WO2020256868A1 - Immune system modulators for the treatment of squamous lung premalignancy - Google Patents

Immune system modulators for the treatment of squamous lung premalignancy Download PDF

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WO2020256868A1
WO2020256868A1 PCT/US2020/033191 US2020033191W WO2020256868A1 WO 2020256868 A1 WO2020256868 A1 WO 2020256868A1 US 2020033191 W US2020033191 W US 2020033191W WO 2020256868 A1 WO2020256868 A1 WO 2020256868A1
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inhibitors
immune response
response regulator
expression
subject
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WO2020256868A9 (en
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Jennifer E. Beane-Ebel
Marc LENBURG
Carter MERENSTEIN
Boting NING
Avrum Spira
Mary Reid
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Trustees Of Boston University
Health Research, Inc.
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    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/26—Infectious diseases, e.g. generalised sepsis
    • 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
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/20—Supervised data analysis
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B40/00—ICT specially adapted for biostatistics; ICT specially adapted for bioinformatics-related machine learning or data mining, e.g. knowledge discovery or pattern finding
    • G16B40/30—Unsupervised data analysis

Definitions

  • the field of the invention relates to methods for detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by detecting the level of expression of certain immune response regulators described herein and/or by therapeutically increasing the level of immune response regulators and/or by detecting by detecting the level of expression of certain immune response regulators described herein and/or by
  • the immune system is a collection of organs, cells and specialized tissues that work together to defend the body against foreign invaders and diseased cells.
  • a healthy immune system can recognize foreign or aberrant cells and target them for destruction.
  • disorders such as infectious disease, autoimmune disease, and cancer can wreak havoc when the immune system cannot adequately counter the diseased cells.
  • Lung squamous cell cancer develops from non-cancerous lesions in the airway known as bronchial premalignant lesions.
  • bronchial premalignant lesions The presence of persistent or progressive dysplastic bronchial premalignant lesions is a marker of increased lung cancer risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung.
  • PMLs bronchial premalignant lesions
  • LUSC Lung Squamous Cell Carcinoma
  • the inventions described herein relate to the discovery that the level of certain immune regulators that can be used to diagnose, detect and/or treat conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer).
  • infectious disease e.g. infectious disease, autoimmune disease, and cancer.
  • PMLs bronchial premalignant lesions
  • the inventors describe a novel set of immune regulators that contribute to immune suppression in bronchial premalignant lesions that persist or progress to higher- grade lesions or invasive lung cancer.
  • Previous targets for immune activation in the context of cancer have focused primarily on late stage tumors and have focused on T cell mediated immunity. Genes that have been discovered to mediate this immune pathway include PD1, PDL1, and CTLA4.
  • the immune regulators identified herein modulate the activity of MHC class I and II antigen presentation, interferon signaling, and B cell related immunity.
  • the immune regulators described herein regulate immune activation and suppression before tumors even form. Accordingly, the inventors have developed methods for determining if the bronchial premalignant lesions are severe and if they are likely to progress to cancer. Additionally, the inventors have developed new therapies for bronchial premalignant lesions that target the underlying molecular changes associated with immune suppression.
  • the inventors discovered that the level of these genes can be used to diagnose, detect and/or treat bronchial BPML and/or lung cancer. Accordingly, the inventors have developed methods for the detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by detecting the level of expression of certain immune regulators described herein and/or by therapeutically modulating the level of those immune regulators.
  • any of the embodiments is a method for treating or preventing a condition caused by or associated with immunosuppressed aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject.
  • a method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
  • a method of treating bronchial premalignant lesions in a subject in need thereof comprising administering at least one of:
  • a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; iii. at least one immune stimulating drug; and/or
  • a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
  • a method of treating bronchial premalignant lesions in a subject in need thereof comprising:
  • an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB 1, HLA-DPA1, and HLA-DRA
  • at least one immune response regulator different from the group must also show a change.
  • immune response regulator selected from Table 1 or Table 2.
  • the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
  • composition formulated for the treatment or prevention of a condition caused by or associated with autoimmune aberrant immune system activity, comprising:
  • the pharmaceutical composition comprises an agonist to the miRs of Table 2A, when 2..., 4... , 6..., 7... miR’ s from Table 2A is underexpressed.
  • condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease, or an infectious disease such as COVID-19.
  • a method of determining the risk of progression of bronchial premalignant lesions to lung cancer or an infectious disease in a subject comprising:
  • compositions that target these immune regulators can serve to inhibit the immune suppressor genes or enhance the immune activator genes and thereby be effective in delaying or preventing the development of conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer).
  • aberrant immune system activity e.g. infectious disease, autoimmune disease, and cancer.
  • FIG. 1 A-FIG. IF depict a schemata of carcinogenesis (FIG.1 A), and squamous lung carcinogenesis (FIG. IB), and some example images of bronchial premalignant lesions along the progression toward squamous lung cancer (FIG. 1C, ID, IE, IF).
  • FIG.2 shows the study design of the Inventor’s study design investigating Bronchial Premalignant Lesions using the pre-cancer genome atlas (PCGA).
  • FIG. 3 shows that endobronchial biopsies divide into four distinct molecular subtypes based on distinct patterns of gene co-expression.
  • FIG. 4 shows a schemata of the Inventor’s study design.
  • FIG. 5 shows that six immune phenotypes were identified in the bronchial PMLs from the
  • Phenotypes with redundant biology or intederminate enrichment were merged with related phenotypes.
  • Samples received a score for each phenotype on a representative gene set. When a phenotype was previously found, the same representative gene set was used. Otherwise, the gene set most correlated to module a gene was used.
  • FIG. 8A - Fig. 8C depicts the mediation analysis. All genes significantly correlated to the three immune phenotypes, Antigen Presentation, B cells, Interferon, were identified, as were genes correlated to progression/regression status. A regression model was used to identify the subset of genes completely mediating the associations between genes, phenotypes, and progression/regression outcome.
  • FIG. 9 depicts that the mediation analysis identified 13 genes completely mediate immune phenotypes, and 2 genes completely mediated by immune phenotypes.
  • FIGs. 10A-10D shows that GSTP1 mediates the effect of antigen presentation on progression.
  • Antigen presentation, B cells, and Interferon are key phenotypes distinguishing progressing and regressing bronchial PMLs.
  • Mediation analysis identified 11 genes that appear to modulate these immune phenotypes, 2 which down-regulates immune response in progressing lesions. It also identified 2 genes significantly up regulated in progressing lesions that are completely mediated by one or more immune phenotypes. These immune modulators represent targets for immuno-prevention of lung squamous cell carcinoma.
  • FIGs. 10B- IOC the first series is progressing/stable lesions and the second series is regressing lesions.
  • FIG. 10D depicts specific immune signaling pathways anticorrelated with GSTP1 expression via Gene Set Enrichment Analysis, including STAT5 signaling, STAT3 signaling and TNFA signaling via NFKB.
  • FIG. 12 shows four miRNAs that were identified which regulate Module 9 gene expression based on the method described in FIG. 11.
  • FIG. 13A-FIG. 13B show that miR-149-5p is significantly upregulated in the progressing/persistent PMLs within the proliferative subtype, while its target genes are down- regulated.
  • FIG. 13B shows that miR-149-5p expression was enriched among epithelial cell type samples and that it was depleted among lymphocyte and myeloid samples. (* p-value ⁇ 0.05). The association between miRNA or mRNA expression levels and lesion progression status was determined using a mixed-effect model, adjusting for batch and patient as a random effect.
  • FIG. 13B shows that miR-149-5p expression is enriched among epithelial cell type samples and that it is depleted among lymphocyte and myeloid samples using FANTOM5 Consortium data.
  • the first series is
  • FIG. 16 shows the expression pattern of NLRC5 target genes. All genes belonged to Module 9 and were significantly down-regulated in the progressive/persistent lesions based on a mixed-effect model, adjusting for batch and patient as a random effect.
  • the first series is
  • FIG. 17 shows the correlation between Module 9 and GPC1 in different cell types based on single-cell RNA-sequencing data containing -4000 cells from 12 samples of 11 patients.
  • GSVA score for Module9 was calculated for each cell.
  • GPC1 is the host gene for miR-149-5p and shares the same transcriptional start site.
  • FIG. 21A and FIG. 2B depict tables listing the subject demographic and clinical annotation in the discovery and validation cohorts.
  • FIG. 22A-FIG. 22E show endobronchial biopsies divide into four distinct molecular subtypes that correlate with clinical and molecular phenotypes.
  • the heatmap shows semi-supervised hierarchal clustering of z-score -normalized gene expression across the 3936 genes and 190 DC biopsies.
  • Bubbleplots showing significant associations (p ⁇ 0.01, two-sided Fisher’s exact test) between the molecular subtypes and genomic smoking status, biopsy histological grade, and the predicted LUSC tumor molecular subtypes.
  • the columns represent the four molecular subtypes (Proliferative, Inflammatory, Secretory, and Normal-like) and the diameter of the circle is proportional to the number of samples within each subtype that have the row phenotype.
  • FIG. 22E Immunofluore scent staining demonstrating the increased MKI67 and KRT5 staining and reduced TUB1A1 staining in the Proliferative subtype.
  • the representative samples shown for the Proliferative and Inflammatory subtypes have dysplasia histology, whereas the samples shown for the Secretory and Normal-like subtypes have normal histology (Magnification c 200).
  • the upper and lower hinges correspond to the first and third quartile, center line represents the median, and whiskers extend from the hinge to the largest or smallest value at most 1.5 times the distance between the quartiles.
  • FIG. 23 depicts a table listing the molecular subtype characteristics in the discovery cohort.
  • FIG. 24A-24C Phenotypic associations with the molecular subtypes are confirmed in an independent sample set.
  • FIG. 24A The 190 DC biopsies and the 3936 genes were used to build a 22- gene nearest centroid molecular subtype classifier.
  • the heatmap shows semi-supervised hierarchal clustering of z-score normalized gene expression across the 22 classifier genes and 190 DC biopsies training samples.
  • FIG. 24B The 22-gene nearest centroid molecular subtype classifier was used to predict the molecular subtypes of the 105 VC biopsies.
  • the heatmap shows semi-supervised hierarchal clustering of z-score normalized gene expression across 22 genes and 105 VC.
  • FIG. 24C Bubbleplots showing significant associations (p ⁇ 0.01 by two-sided Fisher’s exact test) between the VC molecular subtypes and smoking status, biopsy histological grade, and the predicted LUSC tumor molecular subtypes.
  • the columns represent the four molecular subtypes (from left to right: Proliferative, Inflammatory, Secretory, and Normal-like) and the radius of the circle is proportional to the number of samples within each subtype that have the row phenotype.
  • FIG. 25A-FIG. 25B show Normal-appearing bronchial brushes predict the presence of proliferative lesions.
  • FIG. 25A The DC (left) and VC (right) cohorts, showing the number of brushes (y axis) classified as proliferative orange) that have at least one biopsy (y axis) classified as proliferative at the time the brush was sampled. Brushes/biopsies classified as not proliferative are turquoise.
  • FIG. 25B shows the number of brushes (y axis) classified as proliferative orange) that have at least one biopsy (y axis) classified as proliferative at the time the brush was sampled. Brushes/biopsies classified as not proliferative are turquoise.
  • FIG. 25B The DC (left) and VC (right) cohorts, showing the number of brushes (y axis) classified as proliferative orange) that have at least one biopsy (y axis) classified as proliferative at the time the brush was sampled. Brush
  • FIG. 26A-FIG. 26D show Immune alterations are associated with lesion outcome in the Proliferative subtype. Boxplots of Module 9 GSVA scores across DC FIG.26A and VC biopsies FIG. 26B within the Proliferative subtype. There is a significant difference between the
  • FIG. 26C Top: heatmap of z-score-normalized gene expression across the 112 genes in Module 9 in the DC biopsies (left) and the VC biopsies (right). Each heatmap is supervised by Module 9 GSVA scores. Top color bars indicate the histological grade of the biopsies and their progression status. Bottom: heatmap of xCell results indicating the relative abundance of immune cell types across the DC biopsies (left) and the VC biopsies (right). Immune cell types displayed are significantly associated with lesion progression/persistence (FDR ⁇ 0.05 in both the DC and VC, linear model).
  • FIG. 26D Representative histology where the dashed yellow line denotes the separation of epithelium and stromal compartments.
  • Top panels a progressive severe dysplasia has reduced presence of immune cells demonstrated by the marked reduction in expression of M2 macrophages (CD68/163 staining, double-positive cells indicated by the yellow arrows) and CD8 T cells (sample corresponds to *P in c).
  • Bottom panels a regressive moderate dysplasia has increased presence of immune cells including M2 macrophages (CD68/163 staining double-positive cells indicated by the yellow arrows) and CD8 T cells (samples correspond to *R in c).
  • FIG. 26E Representative histology where the dashed yellow line denotes the separation of epithelium and stromal compartments.
  • Top panels a progressive severe dysplasia has reduced presence of immune cells demonstrated by the marked reduction in expression of M2 macrophages (CD68/163 staining, double-positive cells indicated by the yellow arrow
  • FIG. 27A-FIG. 27B depict gene expression level TIMM 13 (translocase of inner mitochondrial membrane 13). This gene is expressed more highly in lesions that progress than those the regress. It is negatively correlated to the expression of a gene set representing B Cell presence, indicating that it can prevent recruitment or activity of B Cells to pre-malignant lesions. TIMM 13 has not been previously associated with B Cell activity, nor has it been implicated in the formation of cancer.
  • FIG. 28A-28B depict gene expression levels of TMEM63C: transmembrane protein 63C This gene is expressed more highly in lesions that progress than those that regress. It is negatively correlated to the expression of a gene set representing MCHII Antigen Presentation, indicating that it can prevent recruitment or activity of antigen presenting cells. This gene has not been previously associated with antigen presentation, nor has it been implicated in the formation of cancer.
  • FIG. 29A-FIG. 29C depict gene expression level of GSTP1 (glutathione S-transferase pi 1).
  • GSTP1 is known to be upregulated in certain cancers, including Lung Squamous Cell Carcinoma, which we have confirmed in data from The Cancer Genome Atlas. This gene is also hypermethylated in B Cell Lymphomas, further indicating it plays a role in preventing B Cell proliferation (Rossi et ah, 2004).
  • FIG. 30A-FIG. 30B depicts gene expression levels of QPRT (quinolinate phosphoribosyltransferase). This gene is upregulated in lesions that regress compared to those the progress. It is positively correlated with expression of our B Cells gene set (FIG. 3 OB), indicating it plays a role in B Cell mediated immunity in premalignant lesions. QPRT has previously been associated with a pro-inflammatory response in monocytes (Jones et al., 2015), but has not been implicated previously in B cell immunity. This gene was found to be anti-apoptotic in leukemic K562 cells, but whether it has this role in differentiated leukocytes is yet unstudied (Ullmark et al., 2017).
  • QPRT quinolinate phosphoribosyltransferase
  • FIG. 31A-FIG. 3 IB depict gene expression level of CPQ: carboxypeptidase Q
  • This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. No previous evidence implicates CPQ in immune activation.
  • FIG. 32A-FIG. 32B depict gene expression level of MRAS (muscle RAS oncogene homolog). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Previous work has demonstrated that M-Ras knockout mice have reduced B Cell abundance and function, indicating that M-Ras is important for proper B Cell activity (Freyer et al., 2012). This association between MRAS, B Cell development, and lung cancer has not been previously identified.
  • MRAS muscle RAS oncogene homolog
  • FIG. 33A-FIG. 33B depict gene expression level of RCAN1 (regulator of calcineurin 1). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Previous work has identified RCAN1 as both a suppressor of the NF-kB pathway and also a target of NF-kB transcriptional regulation (Liu et al., 2015; Zheng et al, 2014).
  • RCAN1 has been previously identified as pro- inflammatory and an important part of both macrophage and T cell mediated immunity (Bhoiwala et al., 2011; Han et al., 2017). Therefore, our work indicates a potentially novel role for RCAN1 in our unique context.
  • FIG. 34A-FIG. 34B depict gene expression level of SERPINIl (serpin peptidase inhibitor, clade I (neuroserpin), member 1). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it can play a role in B Cell mediated immunity in premalignant lesions. No previous evidence implicates SERPINIl in immune activation.
  • FIG. 35A-FIG. 35B depict gene expression level of B2M (beta-2 -macroglobulin).
  • This gene is more highly expressed in regressing lesions than progressing lesions, and is positively correlated to the expression of an Interferon Response gene set.
  • B2M is involved in presentation of antigens via the MCH class I genes, making it an essential component of adaptive immune activation. Interferon is known to upregulate B2M and induce expression of MCH class I genes (Fellous et al., 1979).
  • FIG. 36A-FIG. 361 depict gene expression level of HLA-DRB1, HLA-DPA1, HLA-DRA (Major Histocompatibility Class II genes). All of these genes are more highly expressed in regressing lesions than progressing lesions, and are positively correlated to either both Interferon Response, and B Cell immunity. These genes are highly expressed in B Cells and are necessary for presenting antigens for activation of adaptive immunity.
  • FIG. 37A-FIG. 37B depict gene expression level of MSC (Musculin). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Musculin has been previously implicated in regulation of T lymphocytes (Thl7 cells) (Santarlasci et al, 2017), but has not been studied in B cell activity.
  • MSC Malculin
  • FIG. 38A-FIG. 38B depict gene expression level of SMURF 1 (SMAD specific E3 ubiquitin protein ligase 1). This gene is expressed more highly in lesions that progress than those that regress. It is negatively correlated to the expression of our B Cell gene set. SMURF 1 has been shown to target TRAF proteins for ubiquitination, reducing activity of the NF-kB pathway, an essential pathway in B Cell survival (Ui et al., 2010).
  • SMURF 1 SAD specific E3 ubiquitin protein ligase 1
  • FIG. 39A-FIG. 39B depict gene expression level of SUC5A8 (solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of the B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. SUC5A8 has been identified as a tumor suppressor in both colon and head and neck cancer (Bennett et al., 2008; Gurav et al., 2015), but its function in B cell immunity and lung premalignancy has not yet been reported.
  • SUC5A8 solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8. This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of the B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. SUC5A8 has been
  • FIG. 40 depicts the prediction of Progression/Regression in Proliferative lesions from validation cohorts, using the top two genes from mediation analysis (using binomial glm trained in discovery cohort, genes: SERPINIl and GSTP1).
  • the inventors have discovered a group in immune regulators that control early-stage immune responses, e.g., at the time of tumor formation, disease prevention.
  • provided herein are methods relating to the level of these immune regulators that can be used to diagnose, detect and/or treat conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer). Additionally, the inventors demonstrate that modulating the level of these genes can be used to diagnose, detect and/or treat conditions caused by or exacerbated by aberrant immune system activity, e.g., lung cancer. Accordingly, provided herein are methods for the detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by therapeutically modulating the level of those immune response regulators.
  • aberrant immune system activity e.g. infectious disease, autoimmune disease, and cancer
  • the inventors demonstrate that modulating the level of these genes can be used to diagnose, detect and/or treat conditions caused by or exacerbated by aberrant immune system activity, e.g., lung cancer. Accordingly, provided herein are methods for the detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses
  • PMLs bronchial premalignant lesions
  • the inventors describe a novel set of immune regulators that are responsible for immune suppression in bronchial premalignant lesions that are likely to persist or progress to higher-grade lesion or invasive cancer.
  • immune regulators can fight off infection.
  • Previous targets for immune activation in the context of cancer have focused primarily on late stage tumors and have focused on T cell mediated immunity.
  • Genes that have been discovered to mediate this immune pathway include PD1, PDL1, and CTLA4.
  • the immune regulators identified herein modulate the activity of MHC class II antigen presentation, interferon signaling, and B cell related immunity and regulate immune activation and suppression before tumors even form.
  • the inventors have developed methods for determining if the bronchial premalignant lesions are likely to progress to cancer and new therapies for bronchial premalignant lesions which target the underlying molecular changes which characterize the bronchial premalignant lesions.
  • a method for treating or preventing a condition caused by or associated with immunosuppressed aberrant immune system activity in a subject in need thereof comprising administering to the subject a
  • a method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
  • the change of the immune response relative to a normal control should be at least one standard deviation (s), preferably at least two s from the mean.
  • the deviation between a normal control i.e., a population of individuals that have normal levels, would be either an increased level of expression of at least one s greater than the mean, preferably at least two s greater, or a decreased level of expression of at least one s less than the mean, preferably at least two s.
  • differences that are less than one s can be statistically significant in an individual.
  • the two changes can be (a) at least two increases of a negative immune response regulator, (b) at least two decreases of a positive immune response regulator; or a combination of at least one increase in a negative immune response regulator and a decrease of at least one positive immune response regulator.
  • a positive immune regulator is selected from Table 1 and a negative immune regulator is selected from Table 2.
  • a method of treating immunosuppressed aberrant immune system activity in a subject having or at risk of developing a condition associated with immunosuppressed aberrant immune system activity comprising: i) administering a treatment for the condition associated with immunosuppressed aberrant immune system activity to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level of expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
  • a method of treating immunosuppressed aberrant immune system activity in a subject having or at risk of developing a condition associated with immunosuppressed aberrant immune system activity comprising: i) obtaining a sample from the subject, determining the level of expression of at least 1 negative or positive immune response regulator; and iii) administering a treatment for the condition associated with
  • immunosuppressed aberrant immune system activity if the subject is determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level of expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
  • a method of treating autoimmune aberrant immune system activity in a subject having or at risk of developing a condition associated with autoimmune aberrant immune system activity comprising: i)
  • a treatment for the condition associated with autoimmune aberrant immune system activity to a subject determined to have a level of expression of at least 1 negative immune response regulator which is decreased relative to a reference level of expression of at least 1 positive immune response regulator which is increased relative to a reference level of expression.
  • a method of treating autoimmune aberrant immune system activity in a subject having or at risk of developing a condition associated with autoimmune aberrant immune system activity comprising: i) obtaining a sample from the subject, determining the level of expression of at least 1 negative or positive immune response regulator; and iii) administering a treatment for the condition associated with autoimmune aberrant immune system activity if the subject is determined to have a level of expression of at least 1 negative immune response regulator which is decreased relative to a reference level of expression of at least 1 positive immune response regulator which is increased relative to a reference level of expression.
  • a method of treating bronchial premalignant lesions in a subject in need thereof comprising administering at least one of:
  • a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
  • a method of treating bronchial premalignant lesions in a subject in need thereof comprising:
  • At least one immune stimulating drug and/or viii. an agonist of at least one positive immune response regulator and/or an
  • “aberrant immune system activity” refers to one or more immune system responses which are inappropriately over or under-responsive to a disease agent and/or cell of the subject.
  • an“immune response” refers to a response by one or more cells of the immune system to a pathological or pathogenic stimulus.
  • Cells of the immune system include, but are not limited to B cells, regulatory B cells, T cells, regulatory T cells, antigen-presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils and neutrophils.
  • Aberrant immune system activity can include either immunosuppression or autoimmune activity.
  • “immunosuppression” refers to an active process whereby one or more components of the adaptive or innate immune system is or are prevented from acting against a given target.
  • the immune system includes naturally immunosuppressive function mediated by immune inhibitory receptors or cytokines expressed on the surface of an immune cell, and their interactions with their ligands.
  • cytotoxic CD8 T cells can enter a state of“functional exhaustion,” or “unresponsiveness” whereby they express inhibitory receptors that inhibit or prevent antigen-specific responses, such as proliferation and inflammatory cytokine production.
  • an immune response to a persistent infection or to a cancer or tumor that is suppressed, inhibited, or ineffective, can be enhanced or” un-inhibited”.
  • autoimmune activity refers to immune system activity targeted against an individual’s own health tissues or cells.
  • autoimmune disease or "autoimmune disease or disorder” herein is a disease or disorder arising from and directed against an individual's own tissues or cells or manifestation thereof or resulting condition therefrom.
  • Auto immune related diseases and disorders arise from an overactive and/or abnormal immune response of the body against substances (autoantigens) and tissues normally present in the body, otherwise known as self or autologous substance. This dysregulated inflammatory reaction causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and cell death. Subsequent loss of function is associated with inflammatory tissue damage.
  • a condition caused by or associated with aberrant immune system activity is a condition that arises when, or worsens when the activity of the immune system in the subject is aberrant.
  • Such conditions are well known in the art and include cancer, autoimmune disease, and infectious disease.
  • a condition caused by or associated with immunosuppression refers to a condition, disease or disorder in which the function of the immune response is below a desired level, e.g. a level that can treat or prevent at least one symptom of the disease or disorder.
  • a desired level e.g. a level that can treat or prevent at least one symptom of the disease or disorder.
  • inappropriate immune suppression can be associated with certain cancer tumors in which cells of the immune system fail to attack or are prevented from attacking the tumor, such that thus the immune system fails to effectively reduce or prevent tumor growth.
  • Other examples of inappropriate immunosuppression include, for example, immunosuppression immunosuppression- mediated by a pathogenic organism (infectious disease), and autoimmune disease.
  • an“immune response regulator” refers to a gene expression product that can alter one or more of: the activity of MHC class II antigen presentation, interferon signaling, and B cell related immunity.
  • a“tumor formation immune response regulator” is a type of immune response regulator and refers to a gene expression product can regulate one or more of: the activity of MHC class I and II antigen presentation, interferon signaling, and B cell related immunity prior to tumor formation.
  • An immune response regulator can exert its immune regulating activity as a nucleic acid (e.g., a miRNA), or as polypeptide.
  • An immune response regulator can be a positive immune response regulator or a negative immune response regulator.
  • a positive immune response regulator is one which acts to increase immune system activity.
  • Exemplary positive immune response regulators are provided in Table 1 herein.
  • the positive immune response regulator is a gene expression product of Table 1 or a gene expression product of a gene of Table 1.
  • the positive immune response regulator is one or more of: NLR family CARD domain containing 5 (NLRC5), Quinolinate phosphoribosyltransferase (QPRT), Carboxypeptidase O (CPQ), Muscle RAS oncogene homolog (MRAS), Regulator of calcineurin 1 (RCAN1), Serpin family I member 1 (SERPINI1), Beta-2-microglobulin (B2M), Major
  • HLA-DRBl histocompatibility complex
  • HLA-DPA1 DR alpha 1
  • MSC Musculin
  • SLC5A8 solute carrier family 5 member 8
  • a negative immune response regulator is one which acts to decrease immune system activity.
  • Exemplary negative immune response regulators are provided in Table 2 herein.
  • the negative immune response regulator is a gene expression product of Table 2 or a gene expression product of a gene of Table 2.
  • the negative immune response regulator is one or more of: Translocase of inner mitochondrial membrane 13 homolog (TIMM13), Transmembrane protein 63C (TMEM63C), Glutathione S-transferase pi 1 (GSTP1), SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), miR-149-5p.
  • Table 1 Positive immune response regulators. Gene names, NCBI Gene ID, Nucleic acid sequences, Polypeptide sequences and NCBI reference Sequence IDs are also listed.
  • Table 2 Negative immune response regulators. Gene names, NCBI Gene ID, Nucleic acid sequences, Polypeptide sequences and NCBI reference Sequence IDs are also listed.
  • an immune response regulator when the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of NLRC5, B2M, HLA-DRB1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
  • the different immune response regulator is selected from Table 1 or Table 2.
  • the miR when the change in a negative immune response regulator is a miR, the miR is miR-149-5p.
  • the different marker can also include an miRNA from Table 2A.
  • the aforementioned changes in miR’s in Table 2A could be used to predict an individual proliferative subtype (high-grade legions) of a premalignant lesion or a cancer, for example, lung squamous premalignant legions, and then, one could use the markers in Table 1 and/or Table 2 to identify the state of the immune activity.
  • the changes in expression should be biologically significant, for example statistically significant, a change from a prior level of expression from that individual, a standard deviation from a norm.
  • an anti-proliferative drug for example, Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors;
  • Angiotensin receptor antagonists Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; JNK inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors;
  • Monoamine oxidase inhibitors Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
  • the methods and assays described herein relate to detecting the level of expression of at least two miRNAs, at least three miRNAs, at least four miRNAs, at least five miRNAs, at least s i x miRNAs, at least seven miRNAs, at least eight miRNAs, or all of the miRNAs selected from the group consisting of: of miR-548b-5p, miR-642a-5p, miR- 642b-5p, miR-328a-3p, miR-34b-3p, miR-34b-5p, miR-3664-3p, miR-548i, and miR-766-3p.
  • any combination of the miRNAs can be used in each of various embodiments of the aspects described herein.
  • any pairwise combination of the nine miRNAs can be detected, e.g., any combination shown in Table 2B.
  • Fow expression of these markers is indicative of the proliferative subtype. High expression is good.
  • use of agonists to increase expression of these miRNA is one method of treatment.
  • Table 2B Contemplated exemplary combinations of miRNAs associated with
  • miR-149 NCBI Gene ID NO: 406941; NCBI transcript accession number NR_029702.1; SEQ ID NO: 1) and mature sequence miR-149-5p (SEQ ID NO: 2).
  • A“miR-149-5p oligonucleotide” can be the precursor and/or mature form of miR-149-5p (e.g., SEQ ID NO: 2).
  • miR-149- 5p can be human miR-149-5p, e.g., hsa-miR-149-5p. (see, e.g. also Table 3).
  • miR-548b-5p refers to a mature miRNA derived from miR-548b-5p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 548b-5p (NCBI Gene ID NO: 693128; NCBI transcript accession number NR_030315.1; SEQ ID NO: 3) and mature sequence miR-548b-5p (SEQ ID NO: 4).
  • A“miR-548b-5p oligonucleotide” can be the precursor and/or mature form of miR-548b-5p (e.g., SEQ ID NO: 4).
  • miR-548b-5p can be human miR-548b-5p, e.g., hsa- miR-548b-5p.
  • miR-642a-5p refers to a mature miRNA derived from miR-642a-5p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 642a-5p (NCBI Gene ID NO: 693227; NCBI transcript accession number NR_030372.1; SEQ ID NO: 5 ) and mature sequence miR-642a-5p (SEQ ID NO: 6).
  • A“miR-642a-5p oligonucleotide” can be the precursor and/or mature form of miR-642a-5p (e.g., SEQ ID NO: 6).
  • miR-642a-5p can be human miR-642a-5p, e.g., hsa-miR-642a-5p.
  • miR-642b-5p refers to a mature miRNA derived from miR-642b-5p.
  • sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 642b-5p
  • A“miR-642b-5p oligonucleotide” can be the precursor and/or mature form of miR-642b-5p (e.g., SEQ ID NO: 8).
  • miR-642b-5p can be human, e.g., hsa miR-642b-5p.
  • miR-328a-3p refers to a mature miRNA derived from miR-328a-3p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 328a-3p (NCBI Gene ID NO: 442901; NCBI transcript accession number NR_029887.1 ; SEQ ID NO: 9) and mature sequence miR-328a-3p (SEQ ID NO: 10).
  • A“miR-328a-3poligonucleotide” can be the precursor and/or mature form of miR-328a-3p (e.g., SEQ ID NO: 10).
  • miR- 328a-3p e.g., SEQ ID NO: 10
  • hsa- miR-328a-3p can be human, e.g., hsa- miR-328a-3p.
  • miR-34b-5p refers to a mature miRNA derived from miR-34b-5p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 407041; NCBI transcript accession number NR_029839; SEQ ID NO: 11) and mature sequence (SEQ ID NO: 13).
  • A“oligonucleotide” can be the precursor and/or mature form of miR- 34b-5p (e.g., SEQ ID NO: 13).
  • miR-34b-5p can be human, e.g., hsa-miR-34b- 5p.
  • “miR-3664-3p” refers to a mature miRNA derived from miR-3664-3p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 100500844; NCBI transcript accession number NR_037437.1; SEQ ID NO: 14) and mature sequence (SEQ ID NO: 15).
  • A“miR-3664-3p oligonucleotide” can be the precursor and/or mature form of miR-3664-3p (e.g., SEQ ID NO: 15).
  • miR-3664-3p can be human, e.g., hsa-miR-3664-3p.
  • miR-548i refers to a mature miRNA derived from miR-548i.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human miR-548i (NCBI Gene ID NO: 100302204; NCBI transcript accession number NR_031687.1; SEQ ID NO: 16) and mature sequence (SEQ ID NO: 17).
  • A“miR-548i oligonucleotide” can be the precursor and/or mature form of miR-548i (e.g., SEQ ID NO: 17).
  • miR-548i can be human, e.g., hsa-miR-548i.
  • “miR-766-3p” refers to a mature miRNA derived from miR-766-3p.
  • the sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 768218; NCBI transcript accession number NR_030413.1; SEQ ID NO: 18) and mature sequence (SEQ ID NO: 19).
  • A“miR-766-3p oligonucleotide” can be the precursor and/or mature form of (e.g., SEQ ID NO: 19).
  • miR-766-3p can be human, e.g., has- miR-766-3p.
  • Table 3 List of miRNAs including their NCBI gene IDs, NCBI transcript accession number, miRBase accession numbers, and sequences.
  • sequences of expression products for the immune response regulators described herein are known for a number of species, e.g., human. Sequences for various species are readily obtained in online databases, e.g., NCBI.
  • levels of gene expression of immune response regulators can be modulated (e.g., increased or decreased) in certain subjects, e.g., those with or at risk of developing a condition caused by or associated with aberrant immune system activity in a subject in need thereof (e.g. infectious disease, autoimmune disease, or cancer (e.g. premalignant lesions of different subtypes)).
  • a condition caused by or associated with aberrant immune system activity in a subject in need thereof e.g. infectious disease, autoimmune disease, or cancer (e.g. premalignant lesions of different subtypes)
  • the methods described herein comprise administering a treatment described herein to a subject previously determined to have an expression level(s) of one or more immune response regulators as described herein.
  • described herein is a method of treating a condition caused by or associated with aberrant immune system activity in a subject in need thereof, the method comprising: a) first determining the level of expression of the at least one immune response regulator in a sample obtained from a subject; and b) then administering a treatment as described herein to the subject if the level of expression of the at least one immune response regulator is modulated relative to a reference in the manner described herein.
  • described herein is a method of a condition caused by or associated with aberrant immune system activity in a subject in need thereof, the method comprising: a) determining if the subject has a modulation of a level of expression of at least one immune response regulator as described herein and b) instructing or directing that the subject be administered the appropriate treatment described herein for the particular modulation of expression which has been determined.
  • the step of determining if the subject has modulation of an expression level of at least one immune response regulator can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject.
  • the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject.
  • the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise receiving a report, results, or other means of identifying the subject as a subject with a modulation of a level of expression of at least one immune response regulator.
  • the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
  • measurement of the level of a target and/or detection of the level or presence of a target can comprise a transformation.
  • a transformation e.g. of an expression product (nucleic acid or polypeptide of one of the immune response regulators described herein) or a mutation
  • the term“transforming” or“transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance.
  • the transformation can be physical, biological or chemical. Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation.
  • a biological/chemical transformation can involve the action of at least one enzyme and/or a chemical reagent in a reaction.
  • a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase.
  • a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
  • Transformation, measurement, and/or detection of a target molecule can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent.
  • a reagent e.g. a detection reagent
  • the target-specific reagent is detectably labeled.
  • the target-specific reagent is capable of generating a detectable signal.
  • the target-specific reagent generates a detectable signal when the target molecule is present.
  • Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents.
  • a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent.
  • the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.
  • antibodies for the various targets described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels.
  • amino acid sequences for the targets described herein are known and publically available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein.
  • amino acid sequences of the polypeptides described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat.
  • sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art.
  • sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the fding date of this application.
  • immunohistochemistry is the application of immunochemistry to tissue sections
  • ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations.
  • Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules.
  • signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
  • the assay can be a Western blot analysis.
  • proteins can be separated by two-dimensional gel electrophoresis systems.
  • Two- dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material.
  • the analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection.
  • protein samples are analyzed by mass spectroscopy.
  • Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g.
  • FIA fluorescence-linked immunoassay
  • CLIA chemiluminescence immunoassays
  • ELIA electrochemiluminescence immunoassay
  • CIA counting immunoassay
  • LFIA lateral flow tests or immunoassay
  • MIA magnetic immunoassay
  • protein A immunoassays Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available.
  • the immunoassay can be a quantitative or a semi- quantitative immunoassay.
  • An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen.
  • the assay takes advantage of the highly specific binding of an antibody with its antigen.
  • specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex.
  • the complex is then detected by a variety of methods known in the art.
  • An immunoassay also often involves the use of a detection antibody.
  • Enzyme-linked immunosorbent assay also called ELISA, enzyme immunoassay or EIA
  • ELISA enzyme immunoassay
  • EIA enzyme immunoassay
  • an ELISA involving at least one antibody with specificity for the particular desired antigen can also be performed.
  • a known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen.
  • the detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation.
  • the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound.
  • the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
  • Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
  • a competitive ELISA is used.
  • Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface.
  • a second batch of purified antibodies that are not conjugated on any solid support is also needed.
  • These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal.
  • a sample e.g., a blood sample
  • a known amount of desired antigen e.g., a known volume or concentration of a sample comprising a target polypeptide
  • desired antigen e.g., a known volume or concentration of a sample comprising a target polypeptide
  • the mixture is then are added to coated wells to form competitive combination.
  • a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away.
  • the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test.
  • LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample.
  • LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action.
  • LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc.
  • Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested.
  • LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field.
  • LFIA tests can be operated as either competitive or sandwich assays.
  • Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples.
  • the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof.
  • Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples.
  • lateral flow technology It is also possible to apply multiple capture zones to create a multiplex test.
  • Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested.
  • a dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting.
  • the intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide.
  • Levels can be quantified, for example by densitometry.
  • the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofhioresence assay; mass spectroscopy and/or Immunoelectrophoresis assay.
  • Western blot immunoprecipitation
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunological assay
  • FISH fluorescence in situ hybridization
  • immunohistological staining radioimmunometric assay
  • immunofhioresence assay immunofhioresence assay
  • mass spectroscopy and/or Immunoelectrophoresis assay can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay
  • the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of one or more of the immune response regulators described herein.
  • mRNA messenger RNA
  • Such molecules can be isolated, derived, or amplified from a biological sample, such as a blood sample.
  • Techniques for the detection of mRNA expression is known by persons skilled in the art, and can include but not limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc.
  • the PCR procedure describes a method of gene amplification which is comprised of (i) sequence -specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size.
  • the primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified.
  • mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods.
  • RT reverse-transcription
  • QRT-PCR quantitative RT-PCR
  • real-time PCR methods Methods of RT-PCR and QRT-PCR are well known in the art.
  • the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology.
  • a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single - strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized.
  • an adaptor double or single-stranded
  • the sequence can be determined, e.g. by determining the location and pattern of the hybridization of probes, or measuring one or more characteristics of a single molecule as it passes through a sensor (e.g.
  • exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, and the like.
  • nucleic acid sequences of the genes described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat.
  • sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art.
  • sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the filing date of this application. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
  • Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample.
  • freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials
  • heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine
  • proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
  • one or more of the reagents can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product).
  • Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of
  • reagents e.g. antibodies and nucleic acid probes
  • detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means.
  • the detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies).
  • the detectable label can be linked by covalent or non-covalent means to the reagent.
  • a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules.
  • Detectable labels can include, but are not limited to radioisotopes, biolumine scent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
  • the detection reagent is label with a fluorescent compound.
  • a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3TM, Cy5TM, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5TM, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon GreenTM, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AM
  • a detectable label can be a radiolabel including, but not limited to 3 H, 125 1, 35 S, 14 C, 32 P, and 33 P.
  • a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a
  • Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
  • detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • a detectable tag such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin.
  • Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies
  • biotinylated antibody bound to the biomarker is biotinylated.
  • Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromogenic substrate.
  • streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA.
  • a reagent can also be detectably labeled using fluorescence emitting metals such as 152 Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
  • the level of expression is the level in a sample obtained from a subject.
  • sample or“test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or tissue sample from a subject.
  • present invention encompasses several examples of a biological sample.
  • the biological sample is cells, or tissue, or peripheral blood, or bodily fluid.
  • Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc.
  • the term also includes a mixture of the above-mentioned samples.
  • the term“test sample” also includes untreated or pretreated (or pre-processed) biological samples.
  • a test sample can comprise cells from a subject.
  • the sample obtained from a subject can be a biopsy sample.
  • the sample obtained from a subject can be a blood or serum sample.
  • the sample is an endobronchial biopsy, bronchial brushing sample (e.g., bronchial biopsy, endobronchial brushing sample, large airway biopsy, large airway brushing sample), nasal epithelial cells, sputum, and/or blood obtained from the subject.
  • the sample is a bronchial brushing obtained from the right or left mainstem bronchus.
  • the test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).
  • the test sample can be an untreated test sample.
  • the phrase“untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof.
  • the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein.
  • a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein.
  • the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample.
  • a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof.
  • the test sample can be treated with a chemical and/or biological reagent.
  • Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing.
  • biomolecules e.g., nucleic acid and protein
  • One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing.
  • protease inhibitor which is generally used to protect or maintain the stability of protein during processing.
  • the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject.
  • the subject can be a human subject.
  • the subject can be a subject in need of treatment for (e.g. having or diagnosed as having) a condition caused by or associated with aberrant immune system activity in a subject in need thereof or a subject at risk of or at increased risk of developing a condition caused by or associated with aberrant immune system activity in a subject in need thereof as described elsewhere herein.
  • the biopsy or brushing sample comprises morphologically-normal tissues or cells, e.g., the tissues or cells are not from a lesion and display normal morphology for their in vivo location.
  • the inventors have surprisingly found that the modulation of the immune response regulators occurs even in tissues that are not themselves diseased, or which will become diseased.
  • the level of immune response regulators in easily-sampled areas of the body can provide useful information about what is happening or likely to happen in more inaccessible areas of the body.
  • the biopsy or brushing sample consists essentially of morphologically-normal tissues or cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists of morphologically-normal tissues or cells.
  • the biopsy or brushing sample comprises visually-normal tissues or cells, e.g., the tissues or cells are not from a lesion and to the unaided human eye have a normal appearance for their in vivo location.
  • the biopsy or brushing sample consists essentially of visually-normal tissues or cells.
  • the biopsy or brushing sample consists of visually-normal tissues or cells.
  • the biopsy or brushing sample comprises bronchial premalignant lesion cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists essentially of bronchial premalignant lesion cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists of bronchial premalignant lesion cells.
  • a level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.
  • a level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level.
  • a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.
  • the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms a condition caused by or associated with aberrant immune system activity, e.g., a healthy subject.
  • the reference can also be a level of expression of the target molecule in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.
  • the reference can be the level of a target molecule in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject’s sensitivity or response to a given therapy is changing over time or if the subtype of their lesions is changing.
  • the reference can be the level of a target molecule in a sample obtained from the a subject without lesions, e.g., bronchial lesions and/or premalignant lesions.
  • the level of expression products of no more than 200 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 other genes is/are determined.
  • the level of expression products of no more than 200 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 genes is/are determined.
  • the expression level of a given gene can be normalized relative to the expression level of one or more reference genes or reference proteins.
  • the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level of expression is to be determined.
  • test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.
  • the reference level can be a non-proliferative reference level, e.g., the level in a tissue or cell not comprising a proliferative lesion or from a subject who does not have a proliferative lesion.
  • the level can be the level in inflammatory, secretory, or normal-like lesion subtypes or an average or pooling thereof.
  • the level of expression of at least 1 immune response regulator is detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 2 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 3 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 4 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 5 immune response regulators are detected, determined, or measured.
  • the methods described herein relate to detecting or measuring the level of expression of at least 1 miRNA selected from the group consisting of: miR-149-5p, miR-548b-5p, miR-642a-5p, miR-642b-5p, miR-328a-3p, miR-34b-3p, miR-34b-5p, miR-3664-3p, miR-548i, and miR-766-3p.
  • the methods described herein relate to detecting or measuring the level of expression of at least miR-149-5p.
  • the methods described herein relate to administering an inhibitor of at least miR-149-5p.
  • any one of the immune response regulators of Table 1 or 2 can be detected/measured and/or targeted. In some embodiments of any of the aspects, 2 or more immune response regulators can be detected/measured and/or targeted. All possible pairwise or greater combinations are contemplated herein. Tables 4 and 5 depict exemplary but non-limiting examples of pairwise combinations of the immune response regulators which can be detected or targeted in the present methods.
  • Table 4 Contemplated exemplary combinations of the positive immune response regulators NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINI1, B2M, HLA-DRB1, HLA-DPA1, HLA-DRA, MSC, and SLC5A8 are indicated by“X”.
  • Table 5 Contemplated exemplary combinations of the negative immune response regulators are indicated by“X”.
  • a method comprising measuring the level of expression of at least 1 negative immune response regulator described herein and/or the level of expression of at least 1 positive immune response regulator described herein in a sample obtained from a subject.
  • the sample is an endobronchial biopsy, endobronchial brushing sample, large airway biopsy, large airway brushing sample, nasal epithelial cells, sputum, or blood obtained from the subject.
  • the sample is a bronchial brushing obtained from the right or left mainstem bronchus.
  • the sample comprises or consists of morphologically-normal tissues or cells.
  • the change of the immune response relative to a normal control should be at least one standard deviation (s), preferably at least two s from the mean.
  • s standard deviation
  • the deviation between a normal control i.e., a population of individuals that have normal levels, would be either an increased level of expression of at least one s greater than the mean, preferably at least two s greater, or a decreased level of expression of at least one s less than the mean, preferably at least two s.
  • the two changes can be (a) at least two increases of a negative immune response regulator, (b) at least two decreases of a positive immune response regulator; or a combination of at least one increase in a negative immune response regulator and a decrease of at least one positive immune response regulator.
  • the methods and assays described herein can be combined with methods and assays of the prior art, e.g., those that examine other markers of aberrant immune system activity. It is particularly contemplated that the presently described methods and assays can be combined with the methods and assays described in International Patent Publication W02002/0041243. When methods and assays are combined, they can be practiced in series, in parallel, or as part of the same kit or multiplexed method or assay.
  • the immune response regulators can be therapeutically modulated in order to correct aberrant immune system activity and thereby prevent or treat disease. Accordingly, contemplated herein are methods and pharmaceutical compositions that target these immune response regulators to inhibit the negative immune response regulators or agonize the positive immune response regulators and thereby be effective in delaying or preventing the development of conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer).
  • aberrant immune system activity e.g. infectious disease, autoimmune disease, and cancer.
  • the term“agonist” refers to an agent which increases the expression and/or activity of the target by at least 10% or more, e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000 % or more.
  • the efficacy of an agonist e.g. its ability to increase the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and/or the activity of the target. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g.
  • RTPCR with primers can be used to determine the level of RNA, and Western blotting with an antibody can be used to determine the level of a polypeptide.
  • Suitable primers for a given target are readily identified by one of skill in the art, e.g., using software widely available for this purpose (e.g., Primer3 or PrimerBank, which are both available on the world wide web).
  • Antibodies to polypeptide gene expression products of the immune response regulators described herein are commercially available, e.g., from AbCam
  • an agonist of a given polypeptide can be the polypeptide, a nucleic acid encoding the polypeptide, or a small molecule.
  • Non-limiting examples of agonists of a given polypeptide target can include the target polypeptides or variants or functional fragments thereof and nucleic acids encoding the polypeptide or variants or functional fragments thereof.
  • the agonist of a given target is a polypeptide of that target or variants or functional fragment thereof and/or a nucleic acid encoding the polypeptide or variant or functional fragment thereof.
  • the polypeptide agonist can be an engineered and/or recombinant polypeptide.
  • the polypeptide agonist can be a nucleic acid encoding a polypeptide, e.g. a functional fragment thereof.
  • the nucleic acid can be comprised by a vector.
  • a polypeptide agonist can comprise one of the sequences provided herein for each target. In some embodiments of any of the aspects, a polypeptide agonist can consist essentially of one of the sequences provided below herein for each target. In some embodiments of any of the aspects, a polypeptide agonist can consist of one of the sequences provided below herein for each target. In some embodiments of any of the aspects, an agonist can comprise a nucleic acid encoding one of the sequences provided below herein for each target.
  • an agonist can be a polypeptide comprising a reference/wild-type sequence provided herein with at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the reference/wild-type sequence and which retains the activity of the reference/wild-type sequence. In some embodiments of any of the aspects, an agonist can be a polypeptide comprising a reference/wild-type sequence provided herein with at least 95% identity to the reference/wild-type sequence and which retains the activity of the reference/wild-type sequence.
  • the agonist an exogenous polypeptide.
  • the subject is administered exogenous polypeptide, e.g., the polypeptide is produced in vitro and/or synthesized and purified polypeptide is provided to the subject.
  • the agonist is an ectopic polypeptide.
  • the subject is administered ectopic polypeptide, e.g., the polypeptide is produced in vitro and/or synthesized and purified polypeptide is provided to the subject.
  • the agonist can be a nucleic acid encoding a polypeptide (or a variant or functional fragment thereof) and/or a vector comprising a nucleic acid encoding a polypeptide (or a variant or functional fragment thereof).
  • a nucleic acid encoding a polypeptide can be, e.g., an RNA molecule, a plasmid, and/or an expression vector.
  • the nucleic acid encoding a polypeptide can be an mRNA.
  • the nucleic acid encoding a polypeptide can be a modified mRNA.
  • the agonist can be a nucleic acid encoding a polypeptide, e.g., exogenous and/or ectopic polypeptide.
  • the subject is administered the nucleic acid encoding exogenous and/or ectopic polypeptide, e.g., the nucleic acid is transcribed and/or translated after the administering step to provide exogenous and/or ectopic polypeptide to the subject.
  • inhibitor refers to an agent which can decrease the expression and/or activity of a target, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more.
  • the efficacy of an inhibitor of one or more targets, e.g. its ability to decrease the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and/or the activity of the target.
  • the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.
  • An inhibitor of a target described herein can inhibit the activity, expression, or accumulation of the target polypeptide.
  • Inhibitors can include inhibitors that act directly on the target itself (e.g., that bind to the protein or transcript, e.g., direct inhibitors).
  • an inhibitor of a specified target is an antibody, antibody reagent, or antigen-binding fragment thereof, that specifically binds to the target.
  • the agent that inhibits a given target is an inhibitory nucleic acid.
  • inhibitors of the expression of a given gene can be an inhibitory nucleic acid.
  • “inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), miRNAs, amiRNAs, and the like.
  • a polypeptide or nucleic acid as described herein can be engineered.
  • “engineered” refers to the aspect of having been manipulated by the hand of man.
  • a polypeptide is considered to be“engineered” when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature.
  • progeny of an engineered cell are typically still referred to as“engineered” even though the actual manipulation was performed on a prior entity.
  • the agonist and/or inhibitor is administered as a nucleic acid.
  • a nucleic acid encoding the agonist and/or inhibitor is administered.
  • the subject is administered a vector comprising a nucleic acid.
  • Vectors can be, e.g., a DNA or RNA vector.
  • the methods described herein relate to treatment of bronchial premaligant lesions. In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having bronchial premalignant lesions. In some embodiments of any of the aspects, the condition caused by or associated with aberrant immune system activity is a premalignant lesion. In some embodiments of any of the aspects, the condition caused by or associated with aberrant immune system activity is a bronchial premalignant lesion.
  • premalignant lesion refers to an epithelial lesion or dysplasia which is a precursor or can be a precursor to cancer.
  • the basement membrane is intact with no possibility of metastatic spread, as opposed to cancer.
  • a bronchial premalignant lesion is a premalignant lesion present in the bronchial epithelium of a subject. Bronchial premalignant lesions are typically small and can be difficult to visualize using conventional white light bronchoscopy.
  • Subjects having bronchial premalignant lesions can be identified by a physician using current methods of diagnosing bronchial premalignant lesions.
  • Tests that can aid in a diagnosis of, e.g. bronchial premalignant lesions include, but are not limited to, bronchoscopy, autofluorescence bronchoscopy, etc.
  • a family history of lung cancer, prior history of lung cancer, exposure to risk factors for bronchial premalignant lesions (e.g. cigarette smoke), presence of chronic obstructive pulmonary disease (COPD) can also aid in determining if a subject is likely to have bronchial premalignant lesions or in making a diagnosis of bronchial premalignant lesions.
  • COPD chronic obstructive pulmonary disease
  • Standard treatment for subjects at risk of lung cancer, or who have been identified to have bronchial premalignant lesions is annual screening for lung cancer (e.g. a bronchoscopy and/or chest CT scan).
  • lung cancer e.g. a bronchoscopy and/or chest CT scan.
  • a bronchoscopy-based procedure refers to any endoscopic technique that permits examination of the bronchus and/or lungs.
  • Bronchoscopy-based procedures can include white light bronchoscopy, autofluorescence bronchoscopy, flexible bronchoscopy, rigid bronchoscopy, bronchoalveolar lavage, and the like. Bronchoscopy-based procedures can further include biopsy, brushing, or tissue sampling.
  • An immune -stimulating drug is a drug that increases the activity of the immune system, preferably against cancer or dysplastic cells, wherein that is the primary activity of the compound in the relevant context.
  • the term“immune-stimulating drug” is used to describe any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) which can be used stimulate the immune system.
  • immune stimulating drugs can include immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti -viral drugs that improve interferon signaling such as
  • Pegintron Pegasys, referon A, uniferon, multiferon, rebif, avonex, cinnovex, betaseron, actimmune, reiferon, pegetron
  • DNA synthesis inhibitors e.g., TAS-102, NC-6004, ganciclovir
  • CDK inhibitors e.g.
  • ribonucleotide reductase inhibitors e.g., motexafm, hydroxyurea, fludarabine, cladribine, gemcitabine, tezacitabine, triapine, gallium maltolate, gallium nitrate
  • dihydrofolate reductase inhibitors e.g., methotrexate, piritrexam, cycloguanil, JPC-2056
  • topoisomerase inhibitors e.g.
  • pidorubicine doxorubicin, campothecins, indenosioquinolines, indotecan, imdimitecan, amsacrine, etoposide, teniposide, ICRF-193, genistein); FLT3 inhibitors (e.g.
  • IGF-1 inhibitors IGF-1 inhibitors
  • MEK inhibitors e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733
  • aurora kinase inhibitors e.g.,ZM447439, hesperidin, VX-680
  • PKC inhibitors e.g., ruboxistaurin, chelerythrine, miyabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostate 1, tamoxifen
  • RAF inhibitors e.g., vemurafenib, GDC-0879, PLX-4720, sorafenib, dabrafenib, LGX818); PDFGR
  • THM-I-94 vorinostat, givinostat
  • DNA methyltransferase inhibitors e.g., azacytidine, decitabine, zeublarine
  • EZH2 inhibitors DZNep, EPZ005687, Ell, GSK126, UNC1999, EPZ-6438, tazemetostat
  • a single compound may exhibit multiple activities, e.g., depending on the context.
  • agents that can exhibit primarily an anti-inflammatory activity and/or an anti -proliferative activity can include Acetylcholine receptor antagonist,
  • Acetylcholinesterase inhibitors Acetylcholinesterase inhibitors, Adenosine receptor antagonists, Adrenergic receptor antagonists, Angiotensin receptor antagonists, Apoptosis stimulants, Aurora kinase inhibitors, CDK inhibitors, Cyclooxygenase inhibitors, Cytokine production inhibitors, Dehydrogenase inhibitors, Dopamine receptor antagonist, EGFR inhibitors, ERK1 and ERK2 phosphorylation inhibitors, Estrogen receptor agonists, FLT3 inhibitors, Glucocorticoid receptor agonists, Glutamate receptor antagonists, HDAC inhibitors, Histamine receptor antagonists, Histone lysine methyltransferase inhibitors, HSP inhibitors, IKK inhibitors, Ion channel antagonists, KIT inhibitors, Leucine rich repeat kinase inhibitors, MEK inhibitors, MDM inhibitors, Phosphodiesterase inhibitors, Monoamine oxidase inhibitors, MTOR inhibitors,
  • immune stimulating drugs lacking anti-proliferative/inflammatory activity in any context described herein can include immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti-viral drugs that improve interferon signaling); DNA synthesis inhibitors; IMDH inhibitors; ribonucleotide reductase inhibitors; dihydrofolate reductase inhibitors; SRC inhibitors; retinoid receptor agonists; HDAC inhibitors; and DNA methyltransferase inhibitors.
  • immune-checkpoint inhibitors e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3
  • drugs that stimulate interferon signaling e.g. anti-viral drugs that improve interferon signaling
  • DNA synthesis inhibitors IMDH inhibitors
  • ribonucleotide reductase inhibitors ribonucleotide reductase inhibitor
  • anti-inflammatory refers to a compound capable of reducing or inhibiting inflammation, wherein that is the primary activity of the compound in the relevant context.
  • anti-inflammatory drug or“anti-inflammatory agent” is used to describe any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) which can be used reduce or inhibit inflammation.
  • anti-inflammatory drugs can include NFkB pathway inhibitors (e.g.
  • glucocorticoid receptor agonists e.g. dexamethasone, mifepristone,
  • TGF beta receptor inhibitors e.g. LY-364947, GW-755.55, LY-2109761, galunisertib, SB431542, SB-525334.
  • anti-proliferative drugs include Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; Angiotensin receptor antagonists; Apoptosis stimulants; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; Glutamate receptor antagonists; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; IKK inhibitors; Ion channel antagonists; Leucine rich repeat kinase inhibitors; MDM inhibitors;
  • Monoamine oxidase inhibitors Monoamine oxidase inhibitors; nucleophosmin inhibitors; PPAR receptor agonists;
  • anti-inflammatory drugs lacking anti-proliferative activity in any context described herein can include protein synthesis inhibitors and TGF beta receptor inhibitors.
  • the subject with bronchial premalignant lesions is indicated to be at low risk of progression to malignancy, e.g., the level of expression of at least 1 negative immune response regulator is not increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is not decreased relative to a reference level of expression, then the subject can be provided with treatment options appropriate for a subject who will not develop lung cancer or has a low risk of developing lung cancer in the next 6-12 months.
  • Such treatments can include, e.g., a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue without performing a chest CT scan; or at greater than 6 month intervals, either: a) a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue or b) a chest CT scan.
  • Patients in this low-risk group can also be refused or not administered an immune stimulating drug or chemotherapeutic agent.
  • the methods described herein can also relate to treatment or prevention of conditions associated with aberrant immune system activity, e.g., infectious disease, autoimmune conditions, or cancer.
  • the treatment can be treatment of a patient with early or less aggressive forms of the condition, e.g., to reduce the risk or progression to later or more aggressive forms of the disease.
  • the patient could be pre-symptomatic, exposed, have a latent condition, or the like.
  • the treatment can be prophylactic.
  • prophylactic refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
  • Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
  • Methods of identifying and diagnosing the conditions associated with aberrant immune system activity described herein are well known in the art and readily practiced by clinicians.
  • the subject is administered a treatment for the conditions associated with aberrant immune system activity. Such treatments are well known in the art.
  • a subject determined to have or be at risk of having an infectious disease according to the methods described herein, or determined to be at risk of progressing in an infectious disease according to the methods described herein can be administered an antiviral or antibiotic.
  • Such treatments are known in the art, e,g., antivirals and antibiotics.
  • the infectious disease is a bacterial or viral infection.
  • the viral infection is a coronavirus infection, e.g., a SARS-CoV-2 infection. Treatment and/or diagnosis of SARS-CoV-2 is particularly contemplated herein for methods relating to miR-149-5p due to its prevalence in the basal epithelial cell populations affected by SARS-CoV-2.
  • an infectious disease also known as a transmissible disease or communicable disease, is an illness resulting from a chronic and/or latent infection.
  • Non-limiting examples of infectious viral disease include COVID-19, hepatitis B (caused by hepatitis B virus (HBV)) and hepatitis C (caused by hepatitis C virus (HCV)) adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, and human T cell leukemia virus II.
  • Parasitic persistent infections can arise as a result of infection by, for example, Leishmania, Toxoplasma, Trypanosoma, Plasmodium,
  • an infectious disease can be a latent infection.
  • a chronic infection can include periods in which the infection is a latent infection.
  • the infectious agent such as a virus
  • the virus In a latent viral infection, the virus remains in equilibrium with the host for long periods of time before symptoms again appear; however, the actual viruses cannot typically be detected until reactivation of the disease occurs.
  • latent infections include infections caused by herpes simplex virus (HSV)-l (fever blisters), HSV-2 (genital herpes), and varicella zoster virus VZV (chickenpox-shingles).
  • antiviral refers to any chemical or biological agent with therapeutic usefulness in the inhibition of viral transmission, activity, or replication. Categories of antivirals can include, but are not limited to entry inhibitors, uncoating inhibitors, viral synthesis inhbitiors, assembly inhibitors, and release inhibitors.
  • antivirals include enfuvirtide, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine, fomivirsen, rifampicin, zanamivir, oseltamivir, peramivir, abacavir, acyclovir, adefovir, amprenavir, baloxavir marboxil, boceprevir, cobicistat, combivir, daclatasvir, doravirine, etravirine, ganciclovir, ibalizumab, letermovir, rilpivirine, simeprevir, telbivudine, and valciclovir.
  • antibiotic refers to any chemical or biological agent with therapeutic usefulness in the inhibition of bacterial cell growth or in killing bacteria, e.g, those that are bactericidal or bacteriostatic. Categories of antibiotics can include, but are not limited to those that target the bacterial cell wall (e.g., penicillins, cephalosporins), those that target the bacterial cell membrane (e.g., polymyxins), those that target bacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, sulfonamides), protein synthesis inhibitors (e.g., macrolides, lincosamides, and tetracyclines) , aminoglycosides, cyclic lipopeptides, glycyclines, oxazolidinones, beta-lactams, and lipiarmycins.
  • bacterial cell wall e.g., penicillins, cephalosporins
  • those that target the bacterial cell membrane e
  • antibiotics include penicillin, methicilling, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin,
  • bacampicillin metampicillin, talamipicillin, epicillin, cabenicillin, ticaricillin, temocillin, mezlocillin, piperacillin, azolocillin, clavulanic acid, sulbactam, tazobactam, cafadroxil, cephalexin, cefalotin, cefapirin, cefazolin, cefradine, cefaclor, cefonicid, cefprozil, cefuroxime, loracarbef, cefmetazole, cefotetan, cefoxitin, cefotiam, cefdinir, cefixime, cefotaxime, cefovecin, cefpodoxime, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefoperazone, ceftazimdime, latamoxef, cefepime, cefidero
  • a subject determined to have or be at risk of having cancer according to the methods described herein, or determined to be at risk of progressing in cancer according to the methods described herein can be administered a chemotherapeutic.
  • a chemotherapeutic e.g., a subject determined to have increased expression of a negative immune response regulator or decreased expression of a positive immune response regulator
  • the cancer is a squamous cell cancer, e.g., a lung squamous cell cancer.
  • Non-limiting examples of cancer treatments can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI- 103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothec
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin),
  • aminoglutethimide aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • folic acid replenisher such as frolinic acid
  • aceglatone aminoglutethimide, mitotane, trilostane
  • aldophosphamide glycoside aminolevulinic acid
  • eniluracil amsacrine
  • bestrabucil bisantrene
  • edatraxate defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
  • pirarubicin losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton,
  • chloranbucil GEMZAR® gemcitabine
  • 6-thioguanine 6-thioguanine
  • mercaptopurine methotrexate
  • platinum analogs such as cisplatin, oxaliplatin and carboplatin
  • vinblastine platinum
  • platinum etoposide (VP-16);
  • ifosfamide mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)
  • irinotecan including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine;
  • combretastatin combretastatin
  • leucovorin LV
  • oxaliplatin including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • the methods of treatment for cancer can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
  • chemotherapeutic agent of use e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).
  • the condition caused by or associated with aberrant immune system activity is cancer (e.g., lung cancer, small cell lung cancer or non-small cell lung cancer).
  • Subjects having lung cancer can be identified by a physician using current methods of diagnosing lung cancer.
  • Symptoms and/or complications of lung cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, coughing, coughing blood, shortness of breath, chest pain, wheezing, hoarseness, difficulty breathing, unexplained weight loss, bone pain, and headaches.
  • Tests that may aid in a diagnosis of, e.g. lung cancer include, but are not limited to, x-rays, CT scan, sputum cytology, or biopsies.
  • a family history of lung cancer, prior history of lung cancer, presence of other chronic lung disease such as COPD, or exposure to risk factors for lung cancer (e.g. radon or asbestos exposure) can also aid in determining if a subject is likely to have lung cancer or in making a diagnosis of lung cancer.
  • cancer relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems.
  • Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
  • cancer examples include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer;
  • esophageal cancer eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer;
  • a tumor formed by cells that have spread is called a“metastatic tumor” or a“metastasis.”
  • the metastatic tumor contains cells that are like those in the original (primary) tumor.
  • the term“benign” or“non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
  • A“cancer cell” or“tumor cell” refers to an individual cell of a cancerous growth or tissue.
  • a tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
  • a subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
  • the cancer is a lung squamous cell cancer.
  • a lung squamous cell cancer develops from non-cancerous lesions in the airway known as bronchial premalignant lesions.
  • the presence of persistent or progressive dysplastic bronchial premalignant lesions is a marker of increased lung cancer risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung. Not all bronchial premalignant lesions progress to invasive cancer, and those that do, progress at variable rates with variable outcomes.
  • a subject determined to have or be at risk of having an autoimmune condition according to the methods described herein, or determined to be at risk of progressing in an autoimmune condition according to the methods described herein can be administered an immunosuppressive, a steroid, or the like.
  • an immunosuppressive e.g., a subject determined to have decreased expression of a negative immune response regulator or increased expression of a positive immune response regulator
  • the autoimmune condition is rheumatoid arthritis, lupus, and celiac disease.
  • Autoantigens are endogenous proteins or fragments thereof that elicit this pathogenic immune response.
  • Autoantigen can be any substance or a portion thereof normally found within a mammal that, in an autoimmune disease, becomes the primary (or a primary) target of attack by the immune system.
  • the term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals.
  • the term includes peptic subclasses consisting essentially of immunodominant epitopes or immunodominant epitope regions of autoantigens. Immunodominant epitopes or regions in induced autoimmune conditions are fragments of an autoantigen that can be used instead of the entire autoantigen to induce the disease.
  • immunodominant epitopes or regions are fragments of antigens specific to the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
  • Autoantigens that are known to be associated with autoimmune disease include myelin proteins with demyelinating diseases, e.g. multiple sclerosis and experimental autoimmune myelitis; collagens and rheumatoid arthritis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65); islet cell antigen (ICA512; ICA12) with insulin dependent diabetes.
  • Thl type cytokines include interleukin 2 (IL-2), g-interferon, TNFa and IL-12.
  • IL-2 interleukin 2
  • g-interferon IL-12
  • TNFa IL-12
  • IL-12 interleukin 2
  • Such pro-inflammatory cytokines act to stimulate the immune response, in many cases resulting in the destruction of autologous tissue.
  • Cytokines associated with suppression of T cell response are the Th2 type, and include IL-10, IL-4 and TGF-b. It has been found that Thl and Th2 type T cells may use the identical antigen receptor in response to an immunogen; in the former producing a stimulatory response and in the latter a suppressive response.
  • the autoimmune disorder is selected from the group consisting of thyroiditis, type 1 diabetes mellitus, Hashimoto's thyroidits, Graves' disease, celiac disease, multiple sclerolsis, Guillain-Barre syndrome, Addison's disease, and Raynaud's phenomenon, Goodpasture's disease, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile -onset rheumatoid arthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases
  • glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis
  • inflammatory responses immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus
  • autoimmune infertility e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, Sampter's syndrome, Caplan's syndrome, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, e
  • tracheobronchitis inflammatory dermatoses, dermatoses with acute inflammatory components, and autoimmune uveoretinitis (AUR).
  • AUR autoimmune uveoretinitis
  • Subjects having an autoimmune disease can be identified by a physician using current methods of diagnosing an autoimmune disease.
  • Symptoms and/or complications of an autoimmune disease which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, compassionuge, achy muscles, swelling and redness, low-grade fever, numbness aor tingling of the hands or feet, hair loss, and/or skin rash.
  • Tests that may aid in a diagnosis of, e.g. autoimmune disease include, but are not limited to, blood counts, and an antinuclear antibody test (ANA).
  • ANA antinuclear antibody test
  • a family history of autoimmune disease, or having risk factors for autoimmune disease e.g.
  • Treatments for autoimmune disorders are known in the art and can include, but are not limited to Immunosuppressive drugs, e.g., Cyclosporine (Neoral, Sandimmune, Gengraf, and
  • Tacrolimus Prograf, Protopic, Astagraf XL, and Envarsus XR
  • Methotrexate Trexall, Rasuvo, Rheumatrex, and Otrexup (PF)
  • Sirolimus Rasuvo, Rheumatrex, and Otrexup (PF)
  • Sirolimus Rasuvo, Rheumatrex, and Otrexup (PF)
  • Sirolimus Rasuvo, Rheumatrex, and Otrexup (PF)
  • Sirolimus Rammune
  • Mycophenolic acid Myfortic and CellCept
  • Rituximab Rituxan
  • etanercept Enbrel
  • pentostatin Napent
  • ruxolitinib Jakafi
  • Chemotherapies e.g., Methotrexate (Trexall, Rasuvo, Rheumatrex, and Otrexup (PF)), antithymocyte globulin (Atgam, Thymoglobulin); Steroids, e.g,.
  • Prednisone (Deltasone, Rayos, and Prednisone Intensol), Methylprednisolone (Medrol, Solu-Medrol, and Depo-Medrol), budesonide (Entocort EC, Uceris); Antifungal, e.g., Posaconazole (Noxafd); Antiviral drugs, e.g., Acyclovir (Zovirax and Sitavig), Valacyclovir (Valtrex); and Antibiotics, e.g., Sulfamethoxazole / Trimethoprim (Bactrim, Sulfatrim, and Bactrim DS); Protease inhibitors, e.g. alpha 1 -proteinase inhibitor (Zemaira);
  • the methods and biomarker signatures described herein can be applied to methods of predicting the risk of disease or disease progression in a subject and/or determining the efficacy of treatment or need for further treatment. For example, transition from aberrant to normal immune system activity would indicate that a treatment had been effective or that the treatment can be discontinued.
  • compositions and methods for treating or preventing a condition caused by or associated with aberrant immune system activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator and/or an inhibitor of at least 1 negative immune response regulator to the subject.
  • compositions and methods described herein can be administered to a subject having or diagnosed as having a condition caused by or associated with aberrant immune system activity in a subject in need thereof.
  • the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a disease.
  • "alleviating a symptom" of a disease is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art.
  • Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.
  • the agents described herein can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject.
  • the agents described herein can be administered to a subject by any mode of administration that delivers the agent systemically or locally to a desired surface or target, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration.
  • oral administration forms are also contemplated.
  • injection includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
  • phrases“parenteral administration” and“administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection.
  • phrases“systemic administration,”“administered systemically”,“peripheral administration” and “administered peripherally” as used herein refer to the administration of the agents described herein, other than directly into a target site, tissue, or organ, such that it enters the subject’s circulatory system and, thus, is subject to metabolism and other like processes.
  • the subject is a mammalian subject. In some embodiments of any of the aspects, the subject is a mammalian subject. In some
  • the subject is a human subject. In some embodiments of any of the aspects, the subject is a current or former smoker. In some embodiments of any of the aspects, the subject is a smoker. In some embodiments of any of the aspects, the subject is a non-smoker.
  • the methods described herein can prevent, delay, or slow the development of lung cancer, e.g., lung squamous cell carcinoma.
  • the subject treated according to the present methods is not a subject with lung cancer.
  • the subject treated according to the present methods is a subject who does not have lung cancer.
  • the subject treated according to the present methods is a subject who does not have and has not had lung cancer.
  • the subject treated according to the present methods is at risk of lung cancer.
  • the subject is a subject with a bronchial premalignant lesion.
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50.
  • Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e..
  • Efficacy can be assessed in animal models of a condition described herein, for example animal models of cancer, e.g. a murine xenograft model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
  • the methods described herein comprise administering an effective amount of a composition described herein, e.g. an agonist or inhibitor of a nucleic acid and/or polypeptide described herein to a subject in order to alleviate a symptom of a condition caused by or associated with aberrant immune system activity.
  • a composition described herein e.g. an agonist or inhibitor of a nucleic acid and/or polypeptide described herein to a subject in order to alleviate a symptom of a condition caused by or associated with aberrant immune system activity.
  • "alleviating a symptom” is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • a variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous,
  • Administration can be local or systemic.
  • the term“effective amount” as used herein refers to the amount of an agent needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect.
  • the term "therapeutically effective amount” therefore refers to an amount of the agent that is sufficient to provide a particular effect when administered to atypical subject.
  • An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease.
  • an appropriate“effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.
  • an effective dose of a composition comprising an agonist and/or inhibitor of an immune response regulator as described herein can be administered to a patient once.
  • an effective dose of a composition can be administered to a patient repeatedly.
  • subjects can be administered a therapeutic amount of a composition, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
  • the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer.
  • Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. lung cancer by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
  • the dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen.
  • the dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active agent.
  • the desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule.
  • administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months.
  • dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more.
  • a composition can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
  • the dosage ranges for the administration of an agent according to the methods described herein depend upon, for example, the form of the agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor growth.
  • the dosage should not be so large as to cause adverse side effects.
  • the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • an agent described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein can be determined by the skilled clinician.
  • a treatment is considered“effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein.
  • Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size and/or growth rate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g.
  • An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease.
  • Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of lung cancer in a mouse model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. tumor size and/or growth rate.
  • the technology described herein relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agonist or inhibitor of an immune response regulator as described herein, and optionally a pharmaceutically acceptable carrier.
  • the active ingredients of the pharmaceutical composition comprise an agonist or inhibitor of an immune response regulator as described herein.
  • the active ingredients of the pharmaceutical composition consist essentially of an agonist or inhibitor of an immune response regulator as described herein.
  • the active ingredients of the pharmaceutical composition consist of an agonist or inhibitor of an immune response regulator as described herein.
  • Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art.
  • materials which can serve as pharmaceutically -acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such
  • wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation.
  • the terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.
  • the carrier inhibits the degradation of the active agent.
  • a composition as disclosed herein can be administered in a vesicle, in particular a liposome (see Langer, "New Methods of Drug Delivery,” Science 249: 1527- 1533 (1990); Lopez-Berestein, “Treatment of Systemic Fungal Infections with Liposomal- Amphotericin B,” Liposomes in the Therapy of Infectious Disease and Cancer, pp. 317-327 (1989); and Treat et al, "Liposome encapsulated doxorubicin - preliminary results of phase I and phase II trials" Liposomes in the Therapy of Infectious Disease and Cancer, pp. 353-365 (1989).
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to an agonist, inhibitor, or other therapeutic of the present invention, stabilizers, preservatives, excipients and the like.
  • the preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.
  • a composition as disclosed herein can be administered via a nanoparticle or microparticle. See, for example, Allen et al. Biochim. Biophys. Acta 19993 1150:9- 16, Wissing et al. Adv. Drug. Deliv. Rev. 2004 56: 1257-1272, and Tochilin, Nanoparticulates as Drug Carriers, Imperial College Press (2006); the contents of each of which is incorporated by reference herein in its entirety.
  • the pharmaceutical composition as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS ® -type dosage forms and dose-dumping.
  • Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection
  • compositions can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion.
  • Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
  • compositions described herein can be administered by inhalation, e.g., as a vapor or aerosol formulation or by nebulization.
  • a composition described herein can be provided in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • suitable propellants for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants.
  • a composition described herein can also be administered in a non-pressurized form such as in a nebulizer or atomizer.
  • a composition can also be administered directly to the airways in the form of a dry powder, e.g., by use with an inhaler.
  • Aerosols for the delivery to the respiratory tract are known in the art. See for example, Adjei, A. and Garren, J. Pharm. Res., 1 : 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et ah, Am. Rev. Respir.
  • Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like.
  • controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels.
  • controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug.
  • the therapeutic described herein can be administered in a sustained release formulation.
  • Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts.
  • the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time.
  • Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions.
  • Controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • a variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example,
  • the agonist or inhibitor of an immune response regulator is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
  • the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Additional therapies for the conditions described herein are provided above.
  • “decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount.
  • “reduce,”“reduction” or “decrease” or“inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g.
  • “reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a reference level.
  • a decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
  • the terms“increased”,“increase”,“enhance”, or“activate” are all used herein to mean an increase by a statically significant amount.
  • the terms“increased”,“increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.
  • a“increase” is a statistically significant increase in
  • exogenous refers to a substance present in a cell other than its native source.
  • exogenous when used herein can refer to a nucleic acid (e.g., a miRNA or nucleic acid encoding a miRNA) that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism.
  • “ectopic” can refer to a nucleic acid that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels.
  • the term "endogenous” refers to a substance that is native to the biological system or cell.
  • the subject is a human.
  • a "subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • the terms,“individual,”“patient” and “subject” are used interchangeably herein.
  • the subject is a mammal.
  • the mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of, e.g., lung cancer.
  • a subject can be male or female.
  • a subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. lung cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition.
  • a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition.
  • a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
  • A“subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
  • an antibody reagent refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen.
  • An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody.
  • an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen binding domain of a monoclonal antibody.
  • an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL).
  • an antibody in another example, includes two heavy (H) chain variable regions and two light (L) chain variable regions.
  • antibody reagent encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies.
  • antibody refers to immunoglobulin molecules
  • immunologically active portions of immunoglobulin molecules . e.. molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope -binding portion thereof, and/or bifunctional hybrid antibodies.
  • dAb single domain antibody
  • Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain.
  • the heavy chain constant region consists of three domains CHI, CH2 and CH3.
  • Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain.
  • the light chain constant region consists of a CL domain.
  • the VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR).
  • CDRs complementarity-determining regions
  • FR framework regions
  • Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.
  • Antibodies and/or antibody reagents can include an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding portion thereof.
  • the term“nanobody” or single domain antibody (sdAb) refers to an antibody comprising the small single variable domain (VHH) of antibodies obtained from camelids and dromedaries.
  • VHH small single variable domain
  • Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects.
  • IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/ 02214 (WO 94/04678 published 3 Mar. 1994; which is incorporated by reference herein in its entirety).
  • a region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high afiinity for a target, resulting in a low molecular weight antibody-derived protein known as a“camelid nanobody”.
  • a“camelid nanobody” See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B. et al, 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003
  • the camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule and the protein has a physical diameter of only a few nanometers.
  • One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents.
  • a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.
  • the low molecular weight and compact size further result
  • protein and“polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues.
  • protein and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function.
  • Protein and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps.
  • polypeptide proteins and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof.
  • exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.
  • the terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the full length polypeptide.
  • Conservative substitution variants that maintain the activity of wildtype proteins will include a conservative substitution as defined herein.
  • the identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant.
  • Variants, fragments, and/or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of a disease as described herein.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • nucleic acid or“nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof.
  • the nucleic acid can be either single -stranded or double-stranded.
  • a single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA.
  • the nucleic acid can be DNA.
  • nucleic acid can be RNA.
  • Suitable DNA can include, e.g., genomic DNA or cDNA.
  • Suitable RNA can include, e.g., mRNA or miRNA.
  • a nucleic acid as described herein is comprised by a vector.
  • a nucleic acid sequence as described herein, or any module thereof is operably linked to a vector.
  • the term "vector”, as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells.
  • a vector can be viral or non-viral.
  • the term“vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells.
  • a vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc.
  • expression vector refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell.
  • An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification.
  • RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.
  • gene means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences.
  • the gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or “leader” sequences and 3’ UTR or “trailer” sequences, as well as intervening sequences (introns) between individual coding segments (exons).
  • the agonist or inhibitor of a miRNA can be provided or administered on a vector, e.g., a viral vector.
  • the agonist or inhibitor of a miRNA can be provided or administered as a gene therapy.
  • viral vector refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle.
  • the viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes.
  • the vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
  • recombinant vector is meant a vector that includes a heterologous nucleic acid sequence, or“transgene” that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments of any of the aspects, be combined with other suitable compositions and therapies. In some embodiments of any of the aspects, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
  • an agonist and/or inhibitor of an miRNA as described herein can comprise a modified nucleic acid sequence, e.g., it is chemically modified to enhance stability or other beneficial characteristics.
  • the nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases,
  • sugar modifications e.g., at the 2’ position or 4’ position
  • replacement of the sugar e.g., at the 2’ position or 4’ position
  • RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages.
  • RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • the modified RNA will have a phosphorus atom in its intemucleoside backbone.
  • Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and
  • RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • methyleneimino and methylenehydrazino backbones sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— ,—CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone],— CH2— O— N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)- -CH2— and—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as— 0-P-0-CH2-]
  • RNA mimetics suitable or contemplated for use as agonists or inhibitors
  • both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • a peptide nucleic acid PNA
  • PNA compounds the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an
  • nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • a RNA can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation.
  • the addition of locked nucleic acids has been shown to increase RNA stability in serum, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller,
  • Modified RNAs can also contain one or more substituted sugar moieties.
  • the RNAs, e.g., agonists and/or inhibitors, described herein can include one of the following at the 2' position: OH; F; 0-, S-, orN-alkyl; 0-, S-, or N-alkenyl; 0-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl.
  • Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)n0NH2, and 0(CH2)n0N[(CH2)nCH3)]2, where n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of an RNA, and other substituents having similar properties.
  • the modification includes a 2' methoxy ethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below
  • 2'- dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'- DMAEOE
  • 2'-0— CH2— O— CH2— N(CH2)2 also described in examples herein below.
  • RNA modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. RNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • a nucleic acid as described herein can also include nucleobase (often referred to in the art simply as“base”) modifications or substitutions.
  • “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5 -halo, particularly 5-bromo, 5-trifluoromethyl and
  • nucleobases are particularly useful for increasing the binding affinity of the nucleic acids featured in the invention.
  • These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0- methoxy ethyl sugar modifications.
  • nucleic acid featured in the invention involves chemically linking to the nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the RNA.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem.
  • a thioether e.g., beryl-S-tritylthiol (Manoharan et ah, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1990, 18:3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • inhibitory nucleic acid refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like.
  • the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA).
  • RNA interference Double -stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi).
  • the inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA
  • iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
  • iRNA refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway.
  • RISC RNA-induced silencing complex
  • an iRNA as described herein effects inhibition of the expression and/or activity of a target.
  • contacting a cell with the inhibitor e.g.
  • an iRNA results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.
  • administering an inhibitor e.g.
  • an iRNA to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.
  • the iRNA can be a dsRNA.
  • a dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used.
  • One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence.
  • the target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron
  • the other strand includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions.
  • the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive.
  • the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive.
  • the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive.
  • the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule.
  • a“part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).
  • dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
  • Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA,miRNA, and/or amiRNA, which are well known in the art.
  • siRNA, shRNA,miRNA, and/or amiRNA are well known in the art.
  • One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of an immune response regulator, e.g., using publically available design tools.
  • siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).
  • the RNA of an iRNA is chemically modified to enhance stability or other beneficial characteristics.
  • the nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in“Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
  • Modifications include, for example, (a) end modifications, e.g., 5’ end modifications
  • RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages.
  • RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone.
  • modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides.
  • the modified RNA will have a phosphorus atom in its intemucleoside backbone.
  • Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and
  • RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • methyleneimino and methylenehydrazino backbones sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— ,—CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone],— CH2— O— N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)- -CH2-- and—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as— 0-P-0-CH2-]
  • RNA mimetics suitable or contemplated for use in iRNAs
  • both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups.
  • the base units are maintained for hybridization with an appropriate nucleic acid target compound.
  • One such oligomeric compound an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone.
  • the nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
  • RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA).
  • LNA locked nucleic acids
  • a locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This stmcture effectively "locks" the ribose in the 3'-endo structural conformation.
  • the addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in semm, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833- 843; Grunweller, A. et al., (2003) Nucleic Acids Research 31( 12): 3185-3193).
  • Modified RNAs can also contain one or more substituted sugar moieties.
  • the iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and
  • alkynyl alkynyl.
  • exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)n0NH2, and 0(CH2)n0N[(CH2)nCH3)]2, where n and m are from 1 to about 10.
  • dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, 0N02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties.
  • the modification includes a 2' methoxy ethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-MOE) (Martin et ah, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group.
  • 2'-dimethylaminooxyethoxy i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below
  • 2'- dimethylaminoethoxyethoxy also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'- DMAEOE
  • 2'-0— CH2— O— CH2— N(CH2)2 also described in examples herein below.
  • modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'- 5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as“base”) modifications or substitutions.
  • “unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
  • Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5- halo, particularly 5-bromo, 5-trifluor
  • 5-substituted pyrimidines include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
  • 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0- methoxy ethyl sugar modifications.
  • Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem.
  • athioether e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiochole sterol (Oberhauser et al, Nucl.
  • the terms “treat,” “treatment,” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. lung cancer.
  • the term“treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally“effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective” if the progression of a disease is reduced or halted.
  • treatment includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the term“pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry.
  • pharmaceutically acceptable is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a pharmaceutically acceptable carrier can be a carrier other than water.
  • a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment.
  • a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
  • administering refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site.
  • Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
  • compositions, methods, and respective components thereof refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
  • the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
  • Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein.
  • One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
  • the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes.
  • Other terms are defined herein within the description of the various aspects of the invention.
  • immunosuppressed aberrant immune system activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject.
  • immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
  • a method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
  • condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease.
  • a method of treating bronchial premalignant lesions in a subject in need thereof comprising administering at least one of:
  • the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRBl, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
  • At least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, and miR-149-5p.
  • the different immune response regulator can further include a miRNA selected from Table 2A.
  • Acetylcholine receptor antagonist Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; J K inhibitors; KIT inhibitors; Leucine rich repeat kinase
  • the subject is not administered an immune stimulating drug
  • the level of expression of at least 1 negative immune response regulator is not increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is not decreased relative to a reference level of expression; or
  • the subject is a subject determined to have a level of expression of at least 1 negative immune response regulator which is not increased relative to a reference level, or a level of expression of at least 1 positive immune response regulator which is not decreased relative to a reference level of expression.
  • an immune response regulator is an immune response regulator polypeptide, an immune response regulator miRNA, or a nucleic acid encoding the immune response regulator.
  • inhibitor of an immune response regulator is an antibody, antibody reagent, or inhibitory nucleic acid.
  • the administering step comprises the administration of a vector comprising a nucleic acid encoding the agonist and/or inhibitor.
  • the at least one immune stimulating drug is selected from the group consisting of:
  • immune-checkpoint inhibitors e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3; drugs that stimulate interferon signaling (e.g. anti-viral drugs that improve interferon signaling); DNA synthesis inhibitors; IMDH inhibitors; CDK inhibitors; ribonucleotide reductase inhibitors; dihydrofolate reductase inhibitors; topoisomerase inhibitors; FLT3 inhibitors; IGF-1 inhibitors; MEK inhibitors; aurora kinase inhibitors; PKC inhibitors; RAF inhibitors; PDFGR/KIT inhibitors; VEGFR inhibitors; SRC inhibitors; retinoid receptor agonists; HD AC inhibitors; DNA methyltransferase inhibitors; and EZH2 inhibitors.
  • IMDH inhibitors e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3
  • CDK inhibitors e.g. ribonucleotide reduct
  • biopsy or brushing sample comprises morphologically-normal tissues or cells.
  • a pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with immunosuppressed aberrant immune system activity comprising:
  • composition of paragraph 44, wherein the condition caused by or associated with immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
  • composition of paragraph 45, wherein the cancer is a squamous cell cancer or lung squamous cell cancer.
  • composition of paragraph 45, wherein the infectious disease is a bacterial and/or viral infection.
  • composition of paragraph 47, wherein the viral infection is a coronavirus infection.
  • a pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with autoimmune aberrant immune system activity comprising:
  • the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRBl, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
  • At least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, miR- 149-5p.
  • the different immune response regulator can further include a miRNA selected from Table 2A.
  • the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
  • Acetylcholine receptor antagonist Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; JNK inhibitors; KIT inhibitors; Leucine rich repeat kinase
  • biopsy or brushing sample comprises morphologically-normal tissues or cells.
  • EXAMPLE 1 IMMUNE ALTERATIONS ASSOCIATED WITH DISEASE PROGRESSION IN BRONCHIAL PREMALIGNANT LESIONS.
  • PMLs Premalignant Lesions
  • PMLs arise in airway epithelium and are precursors to squamous cell carcinoma (FIG. 1A and FIG. IB).
  • Drugs that target these genes can serve to inhibit the immune suppressor genes or enhance the immune activator genes and thereby be effective in delaying or preventing the development of lung cancer.
  • the inventors identified herein immune pathways active in bronchial PMLs. Further the inventors identified immune pathways associated with the progression and regression of PMLs.
  • the inventors have profiled via RNA sequencing endobronchial biopsies obtained from high-risk smokers undergoing lung cancer screening via autofluoresence bronchoscopy (FIG. 2).
  • the inventors describe herein four molecular subtypes of bronchial premalignant lesions.
  • the Proliferative molecular subtype was enriched for samples with bronchial dysplasia and progression/persistence of Proliferative lesions was associated with a decrease in expression of genes involved in interferon signaling and antigen processing/presentation pathways.
  • Endobronchial biopsies divide into four distinct molecular subtypes based on distinct patterns of gene co-expression (FIG. 3).
  • the inventors describe a novel set of genes that, based on a set of rigorous computational and statistical tests, were found to be responsible for immune suppression in bronchial premalignant lesions that are likely to persist or progress to higher-grade lesion or invasive cancer.
  • the set of genes that define this invention are GSTP1, TIMM 13, SMURF 1, and TMEM63C, as suppressors of immune activation, and B2M, HLA-DRA, HLA-DRB l, HLA-DPA1, MSC, QPRT, CPQ, MRAS, RCAN1, SERPINIl, SLC5A8 as activators of the immune system in bronchial premalignant lesions.
  • the inventors novel computational methods have identified these genes as mediating the immune changes we observed and they are thus potential targets for immunoprevention of lung cancer.
  • HLA-DRA, HLA-DRB l, and HLA-DPA1 are all components of the major histocompatibility class II complex (1), which functions in antigen presenting cells to display foreign antigens.
  • the B2M gene is a component of the MHC class I complex, which is required for presentation of self antigens in all nucleated cells (2).
  • the MSC gene is known as a downstream regulator of B cell receptor activation, but B cell activity has not previously been shown to be important in this context (3).
  • GSTP1 has been shown in vitro to regulate transcription factors STAT3 and NF-kB, effecting a wide array of immune responses, but has not been identified as a driver of immune response in lung premalignancy (4, 5).
  • TMEM63C has not been previously associated with activation or suppression of the immune system, in any context.
  • One future product of this invention will be a therapeutic for the immunoprevention of lung cancer.
  • This could include a drug or antibody to either inhibit the immune-suppressor genes or enhance the expression of immune activator genes that we have identified.
  • these genes will be useful as a diagnostic tool, helping to differentiate between immune cold lesions likely to progress to cancer, and immune hot/active lesions that are likely to regress to less severe histology or disappear.
  • the diagnostic tool has potential utility in lung cancer CT screening where it could be used to reduce false positives or dictate screening intervals. Additional potential clinical utility could be in the identification of high-risk populations for intervention trials and to monitor the efficacy of the intervention in these trials.
  • the identified genes will be useful drug targets for the treatment of early and late stage cancers and immune activation across many types of cancers.
  • Immune phenotypes present in these lesions were characterized using Weighted Gene Coexpression Network Analysis (WGCNA) to group the ssGSEA scores from 104 immune gene sets (gene sets described in reference 6.) into 6 modules.
  • WGCNA Weighted Gene Coexpression Network Analysis
  • the 6 immune modules were associated with the previously described molecular subtypes and lesion progression/persistence.
  • Three immune modules,“Antigen Presentation”,“Interferon,” and“B Cells” were significantly associated with the progression status of lesions with the Proliferative molecular subtype.
  • the inventors identified genes that were associated with the progression status of each lesion within the Proliferative subtype using a mixed effect linear model (including patient as a random effect, smoking status, batch, and transcript integrity as covariates). Potential immune modulator genes were then identified as those with a high degree of correlation with the three significant progression-related immune modules. Finally, the list of immune modulator genes presented here was determined using mediation analysis, to identify 1) genes whose expression completely mediates an immune module-progression association, or 2) genes whose association with progression is completely mediated by an immune module.
  • GSEA Gene Set Enrichment Analysis
  • VIPER Virtual Inference of Protein-activity by Enriched Regulon
  • Table 6 This table lists the identified nucleic acid mediators of the immune phenotype between progressive/persistent versus regressive lesions. See also Example 1.
  • the inventors have discovered methods for identifying individuals who are most likely to develop squamous lung cancer, methods for lung cancer prevention in these same individuals, and methods of treatment for patients with squamous lung cancer. These methods involve measuring airway gene expression to identify individuals that have a pattern of gene expression reflective of what we have previously named Proliferative Squamous Premalignancy. Among individuals having this gene expression pattern, those with elevated expression of miR- 149, decreased expression of NLRC5, or decreased MHC Class I expression are most likely to develop squamous lung cancer.
  • miRNAs that specifically regulates module 9 in Lung PMLs, which is related to interferon-gamma response and antigen presentation/processing pathways.
  • the expression levels of miR149-5p and its negatively correlated genes are significantly associated with progressive PMLs.
  • miR-149-5p is highly expressed in basal epithelial cell populations, rather than immune cells, based on cell type specific miRNA sequencing database FANTOM5 (available on the world wide web at fantom.gsc.riken.jp/5/) and canonical cell type marker correlation analysis.
  • FANTOM5 available on the world wide web at fantom.gsc.riken.jp/5/
  • the downstream effects of dysregulated miR-149-5p may be mediated by its suppression of NLRC5 expression, which in turn down-regulates MHC Class I dependent immune responses (see also FIG. 11-FIG. 15).
  • NLRC5 a critical regulator of MHC Class I related gene expression, is targeted by miR- 149-5p.
  • the inventors have identified seven high confident regulated targets for NLRC5. All seven of these genes belong to Module 9 and are significantly down-regulated within the progressive lesions of Proliferative subtype. The results show that that miR- 149-5p regulate MHC Class I gene expression and the early immune evasion of PML through suppressing NLRC5 level (FIG. 16).
  • RNA sequencing was obtained from six PML biopsy samples from five patients. 11 cell types were identified using Seurat. The GSVA score for Module 9 was calculated for each cell. GPC1, the host gene for miR-149-5p, is significantly negative correlated with Module 9 GSVA score among the epithelial cells, but not in other cell types. Given that we have previously shown miR-149- 5p is highly specifically expressed in the epithelial cells only, the interaction between miR-149-5p and its target genes in Module 9 is also observed in epithelial cells (FIG. 17).
  • Gene module 4 through 7 can differentiate Proliferative PML subtypes from the others. The inventors next sought to explore whether miRNAs specifically targeted these gene modules could improve the classification. Using pamR and the paired miRNA and mRNA data in the discovery cohort, out of the 103 miRNAs that targets gene module 4-7, 10 miRNAs could improve the cross-validation accuracy for predicting Proliferative subtypes (FIG. 20). [00330] Experimental details
  • Bronchial premalignant lesions are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer.
  • the inventors report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high- risk smokers.
  • the Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways.
  • a Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity.
  • Lung cancer is the leading cause of cancer death taking -160,000 US lives each year, more than colorectal, pancreatic, breast, and prostate cancers combined. To decrease mortality, the inventors need innovative strategies to intercept cancer development by diagnosing the disease at its earliest and potentially most curable stage. Development of LC risk biomarkers and interception strategies requires a detailed understanding of the earliest molecular alterations involved in lung carcinogenesis that occur in the respiratory epithelium 1,2. Exposure to cigarette smoke creates a field of injury throughout the entire respiratory tract by inducing a variety of genomic alterations that can lead to an at-risk airway where premalignant lesions (PMLs) and LCs develop.
  • PMLs premalignant lesions
  • Lung squamous cell carcinoma arises in the epithelial layer of the bronchial airways and is often preceded by the development of PMLs through a stepwise histological progression from normal epithelium to hyperplasia, squamous metaplasia, dysplasia (mild, moderate, and severe), carcinoma in situ (CIS), and finally to invasive and then metastatic LUSC3.
  • LUSC Lung squamous cell carcinoma
  • the presence of high-grade persistent or progressive dysplasia is a marker of increased LC risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung, although many dysplastic lesions do have varied outcomes4,5.
  • mRNA sequencing mRNA-seq
  • CT chest computed tomography
  • Proliferative subtype is enriched with bronchial dysplasia, high basal cell and low ciliated cell signals, and expression of proliferation-associated pathways. Genes involved in interferon signaling and T-cell-mediated immunity were down regulated among progressive/persistent lesions within the Proliferative subtype compared with regressive lesions and these pathways correlated with decreases in both innate and adaptive immune cell types. Molecular classification of biopsies into a high-grade/progressive disease group may be used to stratify patients into prevention trials and to monitor efficacy of the treatment. The results also show that personalized lung cancer chemoprevention, targeting specific cancer-related pathways or the immune system may have potential therapeutic benefits.
  • the subjects are predominantly older smokers, many of which have a prior history of lung cancer, chronic obstructive pulmonary disease (COPD), and occupational exposures that confer a high-risk of developing lung cancer.
  • Clinical characteristics reported at the baseline visit such as sex, age, smoking status (ever or never), pack-years, prior history of lung cancer, COPD status, and occupational exposures were not significantly different between the two cohorts (Table 1).
  • the DC had 190 biopsies and 89 brushes, whereas the VC had 105 biopsies and 48 brushes.
  • Ninety-four percent of subjects had at least one lung anatomic location sampled two or more times via endobronchial biopsy.
  • the DC and VC contained 37.9% and 35.2% biopsies with a histological grade of dysplasia or higher and 23.1% and 19.0% had progressive/persistent dysplasia, respectively (Table 2).
  • the inventors used a previously described smoking-associated signature? to predict the smoking status of each sample, as smoking status was only available at baseline. The predicted smoking status was consistent across all procedures for 63% and 70% of the DC and VC subjects, respectively (Supplementary Table 1).
  • the DC had significantly greater total reads, percent uniquely mapping reads, and median transcript integrity number scores8 among the biopsies than the VC, but these differences between cohorts were not reflected in the brushes (Supplementary Table 2).
  • WGCNA weighted gene co-expression network analysis9
  • DC biopsy gene modules that were highly correlated (absolute Pearson correlation coefficient r > 0.85) to at least one other non-DC biopsy module within each of the four data sets were selected. Genes in the selected modules were filtered by requiring that each gene was also present in at least one of the correlated non-DC biopsy modules, resulting in a set of nine gene modules that consisted of 3936 genes in total (Supplementary Table 3).
  • each molecular subtype In order to characterize each molecular subtype, the inventors first focused on identifying biological pathways enriched in each module, as the pattern of gene module expression defines the PML subtypes. Each gene module was found to be associated with distinct epithelial and immune biological processes (Fig. la, Supplementary Tables 3 and 4, Data 1).
  • the Proliferative subtype is specifically characterized by increased expression of genes involved in energy metabolism and cell cycle pathways (Modules 4 and 5).
  • the Secretory and Normal-like subtypes both have increased expression of genes in cilium-associated pathways (Module 6), however, the Normal-like subtype specifically has decreased expression of genes involved in inflammation, regulation of lymphocytes and leukocytes, and antigen processing and presentation pathways (Modules 8 and 9).
  • the Secretory subtype exhibits decreased expression of genes involved in protein translation (Module 7), whereas RNA processing genes (Module 2) are expressed more highly in the Inflammatory subtype.
  • the inventors further characterized our molecular subtypes by their associations with clinical phenotypes and established LUSC tumor molecular subtypesl 1,12. Sample genomic smoking status, the subject from whom the sample was derived, and sample histology demonstrated significant associations with molecular subtype (p ⁇ 0.01, two-sided Fisher’s exact test, Fig. lb, Supplementary Tables 5 and 6, Supplementary Figs. 1, 2, and 3).
  • the Proliferative and Secretory subtypes are enriched for current smokers and the Proliferative subtype is enriched for bronchial dysplasia (Fig. lb).
  • the Proliferative subtype samples also had high concordance with the LUSC-Classical subtype (Fig. lb).
  • the LUSC-Classical subtype was associated with alterations and over expression of REAP 1 and NFE2L2 as well as amplification of 3q26 with over expression of SOX2, TP63, and PIK3CA11.
  • the LUSC-Classical subtype was associated with increased expression of genes involved in energy metabolism, and the inventor’s Proliferative subtype is in part defined by high expression of Module 4, that is enriched with genes involved in oxidative phosphorylation and the electron transport chain.
  • the Inflammatory and Secretory PML subtypes demonstrate enrichment for the LUSC-Secretory subtype.
  • the LUSC-Secretory subtype was associated with the immune response, and the Inflammatory, and Secretory PMLs have the highest expression of Module 8 that is enriched for genes in these same pathways.
  • the inventors wanted to examine the extent to which the inventor’s PML molecular subtypes were driven by differences in epithelial and immune cell type composition by assessing expression of a number of canonical cell type markers.
  • gene expression of MUC5AC a marker of goblet secretory cells
  • FDR 3.4e-5, linear model
  • SCGB 1A1 a marker of club cells
  • the inventors wanted to determine whether the heterogeneity captured in the DC biopsy-derived molecular subtypes was reproducible in the VC.
  • the inventors developed a 22-gene nearest centroid molecular subtype predictor by selecting genes highly correlated with each of the gene module eigengenes.
  • the predictor has 84.7% accuracy across DC biopsies (training set, Fig. 2a and Supplementary Fig. 6) with the following misclassification rates per subtype 5/52 (9.6%) in Proliferative, 7/37 (18.9%) in Inflammatory, 9/61 (14.8%) in Secretory, and 8/40 (20%) in Normal like.
  • the 22-gene classifier was used to predict the molecular subtype of the 105 VC biopsies (Fig. 2b).
  • the VC subtype predictions were evaluated by examining the concordance of Gene Set Variation Analysis (GSVA)13 scores for each of the 9 modules (using the full set of genes for each module) between the predicted VC subtypes compared with the DC subtypes.
  • GSVA Gene Set Variation Analysis
  • the average behavior of PCI across the subtypes was highly similar (Supplementary Fig. 7) with few exceptions (namely, Module 3 that had the fewest genes).
  • FDR 1.67e-7, linear model
  • FDR 3.03e-10, linear model
  • MUC5AC a marker of goblet epithelial cells
  • Airway brushes reflect biopsy Proliferative subtype
  • the inventors have shown that bronchial brushes from normal-appearing areas of the mainstem bronchus could predict the presence of PMLs 14; however, that study lacked biopsies and brushes from the same subjects.
  • the Proliferative subtype represents a distinct subtype of PMLs enriched for dysplastic histology expressing metabolic and proliferative pathways.
  • Biopsies classified as the Proliferative subtype can represent a group of PMLs that need close monitoring and intervention.
  • the inventors sought to explore whether or not the inventors could predict the presence of Proliferative subtype biopsies using the brushes.
  • the Proliferative subtype is defined by the behavior of Modules 4, 5, 6, and 7 (Table 3), and therefore, the inventors used the subset of 8 genes (from the 22 -gene predictor) that correspond to these modules to predict presence/absence of the Proliferative subtype across the DC and VC biopsies and brushes.
  • a prediction of the Proliferative subtype in a brush is specific (91% and 92% in the DC and VC biopsies, respectively), but not sensitive (39% and 32% DC and VC biopsies, respectively) at indicating the presence of at least one Proliferative PML detected at the same time point (Fig. 3a).
  • GSVA scores for Modules 4, 5, 6, and 7 that define the Proliferative subtype in the DC and VC brushes (Fig. 3b).
  • the GSVA scores were significantly different (FDR ⁇ 0.05) in the Proliferative subtype versus all other samples only for Modules 5 and 6, and thus these likely contribute the most heavily to Proliferative subtype classification in the brushes.
  • Module 5 contains genes associated with cell cycle and proliferation
  • Module 6 contains genes associated with cilium assembly and organization. Upregulation of Module 5 and
  • the genes in Module 9 include a number of genes that encode for proteins involved in interferon signaling as well as antigen processing and presentation (SP100, CIITA, CXCL10, SOCS1, GBP1, GBP4, B2M, TAPI, TAPBP, TRIM14, TRIM21, TRIM22, STAT1, PML, OAS2, OAS3, MX1, ADAR, ISG15, IFI35, IFIT3, IFI27, PSMB8, PSMB9, BST2, IRF1, IRF9, CD74, PSME1, PSME2, HLA-DQA1/DPA1/ DPB1/DRA/ DQB2/DRB1/ DQB 1/DMA/DMB/DOA, HLA- A/B/C/E/F) and include the inhibitory receptor LAG3.
  • SP100 CIITA, CXCL10, SOCS1, GBP1, GBP4, B2M, TAPI, TAPBP, TRIM14, TRIM21, TRIM22, STAT1, PML, OAS2,
  • the inventors wanted to evaluate whether or not the presence or absence of innate or adaptive immune cells were associated with Module 9 expression within the Proliferative subtype.
  • the inventors generated GSVA scores using previously described immune cell signatures 15 and scores for 64 different cell types using the xCell algorithm 16, separately for both the DC and VC biopsies.
  • the inventors identified significant (FDR ⁇ 0.05, linear model) associations between the cell type scores and Module 9 that were in common between the DC and VC biopsies and identified eight cell types (via xCell): dendritic cells, activated dendritic cells, plasmacytoid dendritic cells, macrophages, Ml macrophages as well as CD8+ effector memory T cells, CD8+ central memory T cells, and T regulatory cells (Fig. 4c).
  • CD4 T cells were increased (p « 0.001 in the concordant set, linear model and CD8 T cells were decreased (p « 0.001 in the concordant set, linear model) in PMLs that progress/persist.
  • LUSC is the second most common form of lung cancer. LUSC arises in the epithelial layer of the bronchial airways, and is often preceded by the development of lung squamous PMLs.
  • dysplastic persistent and or progressive PMLs is a marker of increased risk for LUSC5.
  • the inventors lack effective tools to identify PMLs at highest risk of progression to invasive carcinoma (6).
  • the development of markers predictive of disease progression will be important in identifying patients at highest risk for LUSC development and in identifying biological pathways exploitable for LUSC chemoprevention.
  • the inventors profile via mRNA-Seq bronchial brushes and endobronchial biopsies obtained from subjects undergoing longitudinal lung cancer screening by chest CT and autofluorescence bronchoscopy.
  • the inventors identify four transcriptionally distinct groups of biopsies, one of these the inventors label Proliferative and find it to be associated with bronchial dysplasia.
  • Proliferative PMLs can also be identified via gene expression measured from cells in the non-involved large airway epithelium.
  • the inventors further find that persistent/progressive Proliferative PMLs are characterized by decreased expression of genes involved in interferon signaling and antigen processing/presentation pathways. Consistent with these gene expression findings the inventors find that progressive/persistent
  • Genomic gains in loci containing SOX2, TP63, EGFR, MYC, CEP3, and CEP5 are also associated with progression of high-grade dysplasia23.
  • the inventors lack a comprehensive PML molecular classification system to complement pathologic examination.
  • the inventors pursued an unsupervised class discovery approach that led to the identification of four distinct molecular PML subtypes (Proliferative, Inflammatory, Secretory, and Normal -like).
  • the transcriptional patterns differentiating the PML subtypes are robust and a 22 -gene panel identified in the DC can be used to distinguish between the molecular subtypes in an independent VC.
  • the Proliferative subtype is enriched with dysplastic PMLs from current smokers and is characterized by up regulation of metabolic (OXPHOS/ETC/TCA) and cell cycle pathways and down regulation of cilia-associated pathways.
  • OXPHOS/ETC/TCA metabolic pathway pathway
  • Previous work shows increases in metabolic pathways in the airways of subjects with dysplastic lesions 14, in PMLs adjacent to LUSC tumor24, and in smokers at high-risk for lung cancer25 as well as increases in proliferation (via Ki67 levels, as mentioned above) that have been utilized as an endpoint in lung cancer chemoprevention26,27.
  • Identification of patients with Proliferative lesions can be useful to enrich lung cancer chemoprevention trials with high-risk subjects or to identify patients who would benefit from more frequent lung cancer screening.
  • the Inflammatory subtype is predominated by PMLs from former smokers, but interestingly is not significantly enriched for dysplasia, despite similarly decreased expression of cilia-associated pathways, showing an abnormal epithelium.
  • the Inflammatory subtype also shows increased expression of a gene module enriched for genes involved in inflammation and regulation of lymphocytes and leukocytes (Module 8). This gene module is also elevated in the Secretory subtype predominated by current smokers and increased expression of goblet cell markers.
  • IL1B is part of this inflammation-related gene module, and inhibition of IL1B has recently been shown to reduce lung cancer incidence28.
  • the molecular profiling of PMLs and the identification of gene co-expression modules also provides an opportunity to identify the molecular determinants of subsequent PML progression.
  • One of the nine gene co-expression modules used to define the molecular subtypes was significantly decreased between biopsies that progress/persist compared to biopsies that regress within the Proliferative subtype in both the discovery and VC.
  • the module contains genes involved in interferon signaling and antigen processing and presentation, and its expression was correlated with the abundance innate and adaptive immune cells via computational prediction.
  • the inventor’s data show that there are subtype-specific transcriptomic alterations predictive of subsequent LUSC PML progression that are the result of a lack of infiltrating immune cells in the lesion microenvironment. These data show that biomarkers for determining PML subtype and assessing immune infiltration can have utility for the detection of aggressive PMLs that require more intensive clinical management and genes altered in these PMLs can serve as lung
  • biomarkers could either be measured directly in PML tissue or a surrogate tissue such as bronchial airway epithelium.
  • a benefit of biomarkers predicting aggressive PML behavior measured in surrogate tissue is the potential that these biomarkers can also predict the behavior of PMLs not directly observed during bronchoscopy. Future studies are needed to address the specific mechanism of impaired immunosurveillance in progressive/persistent lesions in the Proliferative subtype including single-cell sequencing, high coverage DNA sequencing,
  • Endobronchial biopsies and brushings were obtained from high-risk subjects undergoing lung cancer screening at ⁇ l-year intervals by white light and autofluorescence bronchoscopy and computed tomography at Roswell.
  • the bronchoscopy included visualization of the vocal cords, trachea, main carina, and orifices of the sub-segmental bronchi visible without causing trauma to the bronchial wall. All abnormal and suspicious areas are biopsied twice and the lung anatomic location is recorded (Supplementary Fig. 9, Supplementary Table 9).
  • One biopsy was used for routine pathological evaluation and the other for molecular profiling.
  • Eligibility for screening includes either a previous history of aerodigestive cancer and no disease at the time of enrollment or age > 50, a current or previous history of smoking for a minimum exposure of 20 pack-years and at least one additional risk factor including moderate COPD (defined as forced expiratory volume (FEV1) ⁇ 70%), confirmed asbestos related lung disease or a strong family history of lung cancer (at least 1-2 first-degree relatives). All research specimens were stored in RNA Allprotect (Qiagen) and stored at -80°C.
  • FEV1 forced expiratory volume
  • Subjects were selected that had biopsies collected in repeat locations via serial bronchoscopies; however, after RNA isolation, samples from three subjects had a single biopsy and one subject had a single brushing.
  • Biopsy progression/regression was defined for each biopsy based on the histology of the biopsy and the worst histology recorded for the same lung anatomic location in the future. Histology changes between normal, hyperplasia, and metaplasia were classified as“normal stable”, decreases in histological dysplasia grade or changes from dysplastic histology to normal/hyperplasia/metaplasia were classified as“regressive”, lack of future histological data was classified as“unknown”, and everything else was classified as“progressive/persistent.” The
  • Samples were subsequently divided into the discovery and VC (as outlined above) and by tissue type (biopsy or brush). Subsequent sample and gene filtering was conducted separately on each set as follows: first, EdgeR45 was used to compute normalized data (library sizes normalized using TMM, trimmed mean of M-values, and log2 counts per million computed) and genes were excluded that either had an interquartile range equal to zero or a sum across samples equal or ⁇ 1. Samples were excluded based on values > 2 standard deviations from the mean for more than one of the following criteria: (1) mean Pearson correlation with all other samples calculated across all filtered genes (2) the 1st or 2nd principal components calculated using the filtered gene expression matrix (3) transcript integrity number (TIN, computed by RSeQC). After sample filtering, gene filtering was recomputed as described above on the final set of high-quality samples. The data are available from NCBTs Gene Expression Omnibus using the accession
  • LUSC TCGA squamous cell carcinoma
  • mice develop lesions that are histologically and molecularly comparable to human lesions and that progress to LUSC and the samples represent a range of histology (normal, mild dysplasia, moderate dysplasia, severe dysplasia, CIS, and LUSC tumor) (Supplementary Materials and Methods).
  • the mouse data are available from NCBI’s Gene Expression Omnibus using the accession GSE111091. Sample and gene filtering from the TCGA LUSC tumors and the mouse tissue were processed as described in the Supplementary Materials and Methods (available on the world wide web at per at doi. org/10.1038/s41467-019- 09834-2).
  • WGCNA9 was used with default parameters to derive modules of gene co-expression across the four data sets described above. Residual gene expression values adjusting for RNA quality (median TIN) and batch (Illumina flow cell) were used as input for WGCNA for the biopsy and brush data sets. For the mouse dataset, residual gene expression values adjusting for RNA quality (median TIN), mouse strain, and sample type (laser capture microdissected versus whole tissue) were used as input for WGCNA. For TCGA LUSC tumor samples, residual gene expression values adjusting for plate were used as input for WGCNA. Gene sets were created for each co-expression module for each dataset and then combined to create a compendium of gene sets. For each gene set in the
  • the first principal component was calculated across each z-score normalized dataset.
  • PCI principal component
  • thresholds were set as follows: r > 0.85 was set to 1 and r ⁇ 0.85 set to 0.
  • the genes defining the retained biopsy modules were required to be present in the biopsy module and at least in one of the correlated gene sets.
  • Consensus clustering was performed setting k (number of groups) to 10, the number of iterations to 1000, the subsampling to 80%, the clustering algorithm to partitioning around medoids, and the distance metric to Pearson correlation.
  • the optimal value for k was 4 based on the relative change in area under the cumulative distribution function calculated based on the consensus matrix for each k.
  • genes with the highest squared correlation coefficients to the module eigengene were chosen to represent the module in the predictor.
  • the 22 genes resulting from this analysis across the DC biopsy data were used to train a nearest centroid predictor using the pamr package with a threshold of zero and predict the molecular subtype across the VC biopsies. Prior to predicting the molecular subtype of these test sets, the training and test sets were combat47 adjusted and z-score normalized across combined training and test data. Using the methods described above the inventors derived molecular subtypes using consensus clustering across the VC biopsies and compared these to the predicted subtypes.
  • Modules are referred to being increased or decreased in each of the molecular subtypes based on the direction of change of the majority of the genes in the module.
  • the molecular subtypes in the DC biopsies were annotated according to the behavior of each gene module by calculating whether or not module GSVA13 scores were significantly associated (FDR ⁇ 0.05) with a particular molecular subtype versus all other samples (two-level factor) using a linear mixed effects model with patient as a random effect (using the‘duplicateCorrelation’ function) via limma.
  • biological pathways and transcription factors associated with each subtype were identified using GSEA49 and mSigDB50 gene sets using genes ranked by the t-statistic for their association with each subtype.
  • the ranked lists were created using the limma51 and edgeR45 packages to identify differentially expressed genes associated with subtype membership.
  • Each linear model used voom-transformed52 data and included membership in the subtype of interest, batch, and RNA quality (TIN) as covariates and patient as a random effect.
  • Pathways enriched in the ranked lists were used to annotate the molecular subtypes. FDR values for individual genes were derived from this analysis or analogous models using only samples of normal/hyperplasia histology or dysplasia histology.
  • ESTIMATE algorithm53 was used to infer relative epithelial, stromal, and immune cell content. Immune cell type specific signatures from Bindea et al.15 and epithelial cell type specific signatures from Dvorak et al.54 were used to generate GSVA13 scores across samples for each signature. In addition, residual gene expression values calculated using log RPKM values were inputted into the xCelll6 to infer relative abundances of 64 different cell types. The above categorical phenotypes along with additional clinical variables such as biopsy histology, subject, previous lung cancer history, sex, and biopsy progression/regression status were associated with molecular subtype using two-sided Fisher’s exact test.
  • the inventors wanted to quantify the predictive performance of the brush with regards to the presence of a biopsy of the Proliferative subtype.
  • a subset of the 22-gene molecular subtype predictor was used to predict the presence or absence of the Proliferative subtype across the DC and VC brushes and biopsies. Specifically, the inventors used eight genes (out of the 22) that
  • modules 4 through 7 corresponded to modules 4 through 7 (significantly up or down regulated in the Proliferative subtype) to classify samples as Proliferative or not using the same methodology described above for the molecular subtype predictor.
  • Sensitivity and specificity performance metrics were calculated based on the ability of a Proliferative subtype prediction in the DC or VC brushes to show the presence of at least one biopsy of the Proliferative subtype.
  • the inventors analyzed the behavior of the modules that define the
  • Imaging was performed using an Aperio Slide Scanner for scoring and a Carl Zeiss Axio (x20 and c 40 objectives) and a Carl Zeiss LSM 710 NLO confocal microscope for capturing additional images.
  • Digital slides were analyzed with the Definiens Tissue Studio (Definiens Inc.) for the enumeration of immunofluorescence staining.
  • the enumeration of the immunofluorescence scored each stain including DAPI-positive cells.
  • the enumeration was conducted on different regions (independent areas of tissue) present on a slide (1-5 regions/biopsy) for each biopsy.
  • the percentage of positively staining cells for a given protein was calculated by dividing the number of positively stained cells by the total number of DAPI-positive cells.
  • a binomial linear mixed effects model via the lme4 R package was used to assess differences in the percentages of cells staining positive for a given protein in each region between progressive/persistent versus regressive biopsies using the total cells stained in each region as weights and adjusting for the slide number as a random effect.
  • the models were used across samples from the Proliferative subtype and across samples from the Proliferative subtype where the biopsy outcome (progressive/persistent versus regressive) agreed with the Module 9 GSVA score (scores ⁇ 0 are associated with progression/persistence and scores greater than 0 are associated with regression). Each region was also qualitatively scored as either positive or negative for having a distinct CD8 T-cell localization pattern where cells lined and were embedded within the epithelium.
  • RNA sequencing data from human endobronchial biopsies and brushings has been deposited in the NCBI Gene Expression Omnibus under accession code GSE 109743.
  • Source Data file The source data underlying all figures and tables in the main text and supplementary information are provided as a Source Data file. All other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. A reporting summary for this article is available as a Supplementary Information file. Further information on research design is available in the Nature Research Reporting Summary linked to this article (available on the world wide web at doi.org/10.1038/s41467-019- 09834-2). All custom computer code is available at on the world wide web at
  • references [00390] 1. Campbell JD, et al. The case for a Pre-Cancer Genome Atlas (PCGA) Cancer Prev.

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Abstract

The methods and assays described herein relate to detection, diagnosis, and treatment of aberrant immune system activity (e.g., in bronchial premaglinant lesions), e.g., by detecting the level of expression of certain immune regulators described herein and/or by therapeutically modulating the level of those immune regulators.

Description

IMMUNE SYSTEM MODULATORS FOR THE TREATMENT OF SQUAMOUS LUNG
PREMALIGNANCY
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/848,860 filed May 16, 2019 and 62/951,571 filed December 20, 2019, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The field of the invention relates to methods for detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by detecting the level of expression of certain immune response regulators described herein and/or by therapeutically increasing the level of immune response regulators and/or by detecting by detecting the level of expression of certain immune response regulators described herein and/or by
therapeutically increasing the level of those immune response regulators.
GOVERNMENT SUPPORT
[0003] This invention was made with government support under Grant Nos. U01CA196408, T32HL125232, and Grant No. U2CCA233238 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0004] The immune system is a collection of organs, cells and specialized tissues that work together to defend the body against foreign invaders and diseased cells. A healthy immune system can recognize foreign or aberrant cells and target them for destruction. However, disorders such as infectious disease, autoimmune disease, and cancer can wreak havoc when the immune system cannot adequately counter the diseased cells.
[0005] Recent outbreaks of emerging and re-emerging infectious diseases worldwide, such as COVID-19, severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), measles, avian, pandemic influenza, chikungunya virus, Ebola virus disease (EVD), Zika virus disease, have resulted in a renewed focus on infectious diseases. There is a need for constant readiness and preparedness to deal with infectious disease outbreaks including emerging and re-emerging infectious disease threats. Approaches that increase the ability of the immune system to address the infectious agent, rather than directly attacking the infectious agent with drugs (e.g., antibiotics or antivirals) are promising approaches to expanding the therapeutic tools available to health care professionals. [0006] The origins of cancer are also closely tied to immune system dysfunction. According to the World Health Organization, an estimated 9.6 million deaths globally were attributed to cancer in 2018. Moreover, statistics indicate that the cancer incidence rate is on the rise around the globe. World-wide, lung cancer is one of the most frequently-occurring types of cancer and is a leading cause of cancer-related mortality. Over 1.8 million new cases are diagnosed each year, while 1.56 million individual die due to lung cancer annually. Lung cancer remains the leading cause of cancer- related death in the United States and the world due, in large part, to the inability to detect the disease at its earliest and curable stage.
[0007] Lung squamous cell cancer develops from non-cancerous lesions in the airway known as bronchial premalignant lesions. The presence of persistent or progressive dysplastic bronchial premalignant lesions is a marker of increased lung cancer risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung. However, the molecular events involved in the development of bronchial premalignant lesions (PMLs), and their progression to Lung Squamous Cell Carcinoma (LUSC), is not well understood. Not all bronchial premalignant lesions progress to invasive cancer, and those that do, progress at variable rates with variable outcomes. At present, there are no tools available in the clinic to identify which lesions will progress to cancer and which will not. Development of improved diagnostics and therapeutics is critical to providing improved care for lung cancer patients.
[0008] Thus, there a need in the art to uncover novel methods and pharmaceutical compositions for the detection, diagnosis, and treatment of conditions associated with aberrant immune system activity in a subject, including infectious disease, autoimmune disease, and cancer and uses thereof.
SUMMARY
[0009] The inventions described herein relate to the discovery that the level of certain immune regulators that can be used to diagnose, detect and/or treat conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer). By analyzing the molecular events involved in the development of bronchial premalignant lesions (PMLs) molecular subtypes the inventors found that certain co-expressed gene modules are associated with histological severity and progression to invasive cancer. PMLs arise in airway epithelium and are precursors to lung squamous cell carcinoma. The inventors describe a novel set of immune regulators that contribute to immune suppression in bronchial premalignant lesions that persist or progress to higher- grade lesions or invasive lung cancer.
[0010] Previous targets for immune activation in the context of cancer have focused primarily on late stage tumors and have focused on T cell mediated immunity. Genes that have been discovered to mediate this immune pathway include PD1, PDL1, and CTLA4. The immune regulators identified herein modulate the activity of MHC class I and II antigen presentation, interferon signaling, and B cell related immunity. The immune regulators described herein regulate immune activation and suppression before tumors even form. Accordingly, the inventors have developed methods for determining if the bronchial premalignant lesions are severe and if they are likely to progress to cancer. Additionally, the inventors have developed new therapies for bronchial premalignant lesions that target the underlying molecular changes associated with immune suppression.
[0011] The inventors discovered that the level of these genes can be used to diagnose, detect and/or treat bronchial BPML and/or lung cancer. Accordingly, the inventors have developed methods for the detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by detecting the level of expression of certain immune regulators described herein and/or by therapeutically modulating the level of those immune regulators.
[0012] Recent evidence suggests infectious diseases such as COVID-19 have very different effects on people. Differences in mortality between older men and women are expected to be associated with differences in their immune systems. For individuals suspected of having COVID-19, knowing the state of their immune system is important for additional treatment regimes. In addition to treatment as described above, identifying individuals with a weakened immune response permits identification of patients in need of early and extreme care. For example, early treatment with Gilead’s Remdesivir (C27H35N6O8P), plasma with antibodies to COVID-19, or other forms of treatment including ventilator, etc.
[0013] Accordingly, described herein in one aspect of any of the embodiments is a method for treating or preventing a condition caused by or associated with immunosuppressed aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject.
[0014] In one aspect of any of the embodiments, described herein is a method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
[0015] In one aspect of any of the embodiments, described herein is a method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
ii. at periodic intervals, for example, at least every 2 months ... , every 4 months ... every 6 months ... , every 8 months ... , every 9 months ... , every year... , every 18 months, every two years... , one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
[0016] In one aspect of any of the embodiments, described herein is a method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; ii . at least every 2 months ... , every 4 months ... every 6 months ... , every 8 months ... , every 9 months ... , every year... , every 18 months, every two years ... , one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
[0017] In one embodiment of this aspect and all other aspects, if the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB 1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
[0018] In another embodiment of this aspect and all other aspects, described herein are immune response regulator selected from Table 1 or Table 2.
[0019] In another embodiment of this aspect and all other aspects, there is a change in at least 5 immune response regulators.
[0020] In another embodiment of this aspect and all other aspects, there is a change in at least ten immune response regulators. [0021] In another embodiment of this aspect and all other aspects, the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
[0022] In one embodiment of this aspect and all other aspects, described herein is a
pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with autoimmune aberrant immune system activity, comprising:
a. an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator and b. a pharmaceutical acceptable carrier. In one embodiment of this aspect, and all other aspects, the pharmaceutical composition comprises an agonist to the miRs of Table 2A, when 2..., 4... , 6..., 7... miR’ s from Table 2A is underexpressed.
[0023] In another embodiment of this aspect and all other aspects, the condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease, or an infectious disease such as COVID-19.
[0024] In one embodiment of any aspect, one would use an agonist to the miR’s from Table 2A.
[0025] In one embodiment of this aspect and any other aspect, described herein is a method of determining the risk of progression of bronchial premalignant lesions to lung cancer or an infectious disease in a subject the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) determining the subject is at risk of the bronchial premalignant lesions progressing to lung cancer or an infectious disease if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
[0026] Accordingly, pharmaceutical compositions that target these immune regulators can serve to inhibit the immune suppressor genes or enhance the immune activator genes and thereby be effective in delaying or preventing the development of conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer).
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 A-FIG. IF depict a schemata of carcinogenesis (FIG.1 A), and squamous lung carcinogenesis (FIG. IB), and some example images of bronchial premalignant lesions along the progression toward squamous lung cancer (FIG. 1C, ID, IE, IF).
[0028] FIG.2 shows the study design of the Inventor’s study design investigating Bronchial Premalignant Lesions using the pre-cancer genome atlas (PCGA).
[0029] FIG. 3 shows that endobronchial biopsies divide into four distinct molecular subtypes based on distinct patterns of gene co-expression. [0030] FIG. 4 shows a schemata of the Inventor’s study design.
[0031] FIG. 5 shows that six immune phenotypes were identified in the bronchial PMLs from the
Discovery Cohort of PCGA. 104 immune gene sets were scored in PML biospies using single sample GSEA. Gene sets were clustered into non-redundant immune“phenotypes” using WGCNA.
Phenotypes with redundant biology or intederminate enrichment were merged with related phenotypes. Samples received a score for each phenotype on a representative gene set. When a phenotype was previously found, the same representative gene set was used. Otherwise, the gene set most correlated to module a gene was used.
[0032] FIG. 6 depicts graphs of the immune phenotypes in Proliferative samples that are associated with progression (Discovery cohort, n=52). The first series is progressing/stable lesions and the second series is regressing lesions.
[0033] FIG. 7 depicts graphs of the immune phenotypes in Proliferative samples that are associated with progression/persistence (Validation cohort, n=28). The first series is
progressing/persistent lesions and the second series is regressing lesions. FIG. 8A - Fig. 8C depicts the mediation analysis. All genes significantly correlated to the three immune phenotypes, Antigen Presentation, B cells, Interferon, were identified, as were genes correlated to progression/regression status. A regression model was used to identify the subset of genes completely mediating the associations between genes, phenotypes, and progression/regression outcome.
[0034] FIG. 9 depicts that the mediation analysis identified 13 genes completely mediate immune phenotypes, and 2 genes completely mediated by immune phenotypes.
[0035] FIGs. 10A-10D shows that GSTP1 mediates the effect of antigen presentation on progression. Antigen presentation, B cells, and Interferon are key phenotypes distinguishing progressing and regressing bronchial PMLs. Mediation analysis identified 11 genes that appear to modulate these immune phenotypes, 2 which down-regulates immune response in progressing lesions. It also identified 2 genes significantly up regulated in progressing lesions that are completely mediated by one or more immune phenotypes. These immune modulators represent targets for immuno-prevention of lung squamous cell carcinoma. In FIGs. 10B- IOC, the first series is progressing/stable lesions and the second series is regressing lesions. FIG. 10D depicts specific immune signaling pathways anticorrelated with GSTP1 expression via Gene Set Enrichment Analysis, including STAT5 signaling, STAT3 signaling and TNFA signaling via NFKB.
[0036] FIG. 11 depicts the analysis design. 148 longitudinally collected endobronchial biospies from 30 patients were collected and both mRNA and miRNA sequenced. The miRNAs that potentially regulate a specific gene module were identified first by selecting the miRNAs connected to the genes of this module using both the correlation strength and miRNA target information. Pearson correlation was calculated from expression residuals (adjusted for batch and TIN) for each miRNA- mRNA pair and those with significant negative Pearson correlation coefficients (PCCs; FDR <= 0.05) were selected. Target information was obtained by combining both sequence-based prediction databases and experimentally validated databases. A connection between miRNA and target mRNA was defined by both significant negative correlation and evidence that the miRNA targets the mRNA. Next, the Fisher-Z transformed PCC densities of one miRNA with its targets within the module of interest were compared to that with its targets in other modules using a t-test to select for miRNAs that strongly regulate the module of interest (t < 0). Fisher-exact tests were performed to select for miRNA with significantly more connected target genes in the module of interest than in other modules (OR > 1). miRNAs with significant results from both tests (FDR <= 0.25) were kept.
[0037] FIG. 12 shows four miRNAs that were identified which regulate Module 9 gene expression based on the method described in FIG. 11.
[0038] FIG. 13A-FIG. 13B show that miR-149-5p is significantly upregulated in the progressing/persistent PMLs within the proliferative subtype, while its target genes are down- regulated. FIG. 13B shows that miR-149-5p expression was enriched among epithelial cell type samples and that it was depleted among lymphocyte and myeloid samples. (* p-value < 0.05). The association between miRNA or mRNA expression levels and lesion progression status was determined using a mixed-effect model, adjusting for batch and patient as a random effect. FIG. 13B shows that miR-149-5p expression is enriched among epithelial cell type samples and that it is depleted among lymphocyte and myeloid samples using FANTOM5 Consortium data.
[0039] FIG. 14 shows that the expression level of miR-149-5p is correlated with basal cell canonical cell-type markers KRT5 and KRT8 (FDR <= 0.001). The correlation was calculated based on expression residuals, adjusted for RIN, and batch effects.
[0040] FIG. 15 shows that the expression level of MHC Class I genes are decreases in progressive lesions, among the proliferative subtype (*p-value<=0.05). The first series is
progressive/persistent lesions and the second series is regressive lesions.
[0041] FIG. 16 shows the expression pattern of NLRC5 target genes. All genes belonged to Module 9 and were significantly down-regulated in the progressive/persistent lesions based on a mixed-effect model, adjusting for batch and patient as a random effect. The first series is
progressing/stable lesions and the second series is regressing lesions.
[0042] FIG. 17 shows the correlation between Module 9 and GPC1 in different cell types based on single-cell RNA-sequencing data containing -4000 cells from 12 samples of 11 patients. GSVA score for Module9 was calculated for each cell. GPC1 is the host gene for miR-149-5p and shares the same transcriptional start site. A significant negative correlation between Module9 and GPC1 can be observed only in the epithelial cells (top plot; p-value < 0.001) but not in the immune cells (bottom plot; p-value=0.8).
[0043] FIG. 18 shows the correlation between miR-149-5p and NLRC5 in all Epithelial Cells from FANTOM5 Consortium data (p-value=0.01). [0044] FIG. 19 depicts the sensitivity and specificity of Module9 and Module9 plus miR-149-5p for differentiating progressive/persistent from the regressive lesions in the proliferative subtype (n=28).
[0045] FIG. 20 depicts a list of the discovered miRNAs including accuracies, sensitivities, and specificities. These miRNAs improve the prediction of the proliferative subtype among the discovery cohort comparing to using only gene classifier (n=148). Shown are miR-548b-5p, miR-642a-5p, miR- 642b-5p, miR-328a-3p, miR-34b-3p, miR-34b-5p, miR-3664-3p, miR-548i, miR-766-3p.
[0046] FIG. 21A and FIG. 2B depict tables listing the subject demographic and clinical annotation in the discovery and validation cohorts.
[0047] FIG. 22A-FIG. 22E show endobronchial biopsies divide into four distinct molecular subtypes that correlate with clinical and molecular phenotypes. FIG. 22A. Genes (n = 3936) organized into nine gene co-expression modules were used to discover four molecular subtypes (Proliferative, Inflammatory, Secretory, and Normal-like) across the 190 DC biopsies using consensus clustering.
The heatmap shows semi-supervised hierarchal clustering of z-score -normalized gene expression across the 3936 genes and 190 DC biopsies. The top color bar represents the four molecular subtypes: Proliferative (n = 52 samples), Inflammatory (n = 37 samples), Secretory (n = 61 samples), and Normal-like (n = 40 samples). To the left of the heatmap, barplots for each module show the mean module GSVA score for each subtype. To the right of the heatmap, a summary of enriched biological pathways is listed for each module. FIG. 22B. Bubbleplots showing significant associations (p < 0.01, two-sided Fisher’s exact test) between the molecular subtypes and genomic smoking status, biopsy histological grade, and the predicted LUSC tumor molecular subtypes. The columns represent the four molecular subtypes (Proliferative, Inflammatory, Secretory, and Normal-like) and the diameter of the circle is proportional to the number of samples within each subtype that have the row phenotype. FIG. 22C. Boxplot of MKI67 expression values in biopsies with normal or hyperplasia histology (n = 8, 16, 26, 18 in Proliferative, Inflammatory, Secretory, and Normal-like subtypes, respectively). The MKI67 expression levels of the Proliferative subtype are significantly greater than non-Proliferative subtype samples (FDR = 3.4e-10, linear model). FIG. 22D. Boxplot of expression values of MKI67 in biopsies with dysplastic histology (n = 33, 11, 19, 9 in Proliferative, Inflammatory, Secretory, and Normal -like subtypes, respectively). The MKI67 expression levels of the Proliferative subtype are significantly greater than non-Proliferative subtype samples (FDR= 3.1e-8). FIG. 22E. Immunofluore scent staining demonstrating the increased MKI67 and KRT5 staining and reduced TUB1A1 staining in the Proliferative subtype. The representative samples shown for the Proliferative and Inflammatory subtypes have dysplasia histology, whereas the samples shown for the Secretory and Normal-like subtypes have normal histology (Magnification c 200). In the boxplots, the upper and lower hinges correspond to the first and third quartile, center line represents the median, and whiskers extend from the hinge to the largest or smallest value at most 1.5 times the distance between the quartiles. In each of FIGs. 22A-22E, the series or columns are, from left to right, Proliferative (n = 52 samples), Inflammatory (n = 37 samples), Secretory (n = 61 samples), and Normal-like (n = 40 samples).
[0048] FIG. 23 depicts a table listing the molecular subtype characteristics in the discovery cohort.
[0049] FIG. 24A-24C Phenotypic associations with the molecular subtypes are confirmed in an independent sample set. FIG. 24A. The 190 DC biopsies and the 3936 genes were used to build a 22- gene nearest centroid molecular subtype classifier. The heatmap shows semi-supervised hierarchal clustering of z-score normalized gene expression across the 22 classifier genes and 190 DC biopsies training samples. FIG. 24B. The 22-gene nearest centroid molecular subtype classifier was used to predict the molecular subtypes of the 105 VC biopsies. The heatmap shows semi-supervised hierarchal clustering of z-score normalized gene expression across 22 genes and 105 VC. The rows of the heatmap give the gene name and module membership, and the column color bar shows molecular subtype membership. FIG. 24C. Bubbleplots showing significant associations (p < 0.01 by two-sided Fisher’s exact test) between the VC molecular subtypes and smoking status, biopsy histological grade, and the predicted LUSC tumor molecular subtypes. The columns represent the four molecular subtypes (from left to right: Proliferative, Inflammatory, Secretory, and Normal-like) and the radius of the circle is proportional to the number of samples within each subtype that have the row phenotype.
[0050] FIG. 25A-FIG. 25B show Normal-appearing bronchial brushes predict the presence of proliferative lesions. FIG. 25A. The DC (left) and VC (right) cohorts, showing the number of brushes (y axis) classified as proliferative orange) that have at least one biopsy (y axis) classified as proliferative at the time the brush was sampled. Brushes/biopsies classified as not proliferative are turquoise. FIG. 25B. Boxplots of GSVA scores for modules 4, 5, 6, and 7 (y axis) across all brushes (n = 86 in DC and n = 48 in VC) and biopsies (n = 190 in DC and n = 105 in VC) from each cohort classified as Proliferative or not Proliferative (x axis). The red asterisk indicates significant differences between the Proliferative subtype versus all other samples (FDR < 0.05, linear model). In the boxplots, the upper and lower hinges correspond to the first and third quartile, center line represents the median, and whiskers extend from the hinge to the largest or smallest value at most 1.5 times the distance between the quartiles.
[0051] FIG. 26A-FIG. 26D show Immune alterations are associated with lesion outcome in the Proliferative subtype. Boxplots of Module 9 GSVA scores across DC FIG.26A and VC biopsies FIG. 26B within the Proliferative subtype. There is a significant difference between the
progressive/persistent versus regressive biopsies (p = 0.002 (DC) and p = 0.03 (VC), linear models). FIG. 26C Top: heatmap of z-score-normalized gene expression across the 112 genes in Module 9 in the DC biopsies (left) and the VC biopsies (right). Each heatmap is supervised by Module 9 GSVA scores. Top color bars indicate the histological grade of the biopsies and their progression status. Bottom: heatmap of xCell results indicating the relative abundance of immune cell types across the DC biopsies (left) and the VC biopsies (right). Immune cell types displayed are significantly associated with lesion progression/persistence (FDR < 0.05 in both the DC and VC, linear model). FIG. 26D. Representative histology where the dashed yellow line denotes the separation of epithelium and stromal compartments. Top panels: a progressive severe dysplasia has reduced presence of immune cells demonstrated by the marked reduction in expression of M2 macrophages (CD68/163 staining, double-positive cells indicated by the yellow arrows) and CD8 T cells (sample corresponds to *P in c). Bottom panels: a regressive moderate dysplasia has increased presence of immune cells including M2 macrophages (CD68/163 staining double-positive cells indicated by the yellow arrows) and CD8 T cells (samples correspond to *R in c). FIG. 26E. Boxplots of the percentages of CD68 and CD163, CD68, CD163, CD4, and CD8 positively stained cells between progressive/persistent and regressive biopsies (p < 0.001, linear model, for all comparisons). The x axis labels indicate the number of regions (R) enumerated across (P) subjects for each stain and outcome group depicted in the boxplot. Biopsies were included in the analysis if their clinical outcome was concordant with the Module 9 score. In the boxplots, the upper and lower hinges correspond to the first and third quartile, center line represents the median, and whiskers extend from the hinge to the largest or smallest value at most 1.5 times the distance between the quartiles.
[0052] FIG. 27A-FIG. 27B depict gene expression level TIMM 13 (translocase of inner mitochondrial membrane 13). This gene is expressed more highly in lesions that progress than those the regress. It is negatively correlated to the expression of a gene set representing B Cell presence, indicating that it can prevent recruitment or activity of B Cells to pre-malignant lesions. TIMM 13 has not been previously associated with B Cell activity, nor has it been implicated in the formation of cancer.
[0053] FIG. 28A-28B depict gene expression levels of TMEM63C: transmembrane protein 63C This gene is expressed more highly in lesions that progress than those that regress. It is negatively correlated to the expression of a gene set representing MCHII Antigen Presentation, indicating that it can prevent recruitment or activity of antigen presenting cells. This gene has not been previously associated with antigen presentation, nor has it been implicated in the formation of cancer.
[0054] FIG. 29A-FIG. 29C depict gene expression level of GSTP1 (glutathione S-transferase pi 1).
This gene is expressed more highly in lesions that progress than those that regress. It is negatively correlated to both our B Cells and MCHII Antigen Presentation modules. GSTP1 is known to be upregulated in certain cancers, including Lung Squamous Cell Carcinoma, which we have confirmed in data from The Cancer Genome Atlas. This gene is also hypermethylated in B Cell Lymphomas, further indicating it plays a role in preventing B Cell proliferation (Rossi et ah, 2004). Several small molecule inhibitors of GSTP1 exist, which we propose could readily be used to prevent its immune- suppressive activity. Some examples of inhibitors include: Ethacrynic acid and analogues,
Ethacraplatin, NBDHEX, Auranofm, Glutathione analogues, Piperlongumine, and others (Allocati et ah, 2018). [0055] FIG. 30A-FIG. 30B depicts gene expression levels of QPRT (quinolinate phosphoribosyltransferase). This gene is upregulated in lesions that regress compared to those the progress. It is positively correlated with expression of our B Cells gene set (FIG. 3 OB), indicating it plays a role in B Cell mediated immunity in premalignant lesions. QPRT has previously been associated with a pro-inflammatory response in monocytes (Jones et al., 2015), but has not been implicated previously in B cell immunity. This gene was found to be anti-apoptotic in leukemic K562 cells, but whether it has this role in differentiated leukocytes is yet unstudied (Ullmark et al., 2017).
[0056] FIG. 31A-FIG. 3 IB depict gene expression level of CPQ: carboxypeptidase Q
This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. No previous evidence implicates CPQ in immune activation.
[0057] FIG. 32A-FIG. 32B depict gene expression level of MRAS (muscle RAS oncogene homolog). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Previous work has demonstrated that M-Ras knockout mice have reduced B Cell abundance and function, indicating that M-Ras is important for proper B Cell activity (Freyer et al., 2012). This association between MRAS, B Cell development, and lung cancer has not been previously identified.
[0058] FIG. 33A-FIG. 33B depict gene expression level of RCAN1 (regulator of calcineurin 1). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Previous work has identified RCAN1 as both a suppressor of the NF-kB pathway and also a target of NF-kB transcriptional regulation (Liu et al., 2015; Zheng et al, 2014). This role as a suppressor of NF-kB, a transcription factor also essential for B Cell survival, is somewhat in conflict with the positive association between RCAN 1 and B cell activity that we have identified in our premalignant lesions. However, RCAN1 has been previously identified as pro- inflammatory and an important part of both macrophage and T cell mediated immunity (Bhoiwala et al., 2011; Han et al., 2017). Therefore, our work indicates a potentially novel role for RCAN1 in our unique context.
[0059] FIG. 34A-FIG. 34B depict gene expression level of SERPINIl (serpin peptidase inhibitor, clade I (neuroserpin), member 1). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of our B Cells gene set, indicating it can play a role in B Cell mediated immunity in premalignant lesions. No previous evidence implicates SERPINIl in immune activation.
[0060] FIG. 35A-FIG. 35B depict gene expression level of B2M (beta-2 -macroglobulin).
This gene is more highly expressed in regressing lesions than progressing lesions, and is positively correlated to the expression of an Interferon Response gene set. B2M is involved in presentation of antigens via the MCH class I genes, making it an essential component of adaptive immune activation. Interferon is known to upregulate B2M and induce expression of MCH class I genes (Fellous et al., 1979).
[0061] FIG. 36A-FIG. 361 depict gene expression level of HLA-DRB1, HLA-DPA1, HLA-DRA (Major Histocompatibility Class II genes). All of these genes are more highly expressed in regressing lesions than progressing lesions, and are positively correlated to either both Interferon Response, and B Cell immunity. These genes are highly expressed in B Cells and are necessary for presenting antigens for activation of adaptive immunity.
[0062] FIG. 37A-FIG. 37B depict gene expression level of MSC (Musculin). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with expression of our B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. Musculin has been previously implicated in regulation of T lymphocytes (Thl7 cells) (Santarlasci et al, 2017), but has not been studied in B cell activity.
[0063] FIG. 38A-FIG. 38B depict gene expression level of SMURF 1 (SMAD specific E3 ubiquitin protein ligase 1). This gene is expressed more highly in lesions that progress than those that regress. It is negatively correlated to the expression of our B Cell gene set. SMURF 1 has been shown to target TRAF proteins for ubiquitination, reducing activity of the NF-kB pathway, an essential pathway in B Cell survival (Ui et al., 2010).
[0064] FIG. 39A-FIG. 39B depict gene expression level of SUC5A8 (solute carrier family 5 (sodium/monocarboxylate cotransporter), member 8). This gene is upregulated in lesions that regress compared to those that progress. It is positively correlated with the expression of the B Cells gene set, indicating it plays a role in B Cell mediated immunity in premalignant lesions. SUC5A8 has been identified as a tumor suppressor in both colon and head and neck cancer (Bennett et al., 2008; Gurav et al., 2015), but its function in B cell immunity and lung premalignancy has not yet been reported.
[0065] FIG. 40 depicts the prediction of Progression/Regression in Proliferative lesions from validation cohorts, using the top two genes from mediation analysis (using binomial glm trained in discovery cohort, genes: SERPINIl and GSTP1).
DETAILED DESCRIPTION
[0066] As described herein, the inventors have discovered a group in immune regulators that control early-stage immune responses, e.g., at the time of tumor formation, disease prevention.
Accordingly, provided herein are methods relating to the level of these immune regulators that can be used to diagnose, detect and/or treat conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer). Additionally, the inventors demonstrate that modulating the level of these genes can be used to diagnose, detect and/or treat conditions caused by or exacerbated by aberrant immune system activity, e.g., lung cancer. Accordingly, provided herein are methods for the detection, diagnosis, and treatment of a condition associated with aberrant immune system activity in a subject and uses thereof, e.g. by therapeutically modulating the level of those immune response regulators.
[0067] For example, by analyzing the molecular events involved in the development of bronchial premalignant lesions (PMLs) molecular subtypes the inventors found that certain co-expressed gene modules are associated with histological severity and progression to invasive cancer. PMLs arise in airway epithelium and are precursors to squamous cell carcinoma. The inventors found that among proliferative PMLs, the expression levels of genes related to interferon signaling and antigen processing/presentation were decreased in progressive/persistent lesions, showing early immune suppression is related to lesion progression. Accordingly, the inventors describe a novel set of immune regulators that are responsible for immune suppression in bronchial premalignant lesions that are likely to persist or progress to higher-grade lesion or invasive cancer. In other embodiments, such immune regulators can fight off infection. Previous targets for immune activation in the context of cancer have focused primarily on late stage tumors and have focused on T cell mediated immunity. Genes that have been discovered to mediate this immune pathway include PD1, PDL1, and CTLA4. The immune regulators identified herein modulate the activity of MHC class II antigen presentation, interferon signaling, and B cell related immunity and regulate immune activation and suppression before tumors even form. Accordingly, the inventors have developed methods for determining if the bronchial premalignant lesions are likely to progress to cancer and new therapies for bronchial premalignant lesions which target the underlying molecular changes which characterize the bronchial premalignant lesions.
[0068] In one aspect of any of the embodiments, provided herein is a method for treating or preventing a condition caused by or associated with immunosuppressed aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a
therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject. In one aspect of any of the embodiments, provided herein is a method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
[0069] There are numerous methods for determining whether a change in expression level is statistically significant known to the skilled artisan. For example, see the methodology used in the Figures and accompanying examples. One can also take a“snapshot” of a particular individual over time and see changes. In one aspect of any embodiment, the change of the immune response relative to a normal control (the reference level) should be at least one standard deviation (s), preferably at least two s from the mean. In this case, the deviation between a normal control, i.e., a population of individuals that have normal levels, would be either an increased level of expression of at least one s greater than the mean, preferably at least two s greater, or a decreased level of expression of at least one s less than the mean, preferably at least two s. However, differences that are less than one s can be statistically significant in an individual.
[0070] In one embodiment, there must be at least two changes in immune response regulators relative to the reference level. The two changes can be (a) at least two increases of a negative immune response regulator, (b) at least two decreases of a positive immune response regulator; or a combination of at least one increase in a negative immune response regulator and a decrease of at least one positive immune response regulator.
[0071] In one embodiment, there must be at least 2... , at least 3... , at least 5... , at least 7... , at least 10... , at least 15... , at least 20. .. changes in positive immune response regulators, negative immune response regulators, and/or combinations thereof.
[0072] In one embodiment, a positive immune regulator is selected from Table 1 and a negative immune regulator is selected from Table 2.
[0073] In one aspect of any of the embodiments, provided herein is a method of treating immunosuppressed aberrant immune system activity in a subject having or at risk of developing a condition associated with immunosuppressed aberrant immune system activity, the method comprising: i) administering a treatment for the condition associated with immunosuppressed aberrant immune system activity to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level of expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression. In one aspect of any of the embodiments, provided herein is a method of treating immunosuppressed aberrant immune system activity in a subject having or at risk of developing a condition associated with immunosuppressed aberrant immune system activity, the method comprising: i) obtaining a sample from the subject, determining the level of expression of at least 1 negative or positive immune response regulator; and iii) administering a treatment for the condition associated with
immunosuppressed aberrant immune system activity if the subject is determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level of expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
[0074] In one aspect of any of the embodiments, provided herein is a method of treating autoimmune aberrant immune system activity in a subject having or at risk of developing a condition associated with autoimmune aberrant immune system activity, the method comprising: i)
administering a treatment for the condition associated with autoimmune aberrant immune system activity to a subject determined to have a level of expression of at least 1 negative immune response regulator which is decreased relative to a reference level of expression of at least 1 positive immune response regulator which is increased relative to a reference level of expression. In one aspect of any of the embodiments, provided herein is a method of treating autoimmune aberrant immune system activity in a subject having or at risk of developing a condition associated with autoimmune aberrant immune system activity, the method comprising: i) obtaining a sample from the subject, determining the level of expression of at least 1 negative or positive immune response regulator; and iii) administering a treatment for the condition associated with autoimmune aberrant immune system activity if the subject is determined to have a level of expression of at least 1 negative immune response regulator which is decreased relative to a reference level of expression of at least 1 positive immune response regulator which is increased relative to a reference level of expression.
[0075] In one aspect of any of the embodiments, provided herein is a method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
ii. at periodic intervals, for example, at least every 2 months ... , every 4 months ... every 6 months ... , every 8 months ... , every 9 months ... , every year... , every 18 months, every two years... , one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
[0076] In one aspect of any of the embodiments, provided herein is a method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) administering at least one of:
v. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; vi . at least every 2 months ... , every 4 months ... every 6 months ... , every 8 months ... , every 9 months ... , every year... , every 18 months, every two years ... , one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
vii. at least one immune stimulating drug; and/or viii. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
[0077] As used herein,“aberrant immune system activity” refers to one or more immune system responses which are inappropriately over or under-responsive to a disease agent and/or cell of the subject. As used herein, an“immune response” refers to a response by one or more cells of the immune system to a pathological or pathogenic stimulus. Cells of the immune system include, but are not limited to B cells, regulatory B cells, T cells, regulatory T cells, antigen-presenting cells, dendritic cells, monocytes, macrophages, NKT cells, NK cells, basophils, eosinophils and neutrophils.
Aberrant immune system activity can include either immunosuppression or autoimmune activity.
[0078] As used herein,“immunosuppression” refers to an active process whereby one or more components of the adaptive or innate immune system is or are prevented from acting against a given target. The immune system includes naturally immunosuppressive function mediated by immune inhibitory receptors or cytokines expressed on the surface of an immune cell, and their interactions with their ligands. For example, cytotoxic CD8 T cells can enter a state of“functional exhaustion,” or “unresponsiveness” whereby they express inhibitory receptors that inhibit or prevent antigen-specific responses, such as proliferation and inflammatory cytokine production. Accordingly, by inhibiting the activity and/or expression of such inhibitory receptors or cytokines, an immune response to a persistent infection or to a cancer or tumor that is suppressed, inhibited, or ineffective, can be enhanced or” un-inhibited”.
[0079] As used herein,“autoimmune activity” refers to immune system activity targeted against an individual’s own health tissues or cells. As used herein, the term "autoimmune disease" or "autoimmune disease or disorder” herein is a disease or disorder arising from and directed against an individual's own tissues or cells or manifestation thereof or resulting condition therefrom. Auto immune related diseases and disorders arise from an overactive and/or abnormal immune response of the body against substances (autoantigens) and tissues normally present in the body, otherwise known as self or autologous substance. This dysregulated inflammatory reaction causes an exaggerated response by macrophages, granulocytes, and/or T-lymphocytes leading to abnormal tissue damage and cell death. Subsequent loss of function is associated with inflammatory tissue damage.
[0080] A condition caused by or associated with aberrant immune system activity is a condition that arises when, or worsens when the activity of the immune system in the subject is aberrant. Such conditions are well known in the art and include cancer, autoimmune disease, and infectious disease.
[0081] As used herein, the term“a condition caused by or associated with immunosuppression” refers to a condition, disease or disorder in which the function of the immune response is below a desired level, e.g. a level that can treat or prevent at least one symptom of the disease or disorder. As a non-limiting example, inappropriate immune suppression can be associated with certain cancer tumors in which cells of the immune system fail to attack or are prevented from attacking the tumor, such that thus the immune system fails to effectively reduce or prevent tumor growth. Other examples of inappropriate immunosuppression include, for example, immunosuppression immunosuppression- mediated by a pathogenic organism (infectious disease), and autoimmune disease.
[0082] As used herein, an“immune response regulator” refers to a gene expression product that can alter one or more of: the activity of MHC class II antigen presentation, interferon signaling, and B cell related immunity. As used herein, a“tumor formation immune response regulator” is a type of immune response regulator and refers to a gene expression product can regulate one or more of: the activity of MHC class I and II antigen presentation, interferon signaling, and B cell related immunity prior to tumor formation. An immune response regulator can exert its immune regulating activity as a nucleic acid (e.g., a miRNA), or as polypeptide.
[0083] An immune response regulator can be a positive immune response regulator or a negative immune response regulator. A positive immune response regulator is one which acts to increase immune system activity. Exemplary positive immune response regulators are provided in Table 1 herein. In some embodiments of any of the aspects, the positive immune response regulator is a gene expression product of Table 1 or a gene expression product of a gene of Table 1. In some
embodiments of any of the aspects, the positive immune response regulator is one or more of: NLR family CARD domain containing 5 (NLRC5), Quinolinate phosphoribosyltransferase (QPRT), Carboxypeptidase O (CPQ), Muscle RAS oncogene homolog (MRAS), Regulator of calcineurin 1 (RCAN1), Serpin family I member 1 (SERPINI1), Beta-2-microglobulin (B2M), Major
histocompatibility complex, class II, DR beta 1 (HLA-DRBl), Major histocompatibility complex, class II, DP alpha 1 (HLA-DPA1), major histocompatibility complex, class II, DR alpha (HLA-DRA), Musculin (MSC), and/or solute carrier family 5 member 8 (SLC5A8).
[0084] A negative immune response regulator is one which acts to decrease immune system activity. Exemplary negative immune response regulators are provided in Table 2 herein. In some embodiments of any of the aspects, the negative immune response regulator is a gene expression product of Table 2 or a gene expression product of a gene of Table 2. In some embodiments of any of the aspects, the negative immune response regulator is one or more of: Translocase of inner mitochondrial membrane 13 homolog (TIMM13), Transmembrane protein 63C (TMEM63C), Glutathione S-transferase pi 1 (GSTP1), SMAD specific E3 ubiquitin protein ligase 1 (SMURF1), miR-149-5p.
[0085] Table 1: Positive immune response regulators. Gene names, NCBI Gene ID, Nucleic acid sequences, Polypeptide sequences and NCBI reference Sequence IDs are also listed.
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0002
[0086] Table 2: Negative immune response regulators. Gene names, NCBI Gene ID, Nucleic acid sequences, Polypeptide sequences and NCBI reference Sequence IDs are also listed.
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
[0087] Table 2A. miR Associated with Proliferative Subtype
Figure imgf000040_0002
Figure imgf000041_0001
[0088] In one embodiment of any aspect, when the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of NLRC5, B2M, HLA-DRB1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change. In one embodiment of any aspect, the different immune response regulator is selected from Table 1 or Table 2.
[0089] In one embodiment of any aspect, when the change in a negative immune response regulator is a miR, the miR is miR-149-5p.
[0090] In one embodiment of any aspect, the different marker can also include an miRNA from Table 2A.
[0091] In one embodiment of any aspect, there must be at least 2... , at least 3... , at least 5... , at least 7... , at least 10... , at least 15... , at least 20... changes in Table 2A, to indicate proliferation, e.g., a premalignant lesion of the proliferative subtype or a cancer.
[0092] For example, the aforementioned changes in miR’s in Table 2A could be used to predict an individual proliferative subtype (high-grade legions) of a premalignant lesion or a cancer, for example, lung squamous premalignant legions, and then, one could use the markers in Table 1 and/or Table 2 to identify the state of the immune activity. For example, in one embodiment of any aspect, one would look at miR-149-5p expression. In one embodiment of any aspect, there must be at least 2... , at least 3..., at least 5..., at least 7..., at least 10... , at least 15... , at least 20... changes in positive immune response regulators, negative immune response regulators, and/or combinations thereof to indicate the immune activity.
[0093] As discussed above, the changes in expression should be biologically significant, for example statistically significant, a change from a prior level of expression from that individual, a standard deviation from a norm.
[0094] If one identifies that the individual has lesions of the proliferative subtype, one could use an anti-proliferative drug, for example, Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors;
Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; JNK inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors;
Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
[0095] In some embodiments of any of the aspects, the methods and assays described herein relate to detecting the level of expression of at least two miRNAs, at least three miRNAs, at least four miRNAs, at least five miRNAs, at least six miRNAs, at least seven miRNAs, at least eight miRNAs, or all of the miRNAs selected from the group consisting of: of miR-548b-5p, miR-642a-5p, miR- 642b-5p, miR-328a-3p, miR-34b-3p, miR-34b-5p, miR-3664-3p, miR-548i, and miR-766-3p. Where a subset of the 9 foregoing miRNAs is used, any combination of the miRNAs can be used in each of various embodiments of the aspects described herein. For example, when the level of at least two miRNAs is detected, it is specifically contemplated herein that any pairwise combination of the nine miRNAs can be detected, e.g., any combination shown in Table 2B. Fow expression of these markers is indicative of the proliferative subtype. High expression is good. Thuse, use of agonists to increase expression of these miRNA is one method of treatment.
[0096] Table 2B: Contemplated exemplary combinations of miRNAs associated with
Proliferative Subtype are indicated by“X”.
Figure imgf000042_0001
Figure imgf000043_0001
sequences for the precursor and mature form are known for a variety of species, e.g. human miR-149 (NCBI Gene ID NO: 406941; NCBI transcript accession number NR_029702.1; SEQ ID NO: 1) and mature sequence miR-149-5p (SEQ ID NO: 2). A“miR-149-5p oligonucleotide” can be the precursor and/or mature form of miR-149-5p (e.g., SEQ ID NO: 2). In some embodiments, miR-149- 5p can be human miR-149-5p, e.g., hsa-miR-149-5p. (see, e.g. also Table 3).
[0098] As used herein,“miR-548b-5p” refers to a mature miRNA derived from miR-548b-5p. The sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 548b-5p (NCBI Gene ID NO: 693128; NCBI transcript accession number NR_030315.1; SEQ ID NO: 3) and mature sequence miR-548b-5p (SEQ ID NO: 4). A“miR-548b-5p oligonucleotide” can be the precursor and/or mature form of miR-548b-5p (e.g., SEQ ID NO: 4). In some embodiments, miR-548b-5p can be human miR-548b-5p, e.g., hsa- miR-548b-5p.
[0099] As used herein,“miR-642a-5p” refers to a mature miRNA derived from miR-642a-5p. The sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 642a-5p (NCBI Gene ID NO: 693227; NCBI transcript accession number NR_030372.1; SEQ ID NO: 5 ) and mature sequence miR-642a-5p (SEQ ID NO: 6). A“miR-642a-5p oligonucleotide” can be the precursor and/or mature form of miR-642a-5p (e.g., SEQ ID NO: 6). In some embodiments, miR-642a-5p can be human miR-642a-5p, e.g., hsa-miR-642a-5p.
[00100] As used herein,“miR-642b-5p” refers to a mature miRNA derived from miR-642b-5p. The sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 642b-5p
(NCBI Gene ID NO: 100500845; NCBI transcript accession number NR_037512.1; SEQ ID NO: 7) and mature sequence (SEQ ID NO: 8). A“miR-642b-5p oligonucleotide” can be the precursor and/or mature form of miR-642b-5p (e.g., SEQ ID NO: 8). In some embodiments, miR-642b-5p can be human, e.g., hsa miR-642b-5p.
[00101] As used herein,“miR-328a-3p” refers to a mature miRNA derived from miR-328a-3p. The sequences for the precursor and mature form are known for a variety of species, e.g. human miR- 328a-3p (NCBI Gene ID NO: 442901; NCBI transcript accession number NR_029887.1 ; SEQ ID NO: 9) and mature sequence miR-328a-3p (SEQ ID NO: 10). A“miR-328a-3poligonucleotide” can be the precursor and/or mature form of miR-328a-3p (e.g., SEQ ID NO: 10). In some embodiments, miR- 328a-3p
can be human, e.g., hsa- miR-328a-3p.
[00102] As used herein,“miR-34b-5p” refers to a mature miRNA derived from miR-34b-5p. The sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 407041; NCBI transcript accession number NR_029839; SEQ ID NO: 11) and mature sequence (SEQ ID NO: 13). A“oligonucleotide” can be the precursor and/or mature form of miR- 34b-5p (e.g., SEQ ID NO: 13). In some embodiments, miR-34b-5p can be human, e.g., hsa-miR-34b- 5p.
[00103] As used herein,“miR-3664-3p” refers to a mature miRNA derived from miR-3664-3p. The sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 100500844; NCBI transcript accession number NR_037437.1; SEQ ID NO: 14) and mature sequence (SEQ ID NO: 15). A“miR-3664-3p oligonucleotide” can be the precursor and/or mature form of miR-3664-3p (e.g., SEQ ID NO: 15). In some embodiments, miR-3664-3p can be human, e.g., hsa-miR-3664-3p.
[00104] As used herein,“miR-548i” refers to a mature miRNA derived from miR-548i. The sequences for the precursor and mature form are known for a variety of species, e.g. human miR-548i (NCBI Gene ID NO: 100302204; NCBI transcript accession number NR_031687.1; SEQ ID NO: 16) and mature sequence (SEQ ID NO: 17). A“miR-548i oligonucleotide” can be the precursor and/or mature form of miR-548i (e.g., SEQ ID NO: 17). In some embodiments, miR-548i can be human, e.g., hsa-miR-548i.
[00105] As used herein,“miR-766-3p” refers to a mature miRNA derived from miR-766-3p. The sequences for the precursor and mature form are known for a variety of species, e.g. human (NCBI Gene ID NO: 768218; NCBI transcript accession number NR_030413.1; SEQ ID NO: 18) and mature sequence (SEQ ID NO: 19). A“miR-766-3p oligonucleotide” can be the precursor and/or mature form of (e.g., SEQ ID NO: 19). In some embodiments, miR-766-3p can be human, e.g., has- miR-766-3p.
[00106] Table 3: List of miRNAs including their NCBI gene IDs, NCBI transcript accession number, miRBase accession numbers, and sequences.
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
[00107] The sequences of expression products for the immune response regulators described herein are known for a number of species, e.g., human. Sequences for various species are readily obtained in online databases, e.g., NCBI.
[00108] As described herein, levels of gene expression of immune response regulators can be modulated (e.g., increased or decreased) in certain subjects, e.g., those with or at risk of developing a condition caused by or associated with aberrant immune system activity in a subject in need thereof (e.g. infectious disease, autoimmune disease, or cancer (e.g. premalignant lesions of different subtypes)).
[00109] In some embodiments of any of the aspects, the methods described herein comprise administering a treatment described herein to a subject previously determined to have an expression level(s) of one or more immune response regulators as described herein. In some embodiments of any of the aspects, described herein is a method of treating a condition caused by or associated with aberrant immune system activity in a subject in need thereof, the method comprising: a) first determining the level of expression of the at least one immune response regulator in a sample obtained from a subject; and b) then administering a treatment as described herein to the subject if the level of expression of the at least one immune response regulator is modulated relative to a reference in the manner described herein. In one aspect of any of the embodiments, described herein is a method of a condition caused by or associated with aberrant immune system activity in a subject in need thereof, the method comprising: a) determining if the subject has a modulation of a level of expression of at least one immune response regulator as described herein and b) instructing or directing that the subject be administered the appropriate treatment described herein for the particular modulation of expression which has been determined.
[00110] In some embodiments of any of the aspects, the step of determining if the subject has modulation of an expression level of at least one immune response regulator can comprise i) obtaining or having obtained a sample from the subject and ii) performing or having performed an assay on the sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise performing or having performed an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject.
In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise ordering or requesting an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise receiving the results of an assay on a sample obtained from the subject to determine/measure the level of expression of at least one immune response regulator in the subject. In some embodiments of any of the aspects, the step of determining if the subject has a modulation of a level of expression of at least one immune response regulator can comprise receiving a report, results, or other means of identifying the subject as a subject with a modulation of a level of expression of at least one immune response regulator.
[00111] In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results. In some embodiments of any of the aspects, the step of instructing or directing that the subject be administered a particular treatment can comprise providing a report of the assay results and/or treatment recommendations in view of the assay results.
[00112] In some embodiments of any of the aspects, measurement of the level of a target and/or detection of the level or presence of a target, e.g. of an expression product (nucleic acid or polypeptide of one of the immune response regulators described herein) or a mutation can comprise a transformation. As used herein, the term“transforming” or“transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but is not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve the action of at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzymes, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments of any of the aspects, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).
[00113] Transformation, measurement, and/or detection of a target molecule, e.g. an mRNA or polypeptide can comprise contacting a sample obtained from a subject with a reagent (e.g. a detection reagent) which is specific for the target, e.g., a target-specific reagent. In some embodiments of any of the aspects, the target-specific reagent is detectably labeled. In some embodiments of any of the aspects, the target-specific reagent is capable of generating a detectable signal. In some embodiments of any of the aspects, the target-specific reagent generates a detectable signal when the target molecule is present.
[00114] Methods to measure gene expression products are known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a detectable marker whose presence and location in the subject is detected by standard imaging techniques.
[00115] For example, antibodies for the various targets described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels. Alternatively, since the amino acid sequences for the targets described herein are known and publically available at the NCBI website, one of skill in the art can raise their own antibodies against these polypeptides of interest for the purpose of the methods described herein.
[00116] The amino acid sequences of the polypeptides described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat. The sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art. In some embodiments of any of the aspects, the sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the fding date of this application.
[00117] In some embodiments of any of the aspects, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change of color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.
[00118] In some embodiments of any of the aspects, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two- dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.
[00119] Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiments of any of the aspects, the immunoassay can be a quantitative or a semi- quantitative immunoassay.
[00120] An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as blood or serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.
[00121] Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
[00122] In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (e.g., any of the targets as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.
[00123] In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., a blood sample) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then are added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away.
Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3', 5, 5'- tetramethylbenzidene) color development substrate for localization of horseradish peroxidase- conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce significant color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate. [00124] There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in "Methods in
Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.
[00125] In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests used for medical diagnostics, either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tissue samples etc. Strip tests are also known as dip stick tests, the name bearing from the literal action of "dipping" the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments of any of the aspects, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.
[00126] The use of "dip sticks" or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos.
4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U. S. patent applications Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of "dip stick" technology to detect soluble antigens via immunochemical assays include, but are not limited to US Patent Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a "dip stick" which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the "dip stick," prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this "dip stick" technology for the detection of polypeptides using antibody reagents as described herein.
[00127] Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. A dot blot immobilizes a protein sample on a defined region of a support, which is then probed with antibody and labelled secondary antibody as in Western blotting. The intensity of the signal from the detectable label in either format corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.
[00128] In some embodiments of any of the aspects, the level of a target can be measured, by way of non-limiting example, by Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofhioresence assay; mass spectroscopy and/or Immunoelectrophoresis assay.
[00129] In certain embodiments, the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of one or more of the immune response regulators described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a blood sample. Techniques for the detection of mRNA expression is known by persons skilled in the art, and can include but not limited to, PCR procedures, RT-PCR, quantitative RT-PCR Northern blot analysis, differential gene expression, RNAse protection assay, microarray based analysis, next-generation sequencing; hybridization methods, etc.
[00130] In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence -specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.
[00131] In some embodiments of any of the aspects, the level of an mRNA can be measured by a quantitative sequencing technology, e.g. a quantitative next-generation sequence technology.
Methods of sequencing a nucleic acid sequence are well known in the art. Briefly, a sample obtained from a subject can be contacted with one or more primers which specifically hybridize to a single - strand nucleic acid sequence flanking the target gene sequence and a complementary strand is synthesized. In some next-generation technologies, an adaptor (double or single-stranded) is ligated to nucleic acid molecules in the sample and synthesis proceeds from the adaptor or adaptor compatible primers. In some third-generation technologies, the sequence can be determined, e.g. by determining the location and pattern of the hybridization of probes, or measuring one or more characteristics of a single molecule as it passes through a sensor (e.g. the modulation of an electrical field as a nucleic acid molecule passes through a nanopore). Exemplary methods of sequencing include, but are not limited to, Sanger sequencing, dideoxy chain termination, high-throughput sequencing, next generation sequencing, 454 sequencing, SOLiD sequencing, polony sequencing, Illumina sequencing, Ion Torrent sequencing, sequencing by hybridization, nanopore sequencing, Helioscope sequencing, single molecule real time sequencing, RNAP sequencing, and the like.
Methods and protocols for performing these sequencing methods are known in the art, see, e.g.“Next Generation Genome Sequencing” Ed. Michal Janitz, Wiley-VCH;“High-Throughput Next Generation Sequencing” Eds. Kwon and Ricke, Humanna Press, 2011; and Sambrook et ah, Molecular Cloning:
A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); which are incorporated by reference herein in their entireties.
[00132] The nucleic acid sequences of the genes described herein have been assigned NCBI and ENSBL accession numbers for different species such as human, mouse and rat. The sequences for any of the genes described herein can be readily retrieved from either database by one of ordinary skill in the art. In some embodiments of any of the aspects, the sequence of a gene, transcript, or polypeptide described herein is the sequence available in the NCBI or ENSMBL database as of the filing date of this application. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.
[00133] Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).
[00134] In some embodiments of any of the aspects, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of
incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.
[00135] In some embodiments of any of the aspects, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluorescence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, biolumine scent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.
[00136] In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled reagent is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments of any of the aspects, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6- carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2',4',7',4,7- hexachlorofmorescein (HEX), 6-carboxy-4',5'-dichloro-2',7'-dimethoxyfmorescein (JOE or J), N,N,N',N'-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5- carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g.
Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments of any of the aspects, a detectable label can be a radiolabel including, but not limited to 3H, 1251, 35S, 14C, 32P, and 33P. In some embodiments of any of the aspects, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a
chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha- glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments of any of the aspects, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments of any of the aspects, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.
[00137] In some embodiments of any of the aspects, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies
immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromogenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, CA. A reagent can also be detectably labeled using fluorescence emitting metals such as 152Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTP A) or ethylenediaminetetraacetic acid (EDTA).
[00138] In some embodiments of any of the aspects, the level of expression is the level in a sample obtained from a subject. The term“sample” or“test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a blood or tissue sample from a subject. In some embodiments of any of the aspects, the present invention encompasses several examples of a biological sample. In some embodiments of any of the aspects, the biological sample is cells, or tissue, or peripheral blood, or bodily fluid. Exemplary biological samples include, but are not limited to, a biopsy, a tumor sample, biofluid sample; blood; serum; plasma; urine; sperm; mucus; tissue biopsy; organ biopsy; synovial fluid; bile fluid; cerebrospinal fluid; mucosal secretion; effusion; sweat; saliva; and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term“test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments of any of the aspects, a test sample can comprise cells from a subject. In some embodiments of any of the aspects, the sample obtained from a subject can be a biopsy sample. In some embodiments of any of the aspects, the sample obtained from a subject can be a blood or serum sample.
[00139] In some embodiments of any of the aspects, the sample is an endobronchial biopsy, bronchial brushing sample (e.g., bronchial biopsy, endobronchial brushing sample, large airway biopsy, large airway brushing sample), nasal epithelial cells, sputum, and/or blood obtained from the subject. In some embodiments of any of the aspects, the sample is a bronchial brushing obtained from the right or left mainstem bronchus. The test sample can be obtained by removing a sample from a subject, but can also be accomplished by using a previously isolated sample (e.g. isolated at a prior timepoint and isolated by the same or another person).
[00140] In some embodiments of any of the aspects, the test sample can be an untreated test sample. As used herein, the phrase“untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments of any of the aspects, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments of any of the aspects, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments of any of the aspects, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments of any of the aspects, the test sample can be treated with a chemical and/or biological reagent.
Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.
[00141] In some embodiments of any of the aspects, the methods, assays, and systems described herein can further comprise a step of obtaining or having obtained a test sample from a subject. In some embodiments of any of the aspects, the subject can be a human subject. In some embodiments of any of the aspects, the subject can be a subject in need of treatment for (e.g. having or diagnosed as having) a condition caused by or associated with aberrant immune system activity in a subject in need thereof or a subject at risk of or at increased risk of developing a condition caused by or associated with aberrant immune system activity in a subject in need thereof as described elsewhere herein. [00142] In some embodiments of any of the aspects, the biopsy or brushing sample comprises morphologically-normal tissues or cells, e.g., the tissues or cells are not from a lesion and display normal morphology for their in vivo location. As described herein, the inventors have surprisingly found that the modulation of the immune response regulators occurs even in tissues that are not themselves diseased, or which will become diseased. Thus, the level of immune response regulators in easily-sampled areas of the body can provide useful information about what is happening or likely to happen in more inaccessible areas of the body.
[00143] In some embodiments of any of the aspects, the biopsy or brushing sample consists essentially of morphologically-normal tissues or cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists of morphologically-normal tissues or cells.
[00144] In some embodiments of any of the aspects, the biopsy or brushing sample comprises visually-normal tissues or cells, e.g., the tissues or cells are not from a lesion and to the unaided human eye have a normal appearance for their in vivo location. In some embodiments of any of the aspects, the biopsy or brushing sample consists essentially of visually-normal tissues or cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists of visually-normal tissues or cells.
[00145] In some embodiments of any of the aspects, the biopsy or brushing sample comprises bronchial premalignant lesion cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists essentially of bronchial premalignant lesion cells. In some embodiments of any of the aspects, the biopsy or brushing sample consists of bronchial premalignant lesion cells.
[00146] A level which is less than a reference level can be a level which is less by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, or less relative to the reference level. In some embodiments of any of the aspects, a level which is less than a reference level can be a level which is statistically significantly less than the reference level.
[00147] A level which is more than a reference level can be a level which is greater by at least about 10%, at least about 20%, at least about 50%, at least about 60%, at least about 80%, at least about 90%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or more than the reference level. In some embodiments of any of the aspects, a level which is more than a reference level can be a level which is statistically significantly greater than the reference level.
[00148] In some embodiments of any of the aspects, the reference can be a level of the target molecule in a population of subjects who do not have or are not diagnosed as having, and/or do not exhibit signs or symptoms a condition caused by or associated with aberrant immune system activity, e.g., a healthy subject. In some embodiments of any of the aspects, the reference can also be a level of expression of the target molecule in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same. In some embodiments of any of the aspects, the reference can be the level of a target molecule in a sample obtained from the same subject at an earlier point in time, e.g., the methods described herein can be used to determine if a subject’s sensitivity or response to a given therapy is changing over time or if the subtype of their lesions is changing. In some embodiments of any of the aspects, the reference can be the level of a target molecule in a sample obtained from the a subject without lesions, e.g., bronchial lesions and/or premalignant lesions.
[00149] In some embodiments of any of the aspects, the level of expression products of no more than 200 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 other genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 other genes is/are determined.
[00150] In some embodiments of any of the aspects, the level of expression products of no more than 200 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 100 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 20 genes is/are determined. In some embodiments of any of the aspects, the level of expression products of no more than 10 genes is/are determined.
[00151] In some embodiments of the foregoing aspects, the expression level of a given gene can be normalized relative to the expression level of one or more reference genes or reference proteins.
[00152] In some embodiments, the reference level can be the level in a sample of similar cell type, sample type, sample processing, and/or obtained from a subject of similar age, sex and other demographic parameters as the sample/subject for which the level of expression is to be determined.
In some embodiments, the test sample and control reference sample are of the same type, that is, obtained from the same biological source, and comprising the same composition, e.g. the same number and type of cells.
[00153] In some embodiments of any of the aspects, the reference level can be a non-proliferative reference level, e.g., the level in a tissue or cell not comprising a proliferative lesion or from a subject who does not have a proliferative lesion. For example, the level can be the level in inflammatory, secretory, or normal-like lesion subtypes or an average or pooling thereof.
[00154] In some embodiments of any of the aspects, the level of expression of at least 1 immune response regulator is detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 2 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 3 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 4 immune response regulators are detected, determined, or measured. In some embodiments of any of the aspects, the levels of expression of at least 5 immune response regulators are detected, determined, or measured. [00155] In some embodiments of any of the aspects, the methods described herein relate to detecting or measuring the level of expression of at least 1 miRNA selected from the group consisting of: miR-149-5p, miR-548b-5p, miR-642a-5p, miR-642b-5p, miR-328a-3p, miR-34b-3p, miR-34b-5p, miR-3664-3p, miR-548i, and miR-766-3p. In some embodiments of any of the aspects, the methods described herein relate to detecting or measuring the level of expression of at least miR-149-5p. In some embodiments of any of the aspects, the methods described herein relate to administering an inhibitor of at least miR-149-5p.
[00156] In some embodiments of any of the aspects, any one of the immune response regulators of Table 1 or 2 can be detected/measured and/or targeted. In some embodiments of any of the aspects, 2 or more immune response regulators can be detected/measured and/or targeted. All possible pairwise or greater combinations are contemplated herein. Tables 4 and 5 depict exemplary but non-limiting examples of pairwise combinations of the immune response regulators which can be detected or targeted in the present methods.
[00157] Table 4: Contemplated exemplary combinations of the positive immune response regulators NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINI1, B2M, HLA-DRB1, HLA-DPA1, HLA-DRA, MSC, and SLC5A8 are indicated by“X”.
Figure imgf000059_0001
Figure imgf000060_0001
[00158] Table 5: Contemplated exemplary combinations of the negative immune response regulators are indicated by“X”.
Figure imgf000060_0002
Figure imgf000061_0001
[00159] In one aspect of any of the embodiments, provided herein is a method comprising measuring the level of expression of at least 1 negative immune response regulator described herein and/or the level of expression of at least 1 positive immune response regulator described herein in a sample obtained from a subject. In some embodiments of any of the aspects, the sample is an endobronchial biopsy, endobronchial brushing sample, large airway biopsy, large airway brushing sample, nasal epithelial cells, sputum, or blood obtained from the subject. In some embodiments of any of the aspects, the sample is a bronchial brushing obtained from the right or left mainstem bronchus. In some embodiments of any of the aspects, the sample comprises or consists of morphologically-normal tissues or cells.
[00160] The change of the immune response relative to a normal control (the reference level) should be at least one standard deviation (s), preferably at least two s from the mean. In this case, the deviation between a normal control, i.e., a population of individuals that have normal levels, would be either an increased level of expression of at least one s greater than the mean, preferably at least two s greater, or a decreased level of expression of at least one s less than the mean, preferably at least two s.
[00161] In one embodiment, there must be at least two changes in immune response regulators relative to the reference level. The two changes can be (a) at least two increases of a negative immune response regulator, (b) at least two decreases of a positive immune response regulator; or a combination of at least one increase in a negative immune response regulator and a decrease of at least one positive immune response regulator.
[00162] In one embodiment, there must be at least 2... , at least 3... , at least 5... , at least 7... , at least 10... , at least 15... , at least 20... changes in positive immune response regulators, negative immune response regulators, and/or combinations thereof. It is contemplated herein that the methods and assays described herein can be combined with methods and assays of the prior art, e.g., those that examine other markers of aberrant immune system activity. It is particularly contemplated that the presently described methods and assays can be combined with the methods and assays described in International Patent Publication W02002/0041243. When methods and assays are combined, they can be practiced in series, in parallel, or as part of the same kit or multiplexed method or assay.
[00163] As described herein, the immune response regulators can be therapeutically modulated in order to correct aberrant immune system activity and thereby prevent or treat disease. Accordingly, contemplated herein are methods and pharmaceutical compositions that target these immune response regulators to inhibit the negative immune response regulators or agonize the positive immune response regulators and thereby be effective in delaying or preventing the development of conditions associated with aberrant immune system activity (e.g. infectious disease, autoimmune disease, and cancer).
[00164] As used herein, the term“agonist" refers to an agent which increases the expression and/or activity of the target by at least 10% or more, e.g. by 10% or more, 50% or more, 100% or more, 200% or more, 500% or more, or 1000 % or more. The efficacy of an agonist, e.g. its ability to increase the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and/or the activity of the target. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RTPCR with primers can be used to determine the level of RNA, and Western blotting with an antibody can be used to determine the level of a polypeptide. Suitable primers for a given target are readily identified by one of skill in the art, e.g., using software widely available for this purpose (e.g., Primer3 or PrimerBank, which are both available on the world wide web). Antibodies to polypeptide gene expression products of the immune response regulators described herein are commercially available, e.g., from AbCam
(Cambridge, MA). Assays for measuring the activity of the targets described herein are provided elsewhere herein. In some embodiments of any of the aspects, an agonist of a given polypeptide can be the polypeptide, a nucleic acid encoding the polypeptide, or a small molecule.
[00165] Non-limiting examples of agonists of a given polypeptide target, can include the target polypeptides or variants or functional fragments thereof and nucleic acids encoding the polypeptide or variants or functional fragments thereof. In some embodiments of any of the aspects, the agonist of a given target, is a polypeptide of that target or variants or functional fragment thereof and/or a nucleic acid encoding the polypeptide or variant or functional fragment thereof. In some embodiments of any of the aspects, the polypeptide agonist can be an engineered and/or recombinant polypeptide. In some embodiments of any of the aspects, the polypeptide agonist can be a nucleic acid encoding a polypeptide, e.g. a functional fragment thereof. In some embodiments of any of the aspects, the nucleic acid can be comprised by a vector.
[00166] In some embodiments of any of the aspects, a polypeptide agonist can comprise one of the sequences provided herein for each target. In some embodiments of any of the aspects, a polypeptide agonist can consist essentially of one of the sequences provided below herein for each target. In some embodiments of any of the aspects, a polypeptide agonist can consist of one of the sequences provided below herein for each target. In some embodiments of any of the aspects, an agonist can comprise a nucleic acid encoding one of the sequences provided below herein for each target. In some embodiments of any of the aspects, an agonist can be a polypeptide comprising a reference/wild-type sequence provided herein with at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the reference/wild-type sequence and which retains the activity of the reference/wild-type sequence. In some embodiments of any of the aspects, an agonist can be a polypeptide comprising a reference/wild-type sequence provided herein with at least 95% identity to the reference/wild-type sequence and which retains the activity of the reference/wild-type sequence.
[00167] In some embodiments of any of the aspects, the agonist an exogenous polypeptide. In some embodiments of any of the aspects, the subject is administered exogenous polypeptide, e.g., the polypeptide is produced in vitro and/or synthesized and purified polypeptide is provided to the subject. In some embodiments of any of the aspects, the agonist is an ectopic polypeptide. In some embodiments of any of the aspects, the subject is administered ectopic polypeptide, e.g., the polypeptide is produced in vitro and/or synthesized and purified polypeptide is provided to the subject.
[00168] In some embodiments of any of the aspects, the agonist can be a nucleic acid encoding a polypeptide (or a variant or functional fragment thereof) and/or a vector comprising a nucleic acid encoding a polypeptide (or a variant or functional fragment thereof). A nucleic acid encoding a polypeptide can be, e.g., an RNA molecule, a plasmid, and/or an expression vector. In some embodiments of any of the aspects, the nucleic acid encoding a polypeptide can be an mRNA. In some embodiments of any of the aspects, the nucleic acid encoding a polypeptide can be a modified mRNA. In some embodiments of any of the aspects, the agonist can be a nucleic acid encoding a polypeptide, e.g., exogenous and/or ectopic polypeptide. In some embodiments of any of the aspects, the subject is administered the nucleic acid encoding exogenous and/or ectopic polypeptide, e.g., the nucleic acid is transcribed and/or translated after the administering step to provide exogenous and/or ectopic polypeptide to the subject.
[00169] As used herein,“inhibitor” refers to an agent which can decrease the expression and/or activity of a target, e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98 % or more. The efficacy of an inhibitor of one or more targets, e.g. its ability to decrease the level and/or activity of the target can be determined, e.g. by measuring the level of an expression product of the target and/or the activity of the target. In some embodiments of any of the aspects, the inhibitor can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule. An inhibitor of a target described herein can inhibit the activity, expression, or accumulation of the target polypeptide. Inhibitors can include inhibitors that act directly on the target itself (e.g., that bind to the protein or transcript, e.g., direct inhibitors).
[00170] In some embodiments of any of the aspects, an inhibitor of a specified target is an antibody, antibody reagent, or antigen-binding fragment thereof, that specifically binds to the target.
[00171] In some embodiments of any of the aspects, the agent that inhibits a given target is an inhibitory nucleic acid. In some embodiments of any of the aspects, inhibitors of the expression of a given gene can be an inhibitory nucleic acid. As used herein,“inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), miRNAs, amiRNAs, and the like.
[00172] In some embodiments of any of the aspects, a polypeptide or nucleic acid as described herein can be engineered. As used herein,“engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polypeptide is considered to be“engineered" when at least one aspect of the polypeptide, e.g., its sequence, has been manipulated by the hand of man to differ from the aspect as it exists in nature. As is common practice and is understood by those in the art, progeny of an engineered cell are typically still referred to as“engineered" even though the actual manipulation was performed on a prior entity.
[00173] In some embodiments of any of the aspects, the agonist and/or inhibitor is administered as a nucleic acid. In some embodiments of any of the aspects, a nucleic acid encoding the agonist and/or inhibitor is administered. In some embodiments of any of the aspects, the subject is administered a vector comprising a nucleic acid. Vectors can be, e.g., a DNA or RNA vector.
[00174] In some embodiments of any of the aspects, the methods described herein relate to treatment of bronchial premaligant lesions. In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having bronchial premalignant lesions. In some embodiments of any of the aspects, the condition caused by or associated with aberrant immune system activity is a premalignant lesion. In some embodiments of any of the aspects, the condition caused by or associated with aberrant immune system activity is a bronchial premalignant lesion.
[00175] As used herein,“premalignant lesion” refers to an epithelial lesion or dysplasia which is a precursor or can be a precursor to cancer. The basement membrane is intact with no possibility of metastatic spread, as opposed to cancer. A bronchial premalignant lesion is a premalignant lesion present in the bronchial epithelium of a subject. Bronchial premalignant lesions are typically small and can be difficult to visualize using conventional white light bronchoscopy.
[00176] Subjects having bronchial premalignant lesions can be identified by a physician using current methods of diagnosing bronchial premalignant lesions. Tests that can aid in a diagnosis of, e.g. bronchial premalignant lesions include, but are not limited to, bronchoscopy, autofluorescence bronchoscopy, etc. A family history of lung cancer, prior history of lung cancer, exposure to risk factors for bronchial premalignant lesions (e.g. cigarette smoke), presence of chronic obstructive pulmonary disease (COPD) can also aid in determining if a subject is likely to have bronchial premalignant lesions or in making a diagnosis of bronchial premalignant lesions.
[00177] Standard treatment for subjects at risk of lung cancer, or who have been identified to have bronchial premalignant lesions, is annual screening for lung cancer (e.g. a bronchoscopy and/or chest CT scan). When a subject with bronchial premalignant lesions and aberrant immune system active, such treatment is no longer sufficient and the subject should be treated more aggressively, according to the methods described herein.
[00178] As used herein,“a bronchoscopy-based procedure” refers to any endoscopic technique that permits examination of the bronchus and/or lungs. Bronchoscopy-based procedures can include white light bronchoscopy, autofluorescence bronchoscopy, flexible bronchoscopy, rigid bronchoscopy, bronchoalveolar lavage, and the like. Bronchoscopy-based procedures can further include biopsy, brushing, or tissue sampling.
[00179] An immune -stimulating drug is a drug that increases the activity of the immune system, preferably against cancer or dysplastic cells, wherein that is the primary activity of the compound in the relevant context. As used herein, the term“immune-stimulating drug” is used to describe any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) which can be used stimulate the immune system. Non-limiting examples of immune stimulating drugs can include immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti -viral drugs that improve interferon signaling such as
Pegintron, Pegasys, referon A, uniferon, multiferon, rebif, avonex, cinnovex, betaseron, actimmune, reiferon, pegetron); DNA synthesis inhibitors (e.g., TAS-102, NC-6004, ganciclovir); CDK inhibitors (e.g. purvalanol-a, palbociclib, ribociclib, abemaciclib, and olomoucine II); ribonucleotide reductase inhibitors (e.g., motexafm, hydroxyurea, fludarabine, cladribine, gemcitabine, tezacitabine, triapine, gallium maltolate, gallium nitrate); dihydrofolate reductase inhibitors (e.g., methotrexate, piritrexam, cycloguanil, JPC-2056); topoisomerase inhibitors (e.g. pidorubicine, doxorubicin, campothecins, indenosioquinolines, indotecan, imdimitecan, amsacrine, etoposide, teniposide, ICRF-193, genistein); FLT3 inhibitors (e.g. lestaurtinib, TG-101348, gilteritinib, quizartinib, midostaurin, sorafenib, sunitinib); IGF-1 inhibitors; MEK inhibitors (e.g., trametinib, cobimetinib, binimetinib, selumetinib, PD-325901, TAK-733); aurora kinase inhibitors (e.g.,ZM447439, hesperidin, VX-680); PKC inhibitors (e.g., ruboxistaurin, chelerythrine, miyabenol C, myricitrin, gossypol, verbascoside, BIM-1, bryostate 1, tamoxifen); RAF inhibitors (e.g., vemurafenib, GDC-0879, PLX-4720, sorafenib, dabrafenib, LGX818); PDFGR/KIT inhibitors (e.g., imatinib, sunitinib, sorafenib, pazopanib, nilotinib, motesanib, linifenib); VEGFR inhibitors (e.g., axitinib, cabozantinib, lenvatinib, pazopanib, vandetanib); SRC inhibitors (e.g., KX2-391, bosutinib, saracatinib, PP1, PP2, quercetin, dastabinib); retinoid receptor agonists (e.g., alitretinoin, isoretinoin); HDAC inhibitors (e.g. THM-I-94, vorinostat, givinostat);DNA methyltransferase inhibitors (e.g., azacytidine, decitabine, zeublarine); and EZH2 inhibitors (DZNep, EPZ005687, Ell, GSK126, UNC1999, EPZ-6438, tazemetostat).
[00180] It is noted herein that a single compound may exhibit multiple activities, e.g., depending on the context. Non-examples of agents that can exhibit primarily an anti-inflammatory activity and/or an anti -proliferative activity, depending on the context (e.g., the subject or cell being administered/contacted with the agent) can include Acetylcholine receptor antagonist,
Acetylcholinesterase inhibitors, Adenosine receptor antagonists, Adrenergic receptor antagonists, Angiotensin receptor antagonists, Apoptosis stimulants, Aurora kinase inhibitors, CDK inhibitors, Cyclooxygenase inhibitors, Cytokine production inhibitors, Dehydrogenase inhibitors, Dopamine receptor antagonist, EGFR inhibitors, ERK1 and ERK2 phosphorylation inhibitors, Estrogen receptor agonists, FLT3 inhibitors, Glucocorticoid receptor agonists, Glutamate receptor antagonists, HDAC inhibitors, Histamine receptor antagonists, Histone lysine methyltransferase inhibitors, HSP inhibitors, IKK inhibitors, Ion channel antagonists, KIT inhibitors, Leucine rich repeat kinase inhibitors, MEK inhibitors, MDM inhibitors, Phosphodiesterase inhibitors, Monoamine oxidase inhibitors, MTOR inhibitors, NFkB pathway inhibitors, nucleophosmin inhibitors, PARP inhibitors, PI3K inhibitors, PPAR receptor agonist, RAF inhibitors, SIRT inhibitors, Sodium channel blockers, Topoisomerase inhibitors, Tyrosine kinase inhibitors, VEGFR inhibitors, and a Vitamin D receptor agonists.
[00181] In some embodiments, immune stimulating drugs lacking anti-proliferative/inflammatory activity in any context described herein can include immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti-viral drugs that improve interferon signaling); DNA synthesis inhibitors; IMDH inhibitors; ribonucleotide reductase inhibitors; dihydrofolate reductase inhibitors; SRC inhibitors; retinoid receptor agonists; HDAC inhibitors; and DNA methyltransferase inhibitors. [00182] As used herein, the term "anti-inflammatory" refers to a compound capable of reducing or inhibiting inflammation, wherein that is the primary activity of the compound in the relevant context. As used herein, the term“anti-inflammatory drug” or“anti-inflammatory agent” is used to describe any compound (including its analogs, derivatives, prodrugs and pharmaceutically salts) which can be used reduce or inhibit inflammation. Non-limiting examples of anti-inflammatory drugs can include NFkB pathway inhibitors (e.g. 9-methyl-5H-6-thia-4,5-diaza-chrysene-6, 6-dioxide, denosumab, disulfiram, olmesartan, dithiocarbamates, anatabine, BAY 11-7082, palmitoylethanolamide, iguartimod); protein synthesis inhibitors (e.g. chloramphenicol); anti-ILIB antibodies (e.g.,
Canakinumab); glucocorticoid receptor agonists (e.g. dexamethasone, mifepristone,); and TGF beta receptor inhibitors (e.g. LY-364947, GW-755.55, LY-2109761, galunisertib, SB431542, SB-525334). Further non-limiting examples of anti-proliferative drugs include Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; Angiotensin receptor antagonists; Apoptosis stimulants; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; Glutamate receptor antagonists; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; IKK inhibitors; Ion channel antagonists; Leucine rich repeat kinase inhibitors; MDM inhibitors;
Monoamine oxidase inhibitors; nucleophosmin inhibitors; PPAR receptor agonists;
Phosphodiesterase inhibitors; SIRT inhibitors; sodium channel blockers; and Vitamin D receptor agonists. In some embodiments, anti-inflammatory drugs lacking anti-proliferative activity in any context described herein can include protein synthesis inhibitors and TGF beta receptor inhibitors.
[00183] Conversely, if the subject with bronchial premalignant lesions is indicated to be at low risk of progression to malignancy, e.g., the level of expression of at least 1 negative immune response regulator is not increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is not decreased relative to a reference level of expression, then the subject can be provided with treatment options appropriate for a subject who will not develop lung cancer or has a low risk of developing lung cancer in the next 6-12 months. Such treatments can include, e.g., a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue without performing a chest CT scan; or at greater than 6 month intervals, either: a) a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue or b) a chest CT scan. Patients in this low-risk group can also be refused or not administered an immune stimulating drug or chemotherapeutic agent.
[00184] The methods described herein can also relate to treatment or prevention of conditions associated with aberrant immune system activity, e.g., infectious disease, autoimmune conditions, or cancer. In some embodiments of any of the aspects, the treatment can be treatment of a patient with early or less aggressive forms of the condition, e.g., to reduce the risk or progression to later or more aggressive forms of the disease. E.g., the patient could be pre-symptomatic, exposed, have a latent condition, or the like. In some embodiments of any of the aspects, the treatment can be prophylactic. As used herein“prophylactic” refers to the timing and intent of a treatment relative to a disease or symptom, that is, the treatment is administered prior to clinical detection or diagnosis of that particular disease or symptom in order to protect the patient from the disease or symptom.
Prophylactic treatment can encompass a reduction in the severity or speed of onset of the disease or symptom, or contribute to faster recovery from the disease or symptom. In some embodiments of any of the aspects, prophylactic treatment is not prevention of all symptoms or signs of a disease.
[00185] Methods of identifying and diagnosing the conditions associated with aberrant immune system activity described herein are well known in the art and readily practiced by clinicians. In some embodiments of any of the aspects, the subject is administered a treatment for the conditions associated with aberrant immune system activity. Such treatments are well known in the art.
[00186] For example, a subject determined to have or be at risk of having an infectious disease according to the methods described herein, or determined to be at risk of progressing in an infectious disease according to the methods described herein (e.g., a subject determined to have increased expression of a negative immune response regulator or decreased expression of a positive immune response regulator) can be administered an antiviral or antibiotic. Such treatments are known in the art, e,g., antivirals and antibiotics. In some embodiments of any of the aspects, the infectious disease is a bacterial or viral infection. In some embodiments of any of the aspects, the viral infection is a coronavirus infection, e.g., a SARS-CoV-2 infection. Treatment and/or diagnosis of SARS-CoV-2 is particularly contemplated herein for methods relating to miR-149-5p due to its prevalence in the basal epithelial cell populations affected by SARS-CoV-2.
[00187] In some embodiments of these aspects and all such aspects described herein, the subject in need thereof has or has been diagnosed with an infectious disease. An infectious disease, also known as a transmissible disease or communicable disease, is an illness resulting from a chronic and/or latent infection.
[00188] For individuals suspected of having COVID-19, knowing the state of their immune system is important for additional treatment regimes. In addition to treatment as described above, identifying individuals with a weakened immune response permits identification of patients in need of early and extreme care. For example, early treatment with Gilead’s Remdesivir (C27H35N608P) or other forms of treatment including ventilator, etc. One can also do plasma transplants containing antibodies against COVID-19.
[00189] In a“chronic infection,” the infectious agent is present in the subject at all times.
However, the signs and symptoms of the disease can be present or absent for an extended period of time. Non-limiting examples of infectious viral disease include COVID-19, hepatitis B (caused by hepatitis B virus (HBV)) and hepatitis C (caused by hepatitis C virus (HCV)) adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, human herpesvirus 6, varicella-zoster virus, hepatitis B virus, hepatitis D virus, papilloma virus, parvovirus B19, polyomavirus BK, polyomavirus JC, measles virus, rubella virus, human immunodeficiency virus (HIV), human T cell leukemia virus I, and human T cell leukemia virus II. Parasitic persistent infections can arise as a result of infection by, for example, Leishmania, Toxoplasma, Trypanosoma, Plasmodium, Schistosoma, and Encephalitozoon.
[00190] In some embodiments, an infectious disease can be a latent infection. In some embodiments, a chronic infection can include periods in which the infection is a latent infection. In a “latent infection,” the infectious agent (such as a virus) is seemingly inactive and dormant such that the subject does not always exhibit signs or symptoms. In a latent viral infection, the virus remains in equilibrium with the host for long periods of time before symptoms again appear; however, the actual viruses cannot typically be detected until reactivation of the disease occurs. Non-limiting examples of latent infections include infections caused by herpes simplex virus (HSV)-l (fever blisters), HSV-2 (genital herpes), and varicella zoster virus VZV (chickenpox-shingles).
[00191] As used herein,“antiviral” refers to any chemical or biological agent with therapeutic usefulness in the inhibition of viral transmission, activity, or replication. Categories of antivirals can include, but are not limited to entry inhibitors, uncoating inhibitors, viral synthesis inhbitiors, assembly inhibitors, and release inhibitors. Exemplary, non-limiting antivirals include enfuvirtide, amantadine, rimantadine, pleconaril, acyclovir, zidovudine, lamivudine, fomivirsen, rifampicin, zanamivir, oseltamivir, peramivir, abacavir, acyclovir, adefovir, amprenavir, baloxavir marboxil, boceprevir, cobicistat, combivir, daclatasvir, doravirine, etravirine, ganciclovir, ibalizumab, letermovir, rilpivirine, simeprevir, telbivudine, and valciclovir. One of skill in the art can readily identify an antiviral agent of use e.g. see Antiviral Drugs, Wieslaw M. Kazmierski (ed.) Wiley and Sons (2011); Antiviral Drugs, John S. Driscoll. Wiley and Sons (2005); each of which is incorporated by reference herein in its entirety.
[00192] As used herein,“antibiotic” refers to any chemical or biological agent with therapeutic usefulness in the inhibition of bacterial cell growth or in killing bacteria, e.g, those that are bactericidal or bacteriostatic. Categories of antibiotics can include, but are not limited to those that target the bacterial cell wall (e.g., penicillins, cephalosporins), those that target the bacterial cell membrane (e.g., polymyxins), those that target bacterial enzymes (e.g., rifamycins, lipiarmycins, quinolones, sulfonamides), protein synthesis inhibitors (e.g., macrolides, lincosamides, and tetracyclines) , aminoglycosides, cyclic lipopeptides, glycyclines, oxazolidinones, beta-lactams, and lipiarmycins. Exemplary, non-limiting antibiotics include penicillin, methicilling, nafcillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, ampicillin, amoxicillin, pivampicillin, hetacillin,
bacampicillin, metampicillin, talamipicillin, epicillin, cabenicillin, ticaricillin, temocillin, mezlocillin, piperacillin, azolocillin, clavulanic acid, sulbactam, tazobactam, cafadroxil, cephalexin, cefalotin, cefapirin, cefazolin, cefradine, cefaclor, cefonicid, cefprozil, cefuroxime, loracarbef, cefmetazole, cefotetan, cefoxitin, cefotiam, cefdinir, cefixime, cefotaxime, cefovecin, cefpodoxime, ceftibuten, ceftiofur, ceftizoxime, ceftriaxone, cefoperazone, ceftazimdime, latamoxef, cefepime, cefiderocol, cefpriome, rifampicin, rifabutin, rifapentine, rifamixin, fidaxomicin, ciproflaxicin, moxifloxacin, levofloxacin, sulfafurzole, azithromycin, clarithromycin, erythromycin, fidaxomicin, spiramycin, telihtromycin, lincomycin, clindamycin, pirlimycin, tetracycline, eravacycline, sarecycline, omadacycline, doxycycline, kanamycin, tobramycin, gentamicin, neomycin, streptomycin, vancomycin, tigecycline, linezolid, posizolid, tedizolid, radezolid, cycloserine, contezolid, and daptomycin. One of skill in the art can readily identify an antibiotic agent of use e.g. see Antibiotics in Laboratory Medicine, Victor Lorian (ed.) Wolters Kluwer; and Antibotics Manual, David Schlossberg and Rafik Samuel, John Wiley and Sons (2017); each of which is incorporated by reference herein in its entirety.
[00193] As a further example, a subject determined to have or be at risk of having cancer according to the methods described herein, or determined to be at risk of progressing in cancer according to the methods described herein (e.g., a subject determined to have increased expression of a negative immune response regulator or decreased expression of a positive immune response regulator) can be administered a chemotherapeutic. Such treatments are known in the art. In some embodiments of any of the aspects, the cancer is a squamous cell cancer, e.g., a lung squamous cell cancer.
[00194] Non-limiting examples of cancer treatments can include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI- 103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1- TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone;
aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene;
edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet;
pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor- firee, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone -Poulenc Rorer, Antony, France);
chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16);
ifosfamide; mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11)
(including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoids such as retinoic acid; capecitabine;
combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
[00195] In addition, the methods of treatment for cancer can further include the use of radiation or radiation therapy. Further, the methods of treatment can further include the use of surgical treatments.
[00196] One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Physicians' Cancer Chemotherapy Drug Manual 2014, Edward Chu, Vincent T. DeVita Jr., Jones & Bartlett Learning; Principles of Cancer Therapy, Chapter 85 in Harrison's Principles of Internal Medicine, 18th edition; Therapeutic Targeting of Cancer Cells: Era of Molecularly Targeted Agents and Cancer Pharmacology, Chs. 28-29 in Abeloff s Clinical Oncology, 2013 Elsevier; and Fischer D S (ed): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 2003).
[00197] In some embodiments the condition caused by or associated with aberrant immune system activity is cancer (e.g., lung cancer, small cell lung cancer or non-small cell lung cancer). Subjects having lung cancer can be identified by a physician using current methods of diagnosing lung cancer. Symptoms and/or complications of lung cancer which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, coughing, coughing blood, shortness of breath, chest pain, wheezing, hoarseness, difficulty breathing, unexplained weight loss, bone pain, and headaches. Tests that may aid in a diagnosis of, e.g. lung cancer include, but are not limited to, x-rays, CT scan, sputum cytology, or biopsies. A family history of lung cancer, prior history of lung cancer, presence of other chronic lung disease such as COPD, or exposure to risk factors for lung cancer (e.g. radon or asbestos exposure) can also aid in determining if a subject is likely to have lung cancer or in making a diagnosis of lung cancer.
[00198] As used herein, the term“cancer” relates generally to a class of diseases or conditions in which abnormal cells divide without control and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymph systems. There are several main types of cancer. Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood. Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system. Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.
[00199] Examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer;
esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intra-epithelial neoplasm.; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); lymphoma including Hodgkin’s and non-Hodgkin’s lymphoma; melanoma; myeloma; neuroblastoma; oral cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of the respiratory system; salivary gland carcinoma; sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; cancer of the urinary system; vulval cancer; as well as other carcinomas and sarcomas; as well as B-cell lymphoma (including low grade/follicular non-Hodgkin’s lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom’s Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatoses, edema (such as that associated with brain tumors), and Meigs’ syndrome.
[00200] In some embodiments of any of the aspects, the cancer is a primary cancer. In some embodiments of any of the aspects, the cancer is a malignant cancer. As used herein, the term “malignant” refers to a cancer in which a group of tumor cells display one or more of uncontrolled growth (i.e., division beyond normal limits), invasion (i.e.. intrusion on and destruction of adjacent tissues), and metastasis (i.e. , spread to other locations in the body via lymph or blood). As used herein, the term“metastasize” refers to the spread of cancer from one part of the body to another. A tumor formed by cells that have spread is called a“metastatic tumor” or a“metastasis.” The metastatic tumor contains cells that are like those in the original (primary) tumor. As used herein, the term“benign” or“non-malignant” refers to tumors that may grow larger but do not spread to other parts of the body. Benign tumors are self-limited and typically do not invade or metastasize.
[00201] A“cancer cell” or“tumor cell” refers to an individual cell of a cancerous growth or tissue. A tumor refers generally to a swelling or lesion formed by an abnormal growth of cells, which may be benign, pre -malignant, or malignant. Most cancer cells form tumors, but some, e.g., leukemia, do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.
[00202] A subject that has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject’s body. Included in this definition are malignant, actively proliferative cancers, as well as potentially dormant tumors or micrometastatses. Cancers which migrate from their original location and seed other vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.
[00203] In some embodiments of any of the aspects, the cancer is a lung squamous cell cancer. A lung squamous cell cancer develops from non-cancerous lesions in the airway known as bronchial premalignant lesions. The presence of persistent or progressive dysplastic bronchial premalignant lesions is a marker of increased lung cancer risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung. Not all bronchial premalignant lesions progress to invasive cancer, and those that do, progress at variable rates with variable outcomes.
[00204] As a further example, a subject determined to have or be at risk of having an autoimmune condition according to the methods described herein, or determined to be at risk of progressing in an autoimmune condition according to the methods described herein (e.g., a subject determined to have decreased expression of a negative immune response regulator or increased expression of a positive immune response regulator) can be administered an immunosuppressive, a steroid, or the like. Such treatments are known in the art. In some embodiments of any the aspects, the autoimmune condition is rheumatoid arthritis, lupus, and celiac disease.
[00205] Autoantigens, as used herein, are endogenous proteins or fragments thereof that elicit this pathogenic immune response. Autoantigen can be any substance or a portion thereof normally found within a mammal that, in an autoimmune disease, becomes the primary (or a primary) target of attack by the immune system. The term also includes antigenic substances that induce conditions having the characteristics of an autoimmune disease when administered to mammals. Additionally, the term includes peptic subclasses consisting essentially of immunodominant epitopes or immunodominant epitope regions of autoantigens. Immunodominant epitopes or regions in induced autoimmune conditions are fragments of an autoantigen that can be used instead of the entire autoantigen to induce the disease. In humans afflicted with an autoimmune disease, immunodominant epitopes or regions are fragments of antigens specific to the tissue or organ under autoimmune attack and recognized by a substantial percentage (e.g. a majority though not necessarily an absolute majority) of autoimmune attack T-cells.
[00206] Autoantigens that are known to be associated with autoimmune disease include myelin proteins with demyelinating diseases, e.g. multiple sclerosis and experimental autoimmune myelitis; collagens and rheumatoid arthritis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65); islet cell antigen (ICA512; ICA12) with insulin dependent diabetes.
[00207] A common feature in a number of autoimmune related diseases and inflammatory conditions is the involvement of pro-inflammatory CD4+ T cells. These T cells are responsible for the release of inflammatory, Thl type cytokines. Cytokines characterized as Thl type include interleukin 2 (IL-2), g-interferon, TNFa and IL-12. Such pro-inflammatory cytokines act to stimulate the immune response, in many cases resulting in the destruction of autologous tissue. Cytokines associated with suppression of T cell response are the Th2 type, and include IL-10, IL-4 and TGF-b. It has been found that Thl and Th2 type T cells may use the identical antigen receptor in response to an immunogen; in the former producing a stimulatory response and in the latter a suppressive response.
[00208] In one embodiment of any one of the method described, the autoimmune disorder is selected from the group consisting of thyroiditis, type 1 diabetes mellitus, Hashimoto's thyroidits, Graves' disease, celiac disease, multiple sclerolsis, Guillain-Barre syndrome, Addison's disease, and Raynaud's phenomenon, Goodpasture's disease, arthritis (rheumatoid arthritis such as acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen-induced arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, vertebral arthritis, and juvenile -onset rheumatoid arthritis, arthritis chronica progrediente, arthritis deformans, polyarthritis chronica primaria, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis such as plaque psoriasis, gutatte psoriasis, pustular psoriasis, and psoriasis of the nails, atopy including atopic diseases such as hay fever and Job's syndrome, dermatitis including contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular dermatitis, seborrheic dermatitis, non-specific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory diseases, urticaria such as chronic allergic urticaria and chronic idiopathic urticaria, including chronic autoimmune urticaria, myositis, polymyositis/dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (including systemic scleroderma), sclerosis such as systemic sclerosis, multiple sclerosis (MS) such as spino-optical MS, primary progressive MS (PPMS), and relapsing remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, sclerosis disseminata, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (for example, Crohn's disease, autoimmune -mediated gastrointestinal diseases, colitis such as ulcerative colitis, colitis ulcerosa, microscopic colitis, collagenous colitis, colitis polyposa, necrotizing enterocolitis, and transmural colitis, and autoimmune inflammatory bowel disease), bowel inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndrome, including adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, an autoimmune hematological disorder, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage as in meningitis, herpes gestationis, pemphigoid gestationis, pruritis scroti, autoimmune premature ovarian failure, sudden hearing loss due to an autoimmune condition, IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic and/or brainstem encephalitis, uveitis, such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, posterior uveitis, or autoimmune uveitis, glomerulonephritis (GN) with and without nephrotic syndrome such as chronic or acute
glomerulonephritis such as primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN or idiopathic membranous nephropathy, membrano- or membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, proliferative nephritis, autoimmune polyglandular endocrine failure, balanitis including balanitis circumscripta plasmacellularis, balanoposthitis, erythema annulare centrifugum, erythema dyschromicum perstans, eythema multiform, granuloma annulare, lichen nitidus, lichen sclerosus et atrophicus, lichen simplex chronicus, lichen spinulosus, lichen planus, lamellar ichthyosis, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reaction, eczema including allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and vesicular palmoplantar eczema, asthma such as asthma bronchiale, bronchial asthma, and auto-immune asthma, conditions involving infiltration of T cells and chronic
inflammatory responses, immune reactions against foreign antigens such as fetal A-B-0 blood groups during pregnancy, chronic pulmonary inflammatory disease, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, including lupus nephritis, lupus cerebritis, pediatric lupus, non-renal lupus, extra-renal lupus, discoid lupus and discoid lupus erythematosus, alopecia lupus, systemic lupus erythematosus (SLE) such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and lupus erythematosus disseminatus, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), adult onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, diabetic retinopathy, diabetic nephropathy, diabetic large-artery disorder, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, sarcoidosis, granulomatosis including lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitides, including vasculitis, large-vessel vasculitis (including polymyalgia rheumatica and giant-cell (Takayasu's) arteritis), medium-vessel vasculitis (including Kawasaki's disease and polyarteritis nodosa/periarteritis nodosa), microscopic polyarteritis, immunovasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis such as systemic necrotizing vasculitis, and ANCA- associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS) and ANCA-associated small-vessel vasculitis, temporal arteritis, autoimmune aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, hemolytic anemia or immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia (anemia pemiciosa), Addison's disease, pure red cell anemia or aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome such as those secondary to septicemia, trauma or hemorrhage, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, anti phospholipid antibody syndrome, allergic neuritis, Behcet's disease/syndrome, Castleman's syndrome, Goodpasture's syndrome, Reynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid such as pemphigoid bullous and skin pemphigoid, pemphigus (including pemphigus vulgaris, pemphigus foliaceus, pemphigus mucus-membrane pemphigoid, and pemphigus erythematosus), autoimmune polyendocrinopathies, Reiter's disease or syndrome, an immune complex disorder such as immune complex nephritis, antibody-mediated nephritis, polyneuropathies, chronic neuropathy such as IgM polyneuropathies or IgM-mediated neuropathy, and autoimmune or immune-mediated thrombocytopenia such as idiopathic thrombocytopenic purpura (ITP) including chronic or acute ITP, scleritis such as idiopathic cerato-scleritis, episcleritis, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune endocrine diseases including thyroiditis such as autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis, idiopathic hypothyroidism, Grave's disease, polyglandular syndromes such as autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), paraneoplastic syndromes, including neurologic paraneoplastic syndromes such as Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man or stiff-person syndrome, encephalomyelitis such as allergic encephalomyelitis or encephalomyelitis allergica and experimental allergic encephalomyelitis (EAE), myasthenia gravis such as thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, lupoid hepatitis, giant-cell hepatitis, autoimmune chronic active hepatitis, lymphoid interstitial pneumonitis (LIP), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis such as primary biliary cirrhosis and pneumonocirrhosis, autoimmune enteropathy syndrome, Celiac or Coeliac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease such as autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis such as refractory or relapsed or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome/nonsyphilitic interstitial keratitis, Bell's palsy, Sweet's disease/syndrome, rosacea autoimmune, zoster-associated pain, amyloidosis, a non-cancerous lymphocytosis, a primary lymphocytosis, which includes monoclonal B cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies including channelopathies of the CNS, autism, inflammatory myopathy, focal or segmental or focal segmental glomerulosclerosis (FSGS), endocrine opthalmopathy, uveoretinitis, chorioretinitis, autoimmune hepatological disorder, fibromyalgia, multiple endocrine failure, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia), male and female
autoimmune infertility, e.g., due to anti-spermatozoan antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortion, farmer's lung, erythema multiforme, post- cardiotomy syndrome, Cushing's syndrome, bird-fancier's lung, allergic granulomatous angiitis, benign lymphocytic angiitis, Alport's syndrome, alveolitis such as allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reaction, Sampter's syndrome, Caplan's syndrome, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial lung fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum et diutinum, erythroblastosis fetalis, eosinophilic faciitis, Shulman's syndrome, Felty's syndrome, cyclitis such as chronic cyclitis, heterochronic cyclitis, iridocyclitis (acute or chronic), or Fuch's cyclitis, Henoch-Schonlein purpura, SCID, sepsis, endotoxemia, post-vaccination syndromes, Evan's syndrome, autoimmune gonadal failure, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans, thyrotoxicosis, tabes dorsalis, chorioiditis, giant-cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, idiopathic nephritic syndrome, minimal change nephropathy, benign familial and ischemia-reperfusion injury, transplant organ reperfusion, retinal autoimmunity, aphthae, aphthous stomatitis, arteriosclerotic disorders, aspermiogenesis, autoimmune hemolysis, Boeck's disease, enteritis allergica, erythema nodosum leprosum, idiopathic facial paralysis, chronic fatigue syndrome, febris rheumatica, Hamman-Rich's disease, sensoneural hearing loss, ileitis regionalis, leucopenia, transverse myelitis, primary idiopathic myxedema, ophthalmia symphatica, polyradiculitis acuta, pyoderma gangrenosum, acquired spenic atrophy, vitiligo, toxic-shock syndrome, conditions involving infiltration of T cells, leukocyte-adhesion deficiency, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, diseases involving leukocyte diapedesis, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathies, oophoritis, primary myxedema, autoimmune atrophic gastritis, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine failure, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), myocarditis, nephrotic syndrome, primary sclerosing cholangitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, an eosinophil-related disorder such as eosinophilia, pulmonary infiltration eosinophilia, eosinophilia-myalgia syndrome, Loffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, granulomas containing eosinophils, seronegative spondyloarthritides, polyendocrine autoimmune disease, sclerosing cholangitis, sclera, episclera, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia telangiectasia syndrome, angiectasis, autoimmune disorders associated with collagen disease, rheumatism, allergic hypersensitivity disorders,
glomerulonephritides, reperfusion injury, ischemic re-perfusion disorder, lymphomatous
tracheobronchitis, inflammatory dermatoses, dermatoses with acute inflammatory components, and autoimmune uveoretinitis (AUR).
[00209] Subjects having an autoimmune disease can be identified by a physician using current methods of diagnosing an autoimmune disease. Symptoms and/or complications of an autoimmune disease which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, faituge, achy muscles, swelling and redness, low-grade fever, numbness aor tingling of the hands or feet, hair loss, and/or skin rash. Tests that may aid in a diagnosis of, e.g. autoimmune disease include, but are not limited to, blood counts, and an antinuclear antibody test (ANA). A family history of autoimmune disease, or having risk factors for autoimmune disease (e.g. gender, age, ethnicity, and exposure to environmental agents, such as procainamide, hydrolyzine, mercury, gold, or silver) can also aid in determining if a subject is likely to have autoimmune disease or in making a diagnosis of autoimmune disease. [00210] Treatments for autoimmune disorders are known in the art and can include, but are not limited to Immunosuppressive drugs, e.g., Cyclosporine (Neoral, Sandimmune, Gengraf, and
Restasis), Tacrolimus (Prograf, Protopic, Astagraf XL, and Envarsus XR), Methotrexate (Trexall, Rasuvo, Rheumatrex, and Otrexup (PF)), Sirolimus (Rapamune), Mycophenolic acid (Myfortic and CellCept), Rituximab (Rituxan), etanercept (Enbrel), pentostatin (Nipent), ruxolitinib (Jakafi);
Chemotherapies, e.g., Methotrexate (Trexall, Rasuvo, Rheumatrex, and Otrexup (PF)), antithymocyte globulin (Atgam, Thymoglobulin); Steroids, e.g,. Prednisone (Deltasone, Rayos, and Prednisone Intensol), Methylprednisolone (Medrol, Solu-Medrol, and Depo-Medrol), budesonide (Entocort EC, Uceris); Antifungal, e.g., Posaconazole (Noxafd); Antiviral drugs, e.g., Acyclovir (Zovirax and Sitavig), Valacyclovir (Valtrex); and Antibiotics, e.g., Sulfamethoxazole / Trimethoprim (Bactrim, Sulfatrim, and Bactrim DS); Protease inhibitors, e.g. alpha 1 -proteinase inhibitor (Zemaira);
extracorporeal photopheresis; monoclonal antibodies (daclizumab (Zinbryta), basiliximab (Simulect)), Brentuximab vedotin (Adcetris), Alemtuzumab (Campath, Lemtrada), Tocilizumab (Actemra);
infusion of mesenchymal stromal cells.
[00211] In addition to methods of treatment, the methods and biomarker signatures described herein can be applied to methods of predicting the risk of disease or disease progression in a subject and/or determining the efficacy of treatment or need for further treatment. For example, transition from aberrant to normal immune system activity would indicate that a treatment had been effective or that the treatment can be discontinued.
[00212] In some aspects, provided herein are compositions and methods for treating or preventing a condition caused by or associated with aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator and/or an inhibitor of at least 1 negative immune response regulator to the subject.
[00213] The compositions and methods described herein can be administered to a subject having or diagnosed as having a condition caused by or associated with aberrant immune system activity in a subject in need thereof. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein to a subject in order to alleviate a symptom of a disease. As used herein, "alleviating a symptom" of a disease is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic. The agents described herein can be administered to a subject in need thereof by any appropriate route which results in an effective treatment in the subject. [00214] In some embodiments, the agents described herein can be administered to a subject by any mode of administration that delivers the agent systemically or locally to a desired surface or target, and can include, but is not limited to, injection, infusion, instillation, and inhalation administration. To the extent that polypeptide agents can be protected from inactivation in the gut, oral administration forms are also contemplated. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
[00215] The phrases“parenteral administration" and“administered parenterally” as used herein, refer to modes of administration other than enteral and topical administration, usually by injection.
The phrases“systemic administration,"“administered systemically",“peripheral administration" and “administered peripherally" as used herein refer to the administration of the agents described herein, other than directly into a target site, tissue, or organ, such that it enters the subject’s circulatory system and, thus, is subject to metabolism and other like processes.
In some embodiments of any of the aspects, the subject is a mammalian subject. In some
embodiments of any of the aspects, the subject is a human subject. In some embodiments of any of the aspects, the subject is a current or former smoker. In some embodiments of any of the aspects, the subject is a smoker. In some embodiments of any of the aspects, the subject is a non-smoker.
[00216] The methods described herein can prevent, delay, or slow the development of lung cancer, e.g., lung squamous cell carcinoma. In some embodiments of any of the aspects, the subject treated according to the present methods is not a subject with lung cancer. In some embodiments of any of the aspects, the subject treated according to the present methods is a subject who does not have lung cancer. In some embodiments of any of the aspects, the subject treated according to the present methods is a subject who does not have and has not had lung cancer. In some embodiments of any of the aspects, the subject treated according to the present methods is at risk of lung cancer. In some embodiments of any of the aspects, the subject is a subject with a bronchial premalignant lesion.
[00217] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e.. the concentration of the agent, which achieves a half- maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for tumor growth and/or size, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example animal models of cancer, e.g. a murine xenograft model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed.
[00218] In some embodiments, the methods described herein comprise administering an effective amount of a composition described herein, e.g. an agonist or inhibitor of a nucleic acid and/or polypeptide described herein to a subject in order to alleviate a symptom of a condition caused by or associated with aberrant immune system activity. As used herein, "alleviating a symptom” is ameliorating any condition or symptom associated with the disease. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration.
Administration can be local or systemic.
[00219] The term“effective amount" as used herein refers to the amount of an agent needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of the agent that is sufficient to provide a particular effect when administered to atypical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount" . However, for any given case, an appropriate“effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
[00220] In certain embodiments, an effective dose of a composition comprising an agonist and/or inhibitor of an immune response regulator as described herein can be administered to a patient once.
In certain embodiments, an effective dose of a composition can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.
[00221] In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. lung cancer by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80 % or at least 90% or more.
[00222] The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the active agent. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.
[00223] The dosage ranges for the administration of an agent according to the methods described herein depend upon, for example, the form of the agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for tumor growth. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.
[00224] The efficacy of an agent described herein in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. lung cancer) can be determined by the skilled clinician. However, a treatment is considered“effective treatment," as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size and/or growth rate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of lung cancer in a mouse model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. tumor size and/or growth rate.
[00225] In some embodiments, the technology described herein relates to a pharmaceutical composition comprising an agonist or inhibitor of an immune response regulator as described herein, and optionally a pharmaceutically acceptable carrier. In some embodiments, the active ingredients of the pharmaceutical composition comprise an agonist or inhibitor of an immune response regulator as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist essentially of an agonist or inhibitor of an immune response regulator as described herein. In some embodiments, the active ingredients of the pharmaceutical composition consist of an agonist or inhibitor of an immune response regulator as described herein. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically -acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) semm component, such as semm albumin, HDL and LDL; (22) C2- C12 alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as "excipient", "carrier", "pharmaceutically acceptable carrier" or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent.
[00226] In another embodiment, a composition as disclosed herein can be administered in a vesicle, in particular a liposome (see Langer, "New Methods of Drug Delivery," Science 249: 1527- 1533 (1990); Lopez-Berestein, "Treatment of Systemic Fungal Infections with Liposomal- Amphotericin B," Liposomes in the Therapy of Infectious Disease and Cancer, pp. 317-327 (1989); and Treat et al, "Liposome encapsulated doxorubicin - preliminary results of phase I and phase II trials" Liposomes in the Therapy of Infectious Disease and Cancer, pp. 353-365 (1989). As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multi-lamellar hydrated liquid crystals which are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to an agonist, inhibitor, or other therapeutic of the present invention, stabilizers, preservatives, excipients and the like. The preferred lipids are natural and synthetic phospholipids and phosphatidyl cholines (lecithins) used separately or together. Methods to form liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.
[00227] In some embodiments, a composition as disclosed herein can be administered via a nanoparticle or microparticle. See, for example, Allen et al. Biochim. Biophys. Acta 19993 1150:9- 16, Wissing et al. Adv. Drug. Deliv. Rev. 2004 56: 1257-1272, and Tochilin, Nanoparticulates as Drug Carriers, Imperial College Press (2006); the contents of each of which is incorporated by reference herein in its entirety.
[00228] In some embodiments, the pharmaceutical composition as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.
[00229] Suitable vehicles that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, com oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of an agonist and/or inhibitor of a miRNA as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.
[00230] Pharmaceutical compositions can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia PA. (2005).
[00231] In some embodiments of any of the aspects, the compositions described herein can be administered by inhalation, e.g., as a vapor or aerosol formulation or by nebulization. For use as aerosols, a composition described herein can be provided in solution or suspension may be packaged in a pressurized aerosol container together with suitable propellants, for example, hydrocarbon propellants like propane, butane, or isobutane with conventional adjuvants. A composition described herein can also be administered in a non-pressurized form such as in a nebulizer or atomizer. In some embodiments, a composition can also be administered directly to the airways in the form of a dry powder, e.g., by use with an inhaler. Aerosols for the delivery to the respiratory tract are known in the art. See for example, Adjei, A. and Garren, J. Pharm. Res., 1 : 565-569 (1990); Zanen, P. and Lamm, J.-W. J. Int. J. Pharm., 114: 111-115 (1995); Gonda, I. "Aerosols for delivery of therapeutic and diagnostic agents to the respiratory tract," in Critical Reviews in Therapeutic Drug Carrier Systems, 6:273-313 (1990); Anderson et ah, Am. Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for the systemic delivery of peptides and proteins as well (Patton and Platz, Advanced Drug Delivery Reviews, 8: 179-196 (1992)); Timsina et. ah, Int. J. Pharm., 101 : 1-13 (1995); and Tansey, I. P., Spray Technol. Market, 4:26-29 (1994); French, D. L., Edwards, D. A. and Niven, R. W., Aerosol Sci., 27: 769-783 (1996); Visser, J., Powder Technology 58: 1-10 (1989)); Rudt, S. and R. H. Muller, J. Controlled Release, 22: 263-272 (1992); Tabata, Y, and Y. Ikada, Biomed. Mater. Res., 22: 837-858 (1988); Wall, D. A., Drug Delivery, 2: 10 1-20 1995); Patton, J. and Platz, R., Adv. Drug Del. Rev., 8: 179-196 (1992); Bryon, P., Adv. Drug. Del. Rev., 5: 107-132 (1990); Patton, J. S., et ak, Controlled Release, 28: 15 79-85 (1994); Damms, B. and Bains, W., Nature Biotechnology (1996); Niven, R. W., et ak, Pharm. Res., 12(9); 1343-1349 (1995); and Kobayashi, S., et ak, Pharm. Res., 13(1): 80-83 (1996), contents of all of which are herein incorporated by reference in their entirety.
[00232] Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the therapeutic described herein can be administered in a sustained release formulation.
[00233] Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
[00234] Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
[00235] A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591 ,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1 ; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example,
hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions. [00236] In some embodiments of any of the aspects, the agonist or inhibitor of an immune response regulator is administered as a monotherapy, e.g., another treatment for the condition is not administered to the subject.
[00237] In some embodiments of any of the aspects, the methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Additional therapies for the conditions described herein are provided above.
[00238] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.
[00239] For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.
[00240] The terms“decrease”,“reduced”,“reduction”, or“inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments,“reduce,”“reduction" or “decrease" or“inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or“inhibition” does not encompass a complete inhibition or reduction as compared to a reference level.“Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.
[00241] The terms“increased”,“increase”,“enhance”, or“activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms“increased”,“increase”, “enhance”, or“activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a“increase” is a statistically significant increase in such level.
[00242] The term "exogenous" refers to a substance present in a cell other than its native source. The term "exogenous" when used herein can refer to a nucleic acid (e.g., a miRNA or nucleic acid encoding a miRNA) that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is not normally found and one wishes to introduce the nucleic acid or polypeptide into such a cell or organism. Alternatively,“ectopic” can refer to a nucleic acid that has been introduced by a process involving the hand of man into a biological system such as a cell or organism in which it is found in relatively low amounts and one wishes to increase the amount of the nucleic acid or polypeptide in the cell or organism, e.g., to create ectopic expression or levels. In contrast, the term "endogenous" refers to a substance that is native to the biological system or cell.
[00243] In one aspect of any of the embodiments, the subject is a human.
[00244] As used herein, a "subject" means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms,“individual,”“patient” and “subject” are used interchangeably herein.
[00245] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of, e.g., lung cancer. A subject can be male or female.
[00246] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. lung cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for the condition or the one or more complications related to the condition. Alternatively, a subject can also be one who has not been previously diagnosed as having the condition or one or more complications related to the condition. For example, a subject can be one who exhibits one or more risk factors for the condition or one or more complications related to the condition or a subject who does not exhibit risk factors.
[00247] A“subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.
[00248] As used herein, the term“antibody reagent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments of any of the aspects, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments as well as complete antibodies.
[00249] As used herein, the term“antibody” refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, . e.. molecules that contain an antigen binding site that immunospecifically binds an antigen. The term also refers to antibodies comprised of two immunoglobulin heavy chains and two immunoglobulin light chains as well as a variety of forms including full length antibodies and antigen-binding portions thereof; including, for example, an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody (dAb), a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, a functionally active epitope -binding portion thereof, and/or bifunctional hybrid antibodies. Each heavy chain is composed of a variable region of said heavy chain (abbreviated here as HCVR or VH) and a constant region of said heavy chain. The heavy chain constant region consists of three domains CHI, CH2 and CH3. Each light chain is composed of a variable region of said light chain (abbreviated here as LCVR or VL) and a constant region of said light chain. The light chain constant region consists of a CL domain. The VH and VL regions may be further divided into hypervariable regions referred to as complementarity-determining regions (CDRs) and interspersed with conserved regions referred to as framework regions (FR). Each VH and VL region thus consists of three CDRs and four FRs which are arranged from the N terminus to the C terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is well known to those skilled in the art.
[00250] Antibodies and/or antibody reagents can include an immunoglobulin molecule, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a fully human antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual specific antibody, an anti-idiotypic antibody, a bispecific antibody, and a functionally active epitope-binding portion thereof.
[00251] As used herein, the term“nanobody” or single domain antibody (sdAb) refers to an antibody comprising the small single variable domain (VHH) of antibodies obtained from camelids and dromedaries. Antibody proteins obtained from members of the camel and dromedary (Camelus baclrianus and Calelus dromaderius) family including new world members such as llama species (Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. Certain IgG antibodies from this family of mammals as found in nature lack light chains, and are thus structurally distinct from the typical four chain quaternary structure having two heavy and two light chains, for antibodies from other animals. See PCT/EP93/ 02214 (WO 94/04678 published 3 Mar. 1994; which is incorporated by reference herein in its entirety).
[00252] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high afiinity for a target, resulting in a low molecular weight antibody-derived protein known as a“camelid nanobody”. See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B. et al, 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003
Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J. 17: 3512-3520; each of which is incorporated by reference herein in its entirety. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be“humanized”. Thus the natural low antigenicity of camelid antibodies to humans can be further reduced.
[00253] The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody. The low molecular weight and compact size further result
in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. See U.S. patent application 20040161738 published Aug. 19, 2004; which is incorporated by reference herein in its entirety. These features combined with the low antigenicity to humans indicate great therapeutic potential.
[00254] As used herein, the terms“protein" and“polypeptide" are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms "protein", and "polypeptide" refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. "Protein" and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term "peptide" is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms "protein" and "polypeptide" are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing. The terms also refer to fragments or variants of the polypeptide that maintain at least 50% of the activity or effect, of the full length polypeptide.
Conservative substitution variants that maintain the activity of wildtype proteins will include a conservative substitution as defined herein. The identification of amino acids most likely to be tolerant of conservative substitution while maintaining at least 50% of the activity of the wildtype is guided by, for example, sequence alignment with homologs or paralogs from other species. Amino acids that are identical between homologs are less likely to tolerate change, while those showing conservative differences are obviously much more likely to tolerate conservative change in the context of an artificial variant. Similarly, positions with non-conservative differences are less likely to be critical to function and more likely to tolerate conservative substitution in an artificial variant. Variants, fragments, and/or fusion proteins can be tested for activity, for example, by administering the variant to an appropriate animal model of a disease as described herein.
[00255] As used herein, the term“specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
[00256] As used herein, the term“nucleic acid” or“nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single -stranded or double-stranded. A single -stranded nucleic acid can be one nucleic acid strand of a denatured double- stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double -stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, e.g., genomic DNA or cDNA. Suitable RNA can include, e.g., mRNA or miRNA.
[00257] In some embodiments of any of the aspects, a nucleic acid as described herein (e.g. a a miRNA or a nucleic acid encoding an miRNA) is comprised by a vector. In some of the aspects described herein, a nucleic acid sequence as described herein, or any module thereof, is operably linked to a vector. The term "vector", as used herein, refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term“vector” encompasses any genetic element that is capable of replication when associated with the proper control elements and that can transfer gene sequences to cells. A vector can include, but is not limited to, a cloning vector, an expression vector, a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc. [00258] As used herein, the term "expression vector" refers to a vector that directs expression of an RNA or polypeptide from sequences linked to transcriptional regulatory sequences on the vector. The sequences expressed will often, but not necessarily, be heterologous to the cell. An expression vector may comprise additional elements, for example, the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in human cells for expression and in a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in producing RNA and proteins and as appropriate, secreting proteins, including where applicable, but not limited to, for example, transcription, transcript processing, translation and protein folding, modification and processing. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means the nucleic acid sequence which is transcribed (DNA) to RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. The gene may or may not include regions preceding and following the coding region, e.g. 5’ untranslated (5’UTR) or "leader" sequences and 3’ UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).
[00259] In some embodiments, the agonist or inhibitor of a miRNA can be provided or administered on a vector, e.g., a viral vector. In some embodiments, the agonist or inhibitor of a miRNA can be provided or administered as a gene therapy.
[00260] As used herein, the term“viral vector" refers to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding a polypeptide as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo. Numerous forms of viral vectors are known in the art.
[00261] By“recombinant vector” is meant a vector that includes a heterologous nucleic acid sequence, or“transgene” that is capable of expression in vivo. It should be understood that the vectors described herein can, in some embodiments of any of the aspects, be combined with other suitable compositions and therapies. In some embodiments of any of the aspects, the vector is episomal. The use of a suitable episomal vector provides a means of maintaining the nucleotide of interest in the subject in high copy number extra chromosomal DNA thereby eliminating potential effects of chromosomal integration.
[00262] In some embodiments of any of the aspects, an agonist and/or inhibitor of an miRNA as described herein can comprise a modified nucleic acid sequence, e.g., it is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5’ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases,
(c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as
(d) backbone modifications, including modification or replacement of the phosphodiester
linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the aspects, the modified RNA will have a phosphorus atom in its intemucleoside backbone.
[00263] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2'-5' to 5 '-2'. Various salts, mixed salts and free acid forms are also included. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— ,—CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone],— CH2— O— N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)- -CH2— and—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as— 0-P-0-CH2-]
[00264] In other RNA mimetics suitable or contemplated for use as agonists or inhibitors, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
[00265] A RNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids has been shown to increase RNA stability in serum, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833-843; Grunweller,
A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).
[00266] Modified RNAs can also contain one or more substituted sugar moieties. The RNAs, e.g., agonists and/or inhibitors, described herein can include one of the following at the 2' position: OH; F; 0-, S-, orN-alkyl; 0-, S-, or N-alkenyl; 0-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and alkynyl. Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)n0NH2, and 0(CH2)n0N[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, ON02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an RNA, or a group for improving the pharmacodynamic properties of an RNA, and other substituents having similar properties. In some embodiments of any of the aspects, the modification includes a 2' methoxy ethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e., 2'-0— CH2— O— CH2— N(CH2)2, also described in examples herein below.
[00267] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. RNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
[00268] A nucleic acid as described herein can also include nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein,“unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5 -halo, particularly 5-bromo, 5-trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the nucleic acids featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0- methoxy ethyl sugar modifications.
[00269] The preparation of the modified nucleic acids, backbones, and nucleobases described above are well known in the art.
[00270] Another modification of a nucleic acid featured in the invention, e.g., an agonist or inhibitor as described herein, involves chemically linking to the nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the RNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4: 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et ah, Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10: 1111-1118; Kabanov et al., LEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl- rac-glycero-3-phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654; Shea et al, Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937). [00271] As used herein,“inhibitory nucleic acid” refers to a nucleic acid molecule which can inhibit the expression of a target, e.g., double-stranded RNAs (dsRNAs), inhibitory RNAs (iRNAs), and the like. In some embodiments of any of the aspects, the inhibitory nucleic acid can be a silencing RNA (siRNA), microRNA (miRNA), or short hairpin RNA (shRNA).
[00272] Double -stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA
transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.
[00273] As used herein, the term“iRNA” refers to an agent that contains RNA (or modified nucleic acids as described below herein) and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In some embodiments of any of the aspects, an iRNA as described herein effects inhibition of the expression and/or activity of a target. In some embodiments of any of the aspects, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA. In some embodiments of any of the aspects, administering an inhibitor (e.g. an iRNA) to a subject results in a decrease in the target mRNA level in the subject by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the subject without the presence of the iRNA.
[00274] In some embodiments of any of the aspects, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target, e.g., it can span one or more intron
boundaries. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 base pairs in length inclusive, more generally between 18 and 25 base pairs in length inclusive, yet more generally between 19 and 24 base pairs in length inclusive, and most generally between 19 and 21 base pairs in length nucleotides in length, inclusive. In some embodiments of any of the aspects, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a“part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.
[00275] Exemplary embodiments of types of inhibitory nucleic acids can include, e.g,. siRNA, shRNA,miRNA, and/or amiRNA, which are well known in the art. One skilled in the art would be able to design further siRNA, shRNA, or miRNA to target the nucleic acid sequence of an immune response regulator, e.g., using publically available design tools. siRNA, shRNA, or miRNA is commonly made using companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO).
[00276] In some embodiments of any of the aspects, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids described herein may be synthesized and/or modified by methods well established in the art, such as those described in“Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference.
Modifications include, for example, (a) end modifications, e.g., 5’ end modifications
(phosphorylation, conjugation, inverted linkages, etc.) 3’ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2’ position or 4’ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural intemucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In some embodiments of any of the aspects, the modified RNA will have a phosphorus atom in its intemucleoside backbone.
[00277] Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5 '-3' or 2'-5' to 5 '-2'. Various salts, mixed salts and free acid forms are also included. Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; others having mixed N, O, S and CH2 component parts, and oligonucleosides with heteroatom backbones, and in particular— CH2— NH— CH2— ,—CH2—N(CH3)—0—CH2— [known as a methylene (methylimino) or MMI backbone],— CH2— O— N(CH3)— CH2— ,— CH2— N(CH3)— N(CH3)- -CH2-- and—N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as— 0-P-0-CH2-]
[00278] In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the intemucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.
[00279] The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2' and 4' carbons. This stmcture effectively "locks" the ribose in the 3'-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in semm, and to reduce off-target effects (Elmen, J. et al, (2005) Nucleic Acids Research 33(l):439-447; Mook, OR. et al., (2007) Mol Cane Ther 6(3):833- 843; Grunweller, A. et al., (2003) Nucleic Acids Research 31( 12): 3185-3193).
[00280] Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, described herein can include one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Cl to CIO alkyl or C2 to CIO alkenyl and
alkynyl. Exemplary suitable modifications include 0[(CH2)nO] mCH3, 0(CH2).n0CH3, 0(CH2)nNH2, 0(CH2) nCH3, 0(CH2)n0NH2, and 0(CH2)n0N[(CH2)nCH3)]2, where n and m are from 1 to about 10. In some embodiments of any of the aspects, dsRNAs include one of the following at the 2' position: Cl to CIO lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O- aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, S02CH3, 0N02, N02, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments of any of the aspects, the modification includes a 2' methoxy ethoxy (2'-0— CH2CH20CH3, also known as 2'-0-(2- methoxyethyl) or 2'-MOE) (Martin et ah, Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMAOE, as described in examples herein below, and 2'- dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'- DMAEOE), i.e., 2'-0— CH2— O— CH2— N(CH2)2, also described in examples herein below.
[00281] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'- OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'- 5' linked dsRNAs and the 5' position of 5' terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
[00282] An inhibitory nucleic acid can also include nucleobase (often referred to in the art simply as“base”) modifications or substitutions. As used herein,“unmodified” or“natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5- methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5- propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5- halo, particularly 5-bromo, 5-trifluoromethyl and other 5 -substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- daazaadenine and 3-deazaguanine and 3-deazaadenine. Certain of these nucleobases are particularly useful for increasing the binding affinity of the inhibitory nucleic acids featured in the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-0- methoxy ethyl sugar modifications.
[00283] The preparation of the modified nucleic acids, backbones, and nucleobases described above are well known in the art.
[00284] Another modification of an inhibitory nucleic acid featured in the invention involves chemically linking to the inhibitory nucleic acid to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4: 1053-1060), athioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiochole sterol (Oberhauser et al, Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10: 1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et ak, Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium l,2-di-0-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al, Nucleosides &
Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al, Tetrahedron Lett.,
1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.
Exp. Ther., 1996, 277:923-937).
[00285] As used herein, the terms "treat,” "treatment," "treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. lung cancer. The term“treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a condition. Treatment is generally“effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is“effective" if the progression of a disease is reduced or halted. That is,“treatment" includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment). [00286] As used herein, the term“pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and/or ointment. In some embodiments of any of the aspects, a pharmaceutically acceptable carrier can be an artificial or engineered carrier, e.g., a carrier that the active ingredient would not be found to occur in in nature.
[00287] As used herein, the term "administering," refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.
[00288] The term“statistically significant" or“significantly" refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.
[00289] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term“about.” The term“about” when used in connection with percentages can mean ±1%.
[00290] As used herein, the term“comprising” means that other elements can also be present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.
[00291] The term "consisting of' refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.
[00292] As used herein the term "consisting essentially of' refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.
[00293] The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, "e.g." is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation "e.g." is synonymous with the term "for example." [00294] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.
[00295] Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art to which this disclosure belongs. It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN
9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a
Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.
[00296] In some embodiments of any of the aspects, the disclosure described herein does not concern a process for cloning human beings, processes for modifying the germ line genetic identity of human beings, uses of human embryos for industrial or commercial purposes or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes. [00297] Other terms are defined herein within the description of the various aspects of the invention.
[00298] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.
[00299] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[00300] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.
[00301] The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.
[00302] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:
1. A method for treating or preventing a condition caused by or associated with
immunosuppressed aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject.
2. The method of paragraph 1, wherein the condition caused by or associated with
immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
3. The method of paragraph 2, wherein the cancer is a squamous cell cancer or lung squamous cell cancer.
4. The method of paragraph 2, wherein the infectious disease is a bacterial and/or viral infection.
5. The method of paragraph 4, wherein the viral infection is a coronavirus infection.
6. A method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
7. The method of paragraph 6, wherein the condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease.
8. A method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
ii. at periodic intervals, such as at least every 6 months ... , every 8 months
every 9 months ... , every year... , one of a bronchoscopy -based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression. 9. A method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; ii. at least every 6 months, one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
10. The method of paragraphs 8 or 9, wherein if the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB 1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
11. The method of paragraph 10, wherein the different immune response regulator is selected from Table 1 or Table 2.
12. The method of paragraphs 8 or 9, wherein there are at least two changes in immune response regulators relative to a reference level, wherein the changes can be (a) at least two increases of at least two negative immune response regulators, (b) at least two decreases of at least two positive immune response regulators; or a combination of at least one increase in at least one negative immune response regulator and a decrease of at least one positive immune response regulator.
13. The method of paragraph 11, wherein there is a change in at least 5 immune response regulators. 14. The method of paragraph 11, wherein there is a change in at least ten immune response regulators.
15. The method of any of the preceding paragraphs, wherein the positive immune response regulator is selected from Table 1.
16. The method of any of the preceding paragraphs, wherein the negative immune response regulator is selected from Table 2.
17. The method of any of the preceding paragraphs, wherein the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRBl, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
18. The method of any of the preceding paragraphs, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, and miR-149-5p.
19. The method of any of the preceding paragraphs, wherein the different immune response regulator can further include a miRNA selected from Table 2A.
20. The method of any of the preceeding paragraphs, wherein the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
21. The method of paragraph 20, wherein the method further comprises administering at least one anti -proliferative drug.
22. The method of any of the preceeding paragraphs, wherein the at least one anti -proliferative drug is selected from the group consisting of:
Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; J K inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors; Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
23. The method of either of paragraphs 8, 9, or 10 wherein the subject:
i. is administered:
1. a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue but not a chest CT scan; or
2. at greater than 6 month intervals, one of a bronchoscopy -based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; and/or
ii. the subject is not administered an immune stimulating drug,
when either:
the level of expression of at least 1 negative immune response regulator is not increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is not decreased relative to a reference level of expression; or
the subject is a subject determined to have a level of expression of at least 1 negative immune response regulator which is not increased relative to a reference level, or a level of expression of at least 1 positive immune response regulator which is not decreased relative to a reference level of expression.
24. The method of any of the preceding paragraphs, wherein the agonist of an immune response regulator is an immune response regulator polypeptide, an immune response regulator miRNA, or a nucleic acid encoding the immune response regulator.
25. The method of any of the preceding paragraphs, wherein the inhibitor of an immune response regulator is an antibody, antibody reagent, or inhibitory nucleic acid.
26. The method of any of the preceding paragraphs, wherein the administering step comprises the administration of a vector comprising a nucleic acid encoding the agonist and/or inhibitor.
27. The method of any of the preceding paragraphs, wherein the subject is a human.
28. The method of any of the preceding paragraphs, wherein the administration is prophylactic.
29. The method of any of the preceding paragraphs, wherein the subject is a current or former smoker. 30. The method any of the preceding paragraphs, wherein the level of expression is detected for at least 1 negative or positive immune response regulator.
31. The method any of the preceding paragraphs, wherein the level of expression is detected for at least three negative or positive immune response regulators.
32. The method any of the preceding paragraphs, wherein the level of expression is detected for at least four negative or positive immune response regulators.
33. The method of any of the preceding paragraphs, wherein the level of expression is detected for at least five negative or positive immune response regulators.
34. The method of any of the preceding paragraphs, wherein the level of expression is detected for at least miR-149-5p.
35. The method any of the preceding paragraphs, wherein the expression level of no more than 100 genes is detected.
36. The method of any of the preceding paragraphs, wherein the at least one immune stimulating drug is selected from the group consisting of:
immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti-viral drugs that improve interferon signaling); DNA synthesis inhibitors; IMDH inhibitors; CDK inhibitors; ribonucleotide reductase inhibitors; dihydrofolate reductase inhibitors; topoisomerase inhibitors; FLT3 inhibitors; IGF-1 inhibitors; MEK inhibitors; aurora kinase inhibitors; PKC inhibitors; RAF inhibitors; PDFGR/KIT inhibitors; VEGFR inhibitors; SRC inhibitors; retinoid receptor agonists; HD AC inhibitors; DNA methyltransferase inhibitors; and EZH2 inhibitors.
37. The method of any of the preceding paragraphs, wherein a treatment is administered during a bronchoscopy-based procedure.
38. The method of any of the preceding paragraphs, wherein a treatment is administered systemically.
39. The method of any of the preceding paragraphs, wherein a treatment is administered during a bronchoscopy-based procedure and systemically.
40. The method of any of the preceding paragraphs, whereby the development of lung cancer lung squamous cell carcinoma is prevented, delayed, or slowed. 41. The method of any of the preceding paragraphs, wherein the level of expression is the level of expression in an endobronchial biopsy, bronchial brushing sample, nasal brushing sample, sputum, or blood obtained from the subject.
42. The method any of the preceding paragraphs, wherein the level of expression is the level of expression in a bronchial brushing obtained from the right or left mainstem bronchus.
43. The method of any of the preceding paragraphs, wherein the biopsy or brushing sample comprises morphologically-normal tissues or cells.
44. A pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with immunosuppressed aberrant immune system activity, comprising:
a. an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator and
b. a pharmaceutical acceptable carrier.
45. The composition of paragraph 44, wherein the condition caused by or associated with immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
46. The composition of paragraph 45, wherein the cancer is a squamous cell cancer or lung squamous cell cancer.
47. The composition of paragraph 45, wherein the infectious disease is a bacterial and/or viral infection.
48. The composition of paragraph 47, wherein the viral infection is a coronavirus infection.
49. A pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with autoimmune aberrant immune system activity, comprising:
a. an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator and
b. a pharmaceutical acceptable carrier.
50. The method of paragraph 49, wherein the condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease. 51. A method of determining the risk of progression of bronchial premalignant lesions to lung cancer or an infectious disease in a subject the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) determining the subject is at risk of the bronchial premalignant lesions progressing to lung cancer or an infectious disease if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
52. The method of any of paragraph 51, wherein the positive immune response regulator is selected from Table 1 and the negative immune response regulator is selected from Table 2.
53. The method of paragraphs 51 or 52, wherein if the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
54. The method of paragraph 51 or 53 wherein the positive or negative immune response regulator is at least miR-149-5p.
55. The method of any of paragraphs 51-54, wherein the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRBl, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
56. The method of any of paragraphs 51-56, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, miR- 149-5p.
57. The method of any of paragraphs 51-56, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, and miR-149-5p.
58. The method of any of paragraphs 51-56, wherein the different immune response regulator can further include a miRNA selected from Table 2A. 59. The method of any of paragraphs 51-56, wherein the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
60. The method of any of paragraphs 51-56, wherein the method further comprises administering at least one anti -proliferative drug.
61. The method of any of paragraphs 51-56, wherein the at least one anti -proliferative drug is selected from the group consisting of:
Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; JNK inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors; Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
62. The method of any of paragraphs 51-57, wherein the subject at risk is further treated by administering an agonist of the positive immune response regulator if the subject has a decrease of at least one positive immune response regulators, wherein the agonist is an immune response regulator polypeptide, an immune response regulator miRNA, or a nucleic acid encoding the immune response regulator.
63. The method of any of paragraphs 51-61, wherein the subject at risk is further treated by administering an inhibitor of the negative immune response regulator if the subject has an increase of at least one negative immune response regulators, wherein the inhibitor is an antibody, antibody reagent, ligand, mimetic, or inhibitory nucleic acid.
64. The method of any of paragraphs 51-63, wherein the administering step comprises the administration of a vector comprising a nucleic acid encoding the agonist and/or inhibitor. 65. The method of any of paragraphs 51-64, wherein the subject is a human.
66. The method of any of paragraphs 51-65, wherein the administration is prophylactic.
67. The method of any of paragraphs 51-66, wherein the subject is a current or former smoker.
68. The method of any of paragraphs 51-67, wherein the level of expression is detected for at least 10 negative or positive immune response regulators.
69. The method of any of paragraphs 51-68, wherein the level of expression is detected for at least miR-149-5p.
70. The method of any of paragraphs 51-69, wherein the expression level of no more than 100 genes is detected.
71. The method of any of paragraphs 51-70, wherein a treatment is administered during a bronchoscopy-based procedure.
72. The method of any of paragraphs 51-70, wherein a treatment is administered systemically.
73. The method of any of paragraphs 51-72, wherein a treatment is administered during a bronchoscopy-based procedure and systemically.
74. The method of any of paragraphs 51-73, wherein the sample is in an endobronchial biopsy, airway brushing sample, nasal brushing sample, sputum, or blood from the subject.
75. The method of any of paragraphs 51-74, wherein the sample is a bronchial brushing obtained from the right or left mainstem bronchus.
76. The method of any of paragraphs 51-75, wherein the biopsy or brushing sample comprises morphologically-normal tissues or cells.
EXAMPLES
EXAMPLE 1: IMMUNE ALTERATIONS ASSOCIATED WITH DISEASE PROGRESSION IN BRONCHIAL PREMALIGNANT LESIONS.
[00303] Suppression of the immune system is critical for the progression of lung cancer. [00304] This invention describes genes that modulate the immune system in Bronchial
Premalignant Lesions (PMLs). PMLs arise in airway epithelium and are precursors to squamous cell carcinoma (FIG. 1A and FIG. IB). Drugs that target these genes can serve to inhibit the immune suppressor genes or enhance the immune activator genes and thereby be effective in delaying or preventing the development of lung cancer.
[00305] The inventors identified herein immune pathways active in bronchial PMLs. Further the inventors identified immune pathways associated with the progression and regression of PMLs.
Furthermore, the inventors identified genes responsible for mediating immune responses (FIG. 4).
[00306] The inventors have profiled via RNA sequencing endobronchial biopsies obtained from high-risk smokers undergoing lung cancer screening via autofluoresence bronchoscopy (FIG. 2). The inventors describe herein four molecular subtypes of bronchial premalignant lesions. The Proliferative molecular subtype was enriched for samples with bronchial dysplasia and progression/persistence of Proliferative lesions was associated with a decrease in expression of genes involved in interferon signaling and antigen processing/presentation pathways. Endobronchial biopsies divide into four distinct molecular subtypes based on distinct patterns of gene co-expression (FIG. 3). Eight immune phenotypes were identified in the bronchial PMLs from the Discovery Cohort of PCGA (FIG. 5). The inventors therefore sought to understand the role of the immune system in the progression/persistence of Proliferative bronchial premalignant lesions and identify genes that modulate the immune response. Immune evasion has previously been described in the development of lung cancer, but no previous work has identified specific genes that are driving this effect.
[00307] The inventors describe a novel set of genes that, based on a set of rigorous computational and statistical tests, were found to be responsible for immune suppression in bronchial premalignant lesions that are likely to persist or progress to higher-grade lesion or invasive cancer. The set of genes that define this invention are GSTP1, TIMM 13, SMURF 1, and TMEM63C, as suppressors of immune activation, and B2M, HLA-DRA, HLA-DRB l, HLA-DPA1, MSC, QPRT, CPQ, MRAS, RCAN1, SERPINIl, SLC5A8 as activators of the immune system in bronchial premalignant lesions. The inventors novel computational methods have identified these genes as mediating the immune changes we observed and they are thus potential targets for immunoprevention of lung cancer.
[00308] These genes have not been previously shown to be important for progression of lung premalignancy. HLA-DRA, HLA-DRB l, and HLA-DPA1 are all components of the major histocompatibility class II complex (1), which functions in antigen presenting cells to display foreign antigens. The B2M gene is a component of the MHC class I complex, which is required for presentation of self antigens in all nucleated cells (2). The MSC gene is known as a downstream regulator of B cell receptor activation, but B cell activity has not previously been shown to be important in this context (3). GSTP1 has been shown in vitro to regulate transcription factors STAT3 and NF-kB, effecting a wide array of immune responses, but has not been identified as a driver of immune response in lung premalignancy (4, 5). TMEM63C has not been previously associated with activation or suppression of the immune system, in any context.
[00309] Previous targets for immune activation in the context of cancer have focused primarily on late stage tumors and have focused on T cell mediated immunity. Genes that have been discovered to mediate this immune pathway include PD1, PDL1, and CTLA4 (5). The genes identified herein modulate the activity of MHC class II antigen presentation, interferon signaling, and B cell related immunity, all of which represent distinct, novel. The invention here is also unique in that these genes and their effects are being described in a pre-cancer context. Rather than focusing on immune activation to kill late stage tumors, the inventors identified genes responsible for immune activation and suppression before tumors even form. This makes these genes essential in the prevention of lung cancer.
[00310] One future product of this invention will be a therapeutic for the immunoprevention of lung cancer. This could include a drug or antibody to either inhibit the immune-suppressor genes or enhance the expression of immune activator genes that we have identified. Secondarily, these genes will be useful as a diagnostic tool, helping to differentiate between immune cold lesions likely to progress to cancer, and immune hot/active lesions that are likely to regress to less severe histology or disappear. The diagnostic tool has potential utility in lung cancer CT screening where it could be used to reduce false positives or dictate screening intervals. Additional potential clinical utility could be in the identification of high-risk populations for intervention trials and to monitor the efficacy of the intervention in these trials.
[00311] The identified genes will be useful drug targets for the treatment of early and late stage cancers and immune activation across many types of cancers.
[00312] Experimental details
[00313] Gene expression from biopsies of bronchial premalignant lesions was quantified using RNA sequencing (available on the world wide web at
ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE 109743 (NCBI Gene Expression Omnibus,
GSE109743)). Immune phenotypes present in these lesions were characterized using Weighted Gene Coexpression Network Analysis (WGCNA) to group the ssGSEA scores from 104 immune gene sets (gene sets described in reference 6.) into 6 modules. Using linear modeling, the 6 immune modules were associated with the previously described molecular subtypes and lesion progression/persistence. Three immune modules,“Antigen Presentation”,“Interferon,” and“B Cells” were significantly associated with the progression status of lesions with the Proliferative molecular subtype.
[00314] The inventors identified genes that were associated with the progression status of each lesion within the Proliferative subtype using a mixed effect linear model (including patient as a random effect, smoking status, batch, and transcript integrity as covariates). Potential immune modulator genes were then identified as those with a high degree of correlation with the three significant progression-related immune modules. Finally, the list of immune modulator genes presented here was determined using mediation analysis, to identify 1) genes whose expression completely mediates an immune module-progression association, or 2) genes whose association with progression is completely mediated by an immune module.
[00315] The immune activity of these genes was tested computationally using orthogonal methods, such as Gene Set Enrichment Analysis (GSEA) and for miR-149 using in additional Virtual Inference of Protein-activity by Enriched Regulon (VIPER), in both the premalignant lesion data and in an additional set of lung squamous cell carcinoma tumors from The Cancer Genome Atlas (TCGA). Concordant results between separate computational methods and separate datasets lend credibility to the immune modulatory effects of these genes.
[00316] Table 6: This table lists the identified nucleic acid mediators of the immune phenotype between progressive/persistent versus regressive lesions. See also Example 1.
Figure imgf000115_0001
[00317] References: 1. Annu Rev Immunol. 1994;12:259-93.
2. J Immunol. 1987 Nov 1 ; 139(9): 3132-8.
3. Mol Cell Biol. 1998 Jun;18(6):3130-9.
4. Cell Death Dis. 2014 Jan 23;5:el015.
5. J Hematol Oncol. 2018 Dec 21;11(1): 142.
6. Immunity. 2018 Apr 17;48(4):812-830.el4.
EXAMPLE 2: MIRNAS REGULATES THE IMMUNE RESPONSE AND DRIVE THE
PROGRESSION OF THE PREMALIGNANT LESIONS
[00318] The role of miR- 149 in promoting immune suppression and development of lung squamous cell cancer
[00319] The molecular events involved in the development of bronchial premalignant lesions (PMLs), and their progression to Lung Squamous Cell Carcinoma (LUSC), is not well understood. Prior work characterized lung PML molecular subtypes by co-expressed gene modules associated with histological severity and progression to invasive cancer (1). Within these, the proliferative subtype was enriched for dysplastic PMLs with high basal and low ciliated cell signals. Among proliferative PMLs, the expression levels of genes related to interferon signaling and antigen processing/presentation (Module 9) were decreased in progressive/persistent lesions, showing early immune suppression is related to lesion progression. The molecular mechanisms that drive these alterations are unclear. The inventors identified the role of miRNAs in regulating gene expression associated with lesion outcome.
[00320] The inventors investigated the contribution of miRNAs to the progression of lesions to invasive cancer by regulating the immune response in the PMLs. The inventors investigated the following questions: 1) Do any miRNA regulate the immune response in PMLs by targeting genes in Module 9? 2) Are the expression levels of these miRNAs and target-genes associated with the progression status of the lesions? 3) Are these miRNAs expressed and/or exhibit particular functions in certain types?
[00321] The role of miR- 149 in promoting immune suppression and development of lung squamous cell cancer.
[00322] The inventors have discovered methods for identifying individuals who are most likely to develop squamous lung cancer, methods for lung cancer prevention in these same individuals, and methods of treatment for patients with squamous lung cancer. These methods involve measuring airway gene expression to identify individuals that have a pattern of gene expression reflective of what we have previously named Proliferative Squamous Premalignancy. Among individuals having this gene expression pattern, those with elevated expression of miR- 149, decreased expression of NLRC5, or decreased MHC Class I expression are most likely to develop squamous lung cancer. Individuals exhibiting any of these characteristics should both be regularly tested for the presence of lung cancer and/or treated to lower miR- 149 levels and/or activity, elevate NLRC5 levels and/or activity, elevate MHC Class I expression, or with other agents that promote the recruitment of immune cells to premalignant lesions and the clearing of premalignant lesions by the immune system.
[00323] Using a network based approach the inventors discovered four miRNAs that specifically regulates module 9 in Lung PMLs, which is related to interferon-gamma response and antigen presentation/processing pathways. The expression levels of miR149-5p and its negatively correlated genes are significantly associated with progressive PMLs. miR-149-5p is highly expressed in basal epithelial cell populations, rather than immune cells, based on cell type specific miRNA sequencing database FANTOM5 (available on the world wide web at fantom.gsc.riken.jp/5/) and canonical cell type marker correlation analysis. As an early carcinogenic event, the downstream effects of dysregulated miR-149-5p may be mediated by its suppression of NLRC5 expression, which in turn down-regulates MHC Class I dependent immune responses (see also FIG. 11-FIG. 15).
[00324] NLRC5, a critical regulator of MHC Class I related gene expression, is targeted by miR- 149-5p.
[00325] The inventors have identified seven high confident regulated targets for NLRC5. All seven of these genes belong to Module 9 and are significantly down-regulated within the progressive lesions of Proliferative subtype. The results show that that miR- 149-5p regulate MHC Class I gene expression and the early immune evasion of PML through suppressing NLRC5 level (FIG. 16).
[00326] Single-cell RNA sequencing was obtained from six PML biopsy samples from five patients. 11 cell types were identified using Seurat. The GSVA score for Module 9 was calculated for each cell. GPC1, the host gene for miR-149-5p, is significantly negative correlated with Module 9 GSVA score among the epithelial cells, but not in other cell types. Given that we have previously shown miR-149- 5p is highly specifically expressed in the epithelial cells only, the interaction between miR-149-5p and its target genes in Module 9 is also observed in epithelial cells (FIG. 17).
[00327] Using the cell-type specific miRNA and mRNA sequencing data from FANTOM5 cohort, we found that the negative correlation between miR-149-5p and NLRC5 can be found within the epithelial cell samples (FIG. 18).
[00328] Both Module 9 and miR-149-5p expression levels are significantly different between the progressive/stable and the regressive lesions within the Proliferative subtype. Using leave-one-out cross-validation and random -forest model, the inventors found that the AUC for predicting Proliferative lesion progression can be increased to 0.724 when modeling with Module 9 and miR-149-5p, compared to using Module 9 alone (AUC=0.628). The inventor’s result proves the potential of using miRNA to increase clinical diagnosis power (FIG. 19).
[00329] Gene module 4 through 7 can differentiate Proliferative PML subtypes from the others. The inventors next sought to explore whether miRNAs specifically targeted these gene modules could improve the classification. Using pamR and the paired miRNA and mRNA data in the discovery cohort, out of the 103 miRNAs that targets gene module 4-7, 10 miRNAs could improve the cross-validation accuracy for predicting Proliferative subtypes (FIG. 20). [00330] Experimental details
[00331] mRNA and miRNAs were extracted and sequenced from longitudinally collected endobronchial biopsies from patients with PMLs (148 samples, 30 patients). miRNAs targeting a gene co-expression module were identified based on the enrichment of target genes within the module as well as the degree of negative correlation between the expression of the miRNA and its targets within the module. The association between miRNAs and progression status within the proliferative subtype was tested with a mixed effects model adjusting for batch and patient as random effects. TCGA LUSC and GSE114489 PML data were used for validating the miRNA-mRNA network and the differentially expressed genes. Cell type specificity of miRNA was examined based on cell type specific sequencing data from FANTOM5 and cell type marker correlation analysis. Genes regulated by NLRC5 were identified with ChIP-seq data (available on the world wide web at ncbi.nlm.nih.gov/geo/query/acc. cgi?acc=GSE59092).
[00332] References:
1. Nat Commun. 2019 Apr 23; 10(1): 1856.
2. Proc Natl Acad Sci U S A. 2013 Nov 19; 110(47): 18946-51.
3. Nucleic Acids Res. 2018 Jan 4;46(D1):D296-D302.
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5. Elife. 2015 Dec 14;4:e09410.
6. Nat Biotechnol. 2017 Sep;35(9):872-878.
7. Nat Rev Immunol. 2012 Dec; 12(12): 813-20.
EXAMPLE 3: MOLECULAR SUBTYPING REVEALS IMMUNE ALTERATIONS
ASSOCIATED WITH PROGRESSION OF BRONCHIAL PREMALIGNANT LESIONS
[00333] Abstract
[00334] Bronchial premalignant lesions (PMLs) are precursors of lung squamous cell carcinoma, but have variable outcome, and we lack tools to identify and treat PMLs at risk for progression to cancer. Here the inventors report the identification of four molecular subtypes of PMLs with distinct differences in epithelial and immune processes based on RNA-Seq profiling of endobronchial biopsies from high- risk smokers. The Proliferative subtype is enriched with bronchial dysplasia and exhibits up-regulation of metabolic and cell cycle pathways. A Proliferative subtype-associated gene signature identifies subjects with Proliferative PMLs from normal-appearing uninvolved large airway brushings with high specificity. In progressive/persistent Proliferative lesions expression of interferon signaling and antigen processing/presentation pathways decrease and immunofluorescence show a depletion of innate and adaptive immune cells compared with regressive lesions. Molecular biomarkers measured in PMLs or the uninvolved airway enhance histopathological grading and discover immunoprevention strategies for intercepting the progression of PMLs to lung cancer.
[00335] Introduction [00336] Lung cancer (LC) is the leading cause of cancer death taking -160,000 US lives each year, more than colorectal, pancreatic, breast, and prostate cancers combined. To decrease mortality, the inventors need innovative strategies to intercept cancer development by diagnosing the disease at its earliest and potentially most curable stage. Development of LC risk biomarkers and interception strategies requires a detailed understanding of the earliest molecular alterations involved in lung carcinogenesis that occur in the respiratory epithelium 1,2. Exposure to cigarette smoke creates a field of injury throughout the entire respiratory tract by inducing a variety of genomic alterations that can lead to an at-risk airway where premalignant lesions (PMLs) and LCs develop. Lung squamous cell carcinoma (LUSC) arises in the epithelial layer of the bronchial airways and is often preceded by the development of PMLs through a stepwise histological progression from normal epithelium to hyperplasia, squamous metaplasia, dysplasia (mild, moderate, and severe), carcinoma in situ (CIS), and finally to invasive and then metastatic LUSC3. In fact, the presence of high-grade persistent or progressive dysplasia (moderate or severe) is a marker of increased LC risk both at the lesion site (where they are the presumed precursors of squamous cell lung cancer) and elsewhere in the lung, although many dysplastic lesions do have varied outcomes4,5. Currently, however, the inventors lack effective tools to identify PMLs at highest risk of progression to invasive carcinoma6. The development of markers of disease progression would identify patients at high-risk, show novel lung cancer chemoprevention agents, and provide molecular biomarkers for monitoring outcome in lung cancer prevention trials.
[00337] The inventors showd that molecular characterization of bronchial endobronchial biopsies containing a mixture of epithelial and immune cells would allow us to identify transcriptomic alterations associated with high-grade histology and premalignant lesion progression. In this study, the inventors used mRNA sequencing (mRNA-seq) to profile endobronchial biopsies and brushings obtained through serial bronchoscopies from high-risk smokers undergoing lung cancer screening by autofluorescence bronchoscopy and chest computed tomography (CT). Using the bronchial biopsies, the inventors identified four molecular subtypes associated with clinical phenotypes and biological processes. One subtype (Proliferative subtype) is enriched with bronchial dysplasia, high basal cell and low ciliated cell signals, and expression of proliferation-associated pathways. Genes involved in interferon signaling and T-cell-mediated immunity were down regulated among progressive/persistent lesions within the Proliferative subtype compared with regressive lesions and these pathways correlated with decreases in both innate and adaptive immune cell types. Molecular classification of biopsies into a high-grade/progressive disease group may be used to stratify patients into prevention trials and to monitor efficacy of the treatment. The results also show that personalized lung cancer chemoprevention, targeting specific cancer-related pathways or the immune system may have potential therapeutic benefits.
[00338] Results
[00339] Subject population [00340] In this study, the inventors used mRNA-seq to profde endobronchial biopsies and brushings obtained through serial bronchoscopy of high-risk smokers undergoing lung cancer screening by autofluorescence bronchoscopy and chest CT at the Roswell Park Comprehensive Cancer Center (Roswell) in Buffalo, NY. The Discovery Cohort samples were obtained from the Roswell subjects between 2010 and 2012 (DC; n = 29 patients, n = 191 biopsies, n = 91 brushes), and the Validation Cohort samples were obtained between 2012 and 2015 (VC; n = 20 patients, n = 111 biopsies, and 49 brushes). The subjects are predominantly older smokers, many of which have a prior history of lung cancer, chronic obstructive pulmonary disease (COPD), and occupational exposures that confer a high-risk of developing lung cancer. Clinical characteristics reported at the baseline visit such as sex, age, smoking status (ever or never), pack-years, prior history of lung cancer, COPD status, and occupational exposures were not significantly different between the two cohorts (Table 1). After sample filtering based on several quality metrics, the DC had 190 biopsies and 89 brushes, whereas the VC had 105 biopsies and 48 brushes. Ninety-four percent of subjects had at least one lung anatomic location sampled two or more times via endobronchial biopsy. The DC and VC contained 37.9% and 35.2% biopsies with a histological grade of dysplasia or higher and 23.1% and 19.0% had progressive/persistent dysplasia, respectively (Table 2). The inventors used a previously described smoking-associated signature? to predict the smoking status of each sample, as smoking status was only available at baseline. The predicted smoking status was consistent across all procedures for 63% and 70% of the DC and VC subjects, respectively (Supplementary Table 1). In terms of RNA sequencing quality, the DC had significantly greater total reads, percent uniquely mapping reads, and median transcript integrity number scores8 among the biopsies than the VC, but these differences between cohorts were not reflected in the brushes (Supplementary Table 2).
[00341] LUSC PMLs divide into distinct molecular subtypes
[00342] In order to identify gene expression differences associated with LUSC PML histological severity using the endobronchial biopsies, the inventors used a discovery -based approach to identify de novo molecular subtypes based on distinct patterns of gene co-expression (gene modules). The approach was chosen given that there is histological heterogeneity within biopsies and that pathological analyses were conducted using biopsies adjacent to biopsies profiled via mRNA-Seq. First, the inventors sought to select a set of gene modules that are present across different LUSC- related data sets. Using weighted gene co-expression network analysis9 (WGCNA), gene modules were derived in the DC biopsies (n = 190 samples, n = 16653 genes, n = 15 gene modules), the DC brushes (n = 89 samples, n = 16058 genes, n = 47 gene modules), TCGA LUSC tumorslO (n = 471 samples, n = 17887 genes, n = 55 gene modules), and tracheobronchial samples from mice treated with n-nitrosotris-(2-choroethyl)urea (NTCU) (n = 25 samples, n = 14897 genes, n = 40 gene modules). DC biopsy gene modules that were highly correlated (absolute Pearson correlation coefficient r > 0.85) to at least one other non-DC biopsy module within each of the four data sets were selected. Genes in the selected modules were filtered by requiring that each gene was also present in at least one of the correlated non-DC biopsy modules, resulting in a set of nine gene modules that consisted of 3936 genes in total (Supplementary Table 3). These gene modules identified four molecular subtypes within the DC biopsies via consensus clustering: Proliferative (dark blue, n = 52 samples, 27.4%), Inflammatory (dark green, n = 37 samples, 19.5%), Secretory (light blue, n = 61 samples, 32.1%), and Normal-like (light green, n = 40 samples, 21.1%) (Fig. la, Table 3).
[00343] In order to characterize each molecular subtype, the inventors first focused on identifying biological pathways enriched in each module, as the pattern of gene module expression defines the PML subtypes. Each gene module was found to be associated with distinct epithelial and immune biological processes (Fig. la, Supplementary Tables 3 and 4, Data 1). The Proliferative subtype is specifically characterized by increased expression of genes involved in energy metabolism and cell cycle pathways (Modules 4 and 5). The Secretory and Normal-like subtypes both have increased expression of genes in cilium-associated pathways (Module 6), however, the Normal-like subtype specifically has decreased expression of genes involved in inflammation, regulation of lymphocytes and leukocytes, and antigen processing and presentation pathways (Modules 8 and 9). The Secretory subtype exhibits decreased expression of genes involved in protein translation (Module 7), whereas RNA processing genes (Module 2) are expressed more highly in the Inflammatory subtype.
[00344] The inventors further characterized our molecular subtypes by their associations with clinical phenotypes and established LUSC tumor molecular subtypesl 1,12. Sample genomic smoking status, the subject from whom the sample was derived, and sample histology demonstrated significant associations with molecular subtype (p < 0.01, two-sided Fisher’s exact test, Fig. lb, Supplementary Tables 5 and 6, Supplementary Figs. 1, 2, and 3). The Proliferative and Secretory subtypes are enriched for current smokers and the Proliferative subtype is enriched for bronchial dysplasia (Fig. lb). The Proliferative subtype has high expression of genes involved in cell cycle processes including the proliferation marker MKI67, which is significantly up regulated among samples in this subtype compared with samples in other subtypes (false discovery rate; FDR= 1.0e-30, linear model). The gene remained significantly up regulated in Proliferative normal/hyperplasia samples (FDR = 3.4e-10, linear model) and dysplasia samples (FDR = 3. le-8, linear model), and these observations are supported by an increase in protein expression in representative samples (p = 0.02, linear model) (Fig. lc-e, Supplementary Fig. 4, and Supplementary Table 7). The Proliferative subtype samples also had high concordance with the LUSC-Classical subtype (Fig. lb). In the TCGA LUSC tumors, the LUSC-Classical subtype was associated with alterations and over expression of REAP 1 and NFE2L2 as well as amplification of 3q26 with over expression of SOX2, TP63, and PIK3CA11. Similarly, the inventor’s Proliferative PMLs have increased expression of KEAP1, NFE2L2, TP63, and PIK3CA (FDR= 1.4e-6, 4.5e-12, 1.4e-9, and 0.03, respectively, linear model) (Supplementary Fig. 5A).
Furthermore, the LUSC-Classical subtype was associated with increased expression of genes involved in energy metabolism, and the inventor’s Proliferative subtype is in part defined by high expression of Module 4, that is enriched with genes involved in oxidative phosphorylation and the electron transport chain. In contrast, the Inflammatory and Secretory PML subtypes demonstrate enrichment for the LUSC-Secretory subtype. The LUSC-Secretory subtype was associated with the immune response, and the Inflammatory, and Secretory PMLs have the highest expression of Module 8 that is enriched for genes in these same pathways.
[00345] Finally, the inventors wanted to examine the extent to which the inventor’s PML molecular subtypes were driven by differences in epithelial and immune cell type composition by assessing expression of a number of canonical cell type markers. The Inflammatory and Secretory subtypes have higher levels of expression of the white blood cell marker PTPRC (CD45) consistent with enrichment of the LUSC-Secretory subtype (Supplementary Fig. 5B, FDR = 0.12 and 0.01, respectively, linear model). Consistent with the pathways enriched in Module 6, the ciliated cell marker TUB1A1 expression is decreased in the Inflammatory and Proliferative subtypes (FDR= l. le- 4 and 3.5e-19, respectively, linear model), and this is also shown by a decrease in acetylated a-tubulin staining in representative histological samples (Fig. le, Supplementary Fig. 4, and Supplementary Table 7). The Proliferative subtype has the highest expression (FDR = 2.4e-15, linear model) of basal cell marker (KRT5), indicating enrichment of lesions with high-grade histology that tightly correlates with protein expression in representative histology samples (p = 0.01, linear model) (Fig. le, Supplementary Fig. 4, and 5B, Supplementary Table 7). In addition, gene expression of MUC5AC, a marker of goblet secretory cells, is increased in subtypes enriched for current smokers (Proliferative and Secretory) but is the most significantly increased in the Secretory subtype (FDR= 3.4e-5, linear model). In contrast, gene expression of SCGB 1A1, a marker of club cells, is the lowest in the Proliferative subtype (FDR = 6. le-5, linear model). The Normal-like subtype is supported by expression of all epithelial cell types and has the lowest expression of CD45 (FDR = 7.6e-4, linear model, Supplementary Figs. 5B-D). The expression levels of these marker genes agree with cell type deconvolution methods to examine epithelial and immune cell content (Supplementary Figs. 5C-D). The summation of these characterizations highlights epithelial and immune cell associated pathways that are modulated by smoking and PML histology and identifies the Proliferative subtype as a subset of high-grade PMLs that express proliferative and cell cycle-related pathways.
[00346] Molecular subtypes are replicated in the VC
[00347] Next, the inventors wanted to determine whether the heterogeneity captured in the DC biopsy-derived molecular subtypes was reproducible in the VC. The inventors developed a 22-gene nearest centroid molecular subtype predictor by selecting genes highly correlated with each of the gene module eigengenes. The predictor has 84.7% accuracy across DC biopsies (training set, Fig. 2a and Supplementary Fig. 6) with the following misclassification rates per subtype 5/52 (9.6%) in Proliferative, 7/37 (18.9%) in Inflammatory, 9/61 (14.8%) in Secretory, and 8/40 (20%) in Normal like. The 22-gene classifier was used to predict the molecular subtype of the 105 VC biopsies (Fig. 2b). The VC subtype predictions were evaluated by examining the concordance of Gene Set Variation Analysis (GSVA)13 scores for each of the 9 modules (using the full set of genes for each module) between the predicted VC subtypes compared with the DC subtypes. The average behavior of PCI across the subtypes was highly similar (Supplementary Fig. 7) with few exceptions (namely, Module 3 that had the fewest genes). In addition, the inventors compared the VC subtype predictions from the 22-gene classifier to subtypes derived in the VC biopsies using the same methodology used to derive the DC subtypes and found significant concordance (p = 1.Oe-7, two-sided Fisher’s exact test, with the Proliferative subtype having the greatest concordance between predictions, Supplementary Fig. 6).
[00348] The statistical associations between the VC subtypes (via the 22-gene classifier) and clinical and molecular phenotypes across the VC biopsies are analogous to those observed across the DC biopsies (Fig. 2c, Supplementary Tables 5 and 6, Supplementary Figs. 1 and 3). In brief, the Proliferative subtype is enriched for current smokers, biopsies with bronchial dysplasia, and the LUSC-Classical tumor subtype (Fig. 2c, Supplementary Table 5). Epithelial and white blood cell marker gene expression across the VC biopsies reveals higher levels of the white blood cell marker PTPRC (CD45 expression) in the Inflammatory subtype (FDR = 0.002, linear model) consistent with enrichment of the LUSC-Secretory subtype (Supplementary Fig. 5F). The Inflammatory and Proliferative subtypes have reduced ciliated cell marker expression (FOXJ1) consistent with Module 6 (FOXJ1 FDR = 0.0005 and FDR = 2.62e-6 and Module 6 FDR = 5.73e-6 and FDR = 4.34e-10, respectively, linear models). The Proliferative subtype has the highest expression of basal cell marker KRT5 (FDR = 1.67e-7, linear model), proliferation marker MKI67 (FDR= 3.03e-10, linear model), and cell cycle-associated Module 5 (FDR = 1.23e-18, linear model) indicating enrichment of lesions expressing characteristics associated with high-grade histology. Gene expression of SCGB 1A1, a marker of club cells, is the lowest in the Proliferative subtype (FDR= 1.8e-4, linear model). All epithelial cell type markers are expressed in the Normal-like subtype, and CD45 expression is decreased (FDR = 0.14, linear model, Supplementary Figs. 5F-G). Gene expression of MUC5AC, a marker of goblet epithelial cells, was increased in current smokers and most significantly in the Secretory subtype in the DC biopsies; however, in the VC biopsies this trend is not preserved as current smokers are not enriched in the Secretory subtype. The expression levels of these marker genes agree with other deconvolution methods to examine epithelial and immune cell content (Supplementary Figs. 5E-H).
[00349] Airway brushes reflect biopsy Proliferative subtype
[00350] Previously, the inventors have shown that bronchial brushes from normal-appearing areas of the mainstem bronchus could predict the presence of PMLs 14; however, that study lacked biopsies and brushes from the same subjects. Above, in both the DC and the VC biopsies, the Proliferative subtype, represents a distinct subtype of PMLs enriched for dysplastic histology expressing metabolic and proliferative pathways. Biopsies classified as the Proliferative subtype can represent a group of PMLs that need close monitoring and intervention. As a result, the inventors sought to explore whether or not the inventors could predict the presence of Proliferative subtype biopsies using the brushes. The Proliferative subtype is defined by the behavior of Modules 4, 5, 6, and 7 (Table 3), and therefore, the inventors used the subset of 8 genes (from the 22 -gene predictor) that correspond to these modules to predict presence/absence of the Proliferative subtype across the DC and VC biopsies and brushes. A prediction of the Proliferative subtype in a brush is specific (91% and 92% in the DC and VC biopsies, respectively), but not sensitive (39% and 32% DC and VC biopsies, respectively) at indicating the presence of at least one Proliferative PML detected at the same time point (Fig. 3a). In order to understand the classifier’s performance in predicting the Proliferative subtype in brushes, the inventors examined GSVA scores for Modules 4, 5, 6, and 7 that define the Proliferative subtype in the DC and VC brushes (Fig. 3b). In the DC and VC brushes, the GSVA scores were significantly different (FDR < 0.05) in the Proliferative subtype versus all other samples only for Modules 5 and 6, and thus these likely contribute the most heavily to Proliferative subtype classification in the brushes. Module 5 contains genes associated with cell cycle and proliferation, whereas Module 6 contains genes associated with cilium assembly and organization. Upregulation of Module 5 and
downregulation of Module 6 in the brushes specifically predicts the presence of a Proliferative subtype PML; however, the absence of these signals in the airway field of injury does not preclude the development of a Proliferative subtype PML.
[00351] Immune genes associate with PML progression
[00352] Previous studies of bronchial PMLs show that high-grade lesions (which occur more frequently in current smokers) are more likely to progress to invasive carcinoma5. Therefore, the inventors sought to identify molecular alterations associated with subsequent PML
progression/persistence (n = 15) versus regression (n = 15) among the Proliferative subtype DC biopsies, as these can be clinically relevant to identifying appropriate interception strategies. Using GSVA scores calculated across all the DC biopsies for each of the nine modules, the inventors calculated which scores were statistically different between progressive/persistent versus regressive disease in the samples belonging to the Proliferative subtype. The inventors found that the DC biopsy GSVA module scores for Module 9 were significantly higher among regressive Proliferative PMLs (p = 0.002, linear model, Fig. 4a) compared with progressive/persistent Proliferative PMLs. The association between low Module 9 scores and progression/persistence is replicated in the VC biopsies (n = 7 progressive/persistent and n = 13 regressive biopsies; p = 0.03, linear model, Fig. 4b). The ability of the Module 9 GSVA scores to discriminate between regressive versus progressing/persistent biopsies as measured by the area under the receiver operating characteristic was 0.809 and 0.802 in the DC and VC biopsies, respectively.
[00353] The genes in Module 9 include a number of genes that encode for proteins involved in interferon signaling as well as antigen processing and presentation (SP100, CIITA, CXCL10, SOCS1, GBP1, GBP4, B2M, TAPI, TAPBP, TRIM14, TRIM21, TRIM22, STAT1, PML, OAS2, OAS3, MX1, ADAR, ISG15, IFI35, IFIT3, IFI27, PSMB8, PSMB9, BST2, IRF1, IRF9, CD74, PSME1, PSME2, HLA-DQA1/DPA1/ DPB1/DRA/ DQB2/DRB1/ DQB 1/DMA/DMB/DOA, HLA- A/B/C/E/F) and include the inhibitory receptor LAG3. As a result, the inventors wanted to evaluate whether or not the presence or absence of innate or adaptive immune cells were associated with Module 9 expression within the Proliferative subtype. In an effort to deconvolute the potential presence of immune cell types, the inventors generated GSVA scores using previously described immune cell signatures 15 and scores for 64 different cell types using the xCell algorithm 16, separately for both the DC and VC biopsies. The inventors identified significant (FDR < 0.05, linear model) associations between the cell type scores and Module 9 that were in common between the DC and VC biopsies and identified eight cell types (via xCell): dendritic cells, activated dendritic cells, plasmacytoid dendritic cells, macrophages, Ml macrophages as well as CD8+ effector memory T cells, CD8+ central memory T cells, and T regulatory cells (Fig. 4c). Taken together, the
progressive/persistent biopsies in the Proliferative subtype have down-regulated expression of Module 9 compared with regressive biopsies that correlates with reduced signals from both innate and adaptive immune cell populations.
[00354] Immune cell populations are altered in PML progression
[00355] In order to confirm the relationship between the immune cell types associated with Module 9 and histologic progression/persistence of PMLs in the Proliferative subtype,
immunofluore scent staining of macrophages/monocytes (n = 52 regions enumerated from n = 16 subjects), CD4 (n = 50 regions enumerated from n = 17 subjects), and CD8 T cells (n = 47 regions enumerated from n = 16 subjects) was performed (Supplementary Table 7). The results were analyzed across all subjects assayed within the Proliferative subtype and across the subset of subjects where the lesion outcome (progression/persistence versus regression) was concordant with the Module 9 GSVA score (denoted as concordant set). Staining of CD68, a pan macrophage (and tumor-associated macrophage) marker (Ml type macrophages), was increased in progressive/persistent lesions (p « 0.001 in the concordant set). In contrast, staining of CD163 in combination with CD68, thought to be suggestive of M2 type macrophages, were decreased among the progressive/persistent lesions in the Proliferative subtype (p « 0.001 using all subjects and p = 0.0007 in the concordant set, respectively, linear model) (Fig. 4d-e). In addition, CD4 T cells were increased (p « 0.001 in the concordant set, linear model and CD8 T cells were decreased (p « 0.001 in the concordant set, linear model) in PMLs that progress/persist. Interestingly, among progressive/persistent lesions, the CD8 T cells had a distinct localization pattern (p = 0.07 in the concordant set, linear model), where CD8 T cells both lined and were embedded within the epithelium in areas where dysplasia is present (Fig.
4d). The immunofluorescence results did not reach significance, with the exception of CD163, when just the lesion outcome was used without regard to the Module 9 score.
[00356] Discussion
[00357] LUSC is the second most common form of lung cancer. LUSC arises in the epithelial layer of the bronchial airways, and is often preceded by the development of lung squamous PMLs.
The presence of dysplastic persistent and or progressive PMLs is a marker of increased risk for LUSC5. Currently, however, the inventors lack effective tools to identify PMLs at highest risk of progression to invasive carcinoma (6). The development of markers predictive of disease progression will be important in identifying patients at highest risk for LUSC development and in identifying biological pathways exploitable for LUSC chemoprevention. Towards this goal, the inventors profile via mRNA-Seq bronchial brushes and endobronchial biopsies obtained from subjects undergoing longitudinal lung cancer screening by chest CT and autofluorescence bronchoscopy. The inventors identify four transcriptionally distinct groups of biopsies, one of these the inventors label Proliferative and find it to be associated with bronchial dysplasia. Patients with Proliferative PMLs can also be identified via gene expression measured from cells in the non-involved large airway epithelium. The inventors further find that persistent/progressive Proliferative PMLs are characterized by decreased expression of genes involved in interferon signaling and antigen processing/presentation pathways. Consistent with these gene expression findings the inventors find that progressive/persistent
Proliferative PMLs are depleted for CD68+/CD163+ macrophages and CD8 T cells by
immunofluorescence. Collectively, these data show both the potential to identify a subset of patients with progressive/persistent LUSC PMLs, who are at risk for developing invasive lung cancer, on the basis of airway gene expression; as well as the potential to decrease the risk for progression in these patients by augmenting the immune response associated with regression.
[00358] Previous studies show a range of genomic alterations associated with bronchial dysplasia. Increased expression of EGFR and Ki67 staining of epithelial cells is associated with increasing histologic severity and subsequent histologic progressions, 17. Altered protein levels of TP53, CCND1, CCNE1, BAX, and BCL2 have been associated with CIS or lung cancer occurrence independent of histological grade 18. Telomere shortening and maintenance 19 and loss of heterozygosity in regions frequently deleted in lung cancer (3p, 5q, 9p, 13q, 17p) has been observed in early hyperplasia/metaplasia lesions20-22 and found to increase in frequency and size in higher- grade dysplasia. Genomic gains in loci containing SOX2, TP63, EGFR, MYC, CEP3, and CEP5 are also associated with progression of high-grade dysplasia23. Despite the numerous genomic alterations associated with PML histological grade and progression, the inventors lack a comprehensive PML molecular classification system to complement pathologic examination. The inventors pursued an unsupervised class discovery approach that led to the identification of four distinct molecular PML subtypes (Proliferative, Inflammatory, Secretory, and Normal -like). The transcriptional patterns differentiating the PML subtypes are robust and a 22 -gene panel identified in the DC can be used to distinguish between the molecular subtypes in an independent VC. Interestingly, whereas prior lung cancer history may influence airway gene expression and about two-thirds of the subjects have a prior history of lung cancer, the inventors do not detect a significant association between lung cancer history and molecular subtype, and there is a similar diversity of molecular subtypes between biopsies collected from subjects with and without a lung cancer history. This observation is supported by prior work describing gene expression alterations that reflect the presence of bronchial dysplasia using airway brushings in a similar cohort 14. [00359] Among the molecular subtypes, the Proliferative subtype is enriched with dysplastic PMLs from current smokers and is characterized by up regulation of metabolic (OXPHOS/ETC/TCA) and cell cycle pathways and down regulation of cilia-associated pathways. Previous work shows increases in metabolic pathways in the airways of subjects with dysplastic lesions 14, in PMLs adjacent to LUSC tumor24, and in smokers at high-risk for lung cancer25 as well as increases in proliferation (via Ki67 levels, as mentioned above) that have been utilized as an endpoint in lung cancer chemoprevention26,27. Identification of patients with Proliferative lesions can be useful to enrich lung cancer chemoprevention trials with high-risk subjects or to identify patients who would benefit from more frequent lung cancer screening. The Inflammatory subtype is predominated by PMLs from former smokers, but interestingly is not significantly enriched for dysplasia, despite similarly decreased expression of cilia-associated pathways, showing an abnormal epithelium. The Inflammatory subtype also shows increased expression of a gene module enriched for genes involved in inflammation and regulation of lymphocytes and leukocytes (Module 8). This gene module is also elevated in the Secretory subtype predominated by current smokers and increased expression of goblet cell markers. Interestingly, IL1B is part of this inflammation-related gene module, and inhibition of IL1B has recently been shown to reduce lung cancer incidence28.
[00360] The inventor’s prior work has extensively studied gene expression alterations in normal appearing airway epithelium by profding cells obtained via brushing the mainstem bronchus during bronchoscopy7, 14,29-34. In the current study, the inventors have both normal-appearing bronchial brushes and endobronchial biopsies collected during the same procedure, allowing us to identify gene expression differences in brushings, which show the presence of Proliferative subtype PMLs. In both the discovery and VC, applying a Proliferative subtype classifier (based on PML biopsy gene expression) to gene expression data from the brushings resulted in Proliferative subtype predictions that were very specific (91%) but not sensitive (31-38%). Brushes classified as Proliferative have increased expression of cell cycle pathways and decreased expression of cilia-associated genes, showing that they are more similar to squamous metaplasia than normal epithelium. Potentially, a subset of patients can harbor widespread airway damage that serves as a marker for Proliferative bronchial dysplasia leading to modest sensitivity, but high specificity. In other cases, the area of damage that gives rise to these Proliferative PMLs can be more localized, and therefore difficult to detect by brushing, contributing to decreased sensitivity. These findings show that therapeutics to target changes throughout the entire airway epithelium can be necessary in some subjects, whereas, more site-specific ablation can be more effective in certain cases. Another possibility and area of future research, is that a Proliferative subtype brush is a predictor of incident LUSC.
[00361] The molecular profiling of PMLs and the identification of gene co-expression modules also provides an opportunity to identify the molecular determinants of subsequent PML progression. One of the nine gene co-expression modules used to define the molecular subtypes was significantly decreased between biopsies that progress/persist compared to biopsies that regress within the Proliferative subtype in both the discovery and VC. The module contains genes involved in interferon signaling and antigen processing and presentation, and its expression was correlated with the abundance innate and adaptive immune cells via computational prediction. By immunofluorescent staining of formalin-fixed paraformaldehyde-embedded (FFPE) biopsy sections the inventors confirmed that the progressive/persistent Proliferative lesions with low Module 9 GSVA scores had fewer CD 163+ macrophages and CD8+ T cells and the CD8+T cells had a distinct localization pattern. These lesions also contained greater numbers of CD4+T cells, and it will be important in future work to assess if these cells are T regulatory cells promoting an immune suppressive environment.
[00362] The presence of tumor-associated macrophages with the polarized phenotypes (Ml as pro-inflammatory or M2 as anti-inflammatory) has been associated with lung cancer prognosis. The presence of predominantly M2 macrophages, marked by the expression of CD163, has been associated with worse survival. However, in the context of lung PMLs this relationship is not well studied. The inventor’s finding that regressive Proliferative PMLs have more CD 163+ cells and increased expression of genes involved in interferon ( I FN )g signaling is consistent with observations in oral squamous cell carcinoma PMLs where the presence of CD 163+ macrophages with active IFNy signaling is associated with better outcomes35. In addition, the inventors observed fewer CD8+ T cells and lower expression of human leukocyte antigen (HLA) class I genes and B2M in
progressive/persistent lesions within the Proliferative subtype. Disruptions in proper T-cell-mediated immunosurveillance have been described in several studies showing that impaired HLA class I antigen processing and presentation including down regulation or loss of B2M36,37 and interferon signaling38 in lung tumors affects response and acquired resistance to checkpoint inhibitors. Lung tumors lacking an HLA-I complex had lower cytotoxic CD8+ lymphocyte infiltration, and this was also associated with lower levels of PD-L1. In addition, studies have also showed negative impacts on efficacy of checkpoint inhibitors as well as survival in patients with LC that have tumors with increased CD4+ T cells expressing T-regulatory markers (FOXP3, CD25), resulting in
immunosuppressive state showed to hinder the recruitment and effector functions of CD 8+ T cells39,40. Future DNA sequencing data on the PMLs profiled here can show heterozygous or homozygous loss of B2M or mutations in other genes in the interferon and antigen processing and presentation pathways; however, even in the case of acquired resistance, mutations and copy number changes could not explain the down regulation of these pathways across all subjects, showing that other epigenetic alterations or signaling pathways can have a role. Unraveling the mechanisms of innate and adaptive immune downregulation in this subset of PMLs will be important to identifying potential immunoprevention therapies.
[00363] The inventor’s data show that there are subtype-specific transcriptomic alterations predictive of subsequent LUSC PML progression that are the result of a lack of infiltrating immune cells in the lesion microenvironment. These data show that biomarkers for determining PML subtype and assessing immune infiltration can have utility for the detection of aggressive PMLs that require more intensive clinical management and genes altered in these PMLs can serve as lung
chemoprevention candidates. These biomarkers could either be measured directly in PML tissue or a surrogate tissue such as bronchial airway epithelium. A benefit of biomarkers predicting aggressive PML behavior measured in surrogate tissue is the potential that these biomarkers can also predict the behavior of PMLs not directly observed during bronchoscopy. Future studies are needed to address the specific mechanism of impaired immunosurveillance in progressive/persistent lesions in the Proliferative subtype including single-cell sequencing, high coverage DNA sequencing,
characterization of neoepitope burden, assessment of epigenetic alterations, and comprehensive characterization of the identified immune populations. Overall, the inventor’s data support the potential for immunoprevention strategies to intercept the progression of PMLs to lung cancer.
[00364] Methods
[00365] Subject population and sample collection
[00366] Endobronchial biopsies and brushings were obtained from high-risk subjects undergoing lung cancer screening at ~l-year intervals by white light and autofluorescence bronchoscopy and computed tomography at Roswell. The bronchoscopy included visualization of the vocal cords, trachea, main carina, and orifices of the sub-segmental bronchi visible without causing trauma to the bronchial wall. All abnormal and suspicious areas are biopsied twice and the lung anatomic location is recorded (Supplementary Fig. 9, Supplementary Table 9). One biopsy was used for routine pathological evaluation and the other for molecular profiling. In addition, a brushing was obtained from a normal-appearing area of the left or right mainstem bronchus for research. Morphological criteria used to evaluate the biopsies are in accordance with World Health Organization (WHO) guidance41. Eligibility for screening includes either a previous history of aerodigestive cancer and no disease at the time of enrollment or age > 50, a current or previous history of smoking for a minimum exposure of 20 pack-years and at least one additional risk factor including moderate COPD (defined as forced expiratory volume (FEV1) < 70%), confirmed asbestos related lung disease or a strong family history of lung cancer (at least 1-2 first-degree relatives). All research specimens were stored in RNA Allprotect (Qiagen) and stored at -80°C.
[00367] Subjects were selected that had biopsies collected in repeat locations via serial bronchoscopies; however, after RNA isolation, samples from three subjects had a single biopsy and one subject had a single brushing. mRNA-seq was performed on a DC of samples comprising of endobronchial biopsies and brushes collected between 2010 and 2012 (n = 30 subjects, n = 197 biopsies, and n = 91 brushings). mRNA-seq was subsequently performed on a VC of samples comprising of endobronchial biopsies and brushes collected between 2012 and 2015 (n = 20 subjects, n = 111 biopsies, and n = 49 brushings). Biopsy progression/regression was defined for each biopsy based on the histology of the biopsy and the worst histology recorded for the same lung anatomic location in the future. Histology changes between normal, hyperplasia, and metaplasia were classified as“normal stable”, decreases in histological dysplasia grade or changes from dysplastic histology to normal/hyperplasia/metaplasia were classified as“regressive”, lack of future histological data was classified as“unknown”, and everything else was classified as“progressive/persistent.” The
Institutional Review Boards at Boston University Medical Center and Roswell approved the study and all subjects provided written informed consent.
[00368] RNA-Seq library preparation, sequencing, and data processing
[00369] Total RNA was extracted from endobronchial biopsies and bronchial brushings using miRNeasy Mini Kit or AllPrep DNA/RNA/miRNA Universal Kit (Qiagen). Sequencing libraries were prepared from total RNA samples using Illumina TruSeq RNA Kit v2 and multiplexed in groups of four using Illumina TruSeq Paired-End Cluster Kit. Each sample was sequenced on the Illumina HiSeq 2500 to generate paired-end 100-nucleotide reads. Demultiplexing and creation of FASTQ fdes were performed using Illumina CASAVA 1.8.2 or BaseSpace. Samples were aligned using hgl9 and 2-pass STAR42 alignment. Gene and transcript level counts were calculated using RSEM43 using Ensembl v75 annotation. Quality metrics were calculated by STAR and RSeQC8. Samples were excluded were sex annotation did not correlate with gene expression across CYorfl5A, DDX3Y, KDM5D, RPS4Y1, USP9Y, and UTY (n = 4 samples). Sample relatedness within a patient was confirmed using Peddy software44. Samples with a high-rate of heterozygosity (>3 standard deviations above the median) or samples with low relatedness to samples from the same patient (>3 standard deviations below the median) were removed from further analyses (n = 11 samples, two brushes, and nine biopsies). Samples were subsequently divided into the discovery and VC (as outlined above) and by tissue type (biopsy or brush). Subsequent sample and gene filtering was conducted separately on each set as follows: first, EdgeR45 was used to compute normalized data (library sizes normalized using TMM, trimmed mean of M-values, and log2 counts per million computed) and genes were excluded that either had an interquartile range equal to zero or a sum across samples equal or <1. Samples were excluded based on values > 2 standard deviations from the mean for more than one of the following criteria: (1) mean Pearson correlation with all other samples calculated across all filtered genes (2) the 1st or 2nd principal components calculated using the filtered gene expression matrix (3) transcript integrity number (TIN, computed by RSeQC). After sample filtering, gene filtering was recomputed as described above on the final set of high-quality samples. The data are available from NCBTs Gene Expression Omnibus using the accession
GSE 109743.
[00370] Derivation of molecular subtypes
[00371] The DC biopsies (n = 190 samples, n = 16653 genes) and brushes (n = 89 samples, n = 16058 genes) were used to derive the molecular subtypes. Two additional RNA-Seq data sets were used during the derivation of the molecular subtypes: the TCGA squamous cell carcinoma (LUSC) tumorslO (n = 471 samples, n = 17887 genes) and a dataset of tracheobronchial samples from mice treated with NTCU (n = 25 samples, n = 14897 genes). The mice develop lesions that are histologically and molecularly comparable to human lesions and that progress to LUSC and the samples represent a range of histology (normal, mild dysplasia, moderate dysplasia, severe dysplasia, CIS, and LUSC tumor) (Supplementary Materials and Methods). The mouse data are available from NCBI’s Gene Expression Omnibus using the accession GSE111091. Sample and gene filtering from the TCGA LUSC tumors and the mouse tissue were processed as described in the Supplementary Materials and Methods (available on the world wide web at per at doi. org/10.1038/s41467-019- 09834-2).
[00372] WGCNA9 was used with default parameters to derive modules of gene co-expression across the four data sets described above. Residual gene expression values adjusting for RNA quality (median TIN) and batch (Illumina flow cell) were used as input for WGCNA for the biopsy and brush data sets. For the mouse dataset, residual gene expression values adjusting for RNA quality (median TIN), mouse strain, and sample type (laser capture microdissected versus whole tissue) were used as input for WGCNA. For TCGA LUSC tumor samples, residual gene expression values adjusting for plate were used as input for WGCNA. Gene sets were created for each co-expression module for each dataset and then combined to create a compendium of gene sets. For each gene set in the
compendium, the first principal component (PCI) was calculated across each z-score normalized dataset. For each data set, a matrix of absolute Pearson correlation coefficients based on PCI values calculated for each gene sets in the compendium was computed and thresholds were set as follows: r > 0.85 was set to 1 and r < 0.85 set to 0. The four matrices were subsequently summed, and gene sets derived from biopsy co-expression modules that were correlated to another non-biopsy -derived gene set across all data sets were retained (n = 9 modules retained). The genes defining the retained biopsy modules were required to be present in the biopsy module and at least in one of the correlated gene sets.
[00373] The filtering process above yielded a reduced set of genes (n = 3936) that was used to define the molecular subtypes in the biopsy data. The residual gene expression values across the reduced set of genes for the discovery biopsies were used as input for consensus clustering46.
Consensus clustering was performed setting k (number of groups) to 10, the number of iterations to 1000, the subsampling to 80%, the clustering algorithm to partitioning around medoids, and the distance metric to Pearson correlation. The optimal value for k was 4 based on the relative change in area under the cumulative distribution function calculated based on the consensus matrix for each k.
[00374] Molecular subtype predictor
[00375] The DC biopsies across the module genes (n = 3936) were used to derive a molecular subtype predictor. First, squared Pearson correlation coefficients were determined between each gene and the module eigengenes (PCI for each of the nine modules). Genes were retained as part of a module if the squared correlation coefficient was the highest for the module in which it was assigned. The average squared Pearson correlation coefficient of the retained genes to the module eigengene was computed, and the number of genes chosen from each module for the predictor was inversely proportional to this metric. Second, genes with the highest squared correlation coefficients to the module eigengene were chosen to represent the module in the predictor. The 22 genes resulting from this analysis across the DC biopsy data were used to train a nearest centroid predictor using the pamr package with a threshold of zero and predict the molecular subtype across the VC biopsies. Prior to predicting the molecular subtype of these test sets, the training and test sets were combat47 adjusted and z-score normalized across combined training and test data. Using the methods described above the inventors derived molecular subtypes using consensus clustering across the VC biopsies and compared these to the predicted subtypes.
[00376] Identification of gene module biology
[00377] Biological processes and pathways enriched in each of the nine modules used to discover the molecular subtypes in the DC were identified using EnrichR48. Each module was separated into genes positively or negatively correlated with the module eigengene, the Ensembl IDs were converted to Gene Symbols using biomaRt, and the following databases were queried: GO Biological Process 2015, KEGG 2016, WikiPathways 2016, TargetScan microRNA, Transcription Factor PPIs,
TRANSFAC and JASPAR PWMs, OMIM Disease, Reactome 2016, and Biocarta 2016.
Processes/pathways with an FDR < 0.05 were considered to be significantly enriched. Modules are referred to being increased or decreased in each of the molecular subtypes based on the direction of change of the majority of the genes in the module.
[00378] Identification of molecular subtype phenotypes
[00379] The molecular subtypes in the DC biopsies were annotated according to the behavior of each gene module by calculating whether or not module GSVA13 scores were significantly associated (FDR < 0.05) with a particular molecular subtype versus all other samples (two-level factor) using a linear mixed effects model with patient as a random effect (using the‘duplicateCorrelation’ function) via limma. In addition, biological pathways and transcription factors associated with each subtype were identified using GSEA49 and mSigDB50 gene sets using genes ranked by the t-statistic for their association with each subtype. The ranked lists were created using the limma51 and edgeR45 packages to identify differentially expressed genes associated with subtype membership. Each linear model used voom-transformed52 data and included membership in the subtype of interest, batch, and RNA quality (TIN) as covariates and patient as a random effect. Pathways enriched in the ranked lists (FDR < 0.05) were used to annotate the molecular subtypes. FDR values for individual genes were derived from this analysis or analogous models using only samples of normal/hyperplasia histology or dysplasia histology.
[00380] For the DC and VC biopsies, residual gene expression values were used to predict smoking status, FUSC tumor subtype, and the relative abundance of epithelial and immune cells for each sample. Smoking status (current versus former/never) was predicted for each sample as described previously 14. Smoking status was determined at each time point for each subject by calculating the mean of the prediction scores ( >0 for current prediction and <0 for former/never prediction) across all biopsies and brushes sampled. The LUSC tumor subtype was determined as described previouslyl 1 across the genes predictive of the LUSC molecular subtypel2. The
ESTIMATE algorithm53 was used to infer relative epithelial, stromal, and immune cell content. Immune cell type specific signatures from Bindea et al.15 and epithelial cell type specific signatures from Dvorak et al.54 were used to generate GSVA13 scores across samples for each signature. In addition, residual gene expression values calculated using log RPKM values were inputted into the xCelll6 to infer relative abundances of 64 different cell types. The above categorical phenotypes along with additional clinical variables such as biopsy histology, subject, previous lung cancer history, sex, and biopsy progression/regression status were associated with molecular subtype using two-sided Fisher’s exact test.
[00381] In order to characterize the molecular alterations associated with lesion outcome, a linear mixed effects model was used to assess module GSVA score differences between
progressive/persistent versus regressive lesions within each molecular subtype with patient as a random effect via limma. The inventors estimated differences in the immune cell content (separately for xCell and Bindea et al.) between progressive/persistent versus regressive lesions in the
Proliferative subtype via a linear mixed effects model correcting for epithelial cell content
(‘Epithelial’ in xCell and‘Normal mucosa’ in Bindea et al.) and patient as a random effect. The inventors focused on cell types that were significantly different (FDR < 0.05) between
progressive/persistent versus regressive lesions in the Proliferative subtype in both the DC and VC.
[00382] Relationship between the biopsies and brushes
[00383] The inventors wanted to quantify the predictive performance of the brush with regards to the presence of a biopsy of the Proliferative subtype. A subset of the 22-gene molecular subtype predictor was used to predict the presence or absence of the Proliferative subtype across the DC and VC brushes and biopsies. Specifically, the inventors used eight genes (out of the 22) that
corresponded to modules 4 through 7 (significantly up or down regulated in the Proliferative subtype) to classify samples as Proliferative or not using the same methodology described above for the molecular subtype predictor. Sensitivity and specificity performance metrics were calculated based on the ability of a Proliferative subtype prediction in the DC or VC brushes to show the presence of at least one biopsy of the Proliferative subtype. In order to further understand the Proliferative subtype predictions in the brushes, the inventors analyzed the behavior of the modules that define the
Proliferative subtype in the DC biopsies (based on methods above) across the DC and VC brushes.
[00384] Immunofluore scent staining and quantitation
[00385] Standard formalin fixation and embedding techniques were employed at Roswell where five-micron sections were cut from the FFPE samples used for the routine pathological evaluation at Roswell (Supplementary Table 7). Prior to staining, samples were de-waxed with xylene and rehydrated through a graded series of ethanol solutions. AR or citrate buffer was used for antigen retrieval, tissue was incubated with primary antibodies overnight at 4 °C and probed with secondary antibodies with fluorescent conjugates (Invitrogen Alexa Fluor 488, 594, 647) for 1 h at room temperature. Immunostaining was performed using the primary antibodies listed in Supplementary Table 10. Imaging was performed using an Aperio Slide Scanner for scoring and a Carl Zeiss Axio (x20 and c40 objectives) and a Carl Zeiss LSM 710 NLO confocal microscope for capturing additional images. Digital slides were analyzed with the Definiens Tissue Studio (Definiens Inc.) for the enumeration of immunofluorescence staining. The enumeration of the immunofluorescence scored each stain including DAPI-positive cells. The enumeration was conducted on different regions (independent areas of tissue) present on a slide (1-5 regions/biopsy) for each biopsy. For each region, the percentage of positively staining cells for a given protein was calculated by dividing the number of positively stained cells by the total number of DAPI-positive cells. A binomial linear mixed effects model via the lme4 R package was used to assess differences in the percentages of cells staining positive for a given protein in each region between progressive/persistent versus regressive biopsies using the total cells stained in each region as weights and adjusting for the slide number as a random effect. The models were used across samples from the Proliferative subtype and across samples from the Proliferative subtype where the biopsy outcome (progressive/persistent versus regressive) agreed with the Module 9 GSVA score (scores < 0 are associated with progression/persistence and scores greater than 0 are associated with regression). Each region was also qualitatively scored as either positive or negative for having a distinct CD8 T-cell localization pattern where cells lined and were embedded within the epithelium.
[00386] Reporting summary and data availability
[00387] RNA sequencing data from human endobronchial biopsies and brushings has been deposited in the NCBI Gene Expression Omnibus under accession code GSE 109743. RNA (available on the world wide web at ncbi.nlm.nih.gov/geo/query/acc. cgi?acc= GSE109743), sequencing data from mouse lung samples treated with N-nitrosotris-(2-choroethyl) urea has been deposited in the NCBI Gene Expression Omnibus under accession code GSE111091 (available on the world wide web at ncbi.nlm.nih.gov/geo/query/acc. cgi?acc= GSE111091).
[00388] The source data underlying all figures and tables in the main text and supplementary information are provided as a Source Data file. All other data supporting the findings of this study are available within the article and its supplementary information files and from the corresponding author upon reasonable request. A reporting summary for this article is available as a Supplementary Information file. Further information on research design is available in the Nature Research Reporting Summary linked to this article (available on the world wide web at doi.org/10.1038/s41467-019- 09834-2). All custom computer code is available at on the world wide web at
github.com/jbeane0428/Computer-code-for-PML-Molecular-Subtypes. Corresponding input and output data used in the computer code is available on the world wide web at
doi.org/10.6084/m9.figshare.c.443245 l.vl.
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Claims

What is claimed herein is:
1. A method for treating or preventing a condition caused by or associated with
immunosuppressed aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator to the subject.
2. The method of claim 1, wherein the condition caused by or associated with
immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
3. The method of claim 2, wherein the cancer is a squamous cell cancer or lung squamous cell cancer.
4. The method of claim 2, wherein the infectious disease is a bacterial and/or viral infection.
5. The method of claim 4, wherein the viral infection is a coronavirus infection.
6. A method for treating or preventing a condition caused by or associated with autoimmune aberrant immune system activity in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator to the subject.
7. The method of claim 6, wherein the condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease.
8. A method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
ii. at periodic intervals, such as at least every 6 months ... , every 8 months
every 9 months ... , every year... , one of a bronchoscopy -based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
to a subject determined to have a level of expression of at least 1 negative immune response regulator which is increased relative to a reference level or expression of at least 1 positive immune response regulator which is decreased relative to a reference level of expression.
9. A method of treating bronchial premalignant lesions in a subject in need thereof, the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) administering at least one of:
i. both a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; ii. at least every 6 months, one of a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan;
iii. at least one immune stimulating drug; and/or
iv. an agonist of at least one positive immune response regulator and/or an
inhibitor of at least one negative immune response regulator;
if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
10. The method of claims 8 or 9, wherein if the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB 1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
11. The method of claim 10, wherein the different immune response regulator is selected from Table 1 or Table 2.
12. The method of claims 8 or 9, wherein there are at least two changes in immune response regulators relative to a reference level, wherein the changes can be (a) at least two increases of at least two negative immune response regulators, (b) at least two decreases of at least two positive immune response regulators; or a combination of at least one increase in at least one negative immune response regulator and a decrease of at least one positive immune response regulator.
13. The method of claim 11, wherein there is a change in at least 5 immune response regulators.
14. The method of claim 11, wherein there is a change in at least ten immune response regulators.
15. The method of any of the preceding claims, wherein the positive immune response regulator is selected from Table 1.
16. The method of any of the preceding claims, wherein the negative immune response regulator is selected from Table 2.
17. The method of any of the preceding claims, wherein the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRB1, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
18. The method of any of the preceding claims, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, and miR-149-5p.
19. The method of any of the preceding claims, wherein the different immune response regulator can further include a miRNA selected from Table 2A.
20. The method of any of the preceeding claims, wherein the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
21. The method of claim 20, wherein the method further comprises administering at least one anti -proliferative drug.
22. The method of any of the preceeding claims, wherein the at least one anti-proliferative drug is selected from the group consisting of:
Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; J K inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors; Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
23. The method of either of claims 8, 9, or 10 wherein the subject:
i. is administered:
1. a bronchoscopy-based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue but not a chest CT scan; or
2. at greater than 6 month intervals, one of a bronchoscopy -based procedure to survey the central airway wherein the lesions are biopsied to remove abnormal tissue and a chest CT scan; and/or
ii. the subject is not administered an immune stimulating drug,
when either:
the level of expression of at least 1 negative immune response regulator is not increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is not decreased relative to a reference level of expression; or
the subject is a subject determined to have a level of expression of at least 1 negative immune response regulator which is not increased relative to a reference level, or a level of expression of at least 1 positive immune response regulator which is not decreased relative to a reference level of expression.
24. The method of any of the preceding claims, wherein the agonist of an immune response regulator is an immune response regulator polypeptide, an immune response regulator miRNA, or a nucleic acid encoding the immune response regulator.
25. The method of any of the preceding claims, wherein the inhibitor of an immune response regulator is an antibody, antibody reagent, or inhibitory nucleic acid.
26. The method of any of the preceding claims, wherein the administering step comprises the administration of a vector comprising a nucleic acid encoding the agonist and/or inhibitor.
27. The method of any of the preceding claims, wherein the subject is a human.
28. The method of any of the preceding claims, wherein the administration is prophylactic.
29. The method of any of the preceding claims, wherein the subject is a current or former smoker.
30. The method any of the preceding claims, wherein the level of expression is detected for at least 1 negative or positive immune response regulator.
31. The method any of the preceding claims, wherein the level of expression is detected for at least three negative or positive immune response regulators.
32. The method any of the preceding claims, wherein the level of expression is detected for at least four negative or positive immune response regulators.
33. The method of any of the preceding claims, wherein the level of expression is detected for at least five negative or positive immune response regulators.
34. The method of any of the preceding claims, wherein the level of expression is detected for at least miR-149-5p.
35. The method any of the preceding claims, wherein the expression level of no more than 100 genes is detected.
36. The method of any of the preceding claims, wherein the at least one immune stimulating drug is selected from the group consisting of:
immune-checkpoint inhibitors (e.g. inhibitors against, PD-1, PD-L1, CTLA4, and LAG3); drugs that stimulate interferon signaling (e.g. anti-viral drugs that improve interferon signaling); DNA synthesis inhibitors; IMDH inhibitors; CDK inhibitors; ribonucleotide reductase inhibitors; dihydrofolate reductase inhibitors; topoisomerase inhibitors; FLT3 inhibitors; IGF-1 inhibitors; MEK inhibitors; aurora kinase inhibitors; PKC inhibitors; RAF inhibitors; PDFGR/KIT inhibitors; VEGFR inhibitors; SRC inhibitors; retinoid receptor agonists; HD AC inhibitors; DNA methyltransferase inhibitors; and EZH2 inhibitors.
37. The method of any of the preceding claims, wherein a treatment is administered during a bronchoscopy-based procedure.
38. The method of any of the preceding claims, wherein a treatment is administered systemically.
39. The method of any of the preceding claims, wherein a treatment is administered during a bronchoscopy-based procedure and systemically.
40. The method of any of the preceding claims, whereby the development of lung cancer lung squamous cell carcinoma is prevented, delayed, or slowed.
41. The method of any of the preceding claims, wherein the level of expression is the level of expression in an endobronchial biopsy, bronchial brushing sample, nasal brushing sample, sputum, or blood obtained from the subject.
42. The method any of the preceding claims, wherein the level of expression is the level of expression in a bronchial brushing obtained from the right or left mainstem bronchus.
43. The method of any of the preceding claims, wherein the biopsy or brushing sample comprises morphologically-normal tissues or cells.
44. A pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with immunosuppressed aberrant immune system activity, comprising:
a. an agonist of at least 1 positive immune response regulator or an inhibitor of at least 1 negative immune response regulator and
b. a pharmaceutical acceptable carrier.
45. The composition of claim 44, wherein the condition caused by or associated with immunosuppressed aberrant immune system activity is selected from the group consisting of cancer and infectious disease.
46. The composition of claim 45, wherein the cancer is a squamous cell cancer or lung squamous cell cancer.
47. The composition of claim 45, wherein the infectious disease is a bacterial and/or viral infection.
48. The composition of claim 47, wherein the viral infection is a coronavirus infection.
49. A pharmaceutical composition formulated for the treatment or prevention of a condition caused by or associated with autoimmune aberrant immune system activity, comprising:
a. an inhibitor of at least 1 positive immune response regulator or an agonist of at least 1 negative immune response regulator and
b. a pharmaceutical acceptable carrier.
50. The method of claim 49, wherein the condition caused by or associated with autoimmune aberrant immune system activity is selected from the group consisting of rheumatoid arthritis, lupus, and celiac disease.
51. A method of determining the risk of progression of bronchial premalignant lesions to lung cancer or an infectious disease in a subject the method comprising:
(i) obtaining a sample from the subject;
(ii) determining the level of expression of at least 1 negative or positive immune response regulator; and
(iii) determining the subject is at risk of the bronchial premalignant lesions progressing to lung cancer or an infectious disease if the level of expression of at least 1 negative immune response regulator is increased relative to a reference level, or if the level of expression of at least 1 positive immune response regulator is decreased relative to a reference level of expression.
52. The method of any of claim 51, wherein the positive immune response regulator is selected from Table 1 and the negative immune response regulator is selected from Table 2.
53. The method of claims 51 or 52, wherein if the change in an immune response regulator is from an immune response regulator selected from the group of immune response regulators consisting of: NLRC5, B2M, HLA-DRB 1, HLA-DPA1, and HLA-DRA, at least one immune response regulator different from the group must also show a change.
54. The method of claim 51 or 53 wherein the positive or negative immune response regulator is at least miR-149-5p.
55. The method of any of claims 51-54, wherein the at least 1 positive immune response regulator is selected from the group consisting of: NLRC5, QPRT, CPQ, MRAS, RCAN1, SERPINIl, B2M, HLA-DRB l, HLA-DPA1, HLA-DRA, MSC, and SLC5A8.
56. The method of any of claims 51-56, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, miR-149-5p.
57. The method of any of claims 51-56, wherein at least 1 negative immune response regulator is selected from the group consisting of: TIMM 13, TMEM63C, GSTP1, SMURF 1, and miR-149-5p.
58. The method of any of claims 51-56, wherein the different immune response regulator can further include a miRNA selected from Table 2A.
59. The method of any of claims 51-56, wherein the subject is further determined to have a proliferative lesion if the change is an increase in at least five miRNAs, selected from Table 2A.
60. The method of any of claims 51-56, wherein the method further comprises administering at least one anti -proliferative drug.
61. The method of any of claims 51-56, wherein the at least one anti-proliferative drug is selected from the group consisting of:
Acetylcholine receptor antagonist; Acetylcholinesterase inhibitors; Adenosine receptor antagonists; Adrenergic receptor antagonists; AKT inhibitors; Angiotensin receptor antagonists; Apoptosis stimulants; Aurora kinase inhibitors; CDK inhibitors; Cyclooxygenase inhibitors; Cytokine production inhibitors; Dehydrogenase inhibitors; DNA protein kinase inhibitors; focal adhesion inhibitors; Dopamine receptor antagonist; EGFR inhibitors; ERK1 and ERK2 phosphorylation inhibitors; Estrogen receptor agonists; EZH2 inhibitors; FLT3 inhibitors; Glucocorticoid receptor agonists; Glutamate receptor antagonists; HDAC inhibitors; Histamine receptor antagonists; Histone lysine methyltransferase inhibitors; HSP inhibitors; IKK inhibitors; Ion channel antagonists; JAK inhibitors; JNK inhibitors; KIT inhibitors; Leucine rich repeat kinase inhibitors; MDM inhibitors; mediator release inhibitors; MEK inhibitors; MTOR inhibitors; Monoamine oxidase inhibitors; NFkB pathway inhibitors; nucleophosmin inhibitors; PARP inhibitors; PPAR receptor agonists; PI3K inhibitors; tyrosine kinase inhibitors; Phosphodiesterase inhibitors; protein kinase inhibitors; RAF inhibitors; RNA polymerase inhibitors; topoisomerase inhibitors; RNA synthesis inhibitors; SIRT inhibitors; sodium channel blockers; VEGFR inhibitors; and Vitamin D receptor agonists.
62. The method of any of claims 51-57, wherein the subject at risk is further treated by administering an agonist of the positive immune response regulator if the subject has a decrease of at least one positive immune response regulators, wherein the agonist is an immune response regulator polypeptide, an immune response regulator miRNA, or a nucleic acid encoding the immune response regulator.
63. The method of any of claims 51-61, wherein the subj ect at risk is further treated by administering an inhibitor of the negative immune response regulator if the subject has an increase of at least one negative immune response regulators, wherein the inhibitor is an antibody, antibody reagent, ligand, mimetic, or inhibitory nucleic acid.
64. The method of any of claims 51-63, wherein the administering step comprises the administration of a vector comprising a nucleic acid encoding the agonist and/or inhibitor.
65. The method of any of claims 51-64, wherein the subject is a human.
66. The method of any of claims 51-65, wherein the administration is prophylactic.
67. The method of any of claims 51-66, wherein the subject is a current or former smoker.
68. The method of any of claims 51-67, wherein the level of expression is detected for at least 10 negative or positive immune response regulators.
69. The method of any of claims 51-68, wherein the level of expression is detected for at least miR-149-5p.
70. The method of any of claims 51-69, wherein the expression level of no more than 100 genes is detected.
71. The method of any of claims 51-70, wherein a treatment is administered during a bronchoscopy-based procedure.
72. The method of any of claims 51-70, wherein a treatment is administered systemically.
73. The method of any of claims 51-72, wherein a treatment is administered during a bronchoscopy-based procedure and systemically.
74. The method of any of claims 51-73, wherein the sample is in an endobronchial biopsy, airway brushing sample, nasal brushing sample, sputum, or blood from the subject.
75. The method of any of claims 51-74, wherein the sample is a bronchial brushing obtained from the right or left mainstem bronchus.
76. The method of any of claims 51-75, wherein the biopsy or brushing sample comprises morphologically-normal tissues or cells.
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