  • DNAs encoding partial or full-length light and heavy chains, obtained as described above, are inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences.
  • operatively linked is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used.
  • the antibody light chain gene and the antibody heavy chain gene can be inserted into separate vector or, more typically, both genes are inserted into the same expression vector.
  • the antibody genes are inserted into the expression vector by standard methods ⁇ e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present).
  • the expression vector Prior to insertion of the adalimumab, or an adalimumab-related light or heavy chain sequences, the expression vector may already carry antibody constant region sequences.
  • one approach to converting the adalimumab, or an adalimumab-related VH and VL sequences to full-length antibody genes is to insert them into expression vectors already encoding heavy chain constant and light chain constant regions, respectively, such that the VH segment is operatively linked to the CH segment(s) within the vector and the VL segment is operatively linked to the CL segment within the vector.
  • the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell.
  • the antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene.
  • the signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide ⁇ i.e., a signal peptide from a non- immunoglobulin protein).
  • the recombinant expression vectors of the invention carry regulatory sequences that control the expression of the antibody chain genes in a host cell.
  • the term "regulatory sequence” is intended to include promoters, enhancers and other expression control elements ⁇ e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes.
  • Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.
  • CMV cytomegalovirus
  • SV40 Simian Virus 40
  • AdMLP adenovirus major late promoter
  • the recombinant expression vectors used in the invention may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5, 179,017, all by Axel et al.).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced.
  • Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).
  • DHFR dihydrofolate reductase
  • the expression vector(s) encoding the heavy and light chains is transfected into a host cell by standard techniques.
  • the various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like.
  • Preferred mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in R.J. Kaufman and P. A. Sharp (1982) Mol. Biol.
  • the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or, more preferably, secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • Host cells can also be used to produce portions of intact antibodies, such as Fab fragments or scFv molecules. It is understood that variations on the above procedure are within the scope of the present invention. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an antibody of this invention. Recombinant DNA technology may also be used to remove some or all of the DNA encoding either or both of the light and heavy chains that is not necessary for binding to hTNFoc. The molecules expressed from such truncated DNA molecules are also encompassed by the antibodies of the invention.
  • bifunctional antibodies may be produced in which one heavy and one light chain are an antibody of the invention and the other heavy and light chain are specific for an antigen other than hTNFoc by crosslinking an antibody of the invention to a second antibody by standard chemical crosslinking methods.
  • a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection.
  • the antibody heavy and light chain genes are each operatively linked to CMV enhancer/ AdMLP promoter regulatory elements to drive high levels of transcription of the genes.
  • the recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification.
  • the selected transformant host cells are culture to allow for expression of the antibody heavy and light chains and intact antibody is recovered from the culture medium.
  • Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the antibody from the culture medium.
  • nucleic acid, vector and host cell compositions that can be used for recombinant expression of the antibodies and antibody portions used in the invention include nucleic acids, and vectors comprising said nucleic acids, comprising the human TNFoc antibody adalimumab (D2E7).
  • D2E7 human TNFoc antibody adalimumab
  • the nucleotide sequences encoding the adalimumab light and heavy chain variable regions are known in the art, e.g., see U.S. Patent No. 6,090,382.
  • nucleotide sequences encoding adalimumab- related antibodies, or portions thereof can be derived from the nucleotide sequences encoding the adalimumab LCVR and HCVR using the genetic code and standard molecular biology techniques.
  • Antibodies, antibody-portions, and other TNFoc inhibitors for use in the methods of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject.
  • the pharmaceutical composition comprises an antibody, antibody portion, or other TNFoc inhibitor, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the
  • compositions may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody, antibody portion, or other TNFoc inhibitor.
  • auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody, antibody portion, or other TNFoc inhibitor.
  • compositions typically must be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e. , antibody, antibody portion, or other TNFoc inhibitor) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • the invention includes pharmaceutical compositions comprising an effective TNFoc inhibitor and a pharmaceutically acceptable carrier, wherein the effective TNFoc inhibitor may be used to treat IBD.
  • kits for assessing a subject's responsiveness to a TNFa inhibitor for the treatment of IBD e.g., Crohn' s disease or ulcerative colitis
  • kits for treating a subject having an IBD e.g., Crohn' s disease or ulcerative colitis.
  • kits of the invention can be used to determine if a subject with IBD, e.g., Crohn' s disease or ulcerative colitis, will be effectively responsive to a TNFa inhibitor.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a probe that is or can be detectably labeled directed to one of the genes in the gene signature described herein.
  • probe may be an antibody or polynucleotide specific for a protein or gene transcript, respectively.
  • the kit may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter-means, such as a biotin-binding protein, e.g., avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
  • a reporter-means such as a biotin-binding protein, e.g., avidin or streptavidin
  • the kit includes a means for determining the expression level(s) of CSF1R, CSF3R, CCL18, and IL1B; CSF1R, CSF3R, CD36, and IL1B ; CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 and instructions for use of the kit.
  • Means for determining expression levels may include nucleic acid molecules or antibodies, as described in more detail below. Kits of the invention can be used to determine if a subject with Crohn' s disease will be effectively responsive to a TNFoc inhibitor.
  • kits may comprise a carrier means being compartmentalized to receive in close confinement one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a probe that is or can be detectably labeled.
  • probe may be an antibody or polynucleotide specific for a protein or a biomarker (CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or
  • kits containing nucleotide(s) for amplification of the target nucleic acid sequence may also have containers containing nucleotide(s) for amplification of the target nucleic acid sequence and/or a container comprising a reporter- means, such as a biotin-binding protein, e.g., avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, florescent, or radioisotope label.
  • a reporter- means such as a biotin-binding protein, e.g., avidin or streptavidin
  • kit will typically comprise the container described above and one or more other containers comprising materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • a label may be present on the container to indicate that the composition is used for a specific application, and may also indicate directions for either in vivo or in vitro use, such as those described above.
  • kits of the invention have a number of embodiments.
  • a typical embodiment is a kit comprising a container, a label on the container, and a composition contained within the container, wherein the composition includes one or more polynucleotides that hybridize to a complement of the CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or
  • TNFAIP6 under stringent conditions, and the label on the container indicates that the composition can be used to evaluate the expression level(s) of CSFIR, CSF3R, CCL18, and ILIB; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 in a sample, and wherein the kit includes instructions for using the polynucleotide(s) for evaluating the expression level(s) of the CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 RNA or DNA in a particular sample type.
  • kits comprising a container, a label on the container, and a composition contained within the container, wherein the composition includes a primary antibody that binds to a protein or autoantibody biomarker, and the label on the container indicates that the composition can be used to evaluate the presence of such proteins or antibodies in a sample, and wherein the kit includes instructions for using the antibody for evaluating the presence of biomarker proteins in a particular sample type.
  • the kit can further comprise a set of instructions and materials for preparing a sample and applying antibody to the sample.
  • the kit may include both a primary and secondary antibody, wherein the secondary antibody is conjugated to a label, e.g., an enzymatic label.
  • kits include one or more buffers (e.g., block buffer, wash buffer, substrate buffer, etc.), other reagents such as substrate (e.g., chromogen) that is chemically altered by an enzymatic label, epitope retrieval solution, control samples (positive and/or negative controls), control slide(s), etc.
  • Kits can also include instructions for interpreting the results obtained using the kit.
  • the kit can comprise, for example: (1) a first antibody (e.g., attached to a solid support) that binds to a biomarker protein; and, optionally, (2) a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label.
  • a first antibody e.g., attached to a solid support
  • a second, different antibody that binds to either the protein or the first antibody and is conjugated to a detectable label.
  • the kit can comprise, for example: (1) an
  • oligonucleotide e.g., a detectably labeled oligonucleotide, which hybridizes to a nucleic acid sequence encoding a biomarker protein or (2) a pair of primers useful for amplifying a biomarker nucleic acid molecule.
  • the kit can also comprise, e.g., a buffering agent, a preservative, or a protein- stabilizing agent.
  • the kit can further comprise components necessary for detecting the detectable label (e.g., an enzyme or a substrate).
  • the kit can also contain a control sample or a series of control samples that can be assayed and compared to the test sample.
  • Each component of the kit can be enclosed within an individual container, and all of the various containers can be included within a single package, along with instructions for interpreting the results of the assays performed using the kit.
  • kits of the invention may optionally comprise additional components useful for performing the methods of the invention.
  • the kits may comprise means for obtaining a biological sample from a subject, a control sample, e.g., a sample from a subject, one or more sample compartments, an instructional material which describes performance of a method of the invention and specific controls/standards.
  • the instructions can be, for example, printed instructions for performing the assay for evaluating the results.
  • the means for isolating a biological sample from a subject can comprise one or more reagents that can be used to obtain a fluid or tissue from a subject.
  • the means for obtaining a biological sample from a subject may also comprise means for isolating peripheral blood mononuclear cells from a blood sample, for example by positive selection of the monocytes or by negative selection in which all other cell types other than monocytes are removed.
  • kits of the invention may further a TNFoc inhibitor.
  • the kit is designed for use with a human subject.
  • the article of manufacture comprises a container and a label or package insert on or associated with the container.
  • the package insert is on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container holds or contains the antagonist that is effective for treating Crohn's disease and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • At least one active agent in the composition is the B-cell antagonist.
  • the label or package insert indicates that the composition is used for treating IBD, e.g., Crohn's disease or ulcerative colitis, in a subject eligible for treatment with specific guidance regarding dosing amounts and intervals of antagonist and any other medicament being provided.
  • kits and articles of manufacture herein also include information, for example in the form of a package insert or label, indicating that the composition is used for treating IBD, e.g., Crohn's disease or ulcerative colitis, where the expression level(s) of CSFIR, CSF3R, CCL18, and IL1B ; CSFIR, CSF3R, CD36, and IL1B; CLEC4A, CLEC7A, CSTA, CHI3L1, IL1B, IL1RN, TNFAIP6, AND TREM1 ; and/or TNFAIP6 herein are detected in a genetic sample from the patient with the disease.
  • IBD e.g., Crohn's disease or ulcerative colitis
  • the insert or label may take any form, such as paper or electronic media, for example, a magnetically recorded medium (e.g., floppy disk) or a CD- ROM.
  • the label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture.
  • the following information regarding the antagonist may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, and patent
  • an article of manufacture comprising, packaged together, a pharmaceutical composition comprising a TNFa inhibitor and a pharmaceutically acceptable carrier and a label stating that the inhibitor or
  • composition for treating patients with IBD, e.g., Crohn' s disease or ulcerative colitis, from which a genetic sample has been obtained showing the expression level(s) of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB ; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6.
  • IBD e.g., Crohn' s disease or ulcerative colitis
  • the invention provides a method for manufacturing a TNFa inhibitor or a pharmaceutical composition thereof comprising combining in a package the TNFa inhibitor or pharmaceutical composition and a label stating that the TNFa inhibitor or pharmaceutical composition is indicated for treating patients with IBD, e.g., Crohn's disease or ulcerative colitis, from which a genetic sample has been obtained showing the expression level(s) of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6.
  • IBD e.g., Crohn's disease or ulcerative colitis
  • the label may further state that this can be shown by assessing genetic expression as a biomarker of CSFIR, CSF3R, CCL18, and ILIB ; CSFIR, CSF3R, CD36, and ILIB; CLEC4A, CLEC7A, CSTA, CHI3L1, ILIB, ILIRN, TNFAIP6, AND TREM1 ; and/or TNFAIP6.
  • each of the genetic markers identified in the invention may be described individually or in combination with one another.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • Ranges provided herein are understood to be shorthand for all of the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • gene signatures that may be used, for example, to predict whether a patient having IBD (e.g., a Crohn's disease patient) will respond or not respond to treatment with a TNFoc inhibitor, e.g., adalimumab.
  • IBD e.g., a Crohn's disease patient
  • TNFoc inhibitor e.g., adalimumab
  • Examples 1 - 5 provide accurate and effective predictors to identify TNF responders and non-responders.
  • the four identified gene signatures can be used as markers to identify patients having IBD (including Crohn's disease or ulcerative colitis) who will be responsive to treatment with a TNFoc inhibitor (such as adalimumab).
  • microarray data were derived from the following three independent cohorts of Crohn's disease (CD) and ulcerative colitis (UC): GSE 16879 (accessible through GEO series accession number GSE16879 at NCBI's Gene Expression Omnibus; dataset for CD and UC; see Arijs et al. (2009)), GSE52746 (accessible through GEO series accession number GSE52746 at NCBI's Gene Expression Omnibus), and a test data set provided by UMASS.
  • CD Crohn's disease
  • UC ulcerative colitis
  • simplified histology score were determined in all patients, in which histology scores were graded 0, 1, 2 and 3 if they were normal, mild, moderate and severe, respectively.
  • CD patients diagnosed with 4 months' disease duration were included in this study and controls with non- inflammatory bowel disease (non-IBD).
  • the clinical data for the CD patients provided by the literature is included in Leal et al. (2014).
  • Non-CD controls were selected for mild gastrointestinal symptoms or a screening colonoscopy for colorectal cancer. All patients were assessed according to the Montreal classification with CD Endoscopic Index of Severity (CDEIS) of >5 (5.4-20.5), CD Activity Index >140 (142- 189), and the presence of large ulcers (>0.5 cm diameter) in at least one of the explored segments.
  • CDEIS CD Endoscopic Index of Severity
  • CD Activity Index >140 142- 189
  • the exclusion categories were: 1. patients with concomitant infections; 2.
  • Adalimumab was given subcutaneously 160/80 mg at weeks 0 and 2 for induction and 40 mg every other week for maintenance.
  • Infliximab was given intravenously (5 mg/kg) at weeks 0, 2 and 6 for induction and every 8 weeks for maintenance.
  • Response to anti-TNFa therapy was assessed by endoscopy at 12 weeks after first drug administration. Responders to anti-TNF therapy were defined as CDEIS ⁇ 5 and an absence of ulcers, whereas patients who did not achieve such improvement were defined as non-responders.
  • Biopsy samples were immediately snap-frozen in RNA later-RNA Stabilization Reagent (QIAGEN) and kept in liquid nitrogen until needed for RNA isolation and/or immunohistochemistry.
  • An additional test data set was a dataset provided by UMASS Medical School,
  • RNA preparation protocol was used to obtain RNA from samples for the bio informatics analysis described in Examples 2-4 for the UMASS dataset.
  • FFPE Paraffin-Embedded
  • the DNase treatment that followed was designed to eliminate all genomic DNA, including very small fragments that could have been present in FFPE samples. Appropriate binding conditions for RNA were created by adding ethanol. The sample was then transferred to a filter tube and spin column to elute RNA with RNase-free water, contaminants being efficiently washed away.
  • RNA concentration was measured using the Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) and RNA purity and integrity was verified using la-on-chip technology (Agilent 2100 Bioanalyzer, Santa Clara, CA, USA). RNA was kept at - 80 °C until needed for microarray analysis.
  • Tissue samples prepared according to the above FFPE protocol were analyzed using microarray analysis to obtain gene expression profiles.
  • the Standard Whole-Genome DASL (cDN A- mediated Annealing, Selection, extension, and Ligation) HT Assay label protocol (WGDASL HT Assay Guide (15018211 D) (Illumina, Inc.) was carried out using the WG- DASL HT Assay Profiling Reagent Kit.
  • WGDASL HT Assay Guide 15018211 D
  • Illumina, Inc. Illumina, Inc.
  • total RNA was converted into biotinylated cDNA through a reverse transcription reaction using biotinylated oligo (dT) and random nonamer primers.
  • biotinylated cDNA was then annealed with as say- specific oligonucleotides (ASO) specially designed for a single contiguous 50 nucleotide sequence on each cDNA (provided with the Illumina Gene Chip kit).
  • ASO oligonucleotides
  • the resulting products were PCR amplified and labeled with a universal fluorescently labeled primer.
  • the resulting PCR products were then hybridized to the single- stranded labeled products were then hybridized on the complementary gene specific sequence bead to Illumina Whole-Genome Gene Expression Human HT-12 v4 BeadChips and scanned with the iScanTM Reader.
  • the bead chips scan generated intensity data files (.idat files) for each sample, each file containing raw intensity data values for every bead in the scanned image.
  • Bead summary intensities were log2-transformed and normalized using quantile normalization imbedded in Illumina BeadStudio v3.1.3 with GeneExpression plugin v3.4.0 and GenomeStudio 2010.1 with GeneExpression plugin vl.O were used to generate reference data (Illumina, San Diego, CA, USA).
  • GSE16879 and GSE52746 were downloaded as compressed *.CEL file packages from the GEO database, and normalized and background-corrected using Robust Multi-array Average (RMA) algorithm.
  • RMA Robust Multi-array Average
  • the data were analyzed for differential gene expression using an empirical Bayes moderated t-test, implemented in the Bioconductor package Linear Models for
  • Microarray Data LIMMA www.bioconductor.org. The results were sorted by the adjusted p- value and fold change of gene expression.
  • EXAMPLE 1 Bioinformatics analysis of anti-TNFoc non-responder using immune cell lineage gene signature
  • Datasets were obtained based on the differential gene expression profiles among multiple human immune cell lineages using a customized gene chip.
  • the generated datasets contained 136 human immune cell samples for 82 cell lineage gene expression datasets.
  • multiple human immune cell lineage gene signatures including T, B, NK, myeloid cells, were obtained using a multivariate comparison test implemented in the Bioconductor package Linear Models for Microarray Data LIMMA. Three fold change cutoff was chosen to select T, B and NK cell lineage signatures while 5-fold change cutoff to generate myeloid cell lineage signature.
  • the final myeloid cell signature contained the most overexpressed 64 genes compared to all other immune cell gene expression.
  • a manually curated gene signature was selected from myeloid cell lineage signature using hierarchical clustering with Euclidean distance metric and selecting the genes with most significant fold changes and statistical difference between non-responders and responders.
  • CLEC4A C-type lectin domain family 4, member A; CHI3L1 : chitinase 3- like 1 (cartilage glycoprotein-39); MMP7: matrix metallopeptidase 7 (matrilysin, uterine); CLEC7A: C-type lectin domain family 7, member A; ILIB: interleukin 1, beta; CSTA: cystatin A (stefin A); IL1RN: interleukin 1 receptor antagonist; TNFAIP6: tumor necrosis factor, alpha- induced protein 6; TREM1 : triggering receptor expressed on myeloid cells 1) were most significantly differentially upregulated in GSE 16879 training dataset between anti-TNF CD non-responders and responders.
  • Examples 2 to 4 describe multivariate data analysis based on gene set enrichment analysis (GSEA) of the 64 genes identified in the examination of human myeloid cells in CD patients.
  • GSEA Gene Set Enrichment Analysis
  • GSEA is a computational method that determines whether an a priori defined set of genes shows statistically significant, concordant differences between two biological states (e.g. , IBD TNFa inhibitor responder phenotype).
  • EXAMPLE 2 Multivariate Data Analysis and Identification of Gene Signature #1
  • the following Example describes the identification of a gene signature (Gene Signature #1) that can separate responders from non-responders to treatment with a TNF inhibitor in IBD patients, specifically Crohn's and ulcerative colitis patients.
  • the approach considered Gene Set Enrichment Analysis (GSEA) pathway enrichment analysis (Subramanian 2005) to the myeloid 62 gene cell signature that was identified based on human immune cell subsets, and described in Example 1.
  • GSEA Gene Set Enrichment Analysis
  • the top pathways were ranked by p-value, followed by application of a Monte- Carlo based logistic regression with lasso to the 2 nd most enriched pathway consisting of 9 input genes to the Crohn's disease before treatment (BT) data (training set, GEO GSE 16879, Arijs et al 2009), as described in Example 1.
  • This analysis resulted in a signature of 4 genes: CSF1R, CSF3R, CCL18, and IL1B.
  • the lasso penalty parameter estimate from the logistic-lasso model (Friedman et al 2010) that controls the feature selection process was refined by a Monte-Carlo resampling procedure.
  • the gene expressions were first normalized by forming the log 2 RMA normalized expression values of the probe sets. This procedure entails generating 50 bootstrap replications of the normalized dataset and refitting the logistic-lasso model to estimate the lasso penalty parameter. The median of all the lasso penalty parameter estimates across the 50 Monte-Carlo replications was then used to finalize the lasso penalty parameter estimate in the logistic regression model, which in turn finalizes the number of features to be used in the signature.
  • the logistic regression coefficients of the final list of features from the above algorithm were then used to create a linear composite score.
  • the linear composite score is a weighted sum of the normalized expressions of CSF1R, CSF3R, CCL18, and IL1B.
  • our composite score can be calculated for Affymetrix Human Genome U133 Plus 2.0 Arrays as: 0.18 * 203104_at (CSF1R) +
  • An AUC of 1 was obtained for the Test Set #1, an AUC of 0.79 was obtained for Test Set #2, and an AUC of 0.86 was obtained for the ulcerative colitis dataset (Test Set #3) was obtained.
  • the gene signature comprising CSFIR, CSF3R, CCL18, and ILIB was determined to be predictive of response to treatment.
  • Figures 1 and 2 illustrate the separation of responders versus non-responders for the training set for normalized gene expressions and for the composite score respectively.
  • Figures 3-11 illustrate the performance in the three independent Test-Sets 1 to 3 (public dataset, private dataset: UMASS, and an ulcerative colitis dataset, respectively).
  • Example 2 the same resampling-based logistic regression with lasso was applied to the most enriched GSEA pathway consisting of 6 input genes to the Crohn's disease before- treatment data (training set, GEO GSE16879, Arijs 2009) to obtain a second signature of 4 genes: CSFIR, CSF3R, CD36, and ILIB.
  • the linear composite score is a weighted sum of the normalized expressions of these four genes: CSFIR, CSF3R, CD36, and ILIB.
  • the composite score (LC2) can be calculated for Affymetrix Human Genome U133 Plus 2.0 Arrays as:2.31 * 203104_at (CSFIR) + 3.05 * 155329? '_ ⁇ _ ⁇ (CSF3R) + 0.39 * 206488_s_at (CD36) + 1.18 * 205067 _at (ILIB).
  • Figures 14-22 illustrate the performance in the three independent Test-Sets 1 to 3 (public dataset, UMASS dataset, and an ulcerative colitis dataset, respectively).
  • the elastic-net method from the R package glmnet (function glmnet) was applied to Crohn's disease before-treatment data (training set, GEO GSE16879, Arijs 2009) focusing on an initial manually curated list of 9 candidate genes: CHI3L1, CLEC4A, CLEC7A, CSTA, ILIB, ILIRN, MMP7, TNFAIP6, and TREMl.
  • the elastic net mixing parameter was set to 1/4 and the penalty coefficient, lambda, was selected by BIC (the Bayesian information criterion).
  • a subset of 8 genes (TNFAIP6, TREMl, ILIRN, CLEC4A, ILIB, CHI3L1, CLEC7A, and CSTA) was selected for model construction.
  • the final signature is a weighted sum of the normalized expressions of the 8 genes.
  • the composite score (LC 3 ) can be calculated for Affymetrix Human Genome U133 Plus 2.0 Arrays as:1.489 * 206025_s_at (TNFAIP6) + 0.949 * 219434_at (TREMl) + 0.536 * 21265? '_s_at (ILIRN) + 1.145 * 219947 _at (CLEC4A) + 0.943 * 205067 _at (ILIB) + 1.400 * 209395 _at (CHI3L1) + 1.537 * 221698_s_at (CLEC7A) + 0.382 * 204971 _at (CSTA).
  • Figures 23-24 illustrate the univariate separation of responders versus non-responders for the training set for normalized gene expressions and for the composite score respectively.
  • Figures 25-33 illustrate the performance in the three independent test sets 1 to 3 (public dataset, private dataset: UMASS, and an ulcerative colitis dataset, respectively).
  • TNFAIP6 was determined to be predictive of response to treatment.
  • Figures 34-35 illustrate the univariate separation of responders versus non-responders for the training set for normalized gene expressions and for the composite score respectively.
  • Figures 36-41 illustrate the performance in three independent test sets 1 to 3 (public dataset, UMASS dataset, and an ulcerative colitis dataset, respectively).

Abstract

L'invention concerne des méthodes et des compositions pour traiter un sujet ou pour prédire la réponse d'un sujet souffrant d'une affection intestinale inflammatoire (IBD) au traitement avec un inhibiteur de TNFα, tel qu'un anticorps anti-TNFα.
PCT/US2016/031145 2015-05-07 2016-05-06 Méthodes et compositions de diagnostic et de traitement de la maladie intestinale inflammatoire WO2016179469A1 (fr)

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WO2020104705A3 (fr) * 2018-11-23 2020-07-16 Katholieke Universiteit Leuven Prédiction d'une réponse à un traitement dans une maladie intestinale inflammatoire
WO2022020755A3 (fr) * 2020-07-24 2022-03-10 The Regents Of The University Of California Biomarqueurs et procédés de sélection et d'utilisation associés
EP4015651A1 (fr) * 2020-12-17 2022-06-22 Koninklijke Philips N.V. Prédiction de traitement et efficacité d'un traitement anti-tnf alpha chez les patients atteints de la maladie intestinale inflammatoire
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WO2019087200A1 (fr) * 2017-11-06 2019-05-09 Rambam Med-Tech Ltd. Méthodes de pronostic pour traitement anti-tnfα
WO2020104705A3 (fr) * 2018-11-23 2020-07-16 Katholieke Universiteit Leuven Prédiction d'une réponse à un traitement dans une maladie intestinale inflammatoire
WO2022020755A3 (fr) * 2020-07-24 2022-03-10 The Regents Of The University Of California Biomarqueurs et procédés de sélection et d'utilisation associés
EP4015651A1 (fr) * 2020-12-17 2022-06-22 Koninklijke Philips N.V. Prédiction de traitement et efficacité d'un traitement anti-tnf alpha chez les patients atteints de la maladie intestinale inflammatoire
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