US 20150330.997A1 (19) United States (2) Patent Application Publication (10) Pub. No.: US 2015/0330997 A1 Paramithiotis et al. (43) Pub. Date: Nov. 19, 2015

(54) TYPE 2 DLABETES BIOMARKERS AND USES Publication Classification THEREOF (51) Int. Cl. (71) Applicants: Caprion Proteomics Inc., Montreal G0IN 33/68 (2006.01) (CA); VAL-CHUM, LIMITED (52) U.S. CI. PARTNERSHIP, Montreal (CA); CPC ...... G0IN33/6893 (2013.01); G0IN 2560/00 ADAERATA, LIMITED (2013.01); G0IN 2570/00 (2013.01); G0IN PARTNERSHIP, Montreal (CA) 2800/042 (2013.01); G0IN 2800/52 (2013.01) (72) Inventors: Eustache Paramithiotis, Boucherville (57) ABSTRACT (CA); Marc Prentki, Mount-Royal (CA); Rémi Rabasa-Lhoret, Montreal The present invention provides biomarkers, methods and kits (CA); Pascal Croteau, Laval (CA); Joel for diagnosing and prognosing the development of impaired Lanoix, Montreal (CA); S.R. Murthy glucose tolerance in a subject and the progression of diabetes Madiraju, Brossard (CA); Erik Joly, in a subject, as well as methods for identifying a compound Blainville (CA) that can inhibit the development of impaired glucose toler ance and/or type 2 diabetes; reduce or slow down the progres (21) Appl. No.: 14/813,344 sion of normal glucose tolerance to impaired fasting glycae mia, to impaired glucose tolerance, and/or to diabetes; and/or (22) Filed: Jul. 30, 2015 reduce or inhibit the development of complications associ ated with the disease in a subject, and methods for inhibiting Related U.S. Application Data the development of impaired glucose tolerance and/or type 2 diabetes; reducing or slowing down the progression of normal (63) Continuation of application No. PCT/IB2014/000426, glucose tolerance to impaired fasting glycaemia, to impaired filed on Jan. 31, 2014. glucose tolerance, and/or to diabetes; and/or reducing or (60) Provisional application No. 61/758,987, filed on Jan. inhibiting the development of complications associated with 31, 2013. the disease in a subject. Patent Application Publication Nov. 19, 2015 US 2015/0330.997 A1

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TYPE 2 DLABETES BIOMARKERS AND USES SUMMARY OF THE INVENTION THEREOF [0006] The present invention is based, at least in part, on the discovery of markers that are associated with the develop RELATED APPLICATIONS ment of impaired glucose tolerance and/or type 2 diabetes and the response of subjects having impaired glucose tolerance [0001] This application is a 35 U.S.C. 111(a) continuation and/or type 2 diabetes to a treatment. Accordingly, the present application, which claims the benefit of priority to PCT/PCT/ invention provides sensitive and facile methods and kits for IB2014/000426, filed on Jan. 31, 2014 and U.S. Provisional predicting whether a subject has or will develop impaired Patent Application Ser. No. 61/758,987, filed on Jan. 31, glucose tolerance, methods and kits for predicting whether a 2013, the entire contents of each of which is incorporated subject has or will develop diabetes, as well as methods for herein by reference. identifying a compound that can slow down the progression of impaired glucose tolerance and/or type 2 diabetes, methods BACKGROUND OF THE INVENTION of monitoring the effectiveness of a therapy in reducing the progression of impaired glucose tolerance and/or type 2 dia [0002] Diabetes mellitus type 2 (also referred to as nonin betes in a subject, and methods for inhibiting progression of sulin-dependent diabetes mellitus (NIDDM) or adult-onset impaired glucose tolerance and/or type 2 in a cell or a subject diabetes) is a metabolic disorder that is characterized by high by measuring and identifying particular markers, or particular blood glucose in the presence of insulin resistance and rela combinations of markers. tive insulin deficiency. Type 2 diabetes is a progressive dis [0007] Accordingly, in one aspect the present invention ease in which the risks of myocardial infarction, stroke, provides methods for determining whether a subject has or microvascular events and mortality are all strongly associated will develop impaired glucose tolerance. The methods with hyperglycaemia. Type 2 diabetes is also a silent disease include determining the level of one or more markers of the with significant declines in fl-cell function and kidney dam invention, e.g., any one or more of the markers listed in any of age often occurring before any symptoms of the disease mani Tables 1-3; USP9X; SEPT3: INS and SERPINB13; PPY and fest. DAG1; INS, CPM, and MMP7; BTC, MMP7, and PPY; PPY, [0003] The progression from normal glucose tolerance SEPT3, and PTPRJ: CPM, INS, MMP7, and LDLR, in a (NGT) to type 2 diabetes involves intermediate stages of sample(s) from the subject; comparing the level of the one or impaired fasting glucose (IFG) and impaired glucose toler more markers in the subject sample(s) with a level of the one ance (IGT), also known as prediabetes. The pathophysiology or more markers in a control sample(s), wherein a difference underlying the development of these glucose metabolic alter in the level of the one or more markers in the subject sample ations is multifactorial and includes, for example, lifestyle (s) as compared to the level of the one or more markers in the and genetic factors. In particular, obesity is thought to be the control sample(s) indicates that the subject has or will primary cause oftype 2 diabetes in people who are genetically develop impaired glucose tolerance. predisposed to the disease and rates of type 2 diabetes have [0008] In another aspect, the present invention provides increased markedly over the last 50 years in parallel with methods for determining whether a subject has or will obesity. As of 2010 there are approximately 285 million develop type 2 diabetes. The methods include determining the people with the disease compared to around 30 million in level of one or more markers of the invention, e.g., any one or 1985. more of the markers listed in any of Tables 1-3; USP9X, SEPT3: INS and SERPINB13; PPY and DAG1; INS, CPM, [0004] Although numerous risk factors, such as age, body and MMP7; BTC, MMP7, and PPY; PPY SEPT3, and mass index (BMI), and ethnicity, have been associated with PTPRJ: CPM, INS, MMP7, and LDLR, in a sample(s) from the development of prediabetes and type 2 diabetes, these are the subject; comparing the level of the one or more markers in not adequate to accurately predict the risk of progression the subject sample(s) with a level of the one or more markers from normal glucose tolerance to impaired glucose tolerance in a control sample(s), wherein a difference in the level of the and/or from impaired glucose tolerance to type 2 diabetes one or more markers in the subject sample(s) as compared to since the development and progression of diabetes is often the level of the one or more markers in the control sample(s) silent with organ damage occurring before the onset of iden indicates that the subject has or will develop type 2 diabetes. tifiable symptoms. In addition, although methods for deter [0009] In another aspect, the present invention provides mining whether a subject has impaired glucose tolerance methods for determining whether a subject will develop a and/or type 2 diabetes are known (e.g., glucose tolerance type 2 diabetes-associated complication. The methods testing), such methods require overnight fasting and multiple include determining the level of one or more markers of the blood draws over several hours and are often associated with invention, e.g., any one or more of the markers listed in any of side effects, such as, nausea, vomiting, abdominal bloating, Tables 1-3; USP9X; SEPT3: INS and SERPINB13; PPY and and/or headache. DAG1; INS, CPM, and MMP7; BTC, MMP7, and PPY; PPY, [0005] Accordingly, as early identification of subjects who SEPT3, and PTPRJ: CPM, INS, MMP7, and LDLR, in a have impaired glucose tolerance and/or type 2 diabetes and/or sample(s) from the subject; comparing the level of the one or who are at risk of developing impaired glucose tolerance more markers in the subject sample(s) with a level of the one and/or type 2 diabetes and/or those that will respond to a or more markers in a control sample(s), wherein a difference particular therapy would decrease short-term and long-term in the level of the one or more markers in the subject sample complications associated with glucose imbalance, there is a (s) as compared to the level of the one or more markers in the need in the art for reliable and accurate methods of determin control sample(s) indicates that the subject will develop a ing which subjects have or will develop impaired glucose type 2 diabetes-associate complication. tolerance and/or type 2 diabetes and/or respond to atherapy to [0010] In yet another aspect, the present invention provides permit early intervention. methods for determining whether a subject having impaired US 2015/0330.997 A1 Nov. 19, 2015 glucose tolerance and/or type 2 diabetes will respond to a flight (MALDI/TOF) mass spectrometry, liquid chromatog therapy. The methods include determining the level of one or raphy quadruple ion trap electrospray (LCQ-MS), or surface more markers of the invention, e.g., any one or more of the enhanced laser desorption ionization/time of flight (SELDI/ markers listed in any of Tables 1-3; USP9X; SEPT3; INS and TOF) mass spectrometry. SERPINB13; PPY and DAG1; INS, CPM, and MMP7; BTC, [0015] In another embodiment the level in the subject MMP7, and PPY; PPY SEPT3, and PTPRJ: CPM, INS, sample(s) is determined by immunoassay. MMP7, and LDLR, in a sample(s) from the subject; compar [0016] The sample(s) from the subject may be a fluid ing the level of the one or more markers in the subject sample sample(s) or a tissue sample(s). (s) with a level of the one or more markers in a control [0017] In one embodiment, the level of the marker is an sample(s), wherein a difference in the level of the one or more expression level and/or activity of the marker. markers in the subject sample(s) as compared to the level of [0018] In one embodiment the subject is at risk of develop the one or more markers in the control sample(s) indicates ing type 2 diabetes. that the subject will respond to the therapy. [0019. In one aspect, the present invention provides kits for [0011] In another aspect, the present invention provides determining whether a subject has or will develop impaired methods for monitoring the effectiveness of a treatment in a glucose tolerance. The kits include reagents for determining subject having impaired glucose tolerance and/or type 2 dia the level of one or more markers, e.g., one or more markers betes. The methods include determining the level of one or listed in any of Tables 1-3; USP9X; SEPT3: INS and SER more markers of the invention, e.g., any one or more of the PINB13; PPY and DAG1; INS, CPM, and MMP7; BTC, markers listed in any of Tables 1-3; USP9X; SEPT3; INS and MMP7, and PPY; PPY, SEPT3, and PTPRJ: CPM, INS, SERPINB13; PPY and DAG1; INS, CPM, and MMP7; BTC, MMP7, and LDLR, in a subject sample(s) and instructions for MMP7, and PPY; PPY SEPT3, and PTPRJ: CPM, INS, use of the kit to determine whether the subject has or will MMP7, and LDLR, in a first sample(s) from the subject prior develop impaired glucose tolerance. to the initiation of the treatment; determining the level of one [0020] In another aspect, the present invention provides kits or more markers in a second sample(s) from the subject after for determining whether a subject has or will develop type 2 at least a portion of the treatment has been administered; diabetes. The lits include reagents for determining the level of comparing the level of the one or more markers in the first one or more markers, e.g., one or more markers listed in any sample(s) with a level of the one or more markers in the of Tables 1-3; USP9X; SEPT3: INS and SERPINB13; PPY second sample(s), wherein a difference in the level of the one and DAG1; INS, CPM, and MMP7; BTC, MMP7, and PPY, or more markers in the first sample(s) as compared to the level PPY SEPT3, and PTPRJ: CPM, INS, MMP7, and LDLR, in of the one or more markers in the second sample(s) indicates a subject sample(s) and instructions for use of the kit to that the subject will respond to the treatment. determine whether the subject has or will develop type 2 [0012] In one aspect, the present invention provides meth diabetes. ods for identifying a compound that can inhibit the develop [0021] In yet another aspect, the present provides kits for ment of impaired glucose tolerance and/or type 2 diabetes, the determining whether a subject has or will develop type 2 method comprising contacting an aliquot of a sample(s) from diabetes complications. The kits include reagents for deter the subject with each member of a library of compounds; mining the level of one or more markers, e.g., one or more determining the effect of a member of the library of com markers listed in any of Tables 1-3; USP9X; SEPT3; INS and pounds on the level and/or activity of one or more markers of SERPINB13; PPY and DAG1; INS, CPM, and MMP7; BTC, the invention, e.g., any one or more of the markers listed in MMP7, and PPY; PPY, SEPT3, and PTPRJ: CPM, INS, any of Tables 1-3; USP9X; SEPT3; INS and SERPINB13; MMP7, and LDLR, in a subject sample(s) and instructions for PPY and DAG1; INS, CPM, and MMP7; BTC, MMP7, and use of the kit to determine whether the subject has or will PPY; PPY SEPT3, and PTPRJ: CPM, INS, MMP7, and develop type 2 diabetes complications. LDLR, in each of the aliquots; and selecting a member of the [0022] In another aspect, the present invention provides kits library of compounds which modulates the level and/or the for determining whether a subject having impaired glucose activity of the one or more marker(s) of the invention in an tolerance and/or type 2 diabetes will respond to a treatment. aliquot as compared to the level and/or activity of the one or The kits include reagents for determining the level of one or more marker(s) of the invention in a control sample, thereby more markers, e.g., one or more markers listed in any of identifying a compound that can inhibit the development of Tables 1-3; USP9X; SEPT3: INS and SERPINB13; PPY and impaired glucose tolerance and/or type 2 diabetes. DAG1; INS, CPM, and MMP7; BTC, MMP7, and PPY; PPY, [0013] In another aspect, the present invention provides SEPT3, and PTPRJ: CPM, INS, MMP7, and LDLR, in a methods for inhibiting the development of impaired glucose subject sample(s) and instructions for use of the kit to deter tolerance and/or type 2 diabetes in a subject. The methods mine whether the subject will respond to the treatment. include administering to the subject an effective amount of an [0023] In yet another aspect, the present invention provides agent that modulates the expression and/or activity of any one kits of monitoring the effectiveness of a treatment in a subject or more of the markers of the invention, e.g., any one or more having impaired glucose tolerance and/or type 2 diabetes. of the markers listed in any of Tables 1-3; USP9X; SEPT3; The uts include reagents for determining the level of one or INS and SERPINB13; PPY and DAG1; INS, CPM, and more markers, e.g., one or more markers listed in any of MMP7; BTC, MMP7, and PPY; PPY SEPT3, and PTPRJ; Tables 1-3; USP9X; SEPT3: INS and SERPINB13; PPY and CPM, INS, MMP7, and LDLR, thereby inhibiting the devel DAG1; INS, CPM, and MMP7; BTC, MMP7, and PPY; PPY, opment of impaired glucose tolerance and/or type 2 diabetes SEPT3, and PTPRJ: CPM, INS, MMP7, and LDLR, in a in the subject. subject sample(s) and instructions for use of the kit to monitor [0014] In one embodiment the level in the subject sample(s) the effectiveness of the treatment. is determined by mass spectrometry. In one embodiment the [0024] In one embodiment, the kits further comprise mass spectrometry is matrix assisted laser desorption/time of reagents for obtaining a sample from a subject. US 2015/0330.997 A1 Nov. 19, 2015

[0025] In one embodiment, the kits further comprise a con genate; SV. secretory vesicle; SC; secretory vesicle contents; trol sample. Mb: membrane; PM; plasma membrane. [0026] In one aspect, the present invention provides meth ods for identifying a type 2 diabetes marker. The methods DETAILED DESCRIPTION OF THE INVENTION include identifying proteins in the secretory vesicles oftwo or more organs from two or more species under steady state [0031] The present invention is based, at least in part, on the conditions; identifying proteins in the secretory vesicles of discovery of markers that are associated with the develop pancreatic f; cells thereby generating a provisional list of ment of impaired glucose tolerance and/or type 2 diabetes, the steady state markers; identifying the markers in the provi progression of type 2 diabetes, and the response of a subject sional list of steady state markers from the two or more organs having impaired glucose tolerance and/or type 2 diabetes to a from the two or more species common to the markers in the treatment. In particular, biomarkers associated with type 2 secretory vesicles of pancreatic f; cells and removing those diabetes have been discovered, prioritized, and validated in markers from the provisional list of steady state markers, multiple in vitro experimental systems. The markers were thereby generating a list off cell mass markers; identifying identified as being expressed, e.g., essentially specifically proteins in the secretory vesicles of pancreatic f; cells under expressed in fl-cells, and/or as being involved, e.g., essen dysfunctional conditions, identifying proteins in the secre tially specifically involved, in fl-cell function, and/or as being tory vesicles of pancreatic f; cells under normal conditions, involved in response to a therapeutic treatment. identifying the proteins that were differentially expressed [0032] Accordingly, the present invention provides sensi under dysfunctional conditions and under normal conditions, tive and facile methods and kits for predicting whether a thereby generating a provisional list off cell function mark subject has or will develop impaired glucose tolerance, meth ers, determining the level of a fl cell mass marker and/or a fl ods and kits for predicting whether a subject has or will cell function marker in a sample(s) form a test sample and a develop diabetes, as well as methods for identifying a com control sample, wherein a difference in the level of a marker pound that can slow down the progression of impaired glu in the control sample as compared to the level in the test cose tolerance and/or type 2 diabetes, methods of monitoring sample identifies the marker as a type 2 diabetes biomarker. the effectiveness of a therapy in reducing the progression of [0027] In one embodiment, the test sample is from a subject impaired glucose tolerance and/or type 2 diabetes in a subject, having impaired glucose tolerance. In another embodiment, and methods for inhibiting progression of impaired glucose the test sample is from a subject having newly diagnosed type tolerance and/or type 2 in a cell or a subject by measuring and 2 diabetes. In yet another embodiment, the test sample is from identifying particular markers, or particular combinations of a subject having established type 2 diabetes. markers. [0028] In one embodiment, the control sample is from a [0033] Various aspects of the invention are described in subject having normal glucose tolerance. In another embodi further detail in the following subsections: ment, the control sample is from a subject having impaired glucose tolerance. In yet another embodiment, the control I. Definitions sample is from a subject having newly diagnosed type 2 [0034] As used herein, each of the following terms has the diabetes. meaning associated with it in this section. [0029] In another aspect, the present invention provides [0035] The articles “a” and “an” are used herein to refer to methods for identifying a type 2 diabetes marker. The meth one or to more than one (i.e. to at least one) of the grammatical ods include identifying proteins differentially expressed in a object of the article. By way of example, “an element” means sample(s) from a subject before and after treatment, thereby one element or more than one element. generating a list of therapeutic efficacy markers; determining [0036] A “marker” or “biomarker” is an organic biomol the level of one or more of the markers in a first sample ecule which is differentially present in a sample taken from a obtained from a subject having type 2 diabetes prior to pro subject of one phenotypic status (e.g., having a disease) as viding at least a portion of a therapy to the subject; and compared with another phenotypic status (e.g., not having the determining the level of a protein in a second sample obtained disease). A biomarker is differentially present between dif from the subject following provision of at least a portion of ferent phenotypic statuses if the mean or median level, e.g., the therapy, wherein a difference in the level of expression of expression level, of the biomarker in the different groups is the one or more markers in the second sample relative to the calculated to be statistically significant. Common tests for first sample identifies the protein as a type 2 diabetes marker. statistical significance include, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. BRIEF DESCRIPTION OF THE DRAWINGS Biomarkers, alone or in combination, provide measures of [0030) FIG. 1 depicts Western blots of proteins identified relative risk that a subject belongs to one phenotypic status or during the process of secreted proteins preparation. Cell or another. As such, they are useful as markers for, e.g., disease tissue homogenates were prepared by mechanical disruption (prognostics and diagnostics), therapeutic effectiveness of a and secretory pathway vesicles isolated by sucrose density drug (theranostics) and of drug toxicity. centrifugation. The resultant vesicles were washed with salt [0037] In some embodiments, the accuracy of a marker(s) to remove loosely attached proteins, opened with alkali, and useful in the compositions and methods of the present inven the secretory protein contents retrieved by high speed cen tion may be characterized by a Receiver Operating Charac trifugation. Shown are western blots of starting materials teristic curve (“ROC curve”). An ROC is a plot of the true (Hom), intermediate (SV) and final product (SC) prepara positive rate against the false positive rate for the different tions from a rat cell line (A) and human primary islets (B). possible cutpoints of a diagnostic marker(s). An ROC curve The western blot markers were against specific intracellular shows the relationship between sensitivity and specificity. compartments and indicate the progressive enrichment of That is, an increase in sensitivity will be accompanied by a secretory proteins during sample preparation. Hom: homo decrease in specificity. The closer the curve follows the left

US 2015/0330.997 A1 Nov. 19, 2015

having been diagnosed with type 2 diabetes with the previous [0054] The term “determining” means methods which 18 months. In one embodiment, the control level of a marker include detecting the presence or absence of marker(s) in the in a sample from a subject is a level of the marker previously sample, quantifying the amount of marker(s) in the sample, determined in a sample(s) from the subject. and/or qualifying the type of biomarker. Measuring can be [0049] In yet another embodiment, the control level of a accomplished by methods known in the art and those further marker is based on the level of the marker in a sample from a described herein. subject(s) prior to the administration of atherapy for impaired [0055] As used herein, the various forms of the term fasting glycaemia, impaired glucose tolerance, and/or type 2 “modulate” are intended to include stimulation (e.g., increas diabetes. In another embodiment, the control level of a ing or upregulating a particular response or activity) and marker is based on the level of the marker in a sample(s) from inhibition (e.g., decreasing or downregulating a particular a subject(s) having impaired fasting glycaemia, impaired glu response or activity). cose tolerance, and/or type 2 diabetes that is not contacted with a test compound. In another embodiment, the control [0056] A kit is any manufacture (e.g. a package or con level of a marker is based on the level of the marker in a tainer) comprising at least one reagent, e.g. a probe, a primer, sample(s) from a subject(s) having normal glucose tolerance or an antibody, for specifically detecting a marker of the that is contacted with a test compound. In one embodiment, invention, the manufacture being promoted, distributed, or the control level of a marker is based on the expression level sold as a unit for performing the methods of the present of the marker in a sample(s) from an animal model of invention. In certain embodiments, a lit may include a sub impaired fasting glycaemia, impaired glucose tolerance, and/ strate, e.g., a substrate comprising a capture reagent for one or or type 2 diabetes, a cell, or a cell line derived from the animal more markers of the invention and/or a capture reagent bound model of impaired fasting glycaemia, impaired glucose tol to one or more markers of the invention. In some embodi erance, and/or type 2 diabetes. ments, such kits comprise instructions for determining the [0050] Alternatively, and particularly as further informa level of a marker(s) using mass spectrometry. tion becomes available as a result of routine performance of the methods described herein, population-average values for II. Markers of the Invention “control” level of expression of a marker may be used. In other embodiments, the “control” level of a marker may be determined by determining the level of a marker in a subject [0057] The present invention is based upon the discovery of sample obtained from a subject before the suspected onset of markers that are essentially specifically expressed in pancre impaired fasting glycaemia, impaired glucose tolerance, and/ atic fl-cells (Table 1), and/or as being essentially specifically or type 2 diabetes in the subject, from archived subject involved in B-cell function (Table 2), and/or as being involved samples, and the like. in response to a therapeutic treatment (Table 3). These mark ers have been shown to be differentially present in samples of [0051] As used herein, the terms “patient” or “subject” subjects having impaired glucose tolerance and control sub refer to human and non-human animals, e.g., veterinary jects, and/or differentially present in samples of subjects hav patients. The term “non-human animal” includes all verte ing impaired glucose tolerance and subjects having newly brates, e.g., mammals and non-mammals, such as non-human diagnosed type 2 diabetes, and/or differentially present in primates, mice, rabbits, sheep, dog, cat, horse, cow, chickens, samples of subjects having impaired glucose tolerance and amphibians, and reptiles. In one embodiment, the subject is a subjects having established type 2 diabetes, and/or differen human. tially present in samples of subjects having newly diagnosed [0052] In some embodiments, a subject has a body mass type 2 diabetes and subjects having established type 2 diabe index (BMI) of less than about 40kg/m (e.g., about 4039,38, tes, and/or differentially expressed in samples of subjects 37, 36,35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21. responsive to treatment with an insulin sensitizer and subjects 20, 19, or about 18 kg/m3). In other embodiments, a subject non-respnsive to an insulin sensitizer, and/or differentially has a body mass index (BMI) of greater than about 40 kg/m3 expressed in samples of subjects responsive to treatment with (e.g., about 41,42, 43,44, 45,46,47, 48,49, 50, 51, 52, 53, 54, an insulin sensitizer and a secretagogue and subjects non 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66.67, 68, 69, 70, 71, respnsive to an insulin sensitizer and a secretagogue, and/or 72, 73, 74, 75, 76, 77, 78, 79, or about 80 kg/m2). differentially expressed in samples of subjects responsive to [0053] The term “sample” as used herein refers to a collec treatment with an insulin sensitizer, a secretagogue, and insu tion of similarcells or tissue isolated from a subject, as well as lin and subjects non-respnsive to an insulin sensitizer, a secre tissues, cells and fluids present within a subject. The term tagogue, and insulin. “sample” includes any body fluid (e.g., blood fluids, lymph, gynecological fluids, cystic fluid, urine, ocular fluids and [0058] Accordingly, the level of any one marker or any fluids collected by bronchial lavage and/or peritoneal rins combination of markers listed in Tables 1-3 found in a test ing), or a cell from a subject. In one embodiment, the tissue or sample compared to a control, or the presence or absence of cell is removed from the subject. In another embodiment, the one marker or combination of markers listed in Tables 1-3 in tissue or cell is present within the subject. Other subject the test sample may be used in the methods and kits of the samples, include teardrops, serum, cerebrospinal fluid, feces, present invention. sputum and cell extracts. In one embodiment, the biological [0059] The markers of the invention are listed in Tables 1-3. sample contains protein molecules from the test subject. In The nucleotide and amino acid sequences of the markers are another embodiment, the biological sample may contain known in the art and may be found in, for example, the mRNA molecules from the test subject or genomic DNA GenBank Accession numbers listed in Tables 1-3, the entire molecules from the test subject. contents of which are incorporated herein by reference. US 2015/0330.997 A1 Nov. 19, 2015

TABLE 1 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION ABCC8 ATP-binding ABCC8 HUMAN Q09428 NP_000343.2. cassette sub-family NM_000352.3. C member 8 ACPP Prostatic acid PPAP HUMAN P15309 NP 001090.2 phosphatase NM_001099.4 NP_001 127666.1 NM_001134194.1 APLP1 Amyloid-like APLP1 HUMAN P51693 NP 001019978.1. protein 1 NM_001024807.1. NP_005157.1. NM_005166.3. APOL2 Apolipoprotein L2 APOL2 HUMAN Q9BQE5 NP 1.12092.1 NM 030882.2 NP_663612.1 NM 145637.1 APP Amyloid beta A4 A4 HUMAN P05067 NP 000475.1 protein NM_000484.3 NP_001.129488.1. NM_001136016.3 NP 001129.601.1. NM_0011361.29.2 NP_001.129.602.1. NM_001136130.2 NP_001.129.603.1. NM_001136131.2 NP_001.191230.1. NM_001204301.1. NP_001.191231.1. NM_001204302.1. NP_001.191232.1. NM_001204303.1. NP 9588.16.1. NM_201413.2. NP 958817.1. NM_201414.2. ATP8A1 Probable AT8A1 HUMAN Q9Y200 NP_001098.999.1. phospholipid- NM_001105529.1. transporting NP_006086.1. ATPase IA NM_006095.2. ATP9A Probable ATP9A HUMAN O75110 NP_006036.1. phospholipid- NM_006045.1. transporting ATPase IIA BET1L. BET1-like protein BET1L HUMAN Q9NYM9 NP_001092257.1. NM_001098.787.1. BMP7 BOne BMP7 HUMAN P18075 NP_001710.1. morphogenetic NM_001719.2. protein 7 BOLA1 Bol/A-like protein 1 BOLA1 HUMAN Q973E2 NP 057158.1. NM_016074.3. BTC Probetacellulin BTC HUMAN P35070 NP_001720.1. NM_001729.2. C12ORF23 UPF0444 CL023 HUMAN Q8WUH6 NP 689474.1. transmembrane NM 152261.2. protein C12Orf23 C6ORF142 Muscular LMNA- MLIP HUMAN Q5VWP3 NP 612636.2. interacting protein NM 138569.2. C9QRF5 Transmembrane TM245 HUMAN Q9H330 NP 1.14401.2. protein 245 NM_032012.3. CADM1 Cell adhesion CADM1 HUMAN Q9BY67 NP 001091987.1. molecule 1 NM_00109851.7.1. NP 055148.3. NM_014333.3. CASC4 Protein CASC4 CASC4 HUMAN Q6P4E1 NP 612432.2. NM 138423.3. NP 816929.1. NM 177974.2. CASR Peripheral plasma CASR HUMAN P41180 NP_000379.2 membrane protein NM_000388.3 CASK NP_001171536.1 NM_001178065.1 US 2015/0330.997 A1 Nov. 19, 2015

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION CBARA1 Calcium uptake MICU1 HUMAN Q9BPX6 NP_001182447.1. protein 1, NM_001 195518.1. mitochondrial NP_001182448.1. NM_001 195519.1. NP_006068.2. NM_006077.3. CCDC115 Coiled-coil domain- CC115 HUMAN Q96NTO NP 1.15733.2. containing protein NM 032357.2. 115 CD47 Leukocyte surface CD47 HUMAN Q08722 NP_001768.1. antigen CD47 NM_001777.3. NP 942088.1. NM_198793.2. CD59 CD59 glycoprotein CD59 HUMAN P13987 NP 000602.1 NM_000611.5 NP 001120695.1 NM_001 127223.1 NP_001 120697.1 NM_001 127225.1 NP 001120698.1 NM_001 1272.26.1 NP_001 120699.1 NM_001 127227.1 NP 976074.1 NM 203329.2 NP 976O75.1 NM 203330.2 NP 976076.1 NM 203331.2 CDCP1 CUB domain- CDCP1 HUMAN Q9H5V8 NP 073753.3. containing NM_022842.3. protein 1 NP 835.488.1. NM_178181.1. CFDP1 Craniofacial CFDP1. HUMAN Q9UEE9 NP_006.315.1. development NM_006324.2. protein 1 CHGB Secretogramin-1 SCG1 HUMAN P05060 NP_001810.2. NM_001819.2. CHKA Choline kinase CHKA HUMAN P35790 NP_001268.2. alpha NM_001277.2. NP 997634.1. NM 212469.1. CLLD6 SPRY domain- SPRY7 HUMAN Q5W111 NP_001.120954.1. containing NM_001 127482.1. protein 7 NP 0651.89.1. NM_020456.2. CNNM2 Metal transporter CNNM2 HUMAN Q9H8M5 NP 0601 19.3. CNNM2 NM_017649.4. NP 95.1058.1. NM 199076.2. NP 95.1059.1. NM 1990.77.2. CNP 2',3'-cyclic- CN37 HUMAN P09543 NP 149124.3. nucleotide 3'- NM_033133.4. phosphodiesterase CNPY4 Protein canopy CNPY4 HUMAN Q8N129 NP 689968.1. homolog 4 NM 152755.1. CNTN1 Contactin-1 CNTN1 HUMAN Q12860 NP_001242992.1 NM_001256063.1 NP_001242993.1 NM_001256064.1 NP 001834.2 NM_001843.3 NP 778.203.1 NM 175038.2 COMMD10 COMM domain- COMDA HUMAN Q976G5 NP 057228.1. containing NM_016144.2. protein 10 CPE Carboxypeptidase E CBPE HUMAN P16870 NP_001864.1 NM_001873.2 US 2015/0330.997 A1 Nov. 19, 2015

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION CSHL1 Chorionic CSHL HUMAN Q14406 NP 072101.1. somatomammotropin NM_022579.1. hormone-like 1 NP 072102.1. NM_022580.1. NP 072103.1. NM_022581.1. CSTF3 Cleavage CSTF3 HUMAN Q12996 NP_001028677.1 stimulation factor NM_001033505.1 subunit 3 NP_001028678.1 NM_001033506.1 NP_001317.1 NM_001.326.2 CYFIP1 Cytoplasmic FMR1- CYFP1 HUMAN Q7L576 NP_001028200.1. interacting NM_001033028.1. protein 1 NP 055423.1. NM_014608.2. CYFIP2 Cytoplasmic FMR1- CYFP2 HUMAN Q96F07 NP_001032409.2. interacting NM_001037332.2. protein 2 NP_001032410.1. NM_001037333.1. NP 05:51.91.2. NM_014376.2. CYTL1 Cytokine-like CYTL1 HUMAN Q9NRR1 NP 061129.1. protein 1 NM_018659.2. CYTSA Cytospin-A CYTSA HUMAN Q69YQ0 NP 056145.3. NM_015.330.3. DAG1 similar to DAG1 HUMAN Q14118 NP 001159400.2 Dystroglycan -NM_001165928.3 precursor NP_001171105. NM_001177634.2 NP 001171106. NM_001177635.2 NP_001171107. NM_001177636.2 NP 001171108. NM_001177637.2 NP_001171109. NM_001177638.2 NP 001171.110. NM_001177639.2 NP_001171111. NM_001177640.2 NP 001171112. NM_001177641.2 NP_001171.113. NM_001177642.2 NP 001171114. NM_001177643.2 NP_001171.115. NM_001177644.2 NP 004384.4 NM_004393.5 DKK2 Dickkopf-related DKK2 HUMAN Q9UBU2 NP 055236.1. protein 2 NM_014421.2. DSCAML1 Down syndrome DSCL1 HUMAN Q8TD84 NP 065744.2. cell adhesion NM_020693.2. molecule-like protein 1 EDIL3 EGF-like repeat and EDIL3 HUMAN O43854 NP_005702.3. discoidin 1-like NM_00571.1.3. domain-containing protein 3 EMB Embigin EMB HUMAN Q6PCB8 NP 940851.1. NM_1984.49.2. ENPP1 Ectonucleotide ENPP1 HUMAN P22413 NP_006199.2. pyrophosphatase/ NM_006.208.2. phosphodiesterase amily member 1 ENPP4 Ectonucleotide ENPP4 HUMAN Q976X5 NP 0557.51.1. pyrophosphatase/ NM_014936.4. phosphodiesterase amily member 4 US 2015/0330.997 A1 Nov. 19, 2015

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION ENTPD3 Ectonucleoside ENTP3 HUMAN O75355 NP_001239.2. triphosphate NM_001248.2. diphosphohydrolase 3 EPN2 Epsin-2 EPN2 HUMAN O95208 NP 055779.2. NM_014964.4. ERO1 LB ERO1-like protein ERO1B HUMAN Q86YB8 NP 063944.3. betà NM_019891.3. ESYT2 Extended ESYT2 HUMAN AOFGR8 NP 065779.1. synaptotagmin-2 NM_020728.2. EXT1 Exostosin-1 EXT1 HUMAN Q16394 NP_000118.2. NM_000127.2. FAM125A Multivesicular body F125A HUMAN Q96EY5 NP 612410.1. subunit 12A NM 138401.2. FAM126A Hyccin HYCCI HUMAN Q9BYI3 NP 115970.2. NM_032581.3. FAM19A4 Protein FAM19A4 F19A4 HUMAN Q96LR4 NP 001005527.1. NM_001005527.2. NP 872.328.1. NM 182522.4. FAM20A Protein FAM20A FA20A HUMAN Q96MK3 NP 001230675.1. NM_001243746.1. NP 060035.2. NM_017565.3. FAM20B Glycosaminoglycan XYLK HUMAN O75063 NP 055679.1. xylosylkinase NM_014864.3. FAM20C Family with DMP4 HUMAN Q8IXL6 NP 0646.08.2 sequence similarity NM_020223.3 20, member C FAM3C Protein FAM3C FAM3C HUMAN Q925.20 NP_001035109.1. NM_00104.0020.1. NP 0557.03.1. NM_014888.2. FAM75A6 Spermatogenesis- S31.A6 HUMAN Q.5VVP1 NP_001138668.1. associated protein NM_001145196.1. 31 A6 FAM83H Protein FAM83H FA83F HUMAN Q8NEG4 NP 612444.2. NM 138435.2. FBXL2 F-box/LRR-repeat FBXL2 HUMAN Q9UKC9 NP 001165184.1. protein 2 NM_001171713.1. NP 036289.3. NM 012157.3. FGF12 Fibroblast growth FGF12 HUMAN P61328 NP_004104.3. actor 12 NM_004113.5. NP 066360.1. NM_021032.4. FGF19 Fibroblast growth FGF19 HUMAN O95750 NP_005108.1. actor 19 NM_005117.2. FKBP11 Peptidyl-prolyl cis- FKB11 HUMAN Q9NYL4 NP_001137253.1. trans isomerase NM_001143781.1. FKBP11 NP 001137254.1. NM_001143782.1. NP 057678.1. NM_016594.2. FREM1 FRAS1-related FREM1 HUMAN Q5H8C1 NP_001171175.1. extracellular matrix NM_0011777.04.1. protein 1 NP 659403.4. NM 144966.5. GALNT2 Polypeptide N- GALT2 HUMAN Q10471 NP_004472.1. acetylgalactosaminyl- NM_004481.3. transferase 2 GAP43 Neuromodulin NEUM HUMAN P17677 NP_001.123536.1. NM_001 130064.1. NP_002036.1. NM_002045.3. GLRX5 Glutaredoxin- GLRX5 HUMAN Q86SX6 NP 05750.1.2. related protein 5, NM_016417.2. mitochondrial GNPDA2 Glucosamine-6- GNPI2 HUMAN Q8TDQ7 NP 001257809.1. phosphate NM_001270880.1. isomerase 2 NP_001257810.1. US 2015/0330.997 A1 Nov. 19, 2015 10

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION NM_001270881.1. NP 612208.1. NM 138335.2. GPR158 Probable G-protein GP158 HUMAN Q5T848 NP 065803.2. coupled receptor NM_020752.2. 158 GPRIN1 G protein- GRIN1 HUMAN Q7Z2K8 NP 443131.2. regulated inducer NM 052899.2. of neurite outgrowth 1 GREM1 Gremlin-1 GREM1 HUMAN O60565 NP_001178.252.1. NM_001 191323.1. NP 037504.1. NM_013372.6. GREM2 Gremlin-2 GREM2 HUMAN Q9H772 NP_071914.3. M_022469.3. GRK5 G protein-coupled GRK5 HUMAN P34947 NP_005299.1. receptor kinase 5 NM_005308.2. GUK1 Guanylate kinase KGUA HUMAN Q16774 NP 000849.1. NM 000858.5. NP_001152862.1. NM_0011593.90.1. NP_001152863.1. NM_0011593.91.1. NP_001229.768.1. NM_001242839.1. HERC4 Probable E3 HERC4 HUMAN Q.5GLZ8 NP 056416.2. ubiquitin-protein NM_015601.3. igase HERC4 NP_071362.1. NM_022079.2. HPCA Neuron-specific HPCA HUMAN P84074 NP 002134.2. calcium-binding NM_00214.3.2. protein hippocalcin HSP90B2P Putative ENPLL HUMAN Q58FF3 AY956768 endoplasmin-like AAX38.255.1. protein HSPA13 Heat shock 70 kDa HSP13 HUMAN P4.8723 NP_008879.3. protein 13 NM_006948.4. DE Insulin-degrading DE HUMAN P14735 NP 001159418.1. enzyme NM_001165946.1. NP_004960.2. NM_004969.3. GF1 Insulin-like growth GF1 HUMAN P05019 NP 000609.1. actor I NM_000618.3. NP_001104754.1. NM_001111284.1. NP 001104755.1. NM_001111285.1. IGFBP7 Insulin-like growth IBP7 HUMAN Q16270 NP 001544.1. factor-binding NM_001553.2. protein 7 INS Insulin-1 INS HUMAN PO1308 NP_000198.1 NM_000207.2 NP 001172026.1 NM_001185097.1 NP_001172027.1 NM_001185098.1 IRS2 Insulin receptor IRS2 HUMAN Q974H2 NP 003740.2. substrate 2 NM_003749.2. ITFG3 Protein ITFG3 ITFG3 HUMAN Q9HOX4 NP 114428.1. NM_032039.2. ITM2B Integral membrane ITM2B. HUMAN Q97.287 NP 068839.1. protein 2B NM_021999.4. ITPKB Inositol- IP3KB. HUMAN P27987 NP_002212.3. trisphosphate 3- NM_002221.3. kinase B KIAA0564 von Willebrand VWA8 HUMAN A3KMH1 NP_001009814.1. factor A domain- NM_001009814.1. containing protein 8 NP 055873.1. NM_015058.1. KIAA1324 UPF0577 protein K1324 HUMAN Q6UXG2 NP_0012.53977.1. KIAA1324 NM_001267048.1. US 2015/0330.997 A1 Nov. 19, 2015 11

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION NP_0012.53978.1. NM_001267049.1. NP 065826.2. NM 020775.4. KIDINS220 Kinase D- KDIS HUMAN Q9ULHO NP 065789.1. interacting NM_020738.2. substrate of 220 kDa LDLR Low-density LDLR HUMAN PO1130 NP 000518.1 lipoprotein NM_000527.4 receptor NP_001182728.1 NM_001 195799.1 NP_001182729.1 NM_001 1958.00.1 NP_001182732.1 NM_001 1958.03.1 LGALS8 Galectin-8 LEG8 HUMAN O00214 NP_006.490.3. NM_006499.4. NP 963.837.1. NM 201543.2. NP 963.838.1. NM_201544.2. NP 9638.39.1. NM 201545.2. LRRC8E Leucine-rich LRC8E HUMAN Q6NSJ5 NP_0012.5521.3.1. repeat-containing NM_001268284.1. protein 8E NP 001255.214.1. NM_001268285.1. NP 0793.37.2. NM_025061.4. LSAMP Limbic system- LSAMP HUMAN Q13449 NP 002329.2. associated NM_002338.3. membrane protein MAP1 B Microtubule- MAP1B. HUMAN P46821 NP_005900.2. associated protein NM_005909.3. 1B MBP Myelin basic MBP HUMAN P026.86 NP_00102025.2.1. protein NM_001025081.1. NP 001020261.1. NM_00102.5090.1. NP_001020263.1. NM_00102.5092.1. NP 001020271.1. NM_001025100.1. NP_001020272.1. NM_001025.101.1. NP 002376.1. NM_002385.2. MCRS1 Microspherule MCRS1 HUMAN Q96EZ8 NP_00101.2300.1. protein 1 NM_00101.2300.1. NP_006328.2. NM_006337.3. MGAT1 Alpha-1,3- MGAT1 HUMAN P26572 NP_001108089. mannosyl- NM_001 114617.1 glycoprotein 2-beta- NP_001108090. acetylglucosaminyl NM_0011146.18.1 transferase NP 001108091. NM_001114619.1 NP_001108092. NM_001 114620.1 NP 002397.2 NM_002406.3 MIA3 Melanoma MLA3 HUMAN Q5JRA6 NP 940953.2. inhibitory activity NM 198551.2. protein 3 MLN Promotilin MOTI HUMAN P12872 NP 001035.198.1. NM_001040109.1. NP_001171627.1. NM_001 184698.1. NP_002409.1. NM_002418.2. US 2015/0330.997 A1 Nov. 19, 2015 12

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION MPP2 MAGUK p55 MPP2 HUMAN Q14168 NP_005365.3. subfamily member 2 NM_005374.3. MTHFD2 Bifunctional MTDC HUMAN P13995 NP_006627.2. methylenetetra- NM_006636.3. hydrofolate dehydrogenase/ cyclohydrolase, mitochondrial MTUS1 Microtubule- MTUS1 HUMAN Q9ULD2 NP_001001924.1. associated tumor NM_001001924.2. suppressor 1 NP_001001925.1. NM_001001925.2. NP_001001931.1. NM_001001931.2. NP_001159865.1. NM_001166393.1. NP 065800.1. NM_020749.4. MUC13 Mucin-13 MUC13 HUMAN Q9H3R2 RefSeq NP 149038.3. NM_033049.3. MXRA7 Matrix-remodeling- MXRA7 HUMAN P84157 NP_001008528.1. associated protein 7 NM_001008528.1. NP_001008529.1. NM_001008529.1. NP 940932.2. NM 198530.2. NAAA N- NAAA HUMAN Q02083 NP 001035861.1. acylethanolamine- NM_001042402.1. hydrolyzing acid NP 0552.50.2. amidase NM_014.435.3. NAGLU Alpha- ANAG HUMAN P54802 NP 000254.2. acetylglucosaminidase NM_000263.3. NCAM1 Neural cell NCAM1 HUMAN P13591 NP 000606.3. adhesion molecule 1 NM_000615.6. NP 0010701:50.1. NM_001076682.3. NP_001229537.1. NM_001242608.1. NP 85.1996.2. NM 181351.4. NECAB2 N-terminal EF-hand NECA2 HUMAN Q7Z6G3 NP 061938.2. calcium-binding NM_01906.5.2. protein 2 NELL1 Protein kinase C- NELL1, HUMAN Q92832 NP_006148.2 binding protein NM_006157.3 NELL1 NP 963.845.1 NM 201551.1 NEO1 Neogenin NEO1 HUMAN Q92859 NP_001166094.1. NM_001 172623.1. NP_002490.2. NM_002499.3. NFASC Neurofascin NFASC HUMAN O94856 NP_001005.388.2. NM_001005388.2. NP 001005389.2. NM_001005389.1. NP_001153803.1. NM_001160331.1. NP 001153804.1. NM_001160332.1. NP_001153805.1. NM_001160333.1. NP 05:5905.2. NM_015090.3. NGRN Neugrin NGRN HUMAN Q9NPE2 NP_001028260.2. NM_001033088.1. NMU Neuromedin U NMU HUMAN P48.645 NP OO6672.1 NM_006681.2 NPTN Neuroplastin NPTN HUMAN Q97639 NP_001154835.1. NM_001161363.1. NP 001154836.1. NM_001161364.1. NP 036560.1. US 2015/0330.997 A1 Nov. 19, 2015 13

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT ENE ANK Name Description UNIPROT ID ACCESSION CCESSION M 012428.3. P_059429.1. M_017455.3. Neuronal NPTX2 HUMAN P47972 P 0025.14.1. pentraxin-2 M_002523.2. NPY Pro-neuropeptide Y NPY HUMAN PO1303 P 000896.1. M_000905.3. NTNG1 Netrin-G1 NTNG1 HUMAN Q97.212 P 001106697.1. M_001113226.1. P 001106699.1. M_001113228.1. P 055732.2. M_014917.2. NXPH1 Neurexophilin-1 NXPH1 HUMAN P58417 P 68.9958.1. M_152745.2. NXPH2 Neurexophilin-2 NXPH2 HUMAN O951.56 P_009157.1. M_007226.2. ODZ.4 Teneurin-4 TEN4 HUMAN P_00109.2286.2. M_001098816.2. Prolyl 4 P4HA2 HUMAN O15460 P_001017973.1. hydroxylase M_001017973.1. subunit alpha-2 P_001017974.1. M_001017974.1. P 001136070.1. M_001142598.1. P 001136071.1. M_001142599.1. P_004190.1. M_004.199.2. PAM Peptidyl-glycine AMD HUMAN P19021 P 000910.2. alpha-amidating M_000919.3. monooxygenase P 001170777.1. M_001177306.1. P 620121.1. M 138766.2. P 620176.1. M_138821.2. P 620177.1. M_138822.2. PAPPA2 Pappalysin-2 PAPP2 HUMAN P 064714.2. M_020318.2. P 068755.2. M_021936.2. PCSK1 Neuroendocrine NEC1 HUMAN P29 120 P 000430.3. convertase 1 M 000439.4. PCSK2 Neuroendocrine NEC2 HUMAN P16519 P 00118.8457.1. convertase 2 M_001201528.1. P 00118.8458.1. M_001201529.1. P 002585.2. M_002594.3. PDYN Proenkephalin-B PDYN HUMAN PO1213 P 001177821.1. M_001 190892.1. P 001177827.1. M_001 190898.2. P 001177828.1. M_001 190899.2. P 0011778.29.1. M_001 190900.1. P 077722.1. M_024411.4. Phosphatidylinositol PI42A HUMAN P48426 P_005019.2. 5-phosphate 4 M_005028.4. kinase type-2 alpha PLBD2 Putative PLBL2 HUMAN P 775813.2. phospholipase B-like 2 M 173542.3. PLCB4 1 PLCB4 HUMAN Q15147 P 000924.3. phosphatidylinositol M_000933.3. 4,5 P 0011661.17.1. bisphosphate M_001 172646.1. phosphodiesterase P 877949.2. beta-4 NM 182797.2. US 2015/0330.997 A1 Nov. 19, 2015 14

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION PLXNC1 Plexin-C1 PLXC1 HUMAN O60486 NP_005752.1. NM_005761.2. PPAP2A Lipid phosphate LPP1 HUMAN O14494 NP 00370.2.2. phosphohydrolase 1 NM 00371.1.2. NP 7957.14.1. NM 176895.1. PPFLA1 Liprin-alpha-1 LIPA1 HUMAN Q13136 NP_003617.1. NM_003626.3. NP 803172.1. NM 177423.2. PPY Pancreatic PAHO HUMAN PO1298 NP_002713. icosapeptide NM_002722.3 PRNP Major prion PRIO HUMAN PO4156 NP_000302.1. protein NM_000311.3. NP_001073590.1. NM_0010801.21.1. NP_001073591.1. NM_0010801.22.1. NP_001073592.1. NM_0010801.23.1. NP 898902.1. NM 183079.2. PRSS3 Trypsin-3 TRY3 HUMAN P35030 NP_001184026.2. NM_001 197097.2. NP_002762.2. NM_002771.3. NP 031369.2. NM_007343.3. PTPRJ Receptor-type PTPRJ HUMAN Q12913 NP_001091973.1 tyrosine-protein NM_0010985.03.1 phosphatase eta NP 002834.3 NM_002843.3 PTPRN Receptor-type PTPRN HUMAN Q16849 NP_001186692.1. tyrosine-protein NM_001 199763.1. phosphatase-like N NP 001186693.1. NM_001 199764.1. NP 002837.1. NM_002846.3. PTPRN2 Receptor-type PTPR2 HUMAN Q92932 NP 002838.2. tyrosine-protein NM_002847.3. phosphatase N2 NP 570857.2. NM 13084.2.2. NP 570858.2. NM 130843.2. PVR Poliovirus receptor PVR HUMAN P15151 NP_001.129240.1. NM_001135768.1. NP_001.129241.1. NM_001135769.1. NP_001.129242.1. NM_001135770.1. NP_006496.3. NM_006505.3. QPCT Glutaminyl-peptide QPCT HUMAN Q16769 NP 036545.1. cyclotransferase NM_01241.3.3. REG3G Regenerating islet- REG3G HUMAN Q6UW15 NP_001008388.1. derived protein 3- NM_001008387.2. gamma NP_0012.56969.1. NM_001270040.1. NP 940850.1. NM_198448.3. RGS7 Regulator of G- RGS7. HUMAN P49.802 NP_002915.3. protein signaling 7 NM_002924.4. RIMBP2 RIMS-binding RIMB2 HUMAN O15034 NP 056162.4. protein 2 NM_015.347.4. SCAMP1 Secretory carrier- SCAM1 HUMAN O15126 NP 004857.4. associated NM_004.866.4. membrane protein 1 SCAMP2 Secretory carrier- SCAM2 HUMAN O15127 NP 005688.2. associated NM_005697.3. membrane protein 2 US 2015/0330.997 A1 Nov. 19, 2015 15

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION SCAMP3 Secretory carrier- SCAM3 HUMAN O14828 NP_005689.2. associated NM_005698.3. membrane protein 3 NP 443069.1. NM 052837.2. SCG2 Secretogramin-2 SCG2 HUMAN P13521 NP_003460.2. NM_003469.4. SCG3 Secretogramin-3 SCG3 HUMAN Q8WXD2 NP_001158729.1. NM_001165257.1. NP 037375.2. NM_013243.3. SCG5 Neuroendocrine 7B2 HUMAN PO5408 NP_001138229.1. protein 7B2 NM_001144757.1. NP_00301.1.1. NM_003020.3. SCGN Secretagogin SEGN HUMAN O76038 NP_008929.2. NM_006998.3. SDK2 Protein sidekick-2 SDK2 HUMAN Q58EX2 NP_001138424.1. NM_001144952.1. SEMA3A Semaphorin-3A SEM3A HUMAN Q14563 NP_006071.1. NM_006080.2. SEMA3C Semaphorin-3C SEM3C HUMAN Q99985 NP_006370.1. NM_006379.3. SEPT3 Neuronal-specific SEPT3 HUMAN Q9UH03 NP 061979.3 septin-3 NM_019106.5 NP 663786.2 NM 145733.2 SERPINB13 Serpin B13 SPB13 HUMAN Q9UIV8 NP 036529.1 NM_01.2397.3 SERPINI1 Neuroserpin NEUS HUMAN Q99574 NP_001116224.1. NM_001 12275.2.1. NP 005016.1. NM_005025.4. SEZ6L2 Seizure 6-like SE6L2 HUMAN Q6UXD5 NP_001107571.1. protein 2 NM_001114099.2. NP 001107572.1. NM_001114100.2. NP_001230261.1. NM_001243332.1. NP_001230262.1. NM_001243333.1. NP 036542.1. NM_012410.3. NP 963869.2. NM 201575.3. SFT2D3 Vesicle transport SFT2C HUMAN Q587I9 NP 116129.3. protein SFT2C NM_032740.3. SHANK2 SH3 and multiple SHAN2 HUMAN Q9UPX8 NP 036441.2. ankyrin repeat NM_012309.3. domains protein 2 SLC2A13 Proton myo- MYCT HUMAN Q96OE2 NP 443117.3. inositol NM_05288.5.3. cotransporter SLC30A1 Zinc transporter 1 ZNT1 HUMAN Q976M5 NP 067017.2. NM_021194.2. SLC39A14 Zinc transporter S39AE HUMAN Q15043 NP_001121903.1. ZIP14 NM_001 128431.2. NP_001.128625.1. NM_001135153.1. NP 001128626.1. NM_001135154.1. NP 056174.2. NM_015.359.4. SLIT2 Slithomolog 3 SLIT3 HUMAN O75094 NP_003053.1 NM_003062.2 SNAP25 Synaptosomal- SNP25. HUMAN P60880 NP 003072.2. associated protein NM_003081.3. 25 NP 570824.1. NM 130811.2. SNAPIN SNARE-associated SNAPN HUMAN O95295 NP 036569.1. protein Snapin NM_012437.5. US 2015/0330.997 A1 Nov. 19, 2015 16

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION SORCS2 VPS10 domain- SORC2 HUMAN Q96PQ0 NP 065828.2. containing receptor NM_020777.2. Sor(DS2 SPARCL1 SPARC-like protein 1 SPRL1 HUMAN Q14515 NP 001121782.1. NM_001 128310.1. NP 004675.3. NM_004684.4. SPCS3 Signal peptidase SPCS3 HUMAN P61009 NP 06874.7.1. complex subunit 3 NM_021928.3. SPOCK1 Testican-1 TICN1 HUMAN Q08629 NP_004589.1. NM_004598.3. STK10 Serine/threonine- STK10 HUMAN O94804 NP 005981.3. protein kinase 10 NM_005990.3. STX1A. Syntaxin-1A STX1A HUMAN Q16623 NP_001159375.1 NM_0011659.03.1 NP 004594.1 NM_004603.3 STX2 Syntaxin-2 STX2 HUMAN P32856 NP_001971.2. NM_001980.3. NP 91.9337.1. NM_194356.2. SV2A Synaptic vesicle SV2A HUMAN Q7LOJ3 NP 055664.3. glycoprotein 2A NM_014849.3. SVIP Small VCP/p07- SVIP HUMAN Q8NHG7 NP_683691.1. interacting protein NM 148893.1. SYN1 Synapsin-1 SYN1 HUMAN P17600 NP_00888.1.2. NM_006950.3. NP 59.8006.1. NM_133499.2. SYNPO Synaptopodin SYNPO HUMAN Q8N3V7 NP_001103444.1. NM_001109974.2. NP_00115968.0.1. NM_001166208.1. NP_001159681.1. NM_001166209.1. NP_009217.3. NM_007286.5. SYT7 Synaptotagmin-7 SYT7 HUMAN O43581 NP_0041.91.2. NM_004200.3. TACSTD2 Tumor-associated TACD2. HUMAN PO9758 NP 002344.2. calcium signal NM_002353.2. transducer 2 TCN2 Transcobalamin-2 TCO2 HUMAN P20062 NP 00034.6.2. NM_000355.3. NP_001171655.1. NM_001184726.1. TLL2 Tolloid-like protein 2 TLL2 HUMAN Q976L7 NP 036597.1. NM_01246.5.3. TM9SF3 Transmembrane 9 TM9S3 HUMAN Q9HD45 NP 064508.3. superfamily NM_020123.3. member 3 TMEM106B Transmembrane T106B HUMAN Q9NUM4 NP_001 127704.1. protein 106B NM_001134232.1. NP 060844.2. NM_018374.3. TMEM119 Transmembrane TM119 HUMAN Q4V9L6 NP 859075.2. protein 119 NM 181724.2. TMEM132A Transmembrane T132A HUMAN Q24JP5 NP 060340.2. protein 132A NM_017870.3. NP 821174.1. NM_178031.2. TMPRSS11F Transmembrane TM11F HUMAN Q6ZWK6 NP 99.7290.2. protease serine 11F NM 207407.2. TNFSF11 Tumor necrosis TNF11 HUMAN O14788 NP_003692.1. actor ligand NM_003701.3. superfamily NP 143026.1. member 11 NM_033012.3. TNFSF4 Tumor necrosis TNFL4 HUMAN P23510 NP_003317.1. actor ligand NM_003326.3. superfamily member 4 US 2015/0330.997 A1 Nov. 19, 2015 17

TABLE 1-continued 3-Cell Mass Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION TTC7B Tetratricopeptide TTC7B HUMAN Q86TV6 NP_001010854.1. repeat protein 7B NM_001010854.1. TXNDC5 Thioredoxin TXND5 HUMAN Q8NBS9 NP_001139021.1. domain-containing NM_001145549.2. protein 5 NP 110437.2. NM 030810.3. |UBL3 Ubiquitin-like UBL3 HUMAN O95164 NP_009037.1. protein 3 NM 007106.3. |UCHL1 Ubiquitin carboxyl- UCHL1 HUMAN PO9936 NP_0041 72.2. terminal hydrolase NM_004181.4. isozyme L1 VAMP4 Vesicle-associated VAMP4 HUMAN O75379 NP_001172056.1. membrane protein 4 NM_0011851.27.1. NP 003753.2. NM_003762.4. VAT1L Synaptic vesicle VAT1L HUMAN Q9HCJ6 NP 065978.1. membrane protein NM_020927.1. VAT-1 homolog-like VAV3 Guanine nucleotide VAV3 HUMAN Q9UKW4 NP_001073343.1. exchange factor NM_001079874.1. VAV3 NP_006104.4. NM_006113.4. VGF Neurosecretory VGF HUMAN O15240 NP 003369.2. protein VGF NM_003378.3. VWASB2 von Willebrand VW5B2 HUMAN Q8N398 NP 612354.1. factor A domain- NM 138345.1. containing protein SB2 WFDC5 WAP four-disulfide WFDC5 HUMAN Q8TCV5 NP 663627.1. core domain NM 145652.3. protein 5 WFS1 Wolframin WFS1 HUMAN O76024 NP 001139325.1. NM_001145853.1. NP_005996.2. NM_006005.3. WNTSA Protein Wnt-5a WNT5A HUMAN P41221 NP_001243034.1. NM_0012.56105.1. NP 003383.2. NM_003392.4. WNTQB Protein Wnt-9b WNT9B HUMAN O14905 NP_003387.1. NM_003396.1.

TABLE 2 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION ABCC9 ATP-binding ABCC9 HUMAN O60706 NP_005682.2. NM_00569.1.2. cassette sub-family NP 064693.2. NM_020297.2. C member 9 ASNS Asparagine ASNS HUMAN P08243 NP 001171546.1. NM_001178.075.1. synthetase NP_001171547.1. NM_001178076.1. [glutamine- NP_001171548.1. NM_001178077.1. hydrolyzing] NP_001664.3. NM_001673.4. NP 59768.0.2. NM 133436.3. NP 899.199.2. NM 183356.3. GATC Glutamyl- GATC HUMAN O43716 NP 789788.1. NM 176818.2. tRNA(Gln) amidotransferase subunit C, mitochondrial MMP7 Matrilysin MMP7 HUMAN PO9237 NP_002414.1. NM_002423.3. OLFM4 Olfactomedin-4 OLFM4 HUMAN Q6UX06 NP_0064.09.3. NM_006418.4. SERPINE1 Plasminogen PAI1 HUMAN P051.21 NP 000593.1. NM_000602.4. activator inhibitor 1 NP_00115888.5.1. NM_001165413.2. US 2015/0330.997 A1 Nov. 19, 2015 18

TABLE 2-continued 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION SMPDL3B Acid ASM3B HUMAN Q92485 NP_001009568.1. NM_001009568.1. sphingomyelinase- NP 055289.2. NM_014474.2. like phosphodiesterase 3b ADAM9 Disintegrin and ADAM9 HUMAN Q13443 NP_003807.1. NM_003816.2. metalloproteinase domain-containing protein 9 C8Orf55 UPF0670 protein THEM6 HUMAN Q8WUY1 NP 057731.1. NM_016647.2. THEM6 CCL20 C-C motif CCL20 HUMAN P78556 NP 001123518.1. NM_001130.046.1. chemokine 20 NP_004.5.8.2.1. NM_004591.2. GDF15 Growth/ GDF15 HUMAN Q99988 NP_004.855.2. NM_004864.2. differentiation factor 15 IL32 Interleukin-32 IL32 HUMAN P24001 NP_00101.2649.1. NM_001012631.1. NP_00101.2650.1. NM_001012632.1. NP_00101.2651.1. NM_00101.2633.1. NP 00101.2652.1. NM_001012634.1. NP_00101.2653.1. NM_00101.2635.1. NP_00101.2736.1. NM_00101.2718.1. NP_004212.4. NM_004221.4. MMP14 Matrix MMP14 HUMAN P50281 NP_004986.1. NM_004995.2. metalloproteinase 4 SERPINB2 Plasminogen PAI2 HUMAN P05120 NP 001137290.1. NM_00114381.8.1. activator inhibitor 2 NP_002566.1. NM_002575.2. SPINT1 Kunitz-type SPIT1 HUMAN O43278 NP_001027539.1. NM_001032367.1. protease inhibitor 1 NP 003701.1. NM_003710.3. NP 857593.1. NM 181642.2. TNFAIP2 Tumor necrosis TNAP2 HUMAN Q03169 NP_006282.2. NM_006.29.1.2. actor alpha induced protein 2 MMP1 Interstitial MMP1 HUMAN PO3956 NP_002412.1. NM_002421.3. collagenase SPINT2 Kunitz-type SPIT2 HUMAN O43,291 NP_0011595.75.1. NM_001166103.1. protease inhibitor 2 NP 066925.1. NM_021102.3. COL3 A1 Collagen alpha- CO3A1 HUMAN PO2461 NP 000081.1. NM 000090.3. 1(III) chain YBX1 Nuclease-sensitive YBOX1 HUMAN P67809 NP_004.550.2. NM_004559.3. element-binding protein 1 GHRL Appetite-regulating GHRL HUMAN Q9UBU3 NP_001.12841.3.1. NM_00113494.1.1. [10] II.1QIlê NP_001.1284.16.1. NM_001134944.1. NP_001.128417.1. NM_00113494.5.1. NP_001.128418.1. NM_001134946.1. NP 057446.1. NM_016362.3. B4GALT1 Beta-1,4- B4GT1 HUMAN P15291 NP_001488.2. NM_001497.3. galactosyltransferase 1 ACP2 Lysosomal acid PPAL HUMAN P11117 NP_001601.1. NM_001610.2. phosphatase ACSL3 Long-chain-fatty- ACSL3 HUMAN O95573 NP_004448.2. NM_004457.3. acid-CoA ligase 3 NP 976251.1. NM 20337.2.1. ATP6AP2 Renin receptor RENR HUMAN O75787 NP_005756.2. NM_005765.2. B3GAT3 Galactosylgalactosyl- B3GA3 HUMAN O94766 NP 036332.2. NM_01.2200.3. xylosylprotein 3 beta glucuronosyltrans erase 3 CA4 Carbonic CAH4 HUMAN P22748 NP 000708.1. NM_000717.3. anhydrase 4 CAPNS1 Calpain small CPNS1 HUMAN PO4632 NP 00100396.2.1. NM_00100396.2.1. subunit 1 NP_001740.1. NM_001749.2. CIB1 Calcium and CIB1 HUMAN Q998.28 NP_006375.2. NM_006384.3. integrin-binding protein 1 CYB5R1 NADH-cytochrome NB5R1 HUMAN Q9UHQ9 NP 057327.2. NM_01624.3.2. b5 reductase 1 EPHB2 Ephrin type-B EPHB2 HUMAN P293.23 NP_004.433.2. NM_004442.6. receptor 2 NP_059145.2. NM_017449.3. US 2015/0330.997 A1 Nov. 19, 2015 19

TABLE 2-continued 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION FUT3 Galactoside 3(4)-L- FUT3 HUMAN P21217 NP_000140.1. NM_000149.3. fucosyltransferase NP_001091 108.1. NM_001097639.1. NP_001091 109.1. NM_001097640.1. NP 001091110.1. NM_001097641.1. FUT6 Alpha-(1,3)- FUT6 HUMAN P51993 NP_000141.1. NM_000150.2. fucosyltransferase NP_00103.5791.1. NM_001040701.1. FXYD2 Sodium/potassium- ATNG HUMAN P54710 NP_001671.2. NM_001680.4. transporting NP 067614.1. NM 021603.3. ATPase subunit gamma HTATIP2 Oxidoreductase HTAI2 HUMAN Q9BUP3 NP_001091990.1. NM_001098520.1. HTATIP2 NP 001091991.1. NM_001098521.1. NP_001091992.1. NM_001098522.1. NP_001091993.1. NM_001098523.1. NP_006401.3. NM_006410.4. LCN2 Neutrophil NGAL HUMAN P80188 NP 005555.2. NM_005564.3. gelatinase associated lipocalin LMAN2 Vesicular integral- LMAN2 HUMAN Q12907 NP_006.807.1. NM_006816.2. membrane protein VIP36 MAN1A2 Mannosyl- MA1A2 HUMAN O60476 NP_006690.1. NM_006699.3. oligosaccharide 1,2-alpha mannosidase IB PLSCR3 Phospholipid PLS3 HUMAN Q9NRY6 NP_001188.505.1. NM_001201576.1. scramblase 3 NP 065093.2. NM_020360.3. PMVK Phosphomevalonate PMVK HUMAN Q15126 NP_006547.1. NM_006556.3. kinase PTTG1 IP Pituitary tumor- PTTG HUMAN P53801 NP 004330.1. NM_004339.3. transforming gene 1 protein interacting protein TMED2 Transmembrane TMED2 HUMAN Q15363 NP_006.806.1. NM_0068.15.3. emp24 domain containing protein 2 VAMP1 Vesicle-associated VAMP1 HUMAN P23763 NP 055046.1. NM_014231.3. membrane protein 1 NP 058439.1. NM_016830.2. NP 954740.1. NM 1992.45.1. VAMP7 Vesicle-associated VAMP7 HUMAN P51809 NP_0011386.21.1. NM_001145149.2. membrane protein 7 NP_001172112.1. NM_00118518.3.1. NP_005629.1. NM_005638.5. ABHD12 Monoacylglycerol ABD12 HUMAN Q8N2KO NP 001035937.1. NM_00104.2472.2. lipase ABHD12 NP 056415.1. NM_015600.4. ALG5 Dolichyl-phosphate ALG5 HUMAN Q97.673 NP_001135836.1. NM_001142364.1. beta- NP 037470.1. NM_013338.4. glucosyltransferase ALOX12B Arachidonate 12- LX12B HUMAN O75342 NP_001130.1. NM_001139.2. lipoxygenase, 12R type AMPD3 AMP deaminase 3 AMPD3 HUMAN Q01432 NP 000471.1. NM_000480.2. NP_001020560.1. NM_001025.389.1. NP_001020561.1. NM_001025390.1. NP 00116590.1.1. NM_001172430.1. NP_001165902.1. NM_001172431.1. API.5 Apoptosis inhibitor 5 API5 HUMAN Q9BZZ5 NP_001136402.1. NM_001142930.1. NP_001136403.1. NM_001142931.1. NP 001230676.1. NM_0012.43747.1. NP_006586.1. NM_006595.3. ARMC10 Armadillo repeat- ARM10 HUMAN Q8N2F6 NP_001154481.1. NM_001161009.2. containing protein NP_001154482.1. NM_001161010.2. 10 NP_001154483.1. NM_00116101.1.2. NP_001154484.1. NM_00116101.2.2. NP_001154485.1. NM_00116101.3.2. NP 114111.2. NM_031905.4. ARMCX3 Armadillo repeat- ARMX3 HUMAN Q9UH62 NP 057691.1. NM 016607.3. containing X-linked NP 808816.1. NM 177947.2. protein 3 NP 808817.1. NM 177948.2. ASPH Aspartyl/asparaginyl ASPH HUMAN Q12797 NP_001158222.1. NM_001164750.1. beta-hydroxylase NP 001158223.1. NM_001164751.1. NP_001158225.1. NM_001164753.1. NP_0011582.27.1. NM_001164755.1. US 2015/0330.997 A1 Nov. 19, 2015 20

TABLE 2-continued 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION NP_001158228.1. NM_001164756.1. NP 004309.2. NM_004318.3. NP 064549.1. NM_020164.4. NP 115855.1. NM 032466.3. NP 115856.1. NM_032467.3. NP 115857.1. NM_032468.4. ATAD3A ATPase family AAA ATD3A HUMAN Q9NVI7 NP_001164006.1. NM_001170535.1. domain-containing NP 001164007.1. NM_001170536.1. protein 3A NP 060658.3. NM_018188.3. ATAD3B ATPase family AAA ATD3B HUMAN Q5T9A4 NP 114127.3. NM_031921.4. domain-containing protein 3B ATAD3C ATPase family AAA ATD3C HUMAN Q5T2N8 NP_001034300.2. NM_00103921.1.2. domain-containing protein 3C BRP44 Mitochondrial MPC2 HUMAN O95563 NP 001137146.1. NM_001143674.2. pyruvate carrier 2 NP 056230.1. NM_01541.5.3. C190rf68 Uncharacterized CSO68 HUMAN Q86XI8 BCO43.386 protein C190rf68 AAH43.386.1. CCDC56 Cytochrome C COA3 HUMAN Q97.2RO NP 001035521.1. NM_001040431.1. oxidase assembly factor 3 homolog, mitochondrial CEACAM7 Carcinoembryonic CEAM7 HUMAN Q14002 NP_008821.1. NM_006890.3. antigen-related cell adhesion molecule 7 CISD2 CDGSH iron-sulfur CISD2 HUMAN Q8N5K1 NP 001008389.1. NM_001008388.4. domain-containing protein 2 CPM Carboxypeptidase M CBPM HUMAN P14384 NP_001005502.1. NM_001005502.2. NP 001865.1. NM_001874.4. NP 938079.1. NM 1983.20.3. CTBP1 C-terminal-binding CTBP1 HUMAN Q13363 NP_001012632.1. NM_00101.2614.1. protein 1 NP_001319.1. NM_001.328.2. CTBP2 C-terminal-binding CTBP2 HUMAN P565.45 NP 001077383.1. NM_001083914.1. protein 2 NP_001.320.1. NM_001.329.2. NP 073713.2. NM_022802.2. CUZD1 CUB and zona CUZD1 HUMAN Q86UP6 NP_071317.2. NM_022034.5. pellucida-like domain-containing protein 1 DDRGK1 DDRGK domain- DDRGK HUMAN Q96HY6 NP 076424.1. NM_023935.1. containing protein 1 DHRS7B Dehydrogenase/ DRS7B HUMAN Q6IANO NP 056325.2. NM_015510.4. reductase SDR family member 7B EDF1 Endothelia EDF1 HUMAN 060869 NP 003783.1. NM 003792.2. differentiation- NP 694880.1. NM 153200.1. related factor 1 ELMOD2 ELMO domain- ELMD2 HUMAN Q8IZ81 NP 714913.1. NM 153702.3. containing protein 2 ENAH Protein enabled ENAH HUMAN Q8N8S7 NP_001008493.1. NM_001008493.1. homolog NP 060682.2. NM_018212.4. FAM174A Membrane protein F174A HUMAN Q8TBP5 NP 940909.1. NM 1985.07.1. FAM174A FAP Seprase SEPR HUMAN Q12884 NP_004451.2. NM_00446.0.2. FER Tyrosine-protein FER HUMAN P16591 NP_005237.2. NM_005246.2. kinase Fer GAD2 Glutamate DCE2 HUMAN Q05329 NP 000809.1. NM_000818.2. decarboxylase 2 NP_001.127838.1. NM_001134.366.1. GAPDHS Glyceraldehyde-3- G3PT HUMAN O14556 NP 05:51.79.1. NM_01436.4.4. phosphate dehydrogenase, estis-specific HK2 Hexokinase-2 HXK2 HUMAN P52789 NP_000180.2. NM_000189.4. HK3 Hexokinase-3 HXK3 HUMAN P52790 NP 002106.2. NM 002115.2. HKDC1 Putative HKDC1 HUMAN Q2TB90 NP 0794.06.3. NM_025.130.3. hexokinase HKDC1 HSD17B2 Estradiol 17-beta- DHB2 HUMAN P37059 NP 002144.1. NM_002153.2. dehydrogenase 2 HSF2BP Heat shock factor HSF2B HUMAN O75031 NP_008962.1. NM_007031.1. 2-binding protein US 2015/0330.997 A1 Nov. 19, 2015 21

TABLE 2-continued 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION FNGR1 Interferon gamma [NGR1 HUMAN P15260 NP 000407.1. NM_000416.2. receptor 1 LF2 Interleukin LF2 HUMAN Q12905 NP_001254738.1. NM_001267809.1. enhancer-binding NP 004506.2. NM_004515.3. actor 2 TGB6 Integrin beta-6 TB6 HUMAN P18564 NP 000879.2. NM_000888.3. KIAA0090 ER membrane EMC1 HUMAN Q8N766 NP_001258356.1. NM_001271427.1. protein complex NP 001258.357.1. NM_001271428.1. subunit 1 NP_0012583.58.1. NM_001271429.1. NP 055862.1. NM_015047.2. KIAA0776 E3 UFM1-protein UFL1, HUMAN O94874 NP 0561.38.1. NM_015323.4. igase 1 KIAA2013 Uncharacterized K2013 HUMAN Q8IYS2 NP 612355.1. NM 13834.6.2. protein KIAA2013 KLRAQ1 Protein PPR21 HUMAN Q6ZMIO NP_001.129.101.1. NM_001135629.2. phosphatase 1 NP 001180404.1. NM_001 193475.1. regulatory subunit NP 6.94539.1. NM 152994.4. 21 LAMTOR1 Ragulator complex LTOR1 HUMAN Q6IAA8 NP 060377.1. NM_017907.2. protein LAMTOR1 LAMTOR2 Ragulator complex LTOR2 HUMAN Q97.205 NP_001138736.1. NM_001145264.1. protein LAMTOR2 NP 054736.1. NM_014017.3. LAMTOR3 Ragulator complex LTOR3 HUMAN Q9UHA4 NP 068805.1. NM_021970.3. protein LAMTOR3 LRRC63 Leucine-rich LRC63 HUMAN Q05C16 CAI12166.2. repeat-containing BC030.276 protein 63 AAH30276.1. MFN2 Mitofusin-2 MFN2 HUMAN O95140 NP_001121132.1. NM_001 127660.1. NP 055689.1. NM_014874.3. MGAT4B Alpha-1,3- MGT4B HUMAN Q9UQ53 NP 05.5090.1. NM_014275.4. mannosyl- NP 4634.59.1. NM 05401.3.3. glycoprotein 4-beta acetylglucosaminyl transferase B MLF2 Myeloid leukemia MLF2 HUMAN Q15773 NP 005430.1. NM_005439.2. factor 2 MOGS Mannosyl- MOGS HUMAN Q13724 NP_001139630.1. NM_001146158.1. oligosaccharide NP_006.293.2. NM_006.302.2. glucosidase MTMR11 Myotubularin- MTMRB HUMAN A4FUO1 NP_001139334.1. NM_001145862.1. related protein 11 NP 870988.2. NM 181873.3. MTX1 Metaxin-1 MTX1 HUMAN Q13505 NP_002446.2. NM_002455.3. NP 94.2584.1. NM 198883.2. NCEH1 Neutral cholesterol NCEH1 HUMAN Q6PIU2 NP_001139748.1. NM_001146276.1. ester hydrolase 1 NP_001139749.1. NM_001146277.1. NP_001139750.1. NM_001146278.1. NP 065843.3. NM_020792.4. OCLAD2 OCIA domain- OCAD2 HUMAN Q56VL3 NP_001014446.1. NM_001014446.1. containing protein 2 NP_6896.11.1. NM 152398.2. PDE8AB High affinity cAMP- PDE8B HUMAN O95263 NP_001025022.1. NM_001029851.2. specific and IBMX- NP_001025.023.1. NM_001029852.2. insensitive 3',5'- NP_001025.024.1. NM_001029853.2. cyclic NP_001025.025.1. NM_0010298.54.2. phosphodiesterase NP 003710.1. NM 003719.3. 8B PFKFB1 6-phosphofructo-2- F261 HUMAN P16118 NP 002616.2. NM_002625.2. kinase/fructose 2,6-bisphosphatase 1 PIGK GPI-anchor GPI& HUMAN Q92643 NP_005473.1. NM_005482.2. transamidase PLEKHH2 Pleckstrin PKHH2 HUMAN Q8IVE3 NP 742066.2. NM 172069.3. homology domain containing family H member 2 PRUNE2 Protein prune PRUN2 HUMAN Q8WUY3 NP 056040.2. NM_01522.5.2. homolog 2 RDH.11 Retinol RDH11 HUMAN Q8TC12 NP 0571 10.3. NM_016026.3. dehydrogenase 11 RIC8A Synembryn-A RIC8A HUMAN Q9NPQ8 NP 068751.4. NM_021932.4. RUFY3 Protein RUFY3 RUFY3 HUMAN Q7L099 NP 00103.2519.1. NM_001037442.2. NP_001.124181.1. NM_001130709.1. NP 055776.1. NM_014961.3. US 2015/0330.997 A1 Nov. 19, 2015 22

TABLE 2-continued 3-Cell Function Markers of the Invention. Marker Protein UNIPROT Name Description UNIPROT ID ACCESSION GENEANK ACCESSION SDK1 Protein sidekick-1 SDK1 HUMAN Q7Z5N4 NP_6899.57.3. NM 152744.3. SORCS3 VPS10 domain- SORC3 HUMAN Q9UPU3 NP 05.5793.1. NM_014978.1. containing receptor Sor(DS3 SPTLC1 Serine SPTC1 HUMAN O15269 NP_0064.06.1. NM_006415.2. palmitoyltransferase 1 NP 847894.1. NM_1783.24.1. STOML3 Stomatin-like STML3 HUMAN Q8TAV4 NP_001137505.1. NM_00114403.3.1. protein 3 NP 660329.1. NM 145286.2. STX1 B Syntaxin-1B STX18 HUMAN P61266 NP 443106.1. NM_052874.3. SYT5 Synaptotagmin-5 SYT5 HUMAN O00445 NP_003171.2. NM_003180.2. TBL2 Transducin beta- TBL2 HUMAN Q974P3 NP 036585.1. NM_01245.3.2. like protein 2 TGOLN2 Trans-Golgi TGON2 HUMAN O43493 NP_001.193769.1. NM_001206840.1. network integral NP_001.193770.1. NM_001206841.1. membrane protein 2 NP_001.193773.1. NM_001206.844.1. NP OO6455.2. NM_006.464.3. THSD7A Thrombospondin THS7A HUMAN Q9UPZ6 NP 056019.1. NM_015204.2. type-1 domain containing protein 7A TMCO1 Transmembrane TMCO1 HUMAN Q9UM00 NP 06.1899.2. NM_019026.4. and coiled-coil omain-containing protein 1 TMEM123 Porimin PORIM HUMAN Q8N131 NP 443164.2. NM_052932.2. TMPRSS13 Transmembrane TMPSD HUMAN Q9BYE2 NP_001.193719.1. NM_001206790.1. protease serine 13 NP_001231924.1. NM_001244995.1. TMX4 Thioredoxin- TMX4 HUMAN Q9H1E5 NP 066979.2. NM_021156.2. related transmembrane protein 4 TNPO2 Transportin-2 TNPO2 HUMAN O14787 NP_001.129.667.1. NM_0011361.95.1. NP_001.1296.68.1. NM_001136196.1. NP 038461.2. NM_013.433.4. TPBG Trophoblast TPBG HUMAN Q13641 NP 001159864.1. NM_001166392.1. glycoprotein NP_006661.1. NM_006670.4. TRIM42 Tripartite motif- TRIA2 HUMAN Q8IWZ5 NP_6898.29.3. NM 152616.4. containing protein 42 TTC37 Tetratricopeptide TTC37 HUMAN Q6PGP7 NP 05.5454.1. NM_014639.3. repeat protein 37 USP9X Probable ubiquitin USP9X HUMAN Q93008 NP_001034679.2. NM_001039590.2. carboxyl-terminal NP 001034680.2. NM_001039591.2. hydrolase FAF-X VAPB Vesicle-associated VAPB HUMAN O95292 NP_001182606.1. NM_001.1956.77.1. membrane protein- NP_004729.1. NM_004738.4. associated protein B/C VNN2 Vascular non- VNN2 HUMAN O95498 NP_001229279.1. NM_001242350.1. inflammatory NP 004656.2. NM_004665.2. molecule 2 NP 51.1043.1. NM_078488.1. VPS26B Vacuolar protein VP26B HUMAN Q4GOF5 NP 443107.1. NM_052875.3. sorting-associated protein 26B YTHDF2 YTH domain family YTHD2 HUMAN Q9Y5A9 NP 001166299.1. NM_001172828.1. protein 2 NP_001166599.1. NM_001173128.1. NP 057342.2. NM_01625.8.2. ZFPL1 Zinc finger protein- ZFPL1 HUMAN O951.59 NP OO6773.2. NM_006.782.3. like 1

TABLE 3 Therapeutic Efficacy Markers of the Invention.

Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION Alpha-2 A2MG HUMAN PO1023 NP 000005.2 macroglobulin NM_000014.4 US 2015/0330.997 A1 Nov. 19, 2015 23

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION ABI3BP Target of Nesh-SH3 TARSH HUMAN Q7Z7GO NP 056244.2 NM_015429.3 ACE Angiotensin- ACE HUMAN P12821 NP 000780.1 converting enzyme NM 000789.3 NP_001171528.1 NM_001178057.1 NP 690043.1 NM 152830.2 ACTN1 Alpha-actinin-1 ACTN1 HUMAN P12814 NP_001093.1 NM_001102.3 NP_001.123476.1 NM_001 130004.1 NP_001.123477.1 NM_00113.0005.1 AFM Afamin AFAM HUMAN P43652 NP_001.124.1 NM_001133.2 AGT Angiotensinogen ANGT HUMAN PO1019 NP 000020.1 NM_000029.3 ALCAM CD166 antigen CD166 HUMAN Q13740 NP_001230209.1 NM_001243280.1 NP_001618.2 NM_001627.3 ALDOB Fructose- ALDOB HUMAN PO5062 NP 000026.2 bisphosphate NM_000035.3 aldolase B AMBP Protein AMBP AMBP HUMAN PO2760 NP_001624.1 NM_001633.3 ANPEP Aminopeptidase N AMPN HUMAN P15144 NP_001141.2 NM_001150.2 AOC3 Membrane primary AOC3 HUMAN Q16853 NP 003725.1 amine oxidase NM 003734.2 APOA1 Apolipoprotein APOA1 HUMAN PO2647 NP 000030.1 A-I NM_000039.1 APOA2 Apolipoprotein APOA2 HUMAN PO2652 NP_001634.1 A-II NM_001643.1 APOA4 Apolipoprotein APOA4 HUMAN PO6727 M13654; ; AAA51744.1; A-IV X13629; CAA31955.1; BCO74764; AAH74764.1; BC113594; AAI13595.1; BC113596; AAI13597.1; M14566; AAA51748.1 APOB Apolipoprotein APOB. HUMAN PO4114 P 000375.2 B-100 M_000384.2 APOC2 Apolipoprotein APOC2 HUMAN PO2655 P 000474.2 C-II M 000483.4 APOC3 Apolipoprotein APOC3, HUMAN PO2656 P 000031.1 C-III M 000040.1 APOC4 Apolipoprotein APOC4 HUMAN P55056 P_001637.1 C-IV M_001646.2 APOE Apolipoprotein APOE HUMAN PO2649 P 000032.1 E M 000041.2 ARHGDIA Rho GDP- GDIR1 HUMAN P52565 P 001172006.1 dissociation M_001185.077.1 inhibitor 1 P 001172007.1 M_001185078.1 P 004300.1 M 004309.4 ARHGDIB Rho GDP- GDIR2 HUMAN P52566 P 001166.3 dissociation M_001175.4 inhibitor 2 ATRN Attractin ATRN HUMAN O75882 P_001.193976.1 M_001207047.1 P 647537.1 M_1393.21.2 P 647538.1 M_139322.2. AZGP1 Zinc-alpha-2- ZA2G HUMAN P25311 P 001176.1 glycoprotein M_001185.3 B2M Beta-2- B2MG HUMAN P61769 P_004039.1 microglobulin M_004048.2 BST1 ADP-ribosyl cyclase BST1 HUMAN Q10588 P 0043.25.2 2 NM_004334.2 US 2015/0330.997 A1 Nov. 19, 2015 24

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION BTD Biotinidase BTD HUMAN P43251 NP 000051.1 NM_000060.2 C1RL Complement C1r C1RL HUMAN Q9NZP8 NP 057630.2 subcomponent-like NM_016546.2 protein C4BPA C4b-binding C4BPA HUMAN PO4003 NP 000706.1 protein alpha chain NM_000715.3 C9 Complement CO9 HUMAN PO2748 NP 001728.1 component C9 NM_001737.3 CA2 Carbonic CAH2 HUMAN P00918 NP 000058.1 anhydrase 2 NM_000067.2 CACNA2D1 Voltage-dependent CA2D1 HUMAN P54289 NP 000713.2 calcium channel NM_000722.2 subunit alpha-2/ delta-1 CAP1 Adenylyl cyclase- CAP1 HUMAN Q01518 NP 001099000.1 associated protein 1 NM_0011055.30.1 NP_006.358.1 NM_006.367.3 CD14 Monocyte CD14 HUMAN P08571 NP 000582.1 differentiation NM_000591.3 antigen CD14 NP_0010351.10.1 NM_0010400.21.2 NP 001167575.1 NM_001174104.1 NP_001167576.1 NM_001 174105.1 CD163 Scavenger receptor C163A HUMAN Q86VB7 NP_004235.4 cysteine-rich type 1 NM_004.244.5 protein M130 NP 98.1961.2 NM 203416.3 CDSL CD5 antigen-like CD5L HUMAN O43866 NP_005885.1 NM_005894.2 CDH5 Cadherin-5 CADH5 HUMAN P331.51 NP_001786.2 NM_001795.3 CFD Complement factor FAD HUMAN P00746 NP_001919.2 D NM_001928.2 CLEC3B Tetranectin TETN HUMAN P05452 NP_0032.69.2 NM_003278.2 CLSTN1 Calsyntenin-1 CSTN1 HUMAN O94985 NP_001009566.1 NM_001009566.1 NP 055759.3 NM_014944.3 CNDP1 Beta-Ala-His CNDP1 HUMAN Q96KN2 NP 116038.4 dipeptidase NM_03.2649.5 CNN2 Calponin-2 CNN2 HUMAN Q99439 NP 004359.1 NM_004368.2 NP 958434.1 NM 2012.77.1 COL6A1 Collagen alpha- CO6A1 HUMAN P12109 NP_001839.2 1(VI) chain NM_001848.2 COL6A3 Collagen alpha- CO6A3 HUMAN P12111 NP 004360.2 3(VI) chain NM_004369.3 NP 476505.3 NM_05.7164.4 NP 4765.08.2 NM 057167.3 CORO1A Coronin-1A COR1A HUMAN P31 146 NP_001180262.1 NM_001 193333.2 NP 009005.1 NM_007074.3 CPB2 Carboxypeptidase CBPB2 HUMAN Q96IY4 NP_001863.2 B2 NM_001872.3 CRP C-reactive protein CRP HUMAN PO2741 NP 000558.2 NM_000567.2 CRTAC1 Cartilage acidic CRAC1 HUMAN Q9NQ79 NP 001.193457.1 protein 1 NM_001206528.2 NP 060528.3 NM_018058.6 CTBS Di--acetylchitobiase DIAC HUMAN Q01459 NP 004379.1 NM_004388.2 US 2015/0330.997 A1 Nov. 19, 2015 25

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION DBH Dopamine beta- DOPO HUMAN PO9172 N P 000778.3 hydroxylase NM 000787.3 DBNL Drebrin-like DBNL HUMAN Q9UJU6 N P_001014436.1 protein NM_001014436.2 N P 001116428.1 NM_001 122956.1 N P 054782.2 NM 014063.6 DPEP2 Dipeptidase 2 DPEP2 HUMAN Q9H4A9 N P_071750.1 NM_022355.3 ECM1 Extracellular matrix ECM1 HUMAN Q16610 N P 00118978.7.1 protein 1 NM_001202858.1 N P_004416.2 NM_004.425.3 N P 073155.2 NM_022664.2 EFEMP1 EGF-containing FBLN3 HUMAN Q12805 N P_001034437.1 fibulin-like NM_001039348.2 extracellular matrix N P_001034438.1 protein 1 NM_001039349.2 ENPP2 Ectonucleotide ENPP2 HUMAN Q13822 NP_001035181.1 pyrophosphatase/ NM_001040092.2 phosphodiesterase NP_001.124335.1 family member 2 NM_001130863.2 NP_006200.3 NM_006209.4 ERP29 Endoplasmic ERP29 HUMAN P30040 NP OO6808.1 reticulum resident NM_006817.3 protein 29 F10 Coagulation factor FA10 HUMAN POO742 NP 000495.1 X NM 000504.3 F11 Coagulation factor FA11. HUMAN PO3951 NP_0001.19.1 XI NM_000128.3 F12 Coagulation factor FA12 HUMAN POO748 NP 000496.2 XII NM 000505.3 F13B Coagulation factor F13B HUMAN P05160 NP_001985.2 XIII B chain NM_001994.2 F9 Coagulation factor FA9 HUMAN P00740 NP_0001 24.1 DX NM_000133.3 FAM3B Protein FAM3B FAM3B HUMAN P58499 NP 478066.3 NM_058.186.3 NP 99.6847.1 NM 206964.1 FBLN1 Fibulin-1 FBLN1 HUMAN P23142 NP_001987.2 NM_001996.3 NP_006476.2 NM_006485.3 NP_006477.2 NM_006486.2 NP_006478.2 NM_006487.2 FCGBP IgGFc-binding FCGBP HUMAN Q976R7 NP_003881.2 protein NM_003890.2 FERMT3 Fermitin family URP2 HUMAN Q86UX7 NP 113659.3 homolog 3 NM 031471.5 NP 848537.1 NM_178443.2 FETUB Fetuin-B FETUB HUMAN Q9UGM5 NP 055.190.2 NM_014375.2 FLNA Filamin-A FLNA HUMAN P21333 NP_001104026.1 NM_001110556.1 NP 001.447.2 NM_001456.3 FN1 Fibronectin FINC HUMAN P02751 NP_002017.1 NM_002026.2 N P. 473375.2 NM 054034.2 N P 997639.1 NM 212474.1 N P 997641.1 NM 212476.1 N P 997643.1 US 2015/0330.997 A1 Nov. 19, 2015 26

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION NM 212478.1 NP 997647.1 NM 212482.1 FTH1 Ferritin heavy chain FRIH HUMAN PO2794 NP 002023.2 NM_002032.2 FTL Ferritin light chain FRIL HUMAN PO2792 NP_000137.2 NM_000146.3 GAPDH Glyceraldehyde-3- G3P HUMAN P04406 NP 001243728.1 phosphate NM_001256799.1 dehydrogenase NP_002037.2 NM_002046.4 GPLD1 Phosphatidylinositol- PHL.D HUMAN P80.108 NP 001.494.2 glycan-specific NM_001503.3 phospholipase D GPX3 Glutathione GPX3. HUMAN P22352 NP_002075.2 peroxidase 3 NM_002084.3 GSN Gelsolin GELS HUMAN PO6396 NP_000168.1 NM_000177.4 NP_001121134.1 NM_001 127662.1 NP_001121135.2 NM_001 127663.1 NP_001121136.1 NM_001 127664.1 NP_001121137.1 NM_001 127665.1 NP 001121138.1 NM_001 127666.1 NP_001121139.1 NM_001 127667.1 NP 001244958.1 NM_001258029.1 NP 937895. NM 198252.2 GSTP1 Glutathione S- GSTP1 HUMAN PO921.1 NP 000843. transferase P NM_000852.3 HABP2 Hyaluronan- HABP2 HUMAN Q14520 NP_001171131.1 binding protein 2 NM_001177660.1 NP_004123. NM_004132.3 HBA1 Hemoglobin HBA HUMAN P69905 NP 000508. and subunit alpha NM_000517.4 HBA2 NP 000549. NM 000558.3 HBD Hemoglobin HBD HUMAN P02042 NP 000510. subunit delta NM_000519.3 HGFAC Hepatocyte growth HGFA HUMAN Q04756 NP 001519. actor activator NM_001528.2 HPR Haptoglobin- HPTR HUMAN POO739 NP 066275.3 related protein NM_020995.3 HSPA8 Heat shock cognate HSP7C HUMAN P11142 NP_006588. 71 kDa protein NM_006597.4 NP 694881. NM 1532.01.2 HSPB1 Heat shock protein HSPB1 HUMAN PO4792 NP 001531. beta-1 NM_001540.3 HSPG2 Basement PGBM HUMAN P98160 NP_005520.4 membrane-specific NM_005529.5 heparan sulfate proteoglycan core protein IGF2 Insulin-like growth IGF2 HUMAN PO1344 NP 000603.1 actor II NM_000612.4 NP_001007140.2 NM_001007139.4 IGF2R. Cation- MPRI HUMAN P11717 NP 000867.2 independent NM_000.876.2 mannose-6 phosphate receptor US 2015/0330.997 A1 Nov. 19, 2015 27

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION IGFALS Insulin-like growth ALS HUMAN P35858 NP_004961.1 actor-binding NM_004970.2 protein complex acid labile subunit IGFBP3 Insulin-like growth IBP3 HUMAN P17936 NP 000589.2 actor-binding NM_000598.4 protein 3 NP_001013416.1 NM_00101.3398.1 IGFBP4 Insulin-like growth P4 HUMAN P22692 NP 001543.2 actor-binding NM_001552.2 protein 4 IGLL5 Immunoglobulin IGLL5 HUMAN B9A064 NP 001171597.1 ambda-like NM_001178126.1 polypeptide 5 IL18BP nterleukin-18- I18BP HUMAN O95998 NP_001034748.1 binding protein NM_001039659.1 NP_001034749.1 NM_001039660.1 NP_001138.527.1 NM_001145055.1 NP_001138529.1 NM_001145057.1 NP_005690.2 NM_005.699.3 NP 766630.2 NM 173042.2 NP 766632.2 NM 173044.2 IL1RAP Interleukin-1 IL1AP HUMAN Q9NPH3 NP_001161400.1 receptor accessory NM_001167928.1 protein NP 001161401.1 NM_001167929.1 NP_001161402.1 NM_001167930.1 NP 001161403.1 NM_001167931.1 NP 002173.1 NM_002182.3 NP 608273.1 NM 134470.3 ILK Integrin-linked ILK HUMAN Q13418 NP_001014794.1. protein kinase NM_001014794.1. NP 001014795.1. NM_001014795.1. NP_004.508.1. NM_004517.2. SLR Immunoglobulin ISLR HUMAN O14498 NP 005536.1 superfamily NM_005545.3 containing leucine- NP 958934.1 rich repeat protein NM 201526.1 TIH3 Inter-alpha-trypsin ITIH3 HUMAN Q06033 NP_002.208.3 inhibitor heavy NM_002217.3 chain H3 TIH4 Inter-alpha-trypsin ITIH3 HUMAN Q14624 NP 002.208.3 inhibitor heavy NM_002217.3 chain H3 LBP Lipopolysaccharide- LBP HUMAN P18428 NP_004130.2 binding protein NM_004139.3 LCAT Phosphatidylcholine- LCAT HUMAN P04180 NP_000220.1 sterol NM_000229.1 acyltransferase LRG1 Leucine-rich alpha- A2GL HUMAN PO2750 NP 443204.1 2-glycoprotein NM_052972.2 LUM Lumican LUM HUMAN P51884 NP 002336.1 NM_002345.3 LYVE1 Lymphatic vessel LYVE1 HUMAN Q975Y7 NP 006682.2 endothelial NM_006691.3 hyaluronic acid receptor 1 MASP1 Mannan-binding MASP1 HUMAN P4.8740 NP 001027019.1 ectin serine NM_001031849.2 protease 1 NP_001870.3 US 2015/0330.997 A1 Nov. 19, 2015 28

TABLE 3-continued Therapeutic Efficacy Markers of the Invention. Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION NM_001879.5 NP 624302.1 NM 1391.25.3 MBL2 Mannose-binding MBL2 HUMAN P11226 NP 000233.1 protein C NM_00024.2.2 MCAM Cell surface MUC18 HUMAN P43121 NP_006491.2 glycoprotein NM_006500.2 MUC18 MINPP1 Multiple inositol MINP1 HUMAN Q9UNW1 NP_001171588.1 polyphosphate NM_0011781.17.1 phosphatase 1 NP_001171589.1 NM_001 1781 18.1 NP_004888.2 NM_004897.4 MST1 Hepatocyte growth HGFL HUMAN P26927 NP 066278.3 factor-like protein NM_020998.3 NID1 Nidogen-1 NID1 HUMAN P14543 NP_002.499.2 NM_0025.08.2 ORM1 Alpha-1-acid A1AG1 HUMAN PO2763 NP 000598.2 glycoprotein 1 NM 000607.2 ORM2 Alpha-1-acid A1AG2 HUMAN P19652 NP 000599.1 glycoprotein 2 NM_000608.2 PCOLCE Procollagen C- PCOC1 HUMAN Q15113 NP_002584.2 endopeptidase NM_002.593.3 enhancer 1 PDIA3 Protein disulfide- PDIA3 HUMAN P30101 NP_005304.3 isomerase A3 NM_005313.4 PDIA6 Protein disulfide- PDIA6 HUMAN Q15084 NP_005733.1 isomerase A6 NM_005742.2 PDLIM1 PDZ and LIM PDLI1 HUMAN OOO151 NP 06627.2.1 domain protein 1 NM_020992.3 PEPD Xaa-Pro PEPD HUMAN P12955 NP_000276.2 dipeptidase NM_000285.3 NP_001159528.1 NM_001166056.1 NP_0011595.29.1 NM_001166057.1 PFN1 Profilin-1 PROF1 HUMAN PO7737 NP_005O13.1 NM_005022.3 PGLYRP2 N-acetylmuramoyl- PGRP2 HUMAN Q96PD5 NP 443122.3 L-alanine amidase NM_052890.3 PKM2 Pyruvate kinase KPYM HUMAN P14618 NP_001.193725.1 isozymes M1/M2 NM_001206796.1 NP_001.193726.1 NM_001206797.1 NP_001.193727.1 NM_001206798.1 NP_001.193728.1 NM_001206799.1 NP 002645.3 NM_002654.4 NP 87.2270.1 NM 182470.2 NP 87.2271.1 NM 182471.2 PLEK Pleckstrin PLEK HUMAN P08567 NP 002655.2 NM_002664.2 PPIA Peptidyl-prolyl cis- PPIA HUMAN P62937 NP 066953.1 trans isomerase A NM_021130.3 PRDX2 Peroxiredoxin-2 PRDX2 HUMAN P32119 NP 00:5800.3 NM_005809.4 NP 859428.1 NM 181738.1 PROCR Endothelial protein EPCR HUMAN Q9UNN8 NP_006.395.2 C receptor NM_006404.3 PROS1 Vitamin K- PROS HUMAN PO7225 NP 000304.2 dependent protein S NM_000313.3 PROZ Vitamin K- PROZ HUMAN P22891 NP_001243063.1 dependent protein Z NM_0012561.34.1 NP_003882.1 NM_003891.2 US 2015/0330.997 A1 Nov. 19, 2015 29

TABLE 3-continued Therapeutic Efficacy Markers of the Invention.

Marker Protein UNIPROT ENE ANK Name Description UNIPROT ID ACCESSION CCESSION QSOX1 Sulfhydryl oxidase 1 QSOX1 HUMAN O00391 P_001004128.1 M_001004128.2 P 002817.2 M 002826.4 RNASE1 Ribonuclease RNAS1 HUMAN PO7998 P 002924.1 pancreatic M_002933.4 P 937875.1 M_198232.2 P 937877.1 M_198234.2 P 937878.1 M 198235.2 S100A9 Protein S100-A9 S10A9 HUMAN PO6702 P 002956.1 M_002965.3 SAA4 Serum amyloid A-4 SAA4 HUMAN P35542 P_006503.2 protein M_006512.3 SELL L-selectin LYAM1 HUMAN P141.51 P 000646.2 M 000655.4 SERPINA1 Alpha-1-antitrypsin A1AT HUMAN PO1009 P 000286.3 M 000295.4 P_001002235. M_00100.2235.2 P_001002236. M_001002236.2 P 001121172. M_001 127700.1 P 001121173. M_001 1277.01.1 P 001121174. M_001 127702.1 P 001121175. M_001 127703.1 P 001121176. M_001 1277.04.1 P 001121177. M_001 127705.1 P 001121178. M_001 127706.1 P 001121179. M_001 127707.1 SERPINA4 Kallistatin KAIN HUMAN P29.622 P_006.206.2 M_006215.2 SERPINA6 Corticosteroid CBG HUMAN P08.185 P 001747.2 binding globulin M_001756.3 SERPINAT Thyroxine-binding THBG HUMAN P05543 P 000345.2 globulin M_000354.5 SERPIND1 Heparin cofactor 2 HEP2 HUMAN P05546 P_000176.2 M_000185.3 4F2 cell-surface 4F2 HUMAN P08195 P_00101.2680.1 antigen heavy M_001012662.2 chain P_001012682.1 M_00101.2664.2 P_001013269.1 M_001013251.2 P 002385.3 M 002394.5 SNCA Alpha-synuclein SYUA HUMAN P37840 P 000336.1 M_000345.3 P 00113.9526.1 M_001146054.1 P 001139527.1 M 001146055.1 P_009292.1 0.07308.2 SOD3 Extracellular SODE HUMAN P08294 P_003093.2 superoxide M 003102.2 dismutase [Cu-Zn] SPP2 Secreted SPP24 HUMAN Q13103 P_008875.1 phosphoprotein 24 M_006944.2 TAGLN2 Transgelin-2 TAGL2 HUMAN P37802 s P 003555.1 NM_003564.1 US 2015/0330.997 A1 Nov. 19, 2015 30

TABLE 3-continued Therapeutic Efficacy Markers of the Invention.

Marker Protein UNIPROT ENE ANK Name Description UNIPROT ID ACCESSION CCESSION Serotransferrin TRFE HUMAN PO2787 P_001054.1 M_00106.3.3 THES1 Thrombospondin-1 TSP1 HUMAN PO7996 P_003237.2 M_003246.2 TLN1 Talin-1 TLN1 HUMAN Q97.490 P_006280.3 M_006.289.3 TNC Tenascin TENA HUMAN P24821 P 002151.2 M 002160.3 Tenascin-X TENX HUMAN P22105 P 061978.6 M 019105.6 P 115859.2 M 032470.3 TPM1 Tropomyosin TPM1 HUMAN PO9493 P 000357.3 alpha-1 chain M_000366.5 P_001018005.1 M_001018005.1 P_001018006.1 M_001018006.1 P_001018007.1 M_00101.8007.1 P_001018008.1 M_001018008.1 TPM3 Tropomyosin TPM3 HUMAN P06753 P_001036816.1 alpha-3 chain M_00104335.1.1 P_001036817.1 M_001043352.1 P 689476.2 M_1.52263.2 P 705935. M_153649.3 TPM4 Tropomyosin TPM4 HUMAN P67936 P 0011386.32.1 alpha-4 chain M_001145160.1 P_003281. M_003290.2 TTR Transthyretin TTHY HUMAN P02766 P 000362.1 M_000371.3 VCAM1 Vascular cell VCAM1 HUMAN P19320 P_001069. adhesion protein 1 M_001078.3 P 001186763.1 M_001 199834.1 P_542413. M 080682.2 VCL Vinculin VINC HUMAN P18206 P 003364. M_003373.3 P 054706. M 014000.2 von Willebrand VWF HUMAN PO4275 P 000543.2 factor M 000552.3 YWHAZ 14-3-3 protein 1433Z HUMAN P63 104 P 001129.171.1 Zeta?delta M_001135699.1 P 001129.172.1 M_001135700.1 P 001129.173.1 M 001135701.1 P 001129.174.1 M_001135702.1 P_003397.1 M_003406.3 P 663723.1 M 145690.2 FGG Fibrinogen gamma FIBG HUMAN PO2679 P 000500.2 chain M 000509.4 P 068656.2 M_021870.2 NEO1 Neogenin NEO1 HUMAN Q92859 P 001166094.1 M_001 172623.1 P 002490.2 M_002499.3 FAM20C Extracellular DMP4 HUMAN P 0646.08.2 serine/threonine NM_020223.3 protein kinase Fam?0C US 2015/0330.997 A1 Nov. 19, 2015

TABLE 3-continued Therapeutic Efficacy Markers of the Invention.

Marker Protein UNIPROT GENEANK Name Description UNIPROT ID ACCESSION ACCESSION NCAM1 Neural cell NCAM1 HUMAN P13591 NP 000606.3 adhesion molecule NM_000615.6 1 NP_0010701:50.1 NM_001076682.3 NP_001229537.1 NM_001242608.1 NP 8.5.1996.2 NM 181351.4 PTPRJ Receptor-type PTPRJ HUMAN Q12913 NP_001091973.1 tyrosine-protein NM_0010985.03.1 phosphatase eta NP 002834.3 NM 002843.3

[0060] In certain aspects of the invention, a single marker embodiment, the markers include PPY SEPT3, and PTPRJ. (e.g., any one of the markers listed in Tables 1-3) may be used In one embodiment, the markers include CPM, INS, MMP7, in the methods and compositions of the invention. For and LDLR. example, in one embodiment, the marker for use in the meth [0063] In some embodiments, the methods may further ods and compositions of the invention is USP9X. In one comprise determining the level of a marker selected from the embodiment, the marker is SEPT3. In one embodiment, the group consisting of the markers listed in Table 1-3. In other markeris DAG1. In one embodiment, the markeris PTPRJ. In embodiments, the methods may further comprise determin one embodiment, the markeris CPM. In one embodiment, the ing the level of a marker selected from the group consisting of marker is SERPINB13. In one embodiment, the marker is CSTF3, NELL1, SLIT3, LAMTOR2, MGAT4B, LDLR. In one embodiment, the marker is MMP7. In one TMPRSS11F, ATAD3B, PTPRN, WNT9B, FUT6, embodiment, the marker is BTC. In one embodiment, the B4GALT1, FAM20C, CNTN1, MGAT1, STX1A, NMU, marker is PPY. In one embodiment, the marker is INS. CD59, CASR, and CPE. For example, in one embodiment, [0061] In some embodiments, the methods may further the markers for use in the methods and compositions of the comprise determining the level of a marker selected from the invention include USP9X, SEPT3, and CSTF3. In one group consisting of the markers listed in Table 1-3. In other embodiment, the markers include USP9X, SEPT3, and NELL1. In one embodiment, the markers include USP9X, embodiments, the methods may further comprise determin SEPT3, and SLIT3. In one embodiment, the markers include ing the level of a marker selected from the group consisting of USP9X, SEPT3, and LAMTOR2. In one embodiment, the CSTF3, NELL1, SLIT3, LAMTOR2, MGAT4B, markers include USP9X, SEPT3, and MGAT4B. In one TMPRSS11F, ATAD3B, PTPRN, WNT9B, FUT6, embodiment, the markers include USP9X, SEPT3, and B4GALT1, FAM20C, CNTN1, MGAT1, STX1A, NMU, TMPRSS11F. In one embodiment, the markers include CD59, CASR, and CPE. USP9X, SEPT3, and, ATAD3B. In one embodiment, the [0062] In other aspects of the invention, more than one markers include USP9X, SEPT3, and PTPRN. In one marker, e.g., a plurality of markers, e.g., two, three, four, five, embodiment, the markers include USP9X, SEPT3, and six, seven, eight, nine, ten, eleven, or more markers, may be WNT9B. In one embodiment, the markers include USP9X, used in the methods and compositions of the invention. For SEPT3, and FUT6. In one embodiment, the markers include USP9X, SEPT3, and B4GALT1. In one embodiment, the example, in one embodiment, the markers for use in the markers include USP9X, SEPT3, and FAM20C. In one methods and compositions of the invention include USP9X embodiment, the markers include USP9X, SEPT3, and and SEPT3. In one embodiment, the markers include USP9X CNTN1. In one embodiment, the markers include USP9X, and INS. In one embodiment, the markers include SEPT3 and SEPT3, and MGAT1. In one embodiment, the markers INS. In one embodiment, the markers include, SERPINB13 include USP9X, SEPT3, and STX1A. In one embodiment, and INS. In one embodiment, the markers include PPY and the markers include USP9X, SEPT3, and NMU. In one DAG1. In one embodiment, the markers include PPY and embodiment, the markers include USP9X, SEPT3, and BTC. In one embodiment, the markers include USP9X, CD59. In one embodiment, the markers include USP9X, SEPT3, and DAG1. In one embodiment, the markers include SEPT3, and CASR. In one embodiment, the markers include USP9X, SEPT3, and PTPRJ. In one embodiment, the mark USP9X, SEPT3, and CPE. In one embodiment, the markers ers include USP9X, SEPT3, and CPM. In one embodiment, include USP9X, INS, and CSTF3. In one embodiment, the the markers include USP9X, SEPT3, and SERPINB13. In markers include USP9X, INS, and NELL1. In one embodi one embodiment, the markers include USP9X, SEPT3, and ment, the markers include USP9X, INS, and SLIT2. In one LDLR. In one embodiment, the markers include USP9X, embodiment, the markers include USP9X, INS, and LAM SEPT3, and MMP7. In one embodiment, the markers include TOR2. In one embodiment, the markers include USP9X, INS, USP9X, SEPT3, and BTC. In one embodiment, the markers and MGAT4B. In one embodiment, the markers include include USP9X, SEPT3, and PPY. In one embodiment, the USP9X, INS, and TMPRSS11F. In one embodiment, the markers include USP9X, SEPT3, and INS. In one embodi markers include USP9X, INS, and, ATAD3B. In one embodi ment, the markers include BTC, MMP7, and PPY. In one ment, the markers include USP9X, INS, and PTPRN. In one US 2015/0330.997 A1 Nov. 19, 2015 32 embodiment, the markers include USP9X, INS, and WNTQB. embodiment, the markers include PPY, DAG1, and In one embodiment, the markers include USP9X, INS, and TMPRSS11F. In one embodiment, the markers include PPY, FUT6. In one embodiment, the markers include USP9X, INS, DAG1, and, ATAD3B. In one embodiment, the markers and B4GALT1. In one embodiment, the markers include include PPY, DAG1, and PTPRN. In one embodiment, the USP9X, INS, and FAM20C. In one embodiment, the markers markers include PPY, DAG1, and WNTQB. In one embodi include USP9X, INS, and CNTN1. In one embodiment, the ment, the markers include PPY, DAG1, and FUT6. In one markers include USP9X, INS, and MGAT1. In one embodi embodiment, the markers include PPY, DAG1, and ment, the markers include USP9X, INS, and STX1A. In one B4GALT1. In one embodiment, the markers include PPY, embodiment, the markers include USP9X, INS, and NMU. In DAG1, and FAM20C. In one embodiment, the markers one embodiment, the markers include USP9X, INS, and include PPY, DAG1, and CNTN1. In one embodiment, the CD59. In one embodiment, the markers include USP9X, INS, markers include PPY, DAG1, and MGAT1. In one embodi and CASR. In one embodiment, the markers include USP9X, ment, the markers include PPY, DAG1, and STX1A. In one INS, and CPE. In one embodiment, the markers include embodiment, the markers include PPY, DAG1, and NMU. In SEPT3, INS, and CSTF3. In one embodiment, the markers one embodiment, the markers include PPY, DAG1, and include SEPT3, INS, and NELL1. In one embodiment, the CD59. In one embodiment, the markers include PPY, DAG1. markers include SEPT3, INS, and SLIT2. In one embodi and CASR. In one embodiment, the markers include PPY, ment, the markers include SEPT3, INS, and LAMTOR2. In DAG1, and CPE. In one embodiment, the markers include one embodiment, the markers include SEPT3, INS, and PPY, BTC, and CSTF3. In one embodiment, the markers MGAT4B. In one embodiment, the markers include SEPT3, include PPY, BTC, and NELL1. In one embodiment, the INS, and TMPRSS11F. In one embodiment, the markers markers include PPY, BTC, and SLIT3. In one embodiment, include SEPT3, INS, and, ATAD3B. In one embodiment, the the markers include PPY, BTC, and LAMTOR2. In one markers include SEPT3, INS, and PTPRN. In one embodi embodiment, the markers include PPY, BTC, and MGAT4B. ment, the markers include SEPT3, INS, and WNTQB. In one In one embodiment, the markers include PPY, BTC, and embodiment, the markers include SEPT3, INS, and FUT6. In TMPRSS11F. In one embodiment, the markers include PPY, one embodiment, the markers include SEPT3, INS, and BTC, and, ATAD3B. In one embodiment, the markers include B4GALT1. In one embodiment, the markers include SEPT3, PPY, BTC, and PTPRN. In one embodiment, the markers INS, and FAM20C. In one embodiment, the markers include include PPY, BTC, and WNTQB. In one embodiment, the SEPT3, INS, and CNTN1. In one embodiment, the markers markers include PPY, BTC, and FUT6. In one embodiment, include SEPT3, INS, and MGAT1. In one embodiment, the the markers include PPY, BTC, and B4GALT1. In one markers include SEPT3, INS, and STX1A. In one embodi embodiment, the markers include PPY, BTC, and FAM20C. ment, the markers include SEPT3, INS, and NMU. In one In one embodiment, the markers include PPY, BTC, and embodiment, the markers include SEPT3, INS, and CD59. In CNTN1. In one embodiment, the markers include PPY, BTC, one embodiment, the markers include SEPT3, INS, and and MGAT1. In one embodiment, the markers include PPY, CASR. In one embodiment, the markers include SEPT3, INS, BTC, and STX1A. In one embodiment, the markers include and CPE. In one embodiment, the markers include SER PPY, BTC, and NMU. In one embodiment, the markers PINB13, INS, and CSTF3. In one embodiment, the markers include PPY, BTC, and CD59. In one embodiment, the mark include SERPINB13, INS, and NELL1. In one embodiment, ers include PPY, BTC, and CASR. In one embodiment, the the markers include SERPINB13, INS, and SLIT2. In one markers include PPY, BTC, and CPE. In one embodiment, the embodiment, the markers include SERPINB13, INS, and markers include USP9X, SEPT3, DAG1, and CSTF3. In one LAMTOR2. In one embodiment, the markers include SER embodiment, the markers include USP9X, SEPT3, DAG1. PINB13, INS, and MGAT4B. In one embodiment, the mark and NELL1. In one embodiment, the markers include ers include SERPINB13, INS, and TMPRSS11F. In one USP9X, SEPT3, DAG1, and SLIT2. In one embodiment, the embodiment, the markers include SERPINB13, INS, and, markers include USP9X, SEPT3, DAG1, and LAMTOR2. In ATAD3B. In one embodiment, the markers include SER one embodiment, the markers include USP9X, SEPT3, PINB13, INS, and PTPRN. In one embodiment, the markers DAG1, and MGAT4B. In one embodiment, the markers include SERPINB13, INS, and WNTQB. In one embodiment, include USP9X, SEPT3, DAG1, and TMPRSS11F. In one the markers include SERPINB13, INS, and FUT6. In one embodiment, the markers include USP9X, SEPT3, DAG1. embodiment, the markers include SERPINB13, INS, and and, ATAD3B. In one embodiment, the markers include B4GALT1. In one embodiment, the markers include SER USP9X, SEPT3, DAG1, and PTPRN. In one embodiment, the PINB13, INS, and FAM20C. In one embodiment, the markers markers include USP9X, SEPT3, DAG1, and WNTQB. In one include SERPINB13, INS, and CNTN1. In one embodiment, embodiment, the markers include USP9X, SEPT3, DAG1. the markers include SERPINB13, INS, and MGAT1. In one and FUT6. In one embodiment, the markers include USP9X, embodiment, the markers include SERPINB13, INS, and SEPT3, DAG1, and B4GALT1. In one embodiment, the STX1A. In one embodiment, the markers include SER markers include USP9X, SEPT3, DAG1, and FAM20C. In PINB13, INS, and NMU. In one embodiment, the markers one embodiment, the markers include USP9X, SEPT3, include SERPINB13, INS, and CD59. In one embodiment, DAG1, and CNTN1. In one embodiment, the markers include the markers include SERPINB13, INS, and CASR. In one USP9X, SEPT3, DAG1, and MGAT1. In one embodiment, embodiment, the markers include SERPINB13, INS, and the markers include USP9X, SEPT3, DAG1, and STX1A. In CPE. In one embodiment, the markers include PPY, DAG1. one embodiment, the markers include USP9X, SEPT3, and CSTF3. In one embodiment, the markers include PPY, DAG1, and NMU. In one embodiment, the markers include DAG1, and NELL1. In one embodiment, the markers include USP9X, SEPT3, DAG1, and CD59. In one embodiment, the PPY, DAG1, and SLIT2. In one embodiment, the markers markers include USP9X, SEPT3, DAG1, and CASR. In one include PPY, DAG1, and LAMTOR2. In one embodiment, embodiment, the markers include USP9X, SEPT3, DAG1. the markers include PPY, DAG1, and MGAT4B. In one and CPE. In one embodiment, the markers include USP9X, US 2015/0330.997 A1 Nov. 19, 2015

SEPT3, PTPRJ, and CSTF3. In one embodiment, the markers embodiment, the markers include USP9X, SEPT3, SER include USP9X, SEPT3, PTPRJ, and NELL1. In one embodi PINB13, and FUT6. In one embodiment, the markers include ment, the markers include USP9X, SEPT3, PTPRJ, and USP9X, SEPT3, SERPINB13, and B4GALT1. In one SLIT3. In one embodiment, the markers include USP9X, embodiment, the markers include USP9X, SEPT3, SER SEPT3, PTPRJ, and LAMTOR2. In one embodiment, the PINB13, and FAM20C. In one embodiment, the markers markers include USP9X, SEPT3, PTPRJ, and MGAT4B. In include USP9X, SEPT3, SERPINB13, and CNTN1. In one one embodiment, the markers include USP9X, SEPT3, embodiment, the markers include USP9X, SEPT3, SER PTPRJ, and TMPRSS11F. In one embodiment, the markers PINB13, and MGAT1. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, and, ATAD3B. In one include USP9X, SEPT3, SERPINB13, and STX1A. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, embodiment, the markers include USP9X, SEPT3, SER and PTPRN. In one embodiment, the markers include PINB13, and NMU. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, and WNTQB. In one embodiment, USP9X, SEPT3, SERPINB13, and CD59. In one embodi the markers include USP9X, SEPT3, PTPRJ, and FUT6. In ment, the markers include USP9X, SEPT3, SERPINB13, and one embodiment, the markers include USP9X, SEPT3, CASR. In one embodiment, the markers include USP9X, PTPRJ, and B4GALT1. In one embodiment, the markers SEPT3, SERPINB13, and CPE. In one embodiment, the include USP9X, SEPT3, PTPRJ, and FAM20C. In one markers include USP9X, SEPT3, LDLR, and CSTF3. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, embodiment, the markers include USP9X, SEPT3, LDLR, and CNTN1. In one embodiment, the markers include and NELL1. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, and MGAT1. In one embodiment, USP9X, SEPT3, LDLR, and SLIT2. In one embodiment, the the markers include USP9X, SEPT3, PTPRJ, and STX1A. In markers include USP9X, SEPT3, LDLR, and LAMTOR2. In one embodiment, the markers include USP9X, SEPT3, one embodiment, the markers include USP9X, SEPT3, PTPRJ, and NMU. In one embodiment, the markers include LDLR, and MGAT4B. In one embodiment, the markers USP9X, SEPT3, PTPRJ, and CD59. In one embodiment, the include USP9X, SEPT3, LDLR, and TMPRSS11F. In one markers include USP9X, SEPT3, PTPRJ, and CASR. In one embodiment, the markers include USP9X, SEPT3, LDLR, embodiment, the markers include USP9X, SEPT3, PTPRJ, and, ATAD3B. In one embodiment, the markers include and CPE. In one embodiment, the markers include USP9X, USP9X, SEPT3, LDLR, and PTPRN. In one embodiment, SEPT3, CPM, and CSTF3. In one embodiment, the markers the markers include USP9X, SEPT3, LDLR, and WNTQB. In include USP9X, SEPT3, CPM, and NELL1. In one embodi one embodiment, the markers include USP9X, SEPT3, ment, the markers include USP9X, SEPT3, CPM, and SLIT2. LDLR, and FUT6. In one embodiment, the markers include In one embodiment, the markers include USP9X, SEPT3, USP9X, SEPT3, LDLR, and B4GALT1. In one embodiment, CPM, and LAMTOR2. In one embodiment, the markers the markers include USP9X, SEPT3, LDLR, and FAM20C. include USP9X, SEPT3, CPM, and MGAT4B. In one In one embodiment, the markers include USP9X, SEPT3, embodiment, the markers include USP9X, SEPT3, CPM, and LDLR, and CNTN1. In one embodiment, the markers include TMPRSS11F. In one embodiment, the markers include USP9X, SEPT3, LDLR, and MGAT1. In one embodiment, USP9X, SEPT3, CPM, and, ATAD3B. In one embodiment, the markers include USP9X, SEPT3, LDLR, and STX1A. In the markers include USP9X, SEPT3, CPM, and PTPRN. In one embodiment, the markers include USP9X, SEPT3, one embodiment, the markers include USP9X, SEPT3, CPM, LDLR, and NMU. In one embodiment, the markers include and WNT9B. In one embodiment, the markers include USP9X, SEPT3, LDLR, and CD59. In one embodiment, the USP9X, SEPT3, CPM, and FUT6. In one embodiment, the markers include USP9X, SEPT3, LDLR, and CASR. In one markers include USP9X, SEPT3, CPM, and B4GALT1. In embodiment, the markers include USP9X, SEPT3, LDLR, one embodiment, the markers include USP9X, SEPT3, CPM, and CPE. In one embodiment, the markers include USP9X, and FAM20C. In one embodiment, the markers include SEPT3, MMP7, and CSTF3. In one embodiment, the markers USP9X, SEPT3, CPM, and CNTN1. In one embodiment, the include USP9X, SEPT3, MMP7, and NELL1. In one embodi markers include USP9X, SEPT3, CPM, and MGAT1. In one ment, the markers include USP9X, SEPT3, MMP7, and embodiment, the markers include USP9X, SEPT3, CPM, and SLIT3. In one embodiment, the markers include USP9X, STX1A. In one embodiment, the markers include USP9X, SEPT3, MMP7, and LAMTOR2. In one embodiment, the SEPT3, CPM, and NMU. In one embodiment, the markers markers include U5P9X, SEPT3, MMP7, and MGAT4B. In include USP9X, SEPT3, CPM, and CD59. In one embodi one embodiment, the markers include USP9X, SEPT3, ment, the markers include USP9X, SEPT3, CPM, and CASR. MMP7, and TMPRSS11F. In one embodiment, the markers In one embodiment, the markers include USP9X, SEPT3, include USP9X, SEPT3, MMP7, and, ATAD3B. In one CPM, and CPE. In one embodiment, the markers include embodiment, the markers include USP9X, SEPT3, MMP7, USP9X, SEPT3, SERPINB13, and CSTF3. In one embodi and PTPRN. In one embodiment, the markers include ment, the markers include USP9X, SEPT3, SERPINB13, and USP9X, SEPT3, MMP7, and WNTQB. In one embodiment, NELL1. In one embodiment, the markers include USP9X, the markers include USP9X, SEPT3, MMP7, and FUT6. In SEPT3, SERPINB13, and SLIT2. In one embodiment, the one embodiment, the markers include USP9X, SEPT3, markers include USP9X, SEPT3, SERPINB13, and LAM MMP7, and B4GALT1. In one embodiment, the markers TOR2. In one embodiment, the markers include USP9X, include USP9X, SEPT3, MMP7, and FAM20C. In one SEPT3, SERPINB13, and MGAT4B. In one embodiment, the embodiment, the markers include USP9X, SEPT3, MMP7, markers include USP9X, SEPT3, SERPINB13, and and CNTN1. In one embodiment, the markers include TMPRSS11F. In one embodiment, the markers include USP9X, SEPT3, MMP7, and MGAT1. In one embodiment, USP9X, SEPT3, SERPINB13, and, ATAD3B. In one the markers include USP9X, SEPT3, MMP7, and STX1A. In embodiment, the markers include USP9X, SEPT3, SER one embodiment, the markers include USP9X, SEPT3, PINB13, and PTPRN. In one embodiment, the markers MMP7, and NMU. In one embodiment, the markers include include USP9X, SEPT3, SERPINB13, and WNTQB. In one USP9X, SEPT3, MMP7, and CD59. In one embodiment, the US 2015/0330.997 A1 Nov. 19, 2015 34 markers include USP9X, SEPT3, MMP7, and CASR. In one WNT9B. In one embodiment, the markers include USP9X, embodiment, the markers include USP9X, SEPT3, MMP7, SEPT3, INS, and FUT6. In one embodiment, the markers and CPE. In one embodiment, the markers include USP9X, include USP9X, SEPT3, INS, and B4GALT1. In one embodi SEPT3, BTC, and CSTF3. In one embodiment, the markers ment, the markers include USP9X, SEPT3, INS, and include USP9X, SEPT3, BTC, and NELL1. In one embodi FAM20C. In one embodiment, the markers include USP9X, ment, the markers include USP9X, SEPT3, BTC, and SLIT2. SEPT3, INS, and CNTN1. In one embodiment, the markers In one embodiment, the markers include USP9X, SEPT3, include USP9X, SEPT3, INS, and MGAT1. In one embodi BTC, and LAMTOR2. In one embodiment, the markers ment, the markers include USP9X, SEPT3, INS, and STX1A. include USP9X, SEPT3, BTC, and MGAT4B. In one In one embodiment, the markers include USP9X, SEPT3, embodiment, the markers include USP9X, SEPT3, BTC, and INS, and NMU. In one embodiment, the markers include TMPRSS11F. In one embodiment, the markers include USP9X, SEPT3, INS, and CD59. In one embodiment, the USP9X, SEPT3, BTC, and, ATAD3B. In one embodiment, markers include USP9X, SEPT3, INS, and CASR. In one the markers include USP9X, SEPT3, BTC, and PTPRN. In embodiment, the markers include USP9X, SEPT3, INS, and one embodiment, the markers include USP9X, SEPT3, BTC, CPE. In one embodiment, the markers include BTC, MMP7, and WNT9B. In one embodiment, the markers include PPY, and CSTF3. In one embodiment, the markers include USP9X, SEPT3, BTC, and FUT6. In one embodiment, the BTC, MMP7, PPY, and NELL1. In one embodiment, the markers include USP9X, SEPT3, BTC, and B4GALT1. In markers include BTC, MMP7, PPY, and SLIT2. In one one embodiment, the markers include USP9X, SEPT3, BTC, embodiment, the markers include BTC, MMP7, PPY, and and FAM20C. In one embodiment, the markers include LAMTOR2. In one embodiment, the markers include BTC, USP9X, SEPT3, BTC, and CNTN1. In one embodiment, the MMP7, PPY, and MGAT4B. In one embodiment, the markers markers include USP9X, SEPT3, BTC, and MGAT1. In one include BTC, MMP7, PPY, and TMPRSS11F. In one embodi embodiment, the markers include USP9X, SEPT3, BTC, and ment, the markers include BTC, MMP7, PPY, and, ATAD3B. STX1A. In one embodiment, the markers include USP9X, In one embodiment, the markers include BTC, MMP7, PPY, SEPT3, BTC, and NMU. In one embodiment, the markers and PTPRN. In one embodiment, the markers include BTC, include USP9X, SEPT3, BTC, and CD59. In one embodi MMP7, PPY, and WNTQB. In one embodiment, the markers ment, the markers include USP9X, SEPT3, BTC, and CASR. include BTC, MMP7, PPY, and FUT6. In one embodiment, In one embodiment, the markers include USP9X, SEPT3, the markers include BTC, MMP7, PPY, and B4GALT1. In BTC, and CPE. In one embodiment, the markers include one embodiment, the markers include BTC, MMP7, PPY, and USP9X, SEPT3, PPY, and CSTF3. In one embodiment, the FAM20C. In one embodiment, the markers include BTC, markers include USP9X, SEPT3, PPY, and NELL1. In one MMP7, PPY, and CNTN1. In one embodiment, the markers embodiment, the markers include USP9X, SEPT3, PPY, and include BTC, MMP7, PPY, and MGAT1. In one embodiment, SLIT3. In one embodiment, the markers include USP9X, the markers include BTC, MMP7, PPY, and STX1A. In one SEPT3, PPY, and LAMTOR2. In one embodiment, the mark embodiment, the markers include BTC, MMP7, PPY, and ers include USP9X, SEPT3, PPY, and MGAT4B. In one NMU. In one embodiment, the markers include BTC, MMP7, embodiment, the markers include USP9X, SEPT3, PPY, and PPY, and CD59. In one embodiment, the markers include TMPRSS11F. In one embodiment, the markers include BTC, MMP7, PPY, and CASR. In one embodiment, the USP9X, SEPT3, PPY, and, ATAD3B. In one embodiment, the markers include BTC, MMP7, PPY, and CPE. In one embodi markers include USP9X, SEPT3, PPY, and PTPRN. In one ment, the markers include PPY SEPT3, PTPRJ, and CSTF3. embodiment, the markers include USP9X, SEPT3, PPY, and In one embodiment, the markers include PPY SEPT3, WNT9B. In one embodiment, the markers include USP9X, PTPRJ, and NELL1. In one embodiment, the markers include SEPT3, PPY, and FUT6. In one embodiment, the markers PPY SEPT3, PTPRJ, and SLITB. In one embodiment, the include USP9X, SEPT3, PPY, and B4GALT1. In one markers include PPY SEPT3, PTPRJ, and LAMTOR2. In embodiment, the markers include USP9X, SEPT3, PPY, and one embodiment, the markers include PPY SEPT3, PTPRJ, FAM20C. In one embodiment, the markers include USP9X, and MGAT4B. In one embodiment, the markers include PPY, SEPT3, PPY, and CNTN1. In one embodiment, the markers SEPT3, PTPRJ, and TMPRSS11F. In one embodiment, the include USP9X, SEPT3, PPY, and MGAT1. In one embodi markers include PPY SEPT3, PTPRJ, and, ATAD3B. In one ment, the markers include USP9X, SEPT3, PPY, and STX1A. embodiment, the markers include PPY SEPT3, PTPRJ, and In one embodiment, the markers include USP9X, SEPT3, PTPRN. In one embodiment, the markers include PPY, PPY, and NMU. In one embodiment, the markers include SEPT3, PTPRJ, and WNTQB. In one embodiment, the mark USP9X, SEPT3, PPY, and CD59. In one embodiment, the ers include PPY SEPT3, PTPRJ, and FUT6. In one embodi markers include USP9X, SEPT3, PPY, and CASR. In one ment, the markers include PPY SEPT3, PTPRJ, and embodiment, the markers include USP9X, SEPT3, PPY, and B4GALT1. In one embodiment, the markers include PPY, CPE. In one embodiment, the markers include USP9X, SEPT3, PTPRJ, and FAM20C. In one embodiment, the mark SEPT3, INS, and CSTF3. In one embodiment, the markers ers include PPY SEPT3, PTPRJ, and CNTN1. In one include USP9X, SEPT3, INS, and NELL1. In one embodi embodiment, the markers include PPY SEPT3, PTPRJ, and ment, the markers include USP9X, SEPT3, INS, and SLIT2. MGAT1. In one embodiment, the markers include PPY, In one embodiment, the markers include USP9X, SEPT3, SEPT3, PTPRJ, and STX1A. In one embodiment, the mark INS, and LAMTOR2. In one embodiment, the markers ers include PPY SEPT3, PTPRJ, and NMU. In one embodi include USP9X, SEPT3, INS, and MGAT4B. In one embodi ment, the markers include PPY SEPT3, PTPRJ, and CD59. In ment, the markers include USP9X, SEPT3, INS, and one embodiment, the markers include PPY SEPT3, PTPRJ, TMPRSS11F. In one embodiment, the markers include and CASR. In one embodiment, the markers include PPY, USP9X, SEPT3, INS, and, ATAD3B. In one embodiment, the SEPT3, PTPRJ, and CPE. In one embodiment, the markers markers include USP9X, SEPT3, INS, and PTPRN. In one include CPM, INS, MMP7, LDLR, and CSTF3. In one embodiment, the markers include USP9X, SEPT3, INS, and embodiment, the markers include CPM, INS, MMP7, LDLR, US 2015/0330.997 A1 Nov. 19, 2015

and NELL1. In one embodiment, the markers include CPM, the one or more markers in the sample(s) from the subject as INS, MMP7, LDLR, and SLIT2. In one embodiment, the compared to the level of the one or more markers in the markers include CPM, INS, MMP7, LDLR, and LAMTOR2. control sample indicates that the subject will develop type 2 In one embodiment, the markers include CPM, INS, MMP7, diabetes. LDLR, and MGAT4B. In one embodiment, the markers [0069] Numerous complications have been associated with include CPM, INS, MMP7, LDLR, and TMPRSS11F. In one impaired glucose tolerance and/or type 2 diabetes, especially embodiment, the markers include CPM, INS, MMP7, LDLR, prolonged impaired glucose tolerance and/or type 2 diabetes. and, ATAD3B. In one embodiment, the markers include For example, such subjects have a two to four times the risk of CPM, INS, MMP7, LDLR, and PTPRN. In one embodiment, cardiovascular disease, including ischemic heart disease and the markers include CPM, INS, MMP7, LDLR, and WNTQB. stroke, a 20-fold increase in lower limb amputations, and In one embodiment, the markers include CPM, INS, MMP7, increased rates of hospitalizations. Type 2 diabetes is also the LDLR, and FUT6. In one embodiment, the markers include largest cause of non-traumatic blindness and nephropathy CPM, INS, MMP7, LDLR, and B4GALT1. In one embodi including kidney failure and has been associated with an ment, the markers include CPM, INS, MMP7, LDLR, and increased risk of cognitive dysfunction and dementia through FAM20C. In one embodiment, the markers include CPM, disease processes such as Alzheimer’s disease and vascular INS, MMP7, LDLR, and CNTN1. In one embodiment, the dementia. Other complications include, for example, neur markers include CPM, INS, MMP7, LDLR, and MGAT1. In opathy, acanthosis nigricans, sexual dysfunction, and fre one embodiment, the markers include CPM, INS, MMP7, quent infections. LDLR, and STX1A. In one embodiment, the markers include [0070] As the markers of the present invention have been CPM, INS, MMP7, LDLR, and NMU. In one embodiment, shown to be differentially expressed in subjects newly diag the markers include CPM, INS, MMP7, LDLR, and CD59. In nosed with type 2 diabetes and those having established type one embodiment, the markers include CPM, INS, MMP7, 2 diabetes, e.g., those subjects having prolonged impaired LDLR, and CASR. In one embodiment, the markers include glucose tolerance and/or type 2 diabetes, the present inven CPM, INS, MMP7, LDLR, and CPE, tion also provides methods for determining whether a subject II. Methods of the Invention will develop a type 2 diabetes-associated complication. The methods include determining the level of one or more mark [0064] A. Diagnostic and Prognostic Methods ers of the invention in a sample(s) from the subject with a level [0065] In certain aspects, the present invention provides of the one or more markers in a control sample(s). A differ diagnostic methods. For example, in one aspect, the present ence in the level (e.g., higher or lower) of the one or more invention provides methods for determining whether a sub markers in the sample(s) from the subject as compared to the ject has impaired glucose tolerance. The methods include level of the one or more markers in the control sample indi determining the level of one or more markers of the invention cates that the subject will respond to a diabetic therapy. in a sample(s) from the subject with a level of the one or more [0071] In another aspect the present invention provides markers in a control sample(s). A difference in the level (e.g., methods for determining whether a subject having impaired higher or lower) of the one or more markers in the sample(s) glucose tolerance and/or type 2 diabetes will respond to a from the subject as compared to the level of the one or more treatment regime. The methods include determining the level markers in the control sample indicates that the subject has of one or more markers of the invention in a sample(s) from impaired glucose tolerance. the subject with a level of the one or more markers in a control [0066] In another aspect, the present invention provides sample(s). A difference in the level (e.g., higher or lower) of methods for determining whether a subject has type 2 diabe the one or more markers in the sample(s) from the subject as tes. The methods include determining the level of one or more compared to the level of the one or more markers in the markers of the invention in a sample(s) from the subject with control sample indicates that the subject will respond to a a level of the one or more markers in a control sample(s). A difference in the level (e.g., higher or lower) of one or more treatment. markers in the sample(s) from the subject as compared to the [0072) Numerous diabetic therapies are known in the art level of the one or more markers in the control sample indi and include, for example, insulin sensitizers, such as bigu cates that the subject has type 2 diabetes. anides (e.g., metformin) and thiazolidinediones (e.g., rosigli [0067] The present invention also provides prognostic tazone, pioglitazone, troglitazone); secretagogues, such as methods. For example, in one aspect, the present invention the sulfonylureas (e.g., glyburide, glipizide, glimepiride, provides methods for determining whether a subject will tolbutamide, acetohexamide, tolazamide, chlorpropamide, develop impaired glucose tolerance. The methods include gliclazide, glycopyamide, gliquidone), the nonsulfonylurea determining the level of one or more markers of the invention secretagogues, e.g., meglitimide derivatives (e.g., repaglinide, in a sample(s) from the subject with a level of the one or more nateglinide); the dipeptidyl peptidase IV inhibitors (e.g., sita markers in a control sample(s). A difference in the level (e.g., gliptin, saxagliptin, linagliptin, vildagliptin, allogliptin, sep higher or lower) of one or more markers in the sample(s) from tagliptin); alpha-glucosidase inhibitors (e.g., acarbose, migli the subject as compared to the level of the one or more tol, Voglibose); amylinomimetics (e.g., pramlintide acetate); markers in the control sample indicates that the subject will incretin mimetics (e.g., exematide, liraglutide, taspoglutide); develop impaired glucose tolerance. insulin and its analogues (e.g., rapid acting, slow acting, and [0068] In another aspect, the present invention provides intermediate acting); bile acid sequestrants (e.g., coleseve methods for determining whether a subject will develop type lam); and dopamine agonists (e.g., bromocriptine), alone or 2 diabetes. The methods include determining the level of one in combinations. or more markers of the invention in a sample(s) from the [0073] In certain embodiments of the invention, the treat subject with a level of the one or more markers in a control ment comprises an insulin sensitizer. In another embodiment, sample(s). A difference in the level (e.g., higher or lower) of the treatment comprises an insulin sensitizer and a secreta US 2015/0330.997 A1 Nov. 19, 2015 36 gogue. In yet another embodiment, the treatment comprises of selectively binding to a specific marker of the invention. an insulin sensitizer, a secretagogue, and insulin. Probes can be synthesized by one of skill in the art, or derived [0074] The methods of the present invention can be prac from appropriate biological preparations. Probes may be spe ticed in conjunction with any other method(s) used by the cifically designed to be labeled. Examples of molecules that skilled practitioner to diagnose, prognose, and/or monitor can be utilized as probes include, but are not limited to, RNA, impaired glucose tolerance and/or type 2 diabetes in a subject DNA, proteins, antibodies, and organic molecules. and/or a type 2 diabetes complication and/or response to [0080) Isolated mRNA can be used in hybridization or treatment. For example, the methods of the invention may be amplification assays that include, but are not limited to, performed in conjunction with any clinical measurement of Southern or Northern analyses, polymerase chain reaction glucose tolerance, obesity, and/or diabetes known in the art (PCR) analyses and probe arrays. One method for the deter including serological, cytological and/or detection (and mination of mRNA levels involves contacting the isolated quantification, if appropriate) of other molecular markers. mRNA with a nucleic acid molecule (probe) that can hybrid [0075] In any of the methods (and kits) of the invention, the ize to a marker mRNA. The nucleic acid probe can be, for level of a marker(s) of the invention in a sample obtained from example, a full-length cDNA, or a portion thereof, such as an a subject may be determined by any of a wide variety of oligonucleotide of at least about 7, 10, 15, 20, 25, 30, 35, 40, well-known techniques and methods, which transform a 45, 50, 100, 250 or about 500 nucleotides in length and marker of the invention within the sample into a moiety that sufficient to specifically hybridize under stringent conditions can be detected and quantified. Non-limiting examples of to marker genomic DNA. such methods include analyzing the sample using immuno [0081] In one embodiment, the mRNA is immobilized on a logical methods for detection of proteins, protein purification solid surface and contacted with a probe, for example by methods, protein function or activity assays, nucleic acid running the isolated mRNA on an agarose geland transferring hybridization methods, nucleic acid reverse transcription the mRNA from the gel to a membrane, such as nitrocellu methods, and nucleic acid amplification methods, immunob lose. In an alternative embodiment, the probe?s) are immobi lotting, Western blotting, Northern blotting, electron micros lized on a solid surface and the mRNA is contacted with the copy, mass spectrometry, e.g., MALDI-TOF and SELDI probe?s), for example, in an Affymetrix gene chip array. A TOF, immunoprecipitations, immunofluorescence, skilled artisan can readily adapt known mRNA detection immunohistochemistry, enzyme linked immunosorbent methods for use in determining the level of a marker of the assays (ELISAs), e.g., amplified ELISA, quantitative blood invention mRNA. based assays, e.g., serum ELISA, quantitative urine based [0082] An alternative method for determining the levelofa assays, flow cytometry, Southern hybridizations, array analy marker of the invention in a sample involves the process of sis, and the like, and combinations or sub-combinations nucleic acid amplification and/or reverse transcriptase (to thereof. prepare cDNA) of for example mRNA in the sample, e.g., by [0076] For example, an mRNA sample may be obtained RT-PCR (the experimental embodiment set forth in Mullis, from the sample from the subject (e.g., bronchial lavage, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany mouth Swab, biopsy, or peripheral blood mononuclear cells, (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sus by standard methods) and expression of mRNA(s) encoding a tained sequence replication (Guatelli et al. (1990) Proc. Natl. marker of the invention in the sample may be detected and/or Acad. Sci. USA 87:1874-1878), transcriptional amplification determined using standard molecular biology techniques, system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA such as PCR analysis. A preferred method of PCR analysis is 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/ reverse transcriptase-polymerase chain reaction (RT-PCR). Technology 6:1197), rolling circle replication (Lizardi et al., Other suitable systems for mRNA sample analysis include U.S. Pat. No. 5,854,033) or any other nucleic acid amplifica microarray analysis (e.g., using Affymetrix’s microarray sys tion method, followed by the detection of the amplified mol tem or Illumina’s BeadArray Technology). ecules using techniques well known to those of skill in the art. [0077] It will be readily understood by the ordinarily These detection schemes are especially useful for the detec skilled artisan that essentially any technical means estab tion of nucleic acid molecules if such molecules are presentin lished in the art for detecting the level a marker of the inven very low numbers. In particular aspects of the invention, the tion at either the nucleic acid or protein level, can be used to level of expression of a marker of the invention is determined determine the level a marker of the invention as discussed by quantitative fluorogenic RT-PCR (i.e., the TaqMan"M Sys herein. tem). Such methods typically utilize pairs of oligonucleotide [0078] In one embodiment, the level of a marker of the primers that are specific for a marker of the invention. Meth invention in a sample is determined by detecting a transcribed ods for designing oligonucleotide primers specific for a polynucleotide, or portion thereof, e.g., mRNA, or cDNA, of known sequence are well known in the art. a marker of the invention gene. RNA may be extracted from [0083] The level of a marker of the invention mRNA may cells using RNA extraction techniques including, for be monitored using a membrane blot (such as used in hybrid example, using acid phenol/guanidine isothiocyanate extrac ization analysis such as Northern, Southern, dot, and the like), tion (RNAzol B: Biogenesis), RNeasy RNA preparation kits or microwells, sample tubes, gels, beads or fibers (or any solid (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical support comprising bound nucleic acids). See U.S. Pat. Nos. assay formats utilizing ribonucleic acid hybridization include 5,770.722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, nuclear run-on assays, RT-PCR, RNase protection assays which are incorporated herein by reference. The determina (Melton et al., Nuc. Acids Res. 12:7035), Northern blotting, in tion of a level of a marker of the invention may also comprise situ hybridization, and microarray analysis. using nucleic acid probes in solution. [0079] In one embodiment, the level of a marker of the [0084] In one embodiment of the invention, microarrays invention is determined using a nucleic acid probe. The term are used to detect the level of a marker of the invention. “probe”, as used herein, refers to any molecule that is capable Microarrays are particularly well suited for this purpose US 2015/0330.997 A1 Nov. 19, 2015 37 because of the reproducibility between different experiments. TABLE 4-continued DNA microarrays provide one method for the simultaneous measurement of the levels of large numbers of genes. Each Commercially Available Antibodies array consists of a reproducible pattern of capture probes Marker attached to a solid support. Labeled RNA or DNA is hybrid Name Company Name Catalog Number ized to complementary probes on the array and then detected Abnova Corporation H00001605-MO1 by laser scanning Hybridization intensities for each probe on ProSci, Inc 48–780 the array are determined and converted to a quantitative value Proteintech Group Inc 1017-1-AP representing relative gene expression levels. See, e.g., U.S. SEPT3 Atlas Antibodies HPA003548 Pat. Nos. 6,040,138, 5,800,992 and 6,020,135, 6,033,860, LifeSpan BioSciences LS-C1 20158 and 6,344,316, which are incorporated herein by reference. Sigma-Aldrich HPA003548–100UL High-density oligonucleotide arrays are particularly useful Abgent AT38148. USCN Life Science, Inc. E95863 Hu for determining the gene expression profile for a large number PTPRJ GeneTex GTX82145 of RNA’s in a sample. Thermo Scientific Pierce Antibodies PA1-27625 [0085] In certain situations it may be possible to assay for Abnova Corporation H000057.95-B01 P the level of a marker of the invention at the protein level, using LifeSpan BioSciences LS-C40932 a detection reagent that detects the protein product encoded Novus Biologicals H000057.95–MO1 CPM MyBioSource.com MBS855861 by the mRNA of a marker of the invention. For example, if an Santa Cruz Biotechnology, Inc. SC-98698 antibody reagent is available that binds specifically to a Abnova Corporation H00001368-B01 P marker of the invention protein product to be detected, and Biorbyt Orb 125616 not to other proteins, then such an antibody reagent can be USCN Life Science, Inc. E92397 Hu used to detect the expression of a marker of the invention in a SERPINB13 Fitzgerald Industries International OR-5733 cellular sample from the subject, or a preparation derived Proteintech Group Inc 8045-1-AP Novus Biologicals NBP2–0.1336 from the cellular sample, using standard antibody-based tech Sigma-Aldrich SAB2104770–50UG niques known in the art, such as FACS analysis, and the like. Abnova Corporation PAB1049 [0086] Other known methods for detecting a marker of the LDLR Atlas Antibodies HPAO09647 invention at the protein level include methods such as elec Santa Cruz Biotechnology, Inc. Sc-20744 trophoresis, capillary electrophoresis, high performance liq Abgent AP8960c Abnova Corporation H00003949–AO1 uid chromatography (HPLC), thin layer chromatography Acris Antibodies GmbH BP5013 (TLC), hyperdiffusion chromatography, and the like, or vari MMP7 GeneTex GTX17854 ous immunological methods such as fluid or gel precipitin GenWay Biotech, Inc. GWEB-5EF98D reactions, immunodiffusion (single or double), immunoelec Abgent AF1674a. trophoresis, radioimmunoassay (RIA), enzyme-linked LifeSpan BioSciences LS-C88495–20 R&D Systems MP700 immunosorbent assays (ELISAs), immunofluorescent BTC LifeSpan BioSciences LS-C10O871–100 assays, and Western blotting. Abgent AP1.1669a [0087] Proteins from samples can be isolated using tech Sigma-Aldrich B2430 niques that are well known to those of skill in the art. The R&D Systems AF-261-NA protein isolation methods employed can, for example, be Creative Diagnostics DEIA089 PPY Abnova Corporation H00005539-B01 those described in Harlow and Lane (Harlow and Lane, 1988, LifeSpan BioSciences LS-C38055-200 Antibodies: A Laboratory Manual, Cold Spring Harbor Labo GenWay Biotech, Inc. GWE-C1 C3DC ratory Press, Cold Spring Harbor, N.Y.). R&D Systems MAB6297 [0088] In one embodiment, antibodies, or antibody frag USCN Life Science, Inc. E91265Hu ments, are used in methods such as Western blots or immun INS Abgent AM1985b ofluorescence techniques to detect the expressed proteins. antibodies-online ABIN237690 GeneTex GTX81555 Antibodies for determining the expression of a marker of the Atlas Antibodies HPA004932 invention are commercially available and one of ordinary EMD Millipore Corp EZHLASF-14K skill in the art can readily identify appropriate antibodies for CSTF3 Atlas Antibodies HPAO401.68 use in the methods of the invention. Exemplary commercially Abnova Corporation H00001479–AO1 available antibodies suitable for use in the claimed methods AbD Serotec MCA3034Z for determining the level of a marker of the invention are Fitzgerald Industries International 70R-4939 Abgent AT1663a. listed in the table below (Table 4). NELL1 GeneTex GTX1038.19 Abnova Corporation H00004745–A01 TABLE 4 LifeSpan BioSciences LS-C139121–100 AbD Serotec MCA5151 Z Commercially Available Antibodies Abcam ab55548 SLIT3 EMD Millipore AB5703P Marker Abnova Corporation H00006586–A01 Name Company Name Catalog Number R&D Systems AF3629 USP9X Fitzgerald Industries International 70R-9746 Sigma-Aldrich WHO006586MA Abnova Corporation H00008239-AO 1 Creative Biomart CAB-468.3 MH LifeSpan BioSciences LS-C143435 LAMTOR2 Atlas Antibodies HPA004126 Bethyl Laboratories A301–350A Sigma-Aldrich HPA004126 Abgent AT4497a Cell Signaling Technology 81.45S DAG1 antibodies-online ABIN502745 Abgent AP13338c GeneTex GTX88089 Novus Biologicals NBP1-71687 US 2015/0330.997 A1 Nov. 19, 2015

TABLE 4-continued TABLE 4-continued Commercially Available Antibodies Commercially Available Antibodies Marker Marker Name Company Name Catalog Number Name Company Name Catalog Number MGAT4B Abnova Corporation H000.11282–D01 Proteintech Group Inc 13710-1-AP Sigma-Aldrich SAB1407130 R&D Systems AF3587 Novus Biologicals H000.11282–B01 P Biorbyt Orb 127922 Creative Biomart CPBT-40309MH Abcam abó7394 TMPRSS11F Atlas Antibodies HPA026911 [0089] It is generally preferable to immobilize either the Sigma-Aldrich HPA026911 Abcam ab59857 antibody or proteins on a solid support for Western blots and Novus Biologicals NBP1-94000 immunofluorescence techniques. Suitable solid phase sup Abnova Corporation PAB21857 ports or carriers include any support capable of binding an ATAD3B Abnova Corporation H00083858-B01 P antigen or an antibody. Well-known supports or carriers Thermo Scientific Pierce Antibodies PA5-21160 Novus Biologicals H00083858-B01 include glass, polystyrene, polypropylene, polyethylene, Sigma-Aldrich SAB1400727 dextran, nylon, amylases, natural and modified celluloses, Abcam ab112.563 polyacrylamides, gabbros, and magnetite. PTPRN Atlas Antibodies HPA007 179 GeneTex GTX82148 [0090] One skilled in the art will know many other suitable Thermo Scientific Pierce Antibodies PA1-27627 carriers for binding antibody or antigen, and will be able to Abnova Corporation MAB2710 adapt such support for use with the present invention. For Novus Biologicals H00005798–B02P example, protein isolated from cells can be run on a polyacry WNTQB Abgent AP16959C Aviva Systems Biology ARP41243 T100 lamide gel electrophoresis and immobilized onto a solid LifeSpan BioSciences LS-C1081,28–100 phase support such as nitrocellulose. The support can then be Fitzgerald Industries International 70R-7246 washed with suitable buffers followed by treatment with the R&D Systems AF3669 detectably labeled antibody. The solid phase support can then FUT6 Fitzgerald Industries International 70R-5379 be washed with the buffer a second time to remove unbound Abgent AP4925c Thermo Scientific Pierce Antibodies PA5-24850 antibody. The amount of bound label on the solid support can Sigma-Aldrich AV48467 then be detected by conventional means. Means of detecting Novus Biologicals H00002528–B01 P proteins using electrophoretic techniques are well known to B4GALT1 Atlas Antibodies HPA010806 GeneTex GTX80958 those of skill in the art (see generally, R. Scopes (1982) Abnova Corporation PAB20512 Protein Purification, Springer-Verlag, N.Y.; Deutscher, LifeSpan BioSciences LS-C36410–100 (1990) Methods in Enzymology Vol. 182: Guide to Protein Biorbyt Orb 126744 Purification, Academic Press, Inc., N.Y.). FAM20C Atlas Antibodies HPAO19823 Santa Cruz Biotechnology, Inc. SC-160322 [0091]. Other standard methods include immunoassay tech Abnova Corporation PAB21246 niques which are well known to one of ordinary skill in the art Fitzgerald Industries International 70R-6353 and may be found in Principles And Practice Of Immunoas LifeSpan BioSciences LS-C82574–50 CNTN1 Fitzgerald Industries International 70R-9772 say, 2nd Edition, Price and Newman, eds., MacMillan (1997) Atlas Antibodies HPAO41 060 and Antibodies, A Laboratory Manual, Harlow and Lane, antibodies-online ABIN?48823 eds., Cold Spring Harbor Laboratory, Ch. 9 (1988), each of LifeSpan BioSciences LS-C1 16852–50 which is incorporated herein by reference in its entirety. Abnova Corporation PAB23744 MGAT1 Atlas Antibodies HPA017432 [0092] Antibodies used in immunoassays to determine the antibodies-online ABIN571229 level of a marker of the invention, may be labeled with a Thermo Scientific Pierce Antibodies PA5-12148 detectable label. The term “labeled”, with regard to the probe Abnova Corporation PAB18956 or antibody, is intended to encompass direct labeling of the LifeSpan BioSciences LS-C99702-100 STX1A. Abgent AP98.13a. probe or antibody by coupling (i.e., physically linking) a Fitzgerald Industries International 70R-10562 detectable substance to the probe or antibody, as well as Acris Antibodies GmbH AP15806PU-M indirect labeling of the probe or antibody by reactivity with LifeSpan BioSciences LS-C89914-100 another reagent that is directly labeled. Examples of indirect Covance, Inc. MMS-619.R-500 NMU Atlas Antibodies HPAO25926 labeling include detection of a primary antibody using a fluo GeneTex GTX87991 rescently labeled secondary antibody and end-labeling of a antibodies-online ABIN461275 DNA probe with biotin such that it can be detected with LifeSpan BioSciences LS-C9258–50 fluorescently labeled streptavidin. Biorbyt Orb 126042 CD59 antibodies-online ABIN94204 [0093] In one embodiment, the antibody is labeled, e.g. a Antigenix America Inc. M590020 radio-labeled, chromophore-labeled, fluorophore-labeled, or GeneTex GTX74620 enzyme-labeled antibody. In another embodiment, an anti Ab|D Serotec MCA 1927T Thermo Scientific Pierce Antibodies MA1-700.58 body derivative (e.g. an antibody conjugated with a substrate CASR Atlas Antibodies HPAO396.86 or with the protein or ligand of a protein-ligand pair {e.g. antibodies-online ABIN460094 biotin-streptavidin}), or an antibody fragment (e.g. a single Spring Bioscience E10624 chain antibody, an isolated antibody hypervariable domain, Abnova Corporation PAB18311 Acris Antibodies GmbH AP20293PU-N etc.) which binds specifically with a marker of the invention. CPE Santa Cruz Biotechnology, Inc. Sc-34321 [0094] In one embodiment of the invention, proteomic LifeSpan BioSciences LS-C1 19819–100 methods, e.g., mass spectrometry, are used. Mass spectrom etry is an analytical technique that consists of ionizing chemi

US 2015/0330.997 A1 Nov. 19, 2015 40

Tables 1-3. The entire contents of each of these Uniprot Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223, records is hereby incorporated by reference. Methods for 409), spores (Ladner U.S. Pat. No. 409), plasmids (Cullet al. determining the effect of a compound on the expression and/ (1992) Proc Natl Acad Sci USA 89:1865-1869) or phage or activity of marker are known in the art and/or described (Scott and Smith (1990) Science 249:386-390; Devlin (1990) herein. Science 249:404–406; Cwirla et al. (1990) Proc. Natl. Acad. [0105] A variety of test compounds can be evaluated using Sci. 87:6378-6382; Felici (1991).J. Mol. Biol. 222:301-310; the screening assays described herein. The term “test com Ladner supra.). pound” includes any reagent or test agent which is employed [0110] Compounds identified in the screening assays can in the assays of the invention and assayed for its ability to be used in methods of modulating one or more of the biologi influence the expression and/or activity of a marker. More cal responses regulated by a marker, e.g., glucose tolerance. It than one compound, e.g., a plurality of compounds, can be will be understood that it may be desirable to formulate such tested at the same time for their ability to modulate the expres compound(s) as pharmaceutical compositions prior to con sion and/or activity of a marker in a screening assay. The term tacting them with cells. “screening assay” preferably refers to assays which test the [0111] Once a test compound is identified by one of the ability of a plurality of compounds to influence the readout of variety of methods described hereinbefore, the selected test choice rather than to tests which test the ability of one com compound (or “compound of interest”) can then be further pound to influence a readout. Preferably, the subject assays evaluated for its effect on cells, for example by contacting the identify compounds not previously known to have the effect compound of interest with cells eitherin vivo (e.g., by admin that is being screened for. In one embodiment, high through istering the compound of interest to a subject or animal put screening can be used to assay for the activity of a com model) or ex vivo (e.g., by isolating cells from the subject or pound. animal model and contacting the isolated cells with the com [0106] Candidate/test compounds include, for example, 1) pound of interest or, alternatively, by contacting the com peptides such as soluble peptides, including Ig-tailed fusion pound of interest with a cell line) and determining the effect peptides and members of random peptide libraries (see, e.g., of the compound of interest on the cells, as compared to an Lam, K. S. et al. (1991) Nature 354:82–84; Houghten, R. et al. appropriate control (such as untreated cells or cells treated (1991) Nature 354:84-86) and combinatorial chemistry-de with a control compound, or carrier, that does not modulate rived molecular libraries made of D- and/or L-configuration the biological response). amino acids; 2) phosphopeptides (e.g., members of random [0112] Computer-based analysis of a marker with a known and partially degenerate, directed phosphopeptide libraries, structure can also be used to identify molecules which will see, e.g., Songyang, Z. et al. (1993) Cell 72:767-778); 3) bind to a marker of the invention. Such methods rank mol antibodies (e.g., polyclonal, monoclonal, humanized, anti ecules based on their shape complementary to a receptor site. idiotypic, chimeric, and single chain antibodies as well as For example, using a 3-D database, a program such as DOCK Fab, F(ab')2, Fab expression library fragments, and epitope can be used to identify molecules which will bind to TLR9. binding fragments of antibodies); 4) small organic and inor See DesJarliasetal. (1988).J. Med. Chem, 31:722; Mengetal. ganic molecules (e.g., molecules obtained from combinato (1992) J. Computer Chem. 13:505; Meng et al. (1993) Pro rial and natural product libraries); 5) enzymes (e.g., teins 17:266; Shoichet et al. (1993) Science 259:1445. In endoribonucleases, hydrolases, nucleases, proteases, syn addition, the electronic complementarity of a molecule to a thatases, isomerases, polymerases, kinases, phosphatases, marker can be analyzed to identify molecules which bind to oxido-reductases and ATPases), 6) mutant forms of marker(s) the marker. This can be determined using, for example, a molecules, e.g., dominant negative mutant forms of the mol molecular mechanics force field as described in Meng et al. ecules, 7) nucleic acids, 8) carbohydrates, and 9) natural (1992) J. Computer Chem. 13:505 and Meng et al. (1993) product extract compounds. Proteins 17:266. Other programs which can be used include [0107] Test compounds can be obtained using any of the CLIX which uses a GRID force field in docking of putative numerous approaches in combinatorial library methods ligands. See Lawrence etal. (1992) Proteins 12:31; Goodford known in the art, including: biological libraries; spatially et al. (1985).J. Med. Chem. 28:849; Boobbyer et al. (1989).J. addressable parallel solid phase or solution phase libraries; Med. Chem. 32:1083. synthetic library methods requiring deconvolution; the ‘one [0113] The instant invention also pertains to compounds bead one-compound’ library method; and synthetic library identified using the foregoing screening assays. methods using affinity chromatography selection. The bio [0114] D. Methods for Modulating the Expression and/or logical library approach is limited to peptide libraries, while Activity of a Biomarker of the Invention the other four approaches are applicable to peptide, non [0115] Yet another aspect of the invention pertains to meth peptide oligomer or small molecule libraries of compounds ods of modulating expression and/or activity of a marker in a (Lam, K. S. (1997) Anticancer Drug Des. 12:145). cell. The modulatory methods of the invention involve con [0108] Examples of methods for the synthesis of molecular tacting the cell with an agent that modulates the expression libraries can be found in the art, for example in: DeWitt et al. and/or activity of a marker such that the expression and/or (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb etal. (1994) activity of a marker in the cell is modulated. In order for the Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. expression and/or activity of a marker to be modulated in a (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science cell, the cell is contacted with a modulatory agent in an 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed, Engl. amount sufficient to modulate the expression and/or activity 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed, Engl. of a marker. 33:2061; and Gallop et al. (1994).J. Med. Chem. 37:1233. [0116] A “modulator” or “modulatory agent” is a com [0109] Libraries of compounds can be presented in solution pound or molecule that modulates, and may be, e.g., an ago (e.g., Houghten (1992) Biotechniques 13:412–421), or on nist, antagonist, activator, stimulator, suppressor, or inhibitor. beads (Lam (1991) Nature 354:82–84), chips (Fodor (1993) As used herein, the term “modulator” refers to any moiety

US 2015/0330.997 A1 Nov. 19, 2015 42 progression of diabetes, for a particular patient, composition, formulated as a solution, microemulsion, liposome, or other and mode of administration, without being toxic to the ordered structure suitable to high drug concentration. The patient. The selected dosage level will depend upon a variety carrier can be a solvent or dispersion medium containing, for of pharmacokinetic factors including the activity of the par example, water, ethanol, polyol (for example, glycerol, pro ticular marker(s) modulator employed, or the ester, salt or pylene glycol, and liquid polyethylene glycol, and the like), amide thereof, the route of administration, the time of admin and suitable mixtures thereof. The proper fluidity can be istration, the rate of excretion of the particular modulator maintained, for example, by the use of a coating such as being employed, the duration of the treatment, other drugs, lecithin, by the maintenance of the required particle size in the compounds and/or materials used in combination with the case of dispersion and by the use of surfactants. In many particular modulator employed, the age, sex, weight, condi cases, it will be preferable to include isotonic agents, for tion, general health and prior medical history of the patient example, sugars, polyalcohols such as mannitol, sorbitol, or being treated, and like factors well known in the medical arts. sodium chloride in the composition. Prolonged absorption of A physician or veterinarian having ordinary skill in the art can the injectable compositions can be brought about by includ readily determine and prescribe the effective amount of the ing an agent that delays absorption, for example, monostear modulator required. For example, the physician or veterinar ate salts and gelatin. ian could start doses of the modulator at levels lower than that [0132) Sterile injectable solutions can be prepared by required in order to achieve the desired therapeutic effect and incorporating the active modulator in the required amount in gradually increase the dosage until the desired effect is an appropriate solvent with one or a combination of ingredi achieved. In general, a suitable daily dose of a marker(s) ents enumerated above, as required, followed by sterilization modulator will be that amount which is the lowest dose effec microfiltration. Generally, dispersions are prepared by incor tive to produce a therapeutic effect. Such an effective dose porating the active compound into a sterile vehicle that con will generally depend upon the factors described above. It is tains a basic dispersion medium and the required other ingre preferred that administration be intravenous, intramuscular, dients from those enumerated above. In the case of sterile intraperitoneal, or subcutaneous, preferably administered powders for the preparation of sterile injectable solutions, the proximal to the site of the target. If desired, the effective daily preferred methods of preparation are vacuum drying and dose of a marker(s) modulator may be administered as two, freeze-drying (lyophilization) that yield a powder of the three, four, five, six or more sub-doses administered sepa active ingredient plus any additional desired ingredient from rately at appropriate intervals throughout the day, optionally, a previously sterile-filtered solution thereof. in unit dosage forms. While it is possible for a marker(s) [0133] Marker(s) modulators that can be used in the meth modulator of the present invention to be administered alone, ods of the present invention include those suitable for oral, it is preferable to administer the modulator as a pharmaceu nasal, topical (including buccal and sublingual), rectal, vagi tical formulation (composition). nal and/or parenteral administration. The formulations may [0128] Dosage regimens are adjusted to provide the opti conveniently be presented in unit dosage form and may be mum desired response (e.g., a therapeutic response). For prepared by any methods known in the art of pharmacy. The example, a single bolus may be administered, several divided amount of active ingredient which can be combined with a doses may be administered over time or the dose may be carrier material to produce a single dosage form will vary proportionally reduced or increased as indicated by the exi depending upon the subject being treated, and the particular gencies of the therapeutic situation. For example, the marker mode of administration. The amount of active ingredient (s) modulators used in the methods of the present invention which can be combined with a carrier material to produce a may be administered once or twice weekly by subcutaneous single dosage form will generally be that amount of the modu injection or once or twice monthly by subcutaneous injection. lator which produces a therapeutic effect. Generally, out of [0129] To administer a marker(s) modulator used in the one hundred percent, this amount will range from about methods of the present invention by certain routes of admin 0.001% to about 90% of active ingredient, preferably from istration, it may be necessary to include the modulator in a about 0.005% to about 70%, most preferably from about formulation suitable for preventing its inactivation. For ().01% to about 30%. example, the marker(s) modulator may be administered to a [0134] The phrases “parenteral administration” and subject in an appropriate carrier, for example, liposomes, or a “administered parenterally”, as used herein, means modes of diluent. Pharmaceutically acceptable diluents include saline administration other than enteral and topical administration, and aqueous buffer solutions. Liposomes include water-in usually by injection, and includes, without limitation, intra oil-in-water CGF emulsions, as well as conventional lipo venous, intramuscular, intraarterial, intrathecal, intracapsu somes (Strejan et al. (1984).J. Neuroimmunol. 7:27). lar, intraorbital, intracardiac, intradermal, intraperitoneal, [0130] Pharmaceutically acceptable carriers include sterile transtracheal, subcutaneous, subcuticular, intraarticular, sub aqueous solutions or dispersions and sterile powders for the capsular, subarachnoid, intraspinal, epidural and intrasternal extemporaneous preparation of sterile injectable solutions or injection and infusion. dispersion. The use of such media and agents for pharmaceu [0135) Examples of suitable aqueous and non-aqueous car tically active substances is known in the art. Except insofar as riers which may be employed along with the marker(s) modu any conventional media or agent is incompatible with the lators utilized in the methods of the present invention include active marker(s) modulator, use thereof in pharmaceutical water, ethanol, polyols (such as glycerol, propylene glycol, compositions is contemplated. Supplementary active com polyethylene glycol, and the like), and suitable mixtures pounds can also be incorporated with the marker(s) modula thereof, vegetable oils, such as olive oil, and injectable tor. organic esters, such as ethyl oleate. Proper fluidity can be [0131] Marker(s) modulators used in the methods of the maintained, for example, by the use of coating materials, such invention typically must be sterile and stable under the con as lecithin, by the maintenance of the required particle size in ditions of manufacture and storage. The modulator can be the case of dispersions, and by the use of surfactants. US 2015/0330.997 A1 Nov. 19, 2015

[0136] Marker(s) modulatos may also be administered with hypervariability, termed complementarity determining adjuvants such as preservatives, wetting agents, emulsifying regions (CDR), interspersed with regions that are more con agents and dispersing agents. Prevention of presence of served, termed framework regions (FR). Each V, and V, is microorganisms may be ensured both by sterilization proce composed of three CDRs and four FRs, arranged from amino dures and by the inclusion of various antibacterial and anti terminus to carboxy-terminus in the following order: FR1, fungal agents, for example, paraben, chlorobutanol, phenol CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions sorbic acid, and the like. It may also be desirable to include of the heavy and light chains contain a binding domain that isotonic agents, such as sugars, sodium chloride, and the like interacts with an antigen. The constant regions of the anti into the compositions. In addition, prolonged absorption of bodies may mediate the binding of the immunoglobulin to the injectable pharmaceutical form may be brought about by host tissues or factors, including various cells of the immune the inclusion of agents which delay absorption such as alu system (e.g., effector cells) and the first component (Cld) of minum monostearate and gelatin. the classical complement system. [0137] When marker(s) modulators used in the methods of [0143] The term “antigen-binding portion” of an antibody the present invention are administered to humans and ani (or simply “antibody portion”), as used herein, refers to one or mals, they can be given alone or as a pharmaceutical modu more fragments of an antibody that retain the ability to spe lator containing, for example, 0.001 to 90% (more preferably, cifically bind to an antigen (e.g., a marker). It has been shown 0.005 to 70%, such as 0.01 to 30%) of active ingredient in that the antigen-binding function of an antibody can be per combination with a pharmaceutically acceptable carrier. formed by fragments of a full-length antibody. Examples of [0138] Marker(s) modulators can be administered with binding fragments encompassed within the term “antigen medical devices known in the art. For example, in a preferred binding portion” of an antibody include (i) a Fab fragment, a embodiment, a modulator can be administered with a needle monovalent fragment consisting of the V, VA, CL and CH1 less hypodermic injection device, such as the devices dis domains; (ii) a F(ab')2 fragment, a bivalent fragment compris closed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335, ing two Fab fragments linked by a disulfide bridge at the hinge 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of region; (iii) a Fd fragment consisting of the VA, and CH1 well-known implants and modules useful in the present domains; (iv) a FV fragment consisting of the V, and VA, invention include: U.S. Pat. No. 4,487,603, which discloses domains of a single arm of an antibody, (v) a dAb including an implantable micro-infusion pump for dispensing medica VH and VL domains; (vi) a dAb fragment (Ward et al. (1989) tion at a controlled rate; U.S. Pat. No. 4,486,194, which Nature 341, 544-546), which consists of a V., domain; (vii) a discloses a therapeutic device for administering medications dAb which consists of a VH or a VL domain; and (viii) an through the skin; U.S. Pat. No. 4,447,233, which discloses a isolated complementarity determining region (CDR) or (ix) a medication infusion pump for delivering medication at a pre combination of two or more isolated CDRs which may cise infusion rate; U.S. Pat. No. 4,447,224, which discloses a optionally be joined by a synthetic linker. Furthermore, variable flow implantable infusion apparatus for continuous although the two domains of the FV fragment, V, and Vº, are drug delivery; U.S. Pat. No. 4,439,196, which discloses an coded for by separate genes, they can be joined, using recom osmotic drug delivery system having multi-chamber com binant methods, by a synthetic linker that enables them to be partments; and U.S. Pat. No. 4,475,196, which discloses an made as a single protein chain in which the V, and VA, regions osmotic drug delivery system. Many other such implants, pair to form monovalent molecules (known as single chain FV delivery systems, and modules are known to those skilled in (scr'v); see e.g., Bird et al. (1988) Science 242, 423-426; and the art. Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5879 [0139| 1. Inhibitory Agents 5883). Such single chain antibodies are also intended to be [0140] According to a modulatory method of the invention, encompassed within the term “antigen-binding portion” of an the expression and/or activity of a marker(s) is inhibited in a antibody. These antibody fragments are obtained using con cell or subject by contacting the cell with (or administering to ventional techniques known to those with skill in the art, and a subject) an inhibitory agent Inhibitory agents of the inven the fragments are screened for utility in the same manner as tion can be, for example, molecules that act to decrease or are intact antibodies. Antigen-binding portions can be pro inhibit the expression and/or activity of the marker(s). duced by recombinant DNA techniques, or by enzymatic or [0141] In one embodiment of the invention, the modula chemical cleavage of intact immunoglobulins. tory, e.g., therapeutic, and diagnostic methods described [0144] The term “antibody”, as used herein, includes poly herein employ an antibody that binds, e.g., directly to or clonal antibodies, monoclonal antibodies, chimeric antibod indirectly to, and inhibits marker(s) activity and/or down ies, humanized antibodies, and human antibodies, and those modulates marker(s) expression. that occur naturally or are recombinantly produced according [0142] The term “antibody” or “immunoglobulin,” as used to methods well known in the art. interchangeably herein, includes whole antibodies and any antigen binding fragment (i.e., “antigen-binding portion”) or [0145] In one embodiment, an antibody for use in the meth single chains thereof. An “antibody” comprises at least two ods of the invention is a bispecific antibody. A “bispecific” or heavy (H) chains and two light (L) chains inter-connected by “bifunctional antibody” is an artificial hybrid antibody hav disulfide bonds. Each heavy chain is comprised of a heavy ing two different heavy/light chain pairs and two different chain variable region (abbreviated herein as V,) and a heavy binding sites. Bispecific antibodies can be produced by a chain constant region. The heavy chain constant region is variety of methods including fusion of hybridomas or linking comprised of three domains, CH1, CH2 and CH3. Each light of Fab' fragments. See, e.g., Songsivilai & Lachmann, (1990) chain is comprised of a light chain variable region (abbrevi Clin. Exp. Immunol. 79, 315-321; Kostelny et al. (1992) J. ated herein as V, ) and a light chain constant region. The light Immunol. 148, 1547-1553. chain constant region is comprised of one domain, CL. The [0146] In another embodiment, an antibody for use in the V, and V, regions can be further subdivided into regions of methods of the invention is a camelid antibody as described US 2015/0330.997 A1 Nov. 19, 2015 44 in, for example, PCT Publication WO 94/04678, the entire of myosin-binding protein H, I-set immunoglobulin domain contents of which are incorporated herein by reference. of telokin, NCAM, twitchin, neuroglian, growth hormone [0147] A region of the camelid antibody that is the small, receptor, erythropoietin receptor, prolactin receptor, inter single variable domain identified as Vaz, can be obtained by feron-gamma receptor, fl-galactosidase/glucuronidase, genetic engineering to yield a small protein having high affin fl-glucuronidase, transglutaminase, T-cell antigen receptor, ity for a target, resulting in a low molecular weight, antibody superoxide dismutase, tissue factor domain, cytochrome F. derived protein known as a “camelid nanobody”. See U.S. green fluorescent protein, GroEL, and thaumatin. Pat. No. 5,759,808; see also Stijlemans et al., 2004.J. Biol. [0153] To generate non-antibody binding molecules, a Chem. 279: 1256-1261; Dumoulin et al., 2003 Nature 424: library of clones is created in which sequences in regions of 783-788; Pleschberger et al., 2003 Bioconjugate Chem. 14. the scaffold protein that form antigen binding surfaces (e.g., 440-448; Cortez-Retamozo et al., 2002 Int. J. Cancer 89: regions analogous in position and structure to CDRs of an 456-62; and Lauwereys, et al., 1998 EMBO.J. 17:3512-3520. antibody variable domain immunoglobulin fold) are random Engineered libraries of camelid antibodies and antibody frag ized Library clones are tested for specific binding to the ments are commercially available, for example, from Ablynx, antigen of interest (e.g., TLR9) and for other functions (e.g., Ghent, Belgium. Accordingly, a feature of the present inven inhibition of biological activity of TLR9). Selected clones tion is a camelid nanobody having high affinity for a marker. can be used as the basis for further randomization and selec [0148] In other embodiments of the invention, an antibody tion to produce derivatives of higher affinity for the antigen. for use in the methods of the invention is a diabody, a single [0154] High affinity binding molecules are generated, for chain diabody, or a di-diabody. example, using the tenth module of fibronectin III ("Frå) as [0149] Diabodies are bivalent, bispecific molecules in the scaffold, described in U.S. Pat. Nos. 6,818,418 and 7,115, which V, and V, domains are expressed on a single polypep 396; Roberts and Szostak, 1997 Proc. Natl. Acad. Sci USA tide chain, connected by a linker that is too short to allow for 94:12297, U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,258,558; pairing between the two domains on the same chain. The VA, and Szostaketal. WO98/31700, the entire contents of each of and V, domains pair with complementary domains of another which are incorporated herein by reference. chain, thereby creating two antigen binding sites (see e.g., [0155] Non-antibody binding molecules can be produced Holliger et al., 1993 Proc. Natl. Acad. Sci. USA 90:6444 as dimers or multimers to increase avidity for the target anti 6448; Poljak et al., 1994 Structure 2:1121-1123). Diabodies gen. For example, the antigen binding domain is expressed as can be produced by expressing two polypeptide chains with a fusion with a constant region (Fc) of an antibody that forms either the structure V1, 1–V, a and Vº-V, 4 (VM-V, configu Fc-Fc dimers. See, e.g., U.S. Pat. No. 7,115,396, the entire ration), or V, 4-Vºz, and Vº-Vºz (V, -V., configuration) contents of which are incorporated herein by reference. within the same cell. Most of them can be expressed in soluble [0156] The therapeutic methods of the invention also may form in bacteria. be practiced through the use of antibody fragments and anti [0150] Single chain diabodies (scL)b) are produced by con body mimetics. As detailed below, a wide variety of antibody necting the two diabody-forming polypeptide chains with fragment and antibody mimetic technologies have now been linker of approximately 15 amino acid residues (see Holliger developed and are widely known in the art. While a number of and Winter, 1997 Cancer Immunol. Immunother, 45(3-4): these technologies, such as domain antibodies, Nanobodies, 128-30; Wu et al., 1996 Immunotechnology, 201):21-36). and UniBodies make use of fragments of, or other modifica scDb can be expressed in bacteria in soluble, active mono tions to, traditional antibody structures, there are also alter meric form (see Holliger and Winter, 1997 Cancer Immunol. native technologies, such as Adnectins, Affibodies, DARPins, Immunother, 45(34): 128-30; Wu et al., 1996 Immunotech Anticalins, Avimers, and Versabodies that employ binding nology, 201):21-36; Pluckthun and Pack, 1997 Immunotech structures that, while they mimic traditional antibody bind nology, 3(2): 83-105; Ridgway et al., 1996 Protein Eng., ing, are generated from and function via distinct mechanisms. 9(7):617–21). Some of these alternative structures are reviewed in Gill and [0151] A diabody can be fused to Fc to generate a “di Damle (2006) 17: 653-658. diabody” (see Luet al., 2004.J. Biol. Chem., 279(4):2856-65). [0157] Domain Antibodies (dAbs) are the smallest func [0152] Marker binding molecules that exhibit functional tional binding units of antibodies, corresponding to the vari properties of antibodies but derive their framework and anti able regions of either the heavy (VH) or light (VL) chains of gen binding portions from other polypeptides (e.g., polypep human antibodies. Domantis has developed a series of large tides other than those encoded by antibody genes or generated and highly functional libraries of fully human VH and VL by the recombination of antibody genes in vivo) may also be dAbs (more than ten billion different sequences in each used in the methods of the present invention. The antigen library), and uses these libraries to select dAbs that are spe binding domains (e.g., marker binding domains) of these cific to therapeutic targets. In contrast to many conventional binding molecules are generated through a directed evolution antibodies, domain antibodies are well expressed in bacterial, process. See U.S. Pat. No. 7,115,396. Molecules that have an yeast, and mammalian cell systems. Further details of domain overall fold similar to that of a variable domain of an antibody antibodies and methods of production thereof may be (an “immunoglobulin-like” fold) are appropriate scaffold obtained by reference to U.S. Pat. Nos. 6,291,158; 6,582,915; proteins. Scaffold proteins suitable for deriving antigen bind 6,593,081; 6,172,197; 6,696,245; U.S. Serial No. 2004/ ing molecules include fibronectinora fibronectin dimer, tena 0110941; European patent application No. 1433846 and scin, N-cadherin, E-cadherin, ICAM, titin, GCSF-receptor, European Patents 0368684 & 0616640; WO05/035572, cytokine receptor, glycosidase inhibitor, antibiotic chro WOO4/101790, WOO4/081026, WOO4/058821, WOO4/ moprotein, myelin membrane adhesion molecule P0, CD8, 003019 and WOO3/002609, the contents of each of which is CD4, CD2, class I MHC, T-cell antigen receptor, CD1, C2 herein incorporated by reference in its entirety. and I-set domains of VCAM-1, I-set immunoglobulin domain [0158] Nanobodies are antibody-derived therapeutic pro of myosin-binding protein C, I-set immunoglobulin domain teins that contain the unique structural and functional prop US 2015/0330.997 A1 Nov. 19, 2015

erties of naturally-occurring heavy-chain antibodies. These vitro, and later transcribed and translated in vitro or in vivo. heavy-chain antibodies contain a single variable domain Alternatively, a natural "Fn3 sequence may be isolated or (VHH) and two constant domains (CH2 and CH3) Impor cloned from the genome using standard methods (as per tantly, the cloned and isolated VHH domain is a perfectly formed, e.g., in U.S. Pat. Application No. 20070082365), and stable polypeptide harboring the full antigen-binding capac then mutated using mutagenesis methods known in the art. ity of the original heavy-chain antibody. Nanobodies have a [0165] An aptamer is another type of antibody-mimetic high homology with the VH domains of human antibodies which may be used in the methods of the present invention. and can be further humanized without any loss of activity. Aptamers are typically small nucleotide polymers that bind to [0159] Nanobodies are encoded by single genes and are specific molecular targets. Aptamers may be single or double efficiently produced in almost all prokaryotic and eukaryotic stranded nucleic acid molecules (DNA or RNA), although hosts, e.g., E. coli (see, e.g., U.S. Pat. No. 6,765,087, which is DNA based aptamers are most commonly double stranded. herein incorporated by reference in its entirety), molds (for There is no defined length for an aptamer nucleic acid; how example Aspergillus or Trichoderma) and yeast (for example ever, aptamer molecules are most commonly between 15 and Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see, 40 nucleotides long. e.g., U.S. Pat. No. 6,838,254, which is herein incorporated by [0166] Aptamers may be generated using a variety of tech reference in its entirety). The production process is scalable niques, but were originally developed using in vitro selection and multi-kilogram quantities of Nanobodies have been pro (Ellington and Szostak. (1990) Nature. 346(6287):818-22) duced. Because Nanobodies exhibit a superior stability com and the SELEX method (systematic evolution of ligands by pared with conventional antibodies, they can beformulated as exponential enrichment) (Schneider et al. 1992. J Mol Biol. a long shelf-life, ready-to-use solution. 228(3):862-9) the contents of which are incorporated herein [0160] The Nanoclone method (see, e.g., WO 06/079372, by reference. Other methods to make and uses of aptamers which is herein incorporated by reference in its entirety) is a have been published including Klussmann. The Aptamer proprietary method for generating Nanobodies against a Handbook: Functional Oligonucleotides and Their Applica desired target, based on automated high-throughout selection tions. ISBN: 978-3-527-31059-3; Ulrich et al. 2006. Comb of B-cells and could be used in the context of the instant Chem High Throughput Screen 9(8):619-32; Cerchia and de invention. Franciscis. 2007. Methods Mol Biol. 361:187-200; Ireson and [0161] UniBodies are another antibody fragment technol Kelland. 2006. Mol Cancer Ther. 2006 5(12):2957-62; U.S. ogy, however this one is based upon the removal of the hinge Pat. Nos. 5,582,981; 5,840,867; 5,756,291; 6,261,783; 6,458, region of IgG4 antibodies. The deletion of the hinge region 559; 5,792,613; 6,111,095; and U.S. patent application Ser. results in a molecule that is essentially half the size of tradi Nos. 11/482,671; 11/102,428; 11/291,610; and 10/627,543 tional IgG4 antibodies and has a univalent binding region which are all incorporated herein by reference. rather than the bivalent binding region of IgG4 antibodies. It [0167] Aptamer molecules made from peptides instead of is also well known that IgG4 antibodies are inert and thus do nucleotides may also be used in the methods of the invention. not interact with the immune system, which may be advanta Peptide aptamers share many properties with nucleotide geous for the treatment of diseases where an immune aptamers (e.g., small size and ability to bind target molecules response is not desired, and this advantage is passed onto with high affinity) and they may be generated by selection UniBodies. Further details of UniBodies may be obtained by methods that have similar principles to those used to generate reference to patent application WO2007/059782, which is nucleotide aptamers, for example Baines and Colas. 2006. herein incorporated by reference in its entirety. Drug Discov Today. 11 (7-8):334-41; and Bickle et al. 2006. [0162] Adnectin molecules are engineered binding pro Nat Protoc. 1(3):1066-91 which are incorporated herein by teins derived from one or more domains of the fibronectin reference. protein. In one embodiment, adnectin molecules are derived [0168] Affibody molecules represent a class of affinity pro from the fibronectin type 2.I domain by altering the native teins based on a 58-amino acid residue protein domain, protein which is composed of multiple beta strands distrib derived from one of the IgG-binding domains of staphylococ uted between two beta sheets. Depending on the originating cal protein.A. This three helix bundle domain has been used as tissue, fibronectin may contain multiple type 2.I domains a scaffold for the construction of combinatorial phagemid which may be denoted, e.g., 'Fn3, *Fn3, *Fn3, etc. Adnectin libraries, from which Affibody variants that target the desired molecules may also be derived from polymers of "Frå molecules can be selected using phage display technology related molecules rather than a simple monomeric "Fn3 (Nord K, et al. Nat Biotechnol 1997; 15:772-7. Ronmark J, et Structure. al., Eur J Biochem 2002; 269:2647-55). Further details of [0163] Although the native "Frå domain typically binds to Affibodies and methods of production thereof may be integrin, "Fn3 proteins adapted to become adnectin mol obtained by reference to U.S. Pat. No. 5,831,012 which is ecules are altered so to bind antigens of interest, e.g., a marker herein incorporated by reference in its entirety. (s). In one embodiment, the alteration to the "Fn3 molecule [0169] DARPins (Designed Ankyrin Repeat Proteins) are comprises at least one mutation to a beta strand. In a preferred one example of an antibody mimetic DRP (Designed Repeat embodiment, the loop regions which connect the beta strands Protein) technology that has been developed to exploit the of the "Fn3 molecule are altered to bind to an antigen of binding abilities of non-antibody polypeptides. Repeat pro interest, e.g., a marker(s). teins such as ankyrin or leucine-rich repeat proteins, are ubiq (0164] The alterations in the "Fn3 may be made by any uitous binding molecules, which occur, unlike antibodies, method known in the art including, but not limited to, error intra- and extracellularly. Their unique modular architecture prone PCR, site-directed mutagenesis, DNA shuffling, or features repeating structural units (repeats), which stack other types of recombinational mutagenesis which have been together to form elongated repeat domains displaying vari referenced herein. In one example, variants of the DNA able and modular target-binding surfaces. Based on this encoding the "Fn3 sequence may be directly synthesized in modularity, combinatorial libraries of polypeptides with US 2015/0330.997 A1 Nov. 19, 2015 46 highly diversified binding specificities can be generated. This Versabodies are small proteins of 3-5 kDa with >15% cys strategy includes the consensus design of self-compatible teines, which form a high disulfide density scaffold, replacing repeats displaying variable surface residues and their random the hydrophobic core that typical proteins have. The replace assembly into repeat domains. ment of a large number of hydrophobic amino acids, com [0170] Additional information regarding DARPins and prising the hydrophobic core, with a small number of disul other DRP technologies can be found in U.S. Patent Appli fides results in a protein that is smaller, more hydrophilic (less cation Publication No. 2004/0132028 and International aggregation and non-specific binding), more resistant to pro Patent Application Publication No. WO 02/20565, both of teases and heat, and has a lower density of T-cell epitopes, which are hereby incorporated by reference in their entirety. because the residues that contribute most to MHC presenta [0171] Anticalins are an additional antibody mimetic tech tion are hydrophobic. All four of these properties are well nology, however in this case the binding specificity is derived known to affect immunogenicity, and together they are from lipocalins, a family of low molecular weight proteins expected to cause a large decrease in immunogenicity. that are naturally and abundantly expressed in human tissues [0178] Additional information regarding Versabodies can and body fluids. Lipocalins have evolved to perform a range be found in U.S. Patent Application Publication No. 2007/ of functions in vivo associated with the physiological trans 0.191272 which is hereby incorporated by reference in its port and storage of chemically sensitive or insoluble com entirety. pounds. Lipocalins have a robust intrinsic structure compris [0179| SMIPs" (Small Modular ImmunoPharmaceuti ing a highly conserved fl-barrel which supports four loops at cals-Trubion Pharmaceuticals) engineered to maintain and one terminus of the protein. These loops form the entrance to optimize target binding, effector functions, in vivo half-life, a binding pocket and conformational differences in this part and expression levels. SMIPS consist of three distinct modu of the molecule account for the variation in binding specific lar domains. First they contain a binding domain which may ity between individual lipocalins. consist of any protein which confers specificity (e.g., cell [0172] Lipocalins are cloned and their loops are subjected surface receptors, single chain antibodies, soluble proteins, to engineering in order to create Anticalins. Libraries of struc etc). Secondly, they contain a hinge domain which serves as a turally diverse Anticalins have been generated and Anticalin flexible linker between the binding domain and the effector display allows the selection and screening of binding func domain, and also helps control multimerization of the SMIP tion, followed by the expression and production of soluble drug. Finally, SMIPS contain an effector domain which may protein for further analysis in prokaryotic or eukaryotic sys be derived from a variety of molecules including Fc domains tems. Studies have successfully demonstrated that Anticalins or other specially designed proteins. The modularity of the can be developed that are specific for virtually any human design, which allows the simple construction of SMIPs with target protein can be isolated and binding affinities in the a variety of different binding, hinge, and effector domains, nanomolar or higher range can be obtained. provides for rapid and customizable drug design. [0173] Anticalins can also be formatted as dual targeting [0180] More information on SMIPs, including examples of proteins, so-called Duocalins. A Duocalin binds two separate how to design them, may be found in Zhao et al. (2007) Blood therapeutic targets in one easily produced monomeric protein 110:2569-77 and the following U.S. Pat. App. Nos. using standard manufacturing processes while retaining tar 20050238646; 20050202534; 20050202028; 20050202023; get specificity and affinity regardless of the structural orien 20050202012; 20050186216; 20050180970; and tation of its two binding domains. 20050175614. [0174] Additional information regarding Anticalins can be [0181] In another aspect, the methods of the present inven found in U.S. Pat. No. 7,250,297 and International Patent tion employ immunoconjugate agents that target a marker(s) Application Publication No. WO99/16873, both of which are and which inhibit or down-modulate the marker(s). Agents hereby incorporated by reference in their entirety. that can be targeted to a marker(s) include, but are not limited [0175] Another antibody mimetic technology useful in the to, cytotoxic agents, anti-inflammatory agents, e.g., a steroi context of the instant invention are Avimers. Avimers are dal or nonsteroidal inflammatory agent, or a cytotoxin anti evolved from a large family of human extracellular receptor metabolites (e.g., methotrexate, 6-mercaptopurine, domains by in vitro exon shuffling and phage display, gener 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alky ating multidomain proteins with binding and inhibitory prop lating agents (e.g., mechlorethamine, thioepa chlorambucil, erties. Linking multiple independent binding domains has melphalan, carmustine (BSNU) and lomustine (CCNU), been shown to create avidity and results in improved affinity cyclothosphamide, busulfan, dibromomannitol, streptozoto and specificity compared with conventional single-epitope cin, mitomycin C, and cis-dichlorodiamine platinum (II) binding proteins. Other potential advantages include simple (DDP) cisplatin), anthracyclines (e.g., daunorubicin (for and efficient production of multitarget-specific molecules in merly daunomycin) and doxorubicin), antibiotics (e.g., dac Escherichia coli, improved thermostability and resistance to tinomycin (formerly actinomycin), bleomycin, mithramycin, proteases. Avimers with sub-nanomolar affinities have been and anthramycin (AMC)), and anti-mitotic agents (e.g., vin obtained against a variety of targets. cristine and vinblastine). [0176). Additional information regarding Avimers can be [0182] In another embodiment, marker(s) modulator found in U.S. Patent Application Publication Nos. 2006/ employed in the methods of the invention are small mol 0286603, 2006/0234299, 2006/0223114, 2006/0177831, ecules. As used herein, the term “small molecule” is a term of 2006/0008844, 2005/0221384, 2005/0164301, 2005/ the art and includes molecules that are less than about 7500, 0089932, 2005/0053973, 2005/00485.12, 2004/0175756, all less than about 5000, less than about 1000 molecular weight of which are hereby incorporated by reference in their or less than about 500 molecular weight, and inhibit marker entirety. (s) activity. Exemplary small molecules include, but are not [0177| Versabodies are another antibody mimetic technol limited to, small organic molecules (e.g., Cane et al. 1998. ogy that could be used in the context of the instant invention. Science 282:63), and natural product extract libraries. In US 2015/0330.997 A1 Nov. 19, 2015 47 another embodiment, the compounds are small, organic non modified nucleotides which can be used to generate the anti peptidic compounds. Like antibodies, these small molecule sense nucleic acid include 5-fluorouracil, 5-bromouracil, inhibitors indirectly or directly inhibit the activity of a marker 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-ace (s). tylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxym [0183] In another embodiment, the marker(s) modulator ethylaminomethyl-2-thiouridine, 5-carboxymethylaminom employed in the methods of the present invention is an anti ethyluracil, dihydrouracil, beta-D-galactosylqueosine, sense nucleic acid molecule that is complementary to a gene inosine, N6-isopentenyladenine, 1-methylguanine, 1-meth encoding a marker(s) or to a portion of that gene, or a recom ylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-meth binant expression vector encoding the antisense nucleic acid ylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, molecule. As used herein, an “antisense” nucleic acid com 7-methylguanine, 5-methylaminomethyluracil, 5-meth prises a nucleotide sequence which is complementary to a oxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, “sense” nucleic acid encoding a protein, e.g., complementary 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-meth to the coding strand of a double-stranded cDNA molecule, ylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), complementary to an mRNA sequence or complementary to wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-me the coding strand of a gene. Accordingly, an antisense nucleic thyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, acid can hydrogen bond to a sense nucleic acid. uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid [0184] The use of antisense nucleic acids to down-modu (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypro late the expression of a particular protein in a cell is well pyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, known in the art (see e.g., Weintraub, H. et al., Antisense RNA the antisense nucleic acid can be produced biologically using as a molecular tool for genetic analysis, Reviews–Trends in an expression vector into which a nucleic acid has been sub Genetics, Vol. 1(1) 1986; Askari, F. K. and McDonnell, W. M. cloned in an antisense orientation (i.e., RNA transcribed from (1996) N. Eng. J. Med. 334:316-318; Bennett, M. R. and the inserted nucleic acid will be of an antisense orientation to Schwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, a target nucleic acid of interest, described further in the fol D. and Cohen, J. S. (1995) Cancer Gene Ther. 2:47-59; Rossi, lowing subsection). J. J. (1995) Br Med. Bull. 51:217-225; Wagner, R. W. (1994) [0187] The antisense nucleic acid molecules that can be Nature 372:333-335). An antisense nucleic acid molecule utilized in the methods of the present invention are typically comprises a nucleotide sequence that is complementary to the administered to a subject or generated in situ such that they coding strand of another nucleic acid molecule (e.g., an hybridize with or bind to cellular mRNA and/or genomic mRNA sequence) and accordingly is capable of hydrogen DNA encoding a marker(s) to thereby inhibit expression by bonding to the coding strand of the other nucleic acid mol inhibiting transcription and/or translation. The hybridization ecule. Antisense sequences complementary to a sequence of can be by conventional nucleotide complementarity to form a an mRNA can be complementary to a sequence found in the stable duplex, or, for example, in the case of an antisense coding region of the mRNA, the 5' or 3' untranslated region of nucleic acid molecule which binds to DNA duplexes, through the mRNA or a region bridging the coding region and an specific interactions in the major groove of the double helix. untranslated region (e.g., at the junction of the 5' untranslated An example of a route of administration of antisense nucleic region and the coding region). Furthermore, an antisense acid molecules includes direct injection at a tissue site. Alter nucleic acid can be complementary in sequence to a regula natively, antisense nucleic acid molecules can be modified to tory region of the gene encoding the mRNA, for instance a target selected cells and then administered systemically. For transcription initiation sequence or regulatory element. Pref example, for systemic administration, antisense molecules erably, an antisense nucleic acid is designed so as to be can be modified such that they specifically bind to receptors complementary to a region preceding or spanning the initia or antigens expressed on a selected cell surface, e.g., by tion codon on the coding strandorin the 3' untranslated region linking the antisense nucleic acid molecules to peptides or of an mRNA. antibodies which bind to cell surface receptors or antigens. [0185] Antisense nucleic acids can be designed according The antisense nucleic acid molecules can also be delivered to to the rules of Watson and Crick base pairing. The antisense cells using vectors well known in the art and described in, for nucleic acid molecule can be complementary to the entire example, US20070111230 the entire contents of which are coding region of marker(s) mRNA, but more preferably is an incorporated herein. To achieve sufficient intracellular con oligonucleotide which is antisense to only a portion of the centrations of the antisense molecules, vector constructs in coding or noncoding region of marker(s) mRNA. For which the antisense nucleic acid molecule is placed under the example, the antisense oligonucleotide can be complemen control of a strong pol II or pol III promoter are preferred. tary to the region surrounding the translation start site of [0188] In yet another embodiment, the antisense nucleic marker(s) mRNA. An antisense oligonucleotide can be, for acid molecule employed by the methods of the present inven example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucle tion can include an O-anomeric nucleic acid molecule. An otides in length. Cº-anomeric nucleic acid molecule forms specific double [0186] An antisense nucleic acid can be constructed using stranded hybrids with complementary RNA in which, con chemical synthesis and enzymatic ligation reactions using trary to the usual fl-units, the strands run parallel to each other procedures known in the art. For example, an antisense (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). nucleic acid (e.g., an antisense oligonucleotide) can be The antisense nucleic acid molecule can also comprise a chemically synthesized using naturally occurring nucleotides 2'-O-methylribonucleotide (Inoue et al. (1987) Nucleic Acids or variously modified nucleotides designed to increase the Res. 15:6131-6148) or a chimeric RNA-DNA analogue (In biological stability of the molecules or to increase the physi oue et al. (1987) FEBS Lett. 215:327-330). cal stability of the duplex formed between the antisense and [0189] In another embodiment, an antisense nucleic acid sense nucleic acids, e.g., phosphorothioate derivatives and used in the methods of the present invention is a compound acridine substituted nucleotides can be used. Examples of that mediates RNAi. RNA interfering agents include, but are US 2015/0330.997 A1 Nov. 19, 2015 48 not limited to, nucleic acid molecules including RNA mol introduced by the technology described in Deiters et al., JAm ecules which are homologous to a marker(s) or a fragment Chem Soc 125:11782-11783, 2003; Wang and Schultz, Sci thereof, “short interfering RNA” (siRNA), “short hairpin” or ence 301:964-967, 2003; Wang et al., Science 292:498–500, “small hairpin RNA” (shBNA), and small molecules which 2001; Zhanget al., Science 303:371-373, 2004 or in U.S. Pat. interfere with or inhibit expression of a target gene by RNA No. 7,083,970. Briefly, some of these expression systems interference (RNAi). RNA interference is a post-transcrip involve site-directed mutagenesis to introduce a nonsense tional, targeted gene-silencing technique that uses double codon, such as an amber TAG, into the open reading frame stranded RNA (dsRNA) to degrade messenger RNA (mRNA) encoding a polypeptide of the invention. Such expression containing the same sequence as the dsRNA (Sharp, P.A. and vectors are then introduced into a host that can utilize a tRNA Zamore, P. D. 287, 2431-2432 (2000); Zamore, P. D., et al. specific for the introduced nonsense codon and charged with Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, the nonnatural amino acid of choice. Particular nonnatural 3.19.1-3197 (1999)). The process occurs when an endogenous amino acids that are beneficial for purpose of conjugating ribonuclease cleaves the longer dsRNA into shorter, 21- or moieties to the polypeptides of the invention include those 22-nucleotide-long RNAs, termed small interfering RNAs or with acetylene and azido side chains. Marker(s) polypeptides siRNAs. The smaller RNA segments then mediate the degra containing these novel amino acids can then be pegylated at dation of the target mRNA. Kits for synthesis of RNAi are these chosen sites in the protein. commercially available from, e.g., New England Biolabs and (0195] 2. Stimulatory Agents Ambion. In one embodiment one or more of the chemistries [0196] According to a modulatory method of the invention, described above for use in antisense RNA can be employed. the expression and/or activity of a marker(s) is stimulated in [0190] In still another embodiment, an antisense nucleic a cell or subject by contacting the cell with (or administering acid is a ribozyme. Ribozymes are catalytic RNA molecules to a subject) a stimulatory agent. Stimulatory agents of the with ribonuclease activity which are capable of cleaving a invention can be, for example, molecules that act to stimulate single-stranded nucleic acid, such as an mRNA, to which they or increase the expression and/or activity of the marker(s). have a complementary region. Thus, ribozymes (e.g., ham [0197) Examples of such stimulatory agents include active merhead ribozymes (described in Haselhoff and Gerlach, marker(s) polypeptide and nucleic acid molecules encoding 1988, Nature 334:585-591) can be used to catalytically cleave the marker(s) that are introduced into the cell to increase marker(s) mRNA transcripts to thereby inhibit translation of expression and/or activity of the marker in the cell. A pre the marker(s) mRNA. ferred stimulatory agent is a nucleic acid molecule encoding (0191] Alternatively, gene expression can be inhibited by a marker(s) polypeptide, wherein the nucleic acid molecule is targeting nucleotide sequences complementary to the regula introduced into the cell in a form suitable for expression of the tory region of a marker(s) (e.g., the promoter and/or enhanc active marker(s) polypeptide in the cell. To express a marker ers) to form triple helical structures that prevent transcription (s) polypeptide in a cell, typically a marker(s)-encoding of the marker(s) gene. See generally, Helene, C., 1991, Anti cDNA (full length or partial clNA sequence) is first intro cancer Drug Des. 6(6):569-84; Helene, C. et al., 1992, Ann. duced into a recombinant expression vector using standard N.Y. Acad. Sci. 660:27-36; and Maher, L. J., 1992, Bioassays molecular biology techniques, and the vector may be trans 14(12):807-15. fected into cells using standard molecular biology tech [0192] In another embodiment, the marker(s) modulator niques. A cljRNA can be obtained, for example, by amplifica used in the methods of the present invention is a fusion protein tion using the polymerase chain reaction (PCR), using or peptidic compound derived from the marker(s) amino acid primers based on the marker(s) nucleotide sequence or by sequence. In particular, the inhibitory compound comprises a screening an appropriate cljRNA library. fusion protein or a portion of a marker(s) (or a mimetic [0198] The nucleic acids for use in the methods of the thereof) that mediates interaction of the marker(s) with a invention can also be prepared, e.g., by standard recombinant target molecule such that contact of the marker(s) with this DNA techniques. A nucleic acid of the invention can also be fusion protein or peptidic compound competitively inhibits chemically synthesized using standard techniques. Various the interaction of the marker(s) with the target molecule. Such methods of chemically synthesizing polydeoxynucleotides fusion proteins and peptidic compounds can be made using are known, including solid-phase synthesis which has been standard techniques known in the art. For example, peptidic automated in commercially available DNA synthesizers (See compounds can be made by chemical synthesis using stan e.g., Itakura et al. U.S. Pat. No. 4,598,049; Caruthers et al. dard peptide synthesis techniques and then introduced into U.S. Pat. No. 4,458,066; and Itakura U.S. Pat. Nos. 4,401,796 cells by a variety of means known in the art for introducing and 4,373,071, incorporated by reference herein). peptides into cells (e.g., liposome and the like). [0199] In one embodiment, a nucleic acid molecule encod [0193] The in vivo half-life of the fusion protein or peptidic ing a marker(s) may be present in an inducible construct. In compounds of the invention can be improved by making another embodiment, a nucleic acid molecule encoding peptide modifications, such as the addition of N-linked gly marker(s) may be present in a construct which leads to con cosylation sites into the marker(s) or conjugating the marker stitutive expression. In one embodiment, a nucleic acid mol (s) to poly(ethylene glycol) (PEG, pegylation), e.g., via ecule encoding marker(s) may be delivered to cells, or to lysine-monopegylation. Such techniques have proven to be subjects, in the absence of a vector. beneficial in prolonging the half-life of therapeutic protein [0200) A nucleic acid molecule encoding marker(s) may be drugs. It is expected that pegylation of marker(s) polypep delivered to cells or to subjects using a viral vector, preferably tides of the invention may result in similar pharmaceutical one whose use for gene therapy is well known in the art. advantages. Techniques for the formation of vectors or virions are gener [0194] In addition, pegylation can be achieved in any part ally described in “Working Toward Human Gene Therapy.” of a polypeptide of the invention by the introduction of a Chapter 28 in Recombinant DNA, 2nd Ed., Watson, J. D. et nonnatural amino acid. Certain nonnatural amino acids can be al., eds., New York: Scientific American Books, pp. 567-581 US 2015/0330.997 A1 Nov. 19, 2015 49

(1992). An overview of suitable viral vectors or virions is visna-maedi, which causes encephalitis (visna) or pneumonia provided in Wilson, J. M., Clin. Exp. Immunol. 107(Suppl. (maedi) in sheep; the caprine arthritis-encephalitis virus, 1):31-32 (1997), as well as Nakanishi, M., Crit. Rev. Thera which causes immune deficiency, arthritis, and encephalopa peu. Drug Carrier Systems 12:263-310 (1995); Robbins, P. thy in goats; equine infectious anemia virus (EIAV), which D., et al., Trends Biotechnol. 16:35-40 (1998); Zhang, J., et causes autoimmune hemolytic anemia, and encephalopathy al., Cancer Metastasis Rev. 15:385-401(1996); and Kramm, in horses; feline immunodeficiency virus (FIV), which causes C.M., et al., Brain Pathology 5:345-381 (1995). Such vectors immune deficiency in cats; bovine immune deficiency virus may be derived from viruses that contain RNA (Vile, R. G., et (BIV), which causes lymphadenopathy, lymphocytosis, and al., Br. Med Bull. 51:12-30 (1995)) or DNA (Ali M., et al., possibly central nervous system infection in cattle; and sim Gene Ther. 1:367-384 (1994)). ian immunodeficiency virus (SIV), which cause immune [0201] Examples of viral vector systems utilized in the deficiency and encephalopathy in sub-human primates. Dis gene therapy art and, thus, suitable for use in the present eases caused by these viruses are characterized by a long invention, include the following: retroviruses (Vile, R. G., incubation period and protracted course. Usually, the viruses supra; U.S. Pat. Nos. 5,741,486 and 5,763,242); adenoviruses latently infect monocytes and macrophages, from which they (Brody, S. L., et al., Ann N.Y. Acad. Sci. 716:90-101 (1994); Heise, C. et al., Nat. Med. 3:639-645 (1997)); adenoviral/ spread to other cells. HIV, FIV, and SIV also readily infect T retroviral chimeras (Bilbao, G., et al., FASEB J. 11:624-634 lymphocytes (i.e., T-cells). In one embodiment of the inven (1997); Feng, M., et al., Nat. Biotechnol. 15:866-870 (1997)); tion, the lentivirus is not HIV. adeno-associated viruses (Flotte, T. R. and Carter, B.J., Gene [0204] As used herein, the term “adenovirus” (“Ad”) refers Ther. 2:357-362 (1995); U.S. Pat. No. 5,756,283); herpes to a group of double-stranded DNA viruses with a linear simplex virus I or II (Latchman, D. S., Mol. Biotechnol. genome of about 36 kb. See, e.g., Berkner et al., Curr. Top. 2:179-195 (1994); U.S. Pat. No. 5,763,217; Chase, M., et al., Microbiol. Immunol., 158: 39-61 (1992). In some embodi Nature Biotechnol. 16:444-448 (1998)); parvovirus (Shaugh ments, the adenovirus-based vector is an Ad-2 or Ad-5 based nessy, E., et al., Semin Oncol. 23:159-171 (1996)); reticu vector. See, e.g., Muzyczka, Curr. Top. Microbiol. Immunol., loendotheliosis virus (Donburg, R., Gene Therap. 2:301-310 158: 97-123, 1992; Aliet al., 1994 Gene Therapy 1: 367-384; (1995)). Extrachromosomal replicating vectors may also be U.S. Pat. Nos. 4,797,368, and 5,399,346. Suitable adenovirus used in the gene therapy methods of the present invention. vectors derived from the adenovirus strain Ad type 5 d1324 or Such vectors are described in, for example, Calos, M. P. other strains of adenovirus (e.g., Ad2, Adž, Ad7 etc.) are well (1996) Trends Genet. 12:463-466, the entire contents of known to those skilled in the art. Recombinant adenoviruses which are incorporated herein by reference. Other viruses that are advantageous in that they do not require dividing cells to can be used as vectors for gene delivery include poliovirus, be effective gene delivery vehicles and can be used to infect a papillomavirus, vaccinia virus, lentivirus, as well as hybrid or wide variety of cell types. Additionally, introduced adenovi chimeric vectors incorporating favorable aspects of two or rus DNA (and foreign DNA contained therein) is not inte more viruses (Nakanishi, M. (1995) Crit. Rev. Therapeu. grated into the genome of a host cell but remains episomal, Drug Carrier Systems 12:263-310; Zhang, J., et al. (1996) thereby avoiding potential problems that can occur as a result Cancer Metastasis Rev. 15:385-401; Jacoby, D. R., et al. of insertional mutagenesis in situations where introduced (1997) Gene Therapy 4:1281-1283). DNA becomes integrated into the host genome (e.g., retrovi [0202] The term “AAV vector” refers to a vector derived ral DNA). Moreover, the carrying capacity of the adenovirus from an adeno-associated virus serotype, including without genome for foreign DNA is large (up to 8 kilobases) relative limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, or to other gene delivery vectors (Haj-Ahmand et al. J. Virol. 57, AAVX7. “rAAV vector” refers to a vector that includes AAV 267-273 [1986]). nucleotide sequences as well as heterologous nucleotide sequences. r?AV vectors require only the 145 base terminal [0205] In one embodiment, an adenovirus is a replication repeats in cis to generate virus. All other viral sequences are defective adenovirus. Most replication-defective adenoviral dispensable and may be supplied in trans (Muzyczka (1992) vectors currently in use have all or parts of the viral E1 and E3 CUM Topics Microbiol. Immunol. 158:97). Typically, the genes deleted but retain as much as 80% of the adenovirus rAAV vector genome will only retain the inverted terminal genetic material. Adenovirus vectors deleted for all viral cod repeat (ITR) sequences so as to maximize the size of the ing regions are also described by Kochanek et al. and Cham transgene that can be efficiently packaged by the vector. The berlain et al. (U.S. Pat. No. 5,985,846 and U.S. Pat. No. ITRs need not be the wild-type nucleotide sequences, and 6,083,750). Such viruses are unable to replicate as viruses in may be altered, e.g., by the insertion, deletion or substitution the absence of viral products provided by a second virus, of nucleotides, as long as the sequences provide for functional referred to as a “helper” virus. rescue, replication and packaging. In particular embodi [0206] In one embodiment, an adenoviral vector is a “gut ments, the AAV vector is an AAV2/5 or AAV2/8 vector. Suit less” vector. Such vectors contain a minimal amount of aden able AAV vectors are described in, for example, U.S. Pat. No. ovirus DNA and are incapable of expressing any adenovirus 7,056,502 and Yan et al. (2002) J. Virology 76(5):2043-2053, antigens (hence the term “gutless”). The gutless replication the entire contents of which are incorporated herein by refer defective Ad vectors provide the significant advantage of €11Cè. accommodating large inserts of foreign DNA while com [0203] As used herein, the term “lentivirus” refers to a pletely eliminating the problem of expressing adenoviral group (or genus) of retroviruses that give rise to slowly devel genes that result in an immunological response to viral pro oping disease. Viruses included within this group include teins when a gutless replication defective Advectoris used in HIV (human immunodeficiency virus; including but not lim gene therapy. Methods for producing gutless replication ited to HIV type 1 and HIV type 2), the etiologic agent of the defective Ad vectors have been described, for example, in human acquired immunodeficiency syndrome (AIDS); U.S. Pat. No. 5,981,225 to Kochaneket al., and U.S. Pat. Nos. US 2015/0330.997 A1 Nov. 19, 2015 50

6,063,622 and 6,451,596 to Chamberlain et al; Parks et al., reagents such as, for example, LIPOFECTINR (Invitrogen PNAS 93:13565 (1996) and Lieber et al., J. Virol. 70.8944 Corp., San Diego, Calif.), LIPOFECTAMINER (Invitrogen), 8960 (1996). FUGENER (Roche Applied Science, Basel, Switzerland), [0207] In another embodiment, an adenoviral vector is a JETPEITM (Polyplus-transfection Inc., New York, N.Y.), “conditionally replicative adenovirus” (“CRAds”). CRAds EFFECTENER (Qiagen, Valencia, Calif.), DREAMFECTTM are genetically modified to preferentially replicate in specific (OZ Biosciences, France) and the like), or electroporation cells by either (i) replacing viral promoters with tissue spe (e.g., in vivo electroporation). Suitable methods for trans cific promoters or (ii) deletion of viral genes important for forming or transfecting host cells can be found in Sambrook, replication that are compensated for by the target cells only. et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., The skilled artisan would be able to identify epithelial cell Cold Spring harbor Laboratory, Cold Spring Harbor Labora specific promoters. tory Press, Cold Spring Harbor, N.Y., 1989), and other labo ratory manuals. [0208] Other art known adenoviral vectors may be used in [0214] In one embodiment, a marker(s) is delivered to a the methods of the invention. Examples include Ad vectors subject or cells in the form of a peptide or protein. In order to with recombinant fiber proteins for modified tropism (as produce such peptides or proteins, recombinant expression described in, e.g., van Beusechem et al., 2000 Gene Ther. 7: vectors of the invention can be designed for expression of one 1940-1946), protease pre-treated viral vectors (as described or more marker(s) proteins, and/or portion(s) thereof in in, e.g., Kuriyama et al., 2000 Hum. Gene Ther. 11: 2219 prokaryotic or eukaryotic cells. For example, one or more 2230), E2a temperature sensitive mutant Ad vectors (as glucose transporter proteins and/or portion(s) thereof can be described in, e.g., Engelhardt et al., 1994 Hum. Gene Ther. 5: expressed in bacterial cells such as E. coli, insect cells (using 1217-1229), and “gutless” Ad vectors (as described in, e.g., baculovirus expression vectors) yeast cells or mammalian Armentano et al., 1997 J. Virol. 71: 2408-2416: Chen et al., cells. Suitable host cells are discussed further in Goeddel, 1997 Proc. Nat. Acad. Sci. USA 94; 1645-1650; Schieder et Gene Expression Technology: Methods in Enzymology 185, al., 1998 Nature Genetics 18: 180-183). Academic Press, San Diego, Calif. (1990). Alternatively, the [0209] The vector will include one or more promoters or recombinant expression vector can be transcribed and trans enhancers, the selection of which will be known to those lated in vitro, for example using T7 promoter regulatory skilled in the art. Suitable promoters include, but are not sequences and T7 polymerase. limited to, the retroviral long terminal repeat (LTR), the SV40 [0215] In one embodiment, the recombinant mammalian promoter, the human cytomegalovirus (CMV) promoter, and expression vector is capable of directing expression of the other viral and eukaryotic cellular promoters known to the nucleic acid preferentially in a particular cell type (e.g., tis skilled artisan. sue-specific regulatory elements are used to express the [0210] Guidance in the construction of gene therapy vec nucleic acid). Tissue-specific regulatory elements are known tors and the introduction thereof into affected subjects for in the art. Non-limiting examples of suitable tissue-specific therapeutic purposes may be obtained in the above-refer promoters include retinal cell-type-specific promoters (e.g., enced publications, as well as in U.S. Pat. Nos. 5,631,236, rhodopsin regulatory sequences, Cabp5, Cralbp, Nrl, Crx, 5,688,773, 5,691,177, 5,670,488, 5,529,774, 5,601,818, and Ndrg4, clusterin, Rax, Hesl and the like (Matsuda and Cepko, PCT Publication No. WO 95/06486, the entire contents of supra)), the albumin promoter (liver-specific, Pinkert et al. which are incorporated herein by reference. (1987) Genes Dev. 1:268), neuron-specific promoters (e.g., [0211] Generally, methods are known in the art for viral the neurofilament promoter; Byrne and Ruddle (1989) Proc. infection of the cells of interest. The virus can be placed in Natl. Acad. Sci. U.S.A. 86:5473). Developmentally-regu contact with the cell of interest or alternatively, can be lated promoters are also encompassed, for example the Cº-fe injected into a subject suffering from a retinal disorder, for toprotein promoter (Campes and Tilghman (1989) Genes example, as described in U.S. Provisional Patent Application Dev. 3:537). No. 61/169,835 and PCT Application No. PCT/US09/ [0216] Application of the methods of the invention for the 053730, the contents of each of which are incorporated by treatment and/or prevention of a retinal disorder can result in reference. curing the disorder, decreasing at least one symptom associ ated with the disorder, either in the long term or short term or [0212| Gene therapy vectors comprising a nucleic acid simply a transient beneficial effect to the subject. Accord molecule encoding a marker(s) can be delivered to a subject ingly, as used herein, the terms “treat,” “treatment” and “treat or a cell by any suitable method in the art, for example, ing” include the application or administration of agents, as intravenous injection, local administration, e.g., application described herein, to a subject who is suffering from a retinal of the nucleic acid in a gel, oil, or cream, (see, e.g., U.S. Pat. disorder, or who is susceptible to such conditions with the No. 5,328,470), stereotactic injection (see, e.g., Chen et al. purpose of curing, healing, alleviating, relieving, altering, (1994) Proc. Natl. Acad. Sci. U.S.A. 91:3054), gene gun, or remedying, ameliorating, improving or affecting such condi by electroporation (see, e.g., Matsuda and Cepko (2007) tions or at least one symptom of such conditions. As used Proc. Natl. Acad. Sci. U.S.A. 104:1027), using lipid-based herein, the condition is also “treated” if recurrence of the transfection reagents, or by any other suitable transfection condition is reduced, slowed, delayed or prevented. method. [0217. A modulatory agent, such as a chemical compound, [0213] As used herein, the terms “transformation” and can be administered to a subject as a pharmaceutical compo “transfection” are intended to refer to a variety of art-recog sition. Such compositions typically comprise the modulatory nized techniques for introducing foreign nucleic acid (e.g., agent and a pharmaceutically acceptable carrier. As used DNA) into a host cell, including calcium phosphate or cal herein the term “pharmaceutically acceptable carrier” is cium chloride co-precipitation, DEAE-dextran-mediated intended to include any and all solvents, dispersion media, transfection, lipofection (e.g., using commercially available coatings, antibacterial and antifungal agents, isotonic and US 2015/0330.997 A1 Nov. 19, 2015 absorption delaying agents, and the like, compatible with second sample relative to the first sample, e.g., a statistically pharmaceutical administration. The use of such media and significant level, identifies the protein as a type 2 diabetes agents for pharmaceutically active substances is well known marker. in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the IV. Kits of the Invention compositions is contemplated. Supplementary active com [0222] The invention also provides kits for determining pounds can also be incorporated into the compositions. Phar whether a subject has or will develop impaired glucose toler maceutical compositions can be prepared as described above. ance and/or whether a subject has or will develop type 2 [0218] E. Methods of Identifying Type 2 Diabetes Biomar diabetes. Kits to determine whether a subject will develop kers type 2 diabetes complications, to determine whether a treat [0219) The present invention further provides methods for ment will be efficacious fortreting a subject having impaired identifying type 2 diabetes biomarkers useful as markers for, glucose tolerance and/or type 2 diabetes and kits for moni e.g., disease (prognostics and diagnostics), therapeutic effec toring the effectiveness of a treatment are also provided. tiveness of a drug (theranostics) and of drug toxicity. For [0223] These kits include means for determining the level example, as described above, the markers described herein of one or more markers of the invention and instructions for and the markers identified using the methods for biomarker use of the kit. discovery are useful for, e.g., determining whether a subject [0224] The kits of the invention may optionally comprise has or will develop impaired glucose tolerance; determining additional components useful for performing the methods of whether a subject has or will develop type 2 diabetes; deter the invention. By way of example, the kits may comprise mining whether a subject having type 2 diabetes will respond reagents for obtaining a biological sample from a subject, a to a diabetic therapy; monitoring the effectiveness of a control sample, one or more sample compartments, a diabetic therapy for inhibiting the development of impaired glucose therapeutic, an instructional material which describes perfor tolerance and/or type 2 diabetes, reducing or slowing down mance of a method of the invention and tissue specific con the progression of normal glucose tolerance to impaired fast trols/standards. ing glycaemia, to impaired glucose tolerance, and/or to dia [0225] The reagents for determining the level of one or betes, and/or reducing or inhibiting the development of com more marker(s) can include, for example, buffers or other plications associated with the disease in a subject; in reagents for use in an assay for evaluating the level of one or screening assays to identify molecules which modulate, e.g., more markers, e.g., expression level (e.g., at either the mRNA decrease or increase, the expression and/or activity of a mark or protein level). The instructions can be, for example, printed er(s) of the invention for e.g., use as therapeutics. instructions for performing the assay for evaluating the level [0220) Methods for identifying a type 2 diabetes marker are of one or more marker(s) of the invention. described in the working examples and include identifying [0226] The reagents for isolating a biological sample from proteins in the secretory vesicles of two or more organs from a subject can comprise one or more reagents that can be used two or more species under steady state conditions, identifying to obtain a fluid or tissue from a subject, such as means for proteins in the secretory vesicles of pancreatic f; cells thereby obtaining a saliva or blood. generating a provisional list of steady state markers, identi [0227] The kits of the invention may further comprise fying the markers in the provisional list of steady state mark reagents for culturing a sample obtained from a subject. ers from the two or more organs from the two or more species [0228] Preferably, the kits are designed for use with a common to the markers in the secretory vesicles of pancreatic human subject. f; cells and removing those markers from the provisional list [0229] The present invention is further illustrated by the of steady state markers, thereby generating a list of 5 cell following examples which should not be construed as further mass markers; identifying proteins in the secretory vesicles of limiting. The contents of all references, patents and published pancreatic f; cells under dysfunctional conditions, identifying patent applications cited throughout this application, as well proteins in the secretory vesicles of pancreatic f; cells under as the Figures, are expressly incorporated herein by reference normal conditions, identifying the proteins that were differ in their entirety. entially expressed under dysfunctional conditions and under normal conditions, thereby generating a provisional list off Examples cell function markers, determining the level of a fl cell mass marker and/or a fl cell function marker in a sample form a Example I control subject, e.g., a having normal glucose tolerance, determining the level of the marker in a test sample from a Biomarker Identification subject having, e.g., impaired glucose tolerance and/or type 2 diabetes. A difference in the level of a marker in the control Materials and Methods sample as compared to the level in the test sample, e.g., a [0230] Candidate biomarkers were identified by evaluating statistically significant level, identifies the marker as a type 2 proteins known or suspected to be secreted by pancreatic beta diabetes biomarker. islets. [0221) A type 2 diabetes marker may also be identified by [0231] Three in vitro systems were used to identify secre determining the level of a protein in a first sample obtained tory protein candidate biomarkers, primary human islets and from a subject having type 2 diabetes prior to providing at 2 pancreatic fl-cell lines. The primary human islets were least a portion of atherapy to the subject, and determining the obtained from donors lacking major medical problems. Table level of a protein in a second sample obtained from the subject 5 lists the characteristics of the donors. The cell lines used following provision of at least a portion of the therapy. A were the rat INS832/13 and the mouse MIN6. The experi difference in the level of expression of the protein in the mental systems were analyzed using two conditions, steady US 2015/0330.997 A1 Nov. 19, 2015 52 state, and during an experimental dysfunctional state acterization of the starting cell line homogenates and secre designed to mimic the pancreatic beta cell dysfunction tory protein final products were done using antibodies against observed in type 2 diabetes. proteins expressed in specific subcellular compartments, TABLE 5

Pancreatic islet donor characteristics.

DOnor VP146 VP149 VP151 VP152 Paraskevas VP157 VP166 VP167

Gender F F M F M F M F Age 43 44 59 35 29 26 59 50 Ethnicity Caucasian African Caucasian Caucasian Caucasian African Caucasian African American American American Ht (cm) 172.5 149.9 cm 175 157.5 1.59 170 157.5 Wt (kg) 104 kg 66.8 kg 84.5 80.6 84 61.8 93 BMI 34.9 29.6 27 31.6 22.1 33 20.7 36.3 Cause Anoxic ICH Head Head Anoxic HT/BI CVA/ CVA/ Of brain träu??lä. träumä brain secondary to ICH ICH death injury injury MVA Smoking IIC) quit 13 Occasional Yes 9 <1 ppd Occasional NO y ago (cigar) (1 ppd) (cigar) EtOH 2-3/wk 1 glass Occasional NO 9 rare, *1 Occasional NO wive/day month Serologies Neg Neg CMV+ CMV+, EBV+ None Neg EBV-- Neg Medicines None None None None None None None None Disease None None None None None None None None

ICH; Intracerebral hemorrhage., HT/BI; Head Trauma/brain injury MVA; Motor vehicle accident, EBV+; Epstein-Barr virus positive, CMV, Cytomegalovirus positive

[0232] For identification of proteins secreted during steady such as the plasma membrane, endoplasmic reticulum (ER), state, the cell lines were cultivated in RPMI containing 5 mM Golgi apparatus, and mitochondria. Both membrane-bound glucose and the primary human islets were kept in saline at 4° and soluble proteins associated with these compartments C. until secretory vesicle sample preparation. For identifica were used, to assess the relative enrichment of potentially tion of proteins secreted during a dysfunctional state, the secreted proteins from the relevant subcellular compartments experimental systems were incubated with 20 mM glucose/ in the preparations. FIG. 1 depicts Western blots of starting 0.4 mM palmitate or with 25 mM glucose/0.4 mM palmitate materials (Hom), intermediate (SV) and final product (SC) (El-Assaad et al. (2010) Endocrinology 151:3061-73) until preparations of secreted proteins from a rat cell line (A) and insulin production was reduced by at least 30% and pro human primary islets (B). grammed cell death was induced, events that typically [0234] An additional set of secretory protein samples were occurred between 16-24 hours for the cell lines and between prepared from a selection of major organs or from organs 36-72 hours for the primary islets. known to become involved in diabetes disease progression [0233] Secretory protein preparations from both steady and complications, using the process described above, sub state and dysfunctionalized islets and cell lines were made stituting more robust mechanical tissue disruption for the using the same process. At least 4 independent replicates were more fibrous organs. To generate the human organ secretome used per experimental system. The cultured cells were har dataset, secretory proteins from lung, breast, kidney, prostate, vested by scraping, centrifuged for 5 minutes at 4°C. at 1400 bladder, and colon were prepared. For the rat dataset, secre rpm to remove debris and resuspended in homogenization tory proteins from heart, liver, kidney, skeletal muscle, sub buffer (250 mM sucrose/10 mM Tris pH 7.4/protease inhibi cutaneous fat, and whole pancreas were prepared. This tor EDTA-free cocktail). The islet or cell line suspensions experiment was done in order to identify the secretory pro were homogenized using a Dounce homogenizer. The homo teins that can also be made by other tissues than the primary genate was adjusted to 1.4M sucrose. A 14 ml SW40Ti ultra islets or the beta cell lines. Secretory proteins that can be clear centrifuge tube (Beckman Coulter #344060) was lay made by multiple tissues would thus likely have relatively ered with homogenate followed by 4 ml of 1.2M sucrose and less tissue specificity, and would thus be de-prioritized as topped with 0.8M sucrose. The samples were centrifuged for biomarker candidates. 2 hours at 155,000 g at 4°C. and vesicles were harvested from [0235] Once the secretory protein samples were generated the 0.8–1.2M interface. The vesicles were washed in 0.5M they were further processed for mass spectrometry data KCl followed by incubation in ammonium carbonate pH11. acquisition and peptide and protein identification. Briefly, the Vesicle content was separated from the vesicle membranes by samples were digested with trypsin to generate peptides. The centrifugation at 112,000 g. Protein yields were measured peptides were then separated by strong cation exchange chro using the BCA Protein Assay (Pierce #23227). In instances matography (SCX) into three fractions. Each of the three where sample was limiting, the entire secretory vesicle was fractions per sample was analyzed by reversed phase liquid processed for mass spectrometry analysis. Western blot char chromatography, coupled by electrospray to a Waters QTOF US 2015/0330.997 A1 Nov. 19, 2015

mass spectrometer (LC-MS). Components were detected and TABLE 6 matched across all samples and compared for relative peak intensity. Peak intensity was normalized to account for small Characteristics of subjects used for verification differences in protein concentration between samples. of BCM/BCF candidate biomarkers ANOVA was then applied to identify peptides that were dif Cohort Samples ferentially expressed between the groups of interest in the Normoglycemic subject: NGT 47 samples derived from dysfunctionalized islets or cell lines. Normoglycemic subject: IGT 17 High stringency thresholds were used to ensure the statistical Long term T1D (insulin - 5 yrs) 19 significance of the identified peptides. All intensity values Long term T2D (insulin - 5 yrs) 28 were log (base e) transformed with values <0 replaced by 0. A subset of the samples was used to create an average sample (i.e., the Reference sample) against which all samples were then normalized. The normalization factors were chosen so TABLE 7 that the median of log ratios between each sample and the Additional subjects used for verification of Reference sample over all the peptides was adjusted to zero. BCM/BCF/TEM candidate biomarkers Peptide identification was done with custom protein database ALL SUBJECTS using Mascot (Matrix Science) software. Candidate biomar Number Age Age BMI BMI ker annotation was done using a combination of manual lit of subjects range median range median erature review and network and pathway analysis (Ingenuity). Controls 50 18–74 40 18–30 24 [0236] Several thousand proteins were identified in the Diabetics High BMI 69 24-66 51 39–74 58 secretomes of the primary islets, cell lines, and organs in the Pre-Diabetics High BMI 79 19-64 40 37-75 60 steady state. The secretory proteins identified in the islets or Diabetics Lower BMI 50 26-71 52 33–40 39 cell lines that were also found in the organ secretomes were Pre-Diabetics Lower BMI 47 30-62 41 32–40 38 removed. The remaining proteins were ordered to identify which subset was expressed eitherin the primary humanislets alone, or also in at least one of the cell lines. A total of 170 Results proteins met these criteria, and these proteins therefore con stituted the initial steady state biomarker dataset. [0239] A. Type 2 Diabetes Biomarker Identification [0237] A similar process was used to identify the initial [0240] Three datasets were generated based on the methods dysfunctionalized biomarker dataset. An additional require described above. The first dataset was an extensive catalog of ment to the two previously described criteria was that any of secretory vesicle content proteins prepared from 6 different the candidate biomarkers also be differentially expressed by human organs. The second dataset contained the correspond at least 1.5-fold in the dysfunctional state compared to con ing list of secretory vesicle content proteins from 6 rat organs. trol. A total of 245 proteins met the criteria and these proteins The third dataset was a catalogue of the steady state secretory therefore constituted the initial dysfunctionalized biomarker vesicle content proteins from each of the 3 experimental dataset. systems. The proteins common to the organ secretome data [0238| Subjects used for the plasma-based biomarker veri base and to any one of the experimental systems were then fication analyses are indicated in Tables 6 and 7. Plasma was removed from the experimental system datasets, leaving the processed using 3 different methods. First, common high secreted proteins more likely to be uniquely expressed by abundance plasma proteins were removed using affinity chro fl-cells or fl-islets. Over two thousand proteins were identified matography methods. Removing the most abundant plasma for each species, and on the order of one thousand proteins proteins allowed less abundant plasma proteins to be more were identified from the secretory vesicle contents of the readily measured. Some of the biomarker candidates, how rodent fl-cell lines or primary human fl-islets. Between half ever, were expected to be present beneath the current level of and 3% of these proteins appeared to be also expressed by at detection of the MRM-MS assays deployed. To measure can least one of the organ secretomes. Removal of these com didates from this low abundance class of biomarkers com monly expressed proteins resulted in the fl-cell mass candi mercially available ELISA kits were used. Lastly, plasma was date biomarkers. These candidates were then examined in processed to enrich for exosomes. Exosomes are small detail to prioritize them for further analysis. vesicles that are secreted whole by numerous cell types under normal and disease conditions. Originally described in [0241] The initial analysis indicated a modest overlap in the immune and central nervous system interactions, exosomes net secretome proteins identified from the 3 experimental have since been described to be produced by multiple tissue systems, suggesting only a partial correspondence between types, and are present in multiple different body fluids includ the cell line systems and the primary islets. While that finding ing plasma. Exosomes and are now understood to be part of a may not have been surprising, a similarly modest overlap general, widely used secretion mechanism. observed between the two cell lines was not expected, and Sequential high speed centrifugation methods were used to may indicate distinct physiological states for the cell lines. enrich the exosomes present in blood (Graner M W et al. [0242] The proteins identified were assessed for biological (2009) FASEB.J. 23:1541), and this method was used to make function and network and pathway connections through exosome preparations from the majority of clinical samples manual literature review and networking software analysis. obtained. Analysis of these preparations was expected to test Relatively stringent criteria were used to denote protein to the performance of biomarker that would not otherwise be protein relationships, such as a known direct link between any detected, including low abundance proteins but also mem two proteins be already established, as well as statistical brane associated proteins not expected to be readily solubi significance that the biological functions or pathways that lized in blood. appear to be over-represented be so by greater than chance US 2015/0330.997 A1 Nov. 19, 2015 54 alone. The dataset subset that met these criteria contained a pies to be included. The number of subjects and their treat considerable number of proteins (152). ment regimes at the time of recruitment are indicated in Table [0243| Additional assessments for candidate biomarkers 8. prioritization were to establish tissue specificity, which was done using histochemical assessment of the expression of the TABLE 8 candidate biomarker proteins in the pancreas and in other Characteristics of subjects used for discovery organs. This analysis suggested that a significant proportion of treatment monitoring candidate biomarkers of the higher ranked candidate biomarkers identified had relatively restricted tissue expression, typically to pancreatic Number of samples islets, or if they were also expressed in other tissues, they were found with typically lesser expression in the central nervous Treatment option Baseline Week 2 Total system. A subset of these markers had also been detected in Metformin initiation 12 12 24 Metformin + Sulfonyurea 12 11 23 human body fluids, indicating that these proteins were also Metformin + Sulfonyurea + DPP4 5 5 10 secreted. At the end of the analysis, 200 proteins were priori inhibitor tized and these candidate biomarkers are listed in Table 1 ([? Metformin + DPP4 inhibitor 4 4 8 cell mass (BCM) markers). Metformin + Sulfonyurea + Insulin 9 9 18 [0244] Proteins secreted by the tissues of interest under Total number of samples 42 41 83 steady state conditions may change under stress or under dysfunctional states. Secretion of particular proteins under these conditions may become upregulated or down regulated. [0248] The plasma samples from these subjects were Furthermore, proteins not normally secreted in steady state depleted of high abundance proteins and analyzed. The dif ferentially expressed proteins identified were then associated may become secreted under stress. Identification of these with the available clinical data to identify protein biomarker changes to define biomarker candidates associated with candidates associated with prediction of response (analysis fl-cell and fl-islet function was also performed. using the pre-dose samples) or monitoring of response [0245] The fl-cell lines and primary human fl-islets were (analysis using the post-treatment initiation samples). The incubated with vehicle or with a glucolipotoxic treatment therapeutic efficacy biomarker (TEM) candidates are listed in (described above) for defined periods till the dysfunction Table 3. described earlier was obtained. Following the treatment, [0249] Approximately 150 proteins were identified that secretory vesicle content sample preparation and proteomic were significantly differentially expressed in at least one data acquisition and analysis was executed as above. Several treatment response comparison. Differences were observed hundred proteins that became differentially expressed after in the pre-dose samples of the eventual responders versus the the glucolipotoxic treatment were identified. Subtraction of eventual non-responders. Furthermore, the differences the proteins in common with the organ secretome left 326 between responders and non-responders appear to become non-redundant proteins that were differentially expressed in magnified during the treatment, as more proteins become any of the three experimental systems. The three experimen differentially expressed in the eventual responders compared tal systems continued to display minimal overlap, even to the eventual non-responders once treatment has begun. though they were each treated with the same glucolipotoxic [0250] These analyses indicated that the changes between treatment and each developed a similar drop in insulin pro responders and non-responders become augmented after duction and induction of apoptosis. After applying the priori treatment began, both in the number of proteins differentially tization strategy described above, 129 proteins were selected. expressed per pathway, but also in the introduction of related The fl-cell function (BCF) candidate biomarker proteins and pathways not induced in the pre-treatment samples. their degree of change after treatment are listed in Table 2. [0251] B. Biomarker Validation [0246] The pathway analysis supported the interpretation [0252] The biomarkers identified as described above were that the 3 experimental systems responded differently to the assessed in blood. Human plasma was processed by the three same stimulus. This indicated that the physiological rel methods described earlier. An aliquot of each subject’s evance of the cell line systems might be insufficient to effec plasma sample was depleted of high abundance proteins by tively model the human primary tissue. The response by the affinity chromatography. The remaining material was primary human islets to select the biomarker candidates asso digested with trypsin and analyzed by a multiplex MRM-MS ciated with fl-cell dysfunction was therefore focused on. assay. Another plasma aliquot was used to prepare plasma [0247] A list of biomarker candidates in human plasma that exosomes by sequential high speed centrifugation. The were associated with response to treatment was also devel recovered material was analyzed using the same multiplex oped. All the subjects recruited for this part of the project had MRM-MS assay used on the depleted plasma. Finally, a third type 2 diabetes, and were about to initiate or switch treatment. aliquot of the plasma was used to assess the performance of Plasma was collected prior to the treatment initiation as well 23 biomarker candidates by ELISA. as 2 weeks after treatment was initiated. The subjects were [0253] The clinical cohorts selected were designed to then followed for at least 5 months to establish treatment describe the spectrum of diabetes disease progression. The response. A responder was defined as a subject who displayed early stages of disease progression were represented by nor by treatment’s end glycated hemoglobin levels less than 7% moglycemic controls, which represent non-diabetic healthy without side effects, or had a 1.5% drop of glycated hemo subjects, and by subjects with impaired glucose tolerance, globin by treatment’s end without side effects. Initially the which corresponds to pre-diabetic individuals not yet for objective was to assess metformin treatment only, which is mally diagnosed with type 2 diabetes. Diabetes disease was the first line treatment for type 2 diabetes. The scope of the represented by subjects that have been diagnosed with type 2 study was later expanded to allow subjects with other thera diabetes within the last 1.5 years or at least 5 years previously. US 2015/0330.997 A1 Nov. 19, 2015

These two groups represent the early stage and advanced an umbrella term used to describe what is likely a variety of stage diabetics, respectively. Long term (>5 years since diag conditions that all have in common metabolic imbalance that nosis) type 1 diabetics have also been included in this study. frequently leads to obesity and is often a precursor to T2D. An Study plasma was tested for insulin using a commercial analysis of these subjects was conducted to evaluate the per ELISA kit. All the subjects had blood draws performed in the formance of the candidate biomarkers in a background of AM, after an overnight fast, and thus the insulin reactivity extreme metabolic syndrome. The same type of analysis for detected most likely represented endogenous levels. An the non-morbidly obese subjects was conducted (see Table 7): increase in resting insulin concentration was observed in the plasma samples were depleted of abundant proteins by chro impaired glucose tolerant, early stage, and advanced diabet matography and analyzed using a multiplex MRM-MS assay. ics compared to the controls, consistent with type 2 diabetes Plasma exosome preparations were also made to assess detec disease progression. tion of biomarker candidates that may have been beneath the [0254] In order to validate the biomarkers, the level of the level of detection of the multiplex MRM-MS assay in biomarkers was determined in samples from subjects. The depleted plasma, and a selection of ELISA assays were per samples for the analysis were described in Table 7. They formed as well. The performance of the candidate biomarkers comprised morbidly obese individuals with metabolic syn is presented in Tables 9-12 which provide the DI value for drome, and candidates for bariatric surgery. A subset of these each marker comparison. If the DI value is above 1 the level subjects have been diagnosed with T2D and were undergoing of the protein is upregulated for that particular comparison. If therapy at the time of the blood sampling, whereas others the DI value is less than 1, the level of the marker is down appeared to be in a pre-diabetic state. Metabolic syndrome is regulated for that particular comparison. TABLE 9

MRMANALYSIS OF HUMAN PLASMA SAMPLES OF BCM/BCF CANDIDATE BIOMARKERS *Differential expression (DE) thresholds: -value < 0.05 | q-value < 0.05

Established T1D vs Control Established T2D vs Control New T2D vs Control PROTEIN DI p-Value q-Value DI p-Value q-Value DI p-Value q-Value

INS HUMAN 0.96 0.638 0.000 .37 0.001 0.000 .22 0.032 0.000 USP9X HUMAN 1.18 0.170 0.000 0.76 0.020 0.000 0.88 0.290 0.000 TRIA2 HUMAN 1.27 0.008 0.000 .61 0.000 0.000 .20 0.035 0.000 B4GT1 HUMAN 0.97 0.495 0.000 .52 0.000 0.000 .09 0.068 0.000 MGAT1 HUMAN 0.86 0.096 0.000 .35 0.001 0.000 .15 0.115 0.000 ANAG HUMAN 0.99 0.866 0.000 0.99 0.878 0.000 .31 0.002 0.000 CHKA HUMAN 1.26 0.019 0.000 .56 0.000 0.000 .27 0.013 0.000 CADM1 HUMAN 1.07 0.447 0.031 .11 0.205 0.031 .14 0.115 0.031 DAG1 HUMAN 1.11 0.272 0.000 .72 0.000 0.000 .07 0.469 ().000 CNTN1 HUMAN 1.05 0.523 0.010 .16 0.035 0.010 .06 0.449 0.010 SPRL1, HUMAN 1.09 0.083 0.000 .15 0.004 0.000 .02 0.714 0.000 NCAM1 HUMAN 0.96 0.484 0.076 .01 0.889 0.076 0.95 0.367 0.076 ITM2B. HUMAN 1.06 0.224 0.007 .12 0.024 0.007 .07 0.188 0.007 DMP4 HUMAN 0.97 0.630 0.000 .15 0.013 0.000 .21 0.001 0.000 CD59 HUMAN 0.99 0.919 0.000 .81 0.000 0.000 .18 0.043 0.000 NEO1 HUMAN 0.99 0.802 0.000 .16 0.008 0.000 .04 0.484 0.000 PTPRJ HUMAN 0.99 0.881 0.004 .06 0.148 0.004 .08 0.053 0.004 CBPM HUMAN 0.97 0.732 0.000 .33 0.000 0.000 .26 0.002 0.000 SPIT1 HUMAN 1.02 0.750 0.006 .12 0.038 0.006 .07 0.175 0.006 PVR HUMAN 0.94 0.268 0.000 .15 0.012 0.000 .06 0.286 0.000 QPCT HUMAN 1.05 0.578 0.000 .33 0.000 0.000 .10 0.245 0.000 SDK1 HUMAN 1.04 0.544 0.002 .15 0.018 0.002 0.99 0.928 0.002 NAAA HUMAN 0.99 0.913 0.020 .09 0.105 0.020 .02 0.735 0.020 GALT2 HUMAN 0.96 0.529 0.000 .29 0.000 0.000 .12 0.073 0.000 LMAN2 HUMAN 1.00 0.958 0.000 .37 0.000 0.000 .11 0.123 0.000 A4 HUMAN 1.15 0.079 0.015 .05 0.534 0.015 .13 0.123 0.015

TABLE 10

ELISA ANALYSIS OF HUMAN PLASMA SAMPLES OF BCM/BCF CANDIDATE BIOMARKERS Significance Thresholds: p-value < 0.05 | q-value < 0.05 IGT vs NGT New T2D VS NGT Est T2D VS NGT New T2D VS IGT PROTEIN DI p-Value DI p-Value DI p-Value DI p-Value

INS 1.82 0.005 2.55 0.000 2.82 0.002 1.40 0.043 PPY 0.89 0.627 2.05 0.000 1.78 0.000 2.29 0.000 FUT6 1.07 0.421 0.76 0.000 0.92 0.241 0.71 0.000 CPM 1.16 0.357 1.72 0.000 1.81 0.001 1.48 0.015 SERPINB13 1.04 0.820 0.38 0.000 0.87 0.696 0.37 0.000 WNTQB 0.99 0.979 2.30 0.004 1.61 0.050 2.31 0.019 STX1A 1.46 0.408 3.38 0.038 1.72 0.228 2.31 0.175 US 2015/0330.997 A1 Nov. 19, 2015 56

TABLE 10-continued

ELISA ANALYSIS OF HUMAN PLASMA SAMPLES OF BCM/BCF CANDIDATE BIOMARKERS Significance Thresholds: p-value < 0.05 | q-value < 0.05 BTC 0.34 0.084 .87 0.044 0.96 0.894 5.47 0.002 SNAP25 0.65 0.094 0.64 0.052 1.15 0.507 0.98 0.954 MMP7 1.09 0.576 .22 0.074 3.07 0.006 1.12 0.396 CCL20 1.57 0.322 .62 0.090 1.98 0.055 1.03 0.923 IGFBP7 1.16 0.583 0.62 0.087 1.34 0.238 0.54 0.021 SEPT3 1.82 0.163 0.69 0.115 0.59 0.018 0.38 0.031 SCG5 1.74 0.121 .81 0.125 2.86 0.089 1.04 0.917 TNFSF11 4.23 0.132 2.66 0.140 1.81 0.522 0.63 0.489 REG3A 0.86 0.560 .37 0.373 1.04 0.909 1.60 0.313 PTPRN 0.86 0.138 .11 0.459 0.79 0.020 1.29 0.199 IAPP 2.90 0.158 .40 0.682 2.07 0.258 0.48 0.349 CPE 1.62 0.063 0.97 0.853 0.87 0.275 0.60 0.044

Est T2D vs IGT Est vs New T2D T1D vs NGT T1D vs IGT PROTEIN DI p-Value DI p-Value DI p-Value DI p-Value

INS .55 0.181 .11 0.598 0.19 0.000 0.10 0.000 PPY .99 0.000 0.87 0.123 .64 0.017 .83 0.022 FUT6 0.86 0.072 .22 0.003 0.86 0.043 0.81 0.013 CPM .56 0.028 .06 0.663 .28 0.081 .10 0.495 SERPINB13 0.83 0.676 2.25 0.112 .56 0.435 .50 0.576 WNTQB .63 0.108 0.70 0.094 2.25 0.011 2.26 0.043 STX1A. .18 0.700 0.51 0.100 2.18 0.199 .49 0.515 BTC 2.82 0.021 0.51 0.009 0.92 0.853 2.69 0.213 SNAP25 77 0.103 1.80 0.028 .05 0.834 .61 0.242 MMP7 2.81 0.041 2.51 0.006 .03 0.850 0.95 0.806 CCL20 .26 0.564 1.22 0.448 .05 0.897 0.67 0.355 IGFBP7 .15 0.598 2.15 0.004 2.19 0.004 .88 0.037 SEPT3 0.33 0.012 0.85 0.447 0.79 0.405 0.43 0.084 SCG5 .65 0.428 1.58 0.308 .42 0.241 0.82 0.559 TNFSF11 0.43 0.306 0.68 0.550 2.90 0.129 0.68 0.594 REG3A .21 0.644 0.76 0.425 .28 0.433 .49 0.311 PTPRN 0.91 0.476 0.71 0.021 0.82 0.185 0.96 0.816 IAPP 0.71 0.583 1.47 0.540 .59 0.520 0.55 0.408 CPE 0.54 0.012 0.89 0.429 0.92 0.455 0.57 0.029

TABLE 1.1

MRMANALYSIS OF HUMAN EXOSOME SAMPLES OF BCM/BCF CANDIDATE BIOMARKERS *Differential expression (DE) thresholds: p-value < 0.05 || q-value < 0.05

T1D-Established vs T2D-Established vs Control Control T2D-New VS Control PROTEIN DI p-value q-value DI p-value q-value DI p-value q-value EDF1 HUMAN 128.37 0.000 0.000 0.24 0.136 0.341 33.68 0.001 0.001 SNAPN HUMAN 34.25 0.000 0.000 0.36 0.116 0.316 8.43 0.009 0.007 NXPH1 HUMAN 31.14 0.000 0.000 0.45 0.324 0.505 5.19 0.080 0.035 CDCP1 HUMAN 18.20 0.000 0.000 5.82 0.01.1 0.047 8.00 0.008 0.007 NGR1 HUMAN 5.94 0.002 0.001 1.03 0.957 0.738 0.71 0.621 0.196 BTC HUMAN 4.60 0.007 0.003 0.75 0.617 0.662 2.49 0.131 0.052 NCAM1 HUMAN 4.13 0.001 0.001 1.07 0.886 0.733 2.20 0.102 0.044 RICBA HUMAN 2.98 0.002 0.001 0.99 0.986 0.742 3.28 0.002 0.002 TM11F HUMAN 2.93 0.000 0.000 1.07 0.588 0.662 2.71 0.000 0.000 MGT4B HUMAN 2.89 0.000 0.000 0.91 0.534 0.662 2.75 0.000 0.000 ERO1 B HUMAN 2.75 0.000 0.000 0.99 0.923 0.733 2.06 0.000 0.000 PDYN HUMAN 2.57 0.000 0.000 0.85 0.237 0.419 2.24 0.000 0.000 LTOR2 HUMAN 2.24 0.000 0.000 0.95 0.669 0.671 2.06 0.000 0.000 NELL1, HUMAN 2.03 0.000 0.000 0.97 0.781 0.733 1.71 0.000 0.000 TCO2 HUMAN 1.96 0.000 0.000 1.12 0.406 0.555 1.42 0.022 0.014 PTPRJ HUMAN 1.84 0.003 0.001 1.26 0.203 0.408 1.98 0.000 0.000 CLLD6 HUMAN 1.78 0.009 0.003 1.11 0.669 0.671 1.34 0.309 0.110 ATD3B HUMAN 1.77 0.000 0.000 0.87 0.204 0.408 2.15 0.000 0.000 NXPH2 HUMAN 1.60 0.036 0.01.1 1.04 0.843 0.733 1.65 0.030 0.017 VAV3 HUMAN 1.51 0.014 0.005 0.34 0.007 0.045 1.43 0.057 0.029 PLXC1 HUMAN 0.45 0.019 0.006 1.12 0.590 0.662 0.53 0.070 0.033 CSTF3 HUMAN 0.34 0.000 0.000 1.04 0.744 0.722 0.71 0.020 0.013 MCRS1 HUMAN 1.00 0.998 0.173 0.38 0.004 0.037 0.87 0.670 0.200 LDLR HUMAN 0.96 0.825 0.151 0.56 0.001 0.037 1.12 0.542 0.181

US 2015/0330.997 A1 Nov. 19, 2015 59

[0258] Additional analyses of the markers identified 30 TABLE 14-continued markers that have individual discrimination power, defined as being able to discriminate between two cohorts with an accu Area Under the Curve (AUC) for Single Markers. racy of 75% or greater. Specifically, and as described above, NGT vs IGT vs samples were obtained from control subjects (e.g., normal Marker IGT mT2D eT2D All T2D mT2D eT2D All T2D glucose tolerant (NGT) subjects, pre-diabetic subjects (e.g., ATAD3B — 0.765 — — 0.751 — - subjects having impaired glucose tolerance), subjects diag PTPRN - — 0.730 — - - - nosed as having type 2 diabetes in the previous 18 months WNTQB – 0.794 – 0.513 – – 0.705 FUT6 – 0.844 – 0.572 0.885 – 0.591 (nT2D) and subjects having type 2 diabetes and a complica B4GALT1 - - – 0.945 – – 0.885 tion associated with type 2 diabetes, such as diabetic neur FAM20C 0.878 opathy, retinopathy, nephropathy, cardiovascular disease CNTN1 0.758 MGAT1 — 0.915 — - - - - (eT2D) and the level of each of the markers listed in Tables STX1A — 0.828 — - - - - 1-3 was determined. Pairwise comparisons of the level of NMU - – 0.782 0.877 – – - each marker in NGT subjects and; IGT subjects; n'T2D sub CD59 - - – 0.980 – – 0.903 CASR - - — 0.898 — — - jects; eT2D; and a combination of nT2D and eT2D subjects CPE 0.590 – - - — 0.850 0.875 (All T2D) were performed and the area under the curve for each marker was calculated. Similarly, pairwise comparisons [0259] The ability of these individual biomarkers to act in of the level of each marker in IGT subjects and; nT2D sub combination, as a panel, was also assessed. This preliminary jects; eT2D; and a combination of nT2D and eT2D subjects panel analysis focused on identifying combinations that improved discrimination accuracy, but also used the smallest (All T2D) were performed and the area under the curve for possible number of biomarkers. As shown in Table 15, small each marker was calculated. The results of these analyses are panels of proteins that were able to accurately discriminate shown in Table 14. Therefore a substantial number of well between each of the disease progression cohorts were suc cessfully identified. The area under the curve (AUC) for vari performing candidates was identified. For most comparison, ous combinations of the markers listed in Tables 1-3 was also multiple biomarker candidates with good performance indi determined. The results of these analyses are shown in Table cators were identified. 15. TABLE 1.5

Area. Under the Curve (AUC) for Marker Combinations.

NGT vs IGT VS Markers Proteins in panel IGT nT2D eT2D All T2D nT2D eT2D All T2D INS: USPX 0.774 INS: SERPINB13 - 0.998 – -

- - - INS: SERPINB13 CPM; INS: MMP7; .

PPY SEPT3; PTPRU 3 - - - PPY, DAG1

TABLE 1.4 [0260] The biomarker candidates associated with pancre atic function and disease progression were also evaluated in Area Under the Curve (AUC) for Single Markers. plasma from morbidly obese type 2 diabetics or pre-diabetics. NGT vs IGT vs Fewer proteins overall (13 vs 30) compared to the initial, less obese, cohorts were found to have acceptable individual dis Marker IGT mT2D eT2D All T2D mT2D eT2D All T2D crimination power. However, the list of candidate biomarkers USP9X 0.718 from both cohorts overlapped, with only 2 of the 13 better DAG1 - - 0.989 - – 0.947 - biomarker candidates from the obese subject dataset were SEPT3 - - 0.732 0.814 – 0.834 0.824 PTPRJ - - - 0.774 — 0.774 0.923 detected only in the obese subjects. This suggests that the bulk CPM - 0.876 0.785 0.814 – 0.742 0.746 of the biomarker candidates identified with good discrimina SERPINB13 - 0.885 0.940 tory power had similar performance in both cohorts. While LDLR - - 0.802 0.835 - - - MMP7 - - 0.884 0.838 – 0.847 - this suggests that these biomarker candidates could be rel BTC - 0.690 - - 0.968 0.833 0.798 evant in multiple populations, there were also important dif PPY - 0.907 0.881 0.923 0.961 0.937 0.945 ferences. One of these appears to be that combinations con INS - 0.983 0.802 0.818 - - - taining more proteins were necessary to separate the diabetic CSTF3 - 0.766 - - - - -

NELL1 - 0.741 - - - - - from the pre-diabetic subjects from the obese cohorts. For SLIT2 - 0.861 - - 0.812 — - example, a combination of 5 proteins was required to gener LAMTOR2 - 0.850 - - 0.813 – - ate a panel able to discriminate morbidly obese diabetics from MGAT4B - 0.826 - - 0.786 – - morbidly obese pre-diabetics with an accuracy of 0.826. By TMPRSS11 F - 0.822 - - 0.741 — - comparison, non-morbidly obese pre-diabetics could be dis US 2015/0330.997 A1 Nov. 19, 2015 60 tinguished from diabetics of comparable BMI with an accu TABLE 16-continued racy of 0.998 using a combination of only 3 proteins. This suggests that it might be more difficult to separate the obese BCMBCFTEM High BMI Diabetics vs Non Diabetics diabetics from the obese pre-diabetics, which is why more PANEL COMPOSITION #PROTEINS AUC panel members were required and even then these additional CD59 || CHKA | MGAT1 | TRIM42 || USP9X 5 0.851 panel members produced an overall less accurate combina CD59 || CNTN1 | PTPRJ | TRIM42 | BTC 5 0.850 tion. Variability among the cohort subjects may be a factor B4GALT1 | CD59 || CNTN1 | PTPRJ | TRIM42 5 0.850 affecting panel performance, since the morbidly obese sub CD59 || CNTN1 | PTPRJ | TRIM42 4 0.850 jects included had widely varying BMI values, ranging from CD59 || CNTN1 | PTPRJ | TRIM42 | CPM 5 0.850 35 to 70. Once the subjects were sorted into two groups, one containing subjects with BMI of up to 40, and the other subjects with BMI above 40, the best 5 protein panel compo Example II sition became different for each of these groups, and the best panel performance rose from 0.826 to 0.843 and 0.889, Determination of the Level of One or More respectively (Table 16). Biomarkers in a Subject Sample TABLE 16 [0261] A biological sample (e.g., serum, saliva) is obtained from a subject and the level of one or more of the markers BCMBCFTEM High BMIDiabetics vs Non Diabetics listed in Tables 1-3 is determined by mass spectrometry to determine (e.g., whether a subject has or will develop type 2 PANEL COMPOSITION #PROTEINS AUC diabetes, whether the subject has or will develop impaired CD59 || CNTN1 || MGAT1 | TRIM42 | USP9X 5 0.889 glucose tolerance, whether the subject will develop a type 2 CD59 || CHKA | CNTN1 | TRIM42 | USP9X 5 0.881 diabetes-associated complication, whether the subject having CD59 || CNTN1 | PTPRJ | TRIM42 | USP9X 5 0.879 B4GALT1 | CD59 || CNTN1 | TRIM42 | USP9X 5 0.874 impaired glucose tolerance and/or type 2 diabetes will CD59 || CNTN1 | TRIM42 || USP9X | BTC 5 0.872 respond to a therapy). Briefly, the sample is digested with CD59 || CNTN1 | TRIM42 || USP9X | CPM 5 0.872 trypsin to generate peptides. The peptides are then separated CD59 || CNTN1 | TRIM42 | USP9X | PPY 5 0.871 by strong cation exchange chromatography (SCX) into three CD59 || CNTN1 | FAM20C | TRIM42 || USP9X 5 0.871 CD59 || CNTN1 | TRIM42 || USP9X 4 0.871 fractions. Each of the three fractions per sample is analyzed CD59 || CNTN1 | TRIM42 || USP9X |INS 5 0.871 by reversed phase liquid chromatography, coupled by elec CNTN1 || MGAT1 | PTPRJ | TRIM42 || USP9X 5 0.868 trospray to a Waters QTOF mass spectrometer (LC-MS). CD59 || CHKA | CNTN1 || MGAT1 | USP9X 5 0.867 Components are detected and matched across all samples and CHKA | CNTN1 || MGAT1 | TRIM42 | USP9X 5 0.867 B4GALT1 | CHKA | CNTN1 | TRIM42 | USP9X 5 0.867 compared for relative peak intensity. Peak intensity is nor B4GALT1 | CNTN1 | PTPRJ | TRIM42 || USP9X 5 0.867 malized. The level of the one or more markers in the sample CNTN1 || MGAT1 | TRIM42 | USP9X | INS 5 0.865 is compared to a level of the one or more markers in a control CHKA | CNTN1 | PTPRJ | TRIM42 | USP9X 5 0.865 sample and a difference in the level of the one or more mark B4GALT1 | CNTN1 || MGAT1 | TRIM42 | USP9X 5 0.863 CNTN1 | FAM20C | MGAT1 || TRIM42 | USP9X 5 0.863 ers in the subject sample as compared to the level of the one CNTN1 || MGAT1 | TRIM42 | USP9X | BTC 5 0.861 or more markers in the control sample indicates that the CNTN1 || MGAT1 | TRIM42 | USP9X | CPM 5 0.860 subject has or will develop impaired glucose tolerance. CD59 || CHKA | CNTN1 | FAM20C | USP9X 5 0.860 CNTN1 || MGAT1 | TRIM42 | USP9X 4 0.860 CD59 || CNTN1 | PTPRJ | TRIM42 |INS 5 0.860 EQUIVALENTS CD59 || CHKA | CNTN1 | PTPRJ | USP9X 5 0.859 CNTN1 || MGAT1 | TRIM42 | USP9X | PPY 5 0.859 [0262] In describing exemplary embodiments, specific ter CD59 || CNTN1 || MGAT1 | PTPRJ | TRIM42 5 0.859 minology is used for the sake of clarity. For purposes of B4GALT1 | CNTN1 | FAM20C | TRIM42 || USP9X 5 0.858 description, each specific term is intended to at least include B4GALT1 | CNTN1 | TRIM42 || USP9X |INS 5 0.857 all technical and functional equivalents that operate in a simi B4GALT1 | CNTN1 | TRIM42 || USP9X | PPY 5 0.857 CD59 || CHKA | CNTN1 | USP9X | PPY 5 0.856 lar manner to accomplish a similar purpose. Additionally, in B4GALT1 | CNTN1 | TRIM42 || USP9X 4 0.856 some instances where a particular exemplary embodiment CHKA | CNTN1 | FAM20C | MGAT1 | USP9X 5 0.855 includes a plurality of system elements or method steps, those CD59 || CHKA | CNTN1 | USP9X | BTC 5 0.855 elements or steps may be replaced with a single element or B4GALT1 | CNTN1 | TRIM42 || USP9X | CPM 5 0.855 CNTN1 | PTPRJ | TRIM42 | USP9X | INS 5 0.855 step. Likewise, a single element or step may be replaced with CD59 || CHKA | CNTN1 | USP9X | CPM 5 0.855 a plurality of elements or steps that serve the same purpose. CD59 || CHKA | CNTN1 | USP9X | INS 5 0.854 Further, where parameters for various properties are specified B4GALT1 | CNTN1 | TRIM42 || USP9X | BTC 5 0.854 herein for exemplary embodiments, those parameters may be B4GALT1 | CD59 || CHKA | CNTN1 || USP9X 5 0.853 CD59 || CHKA | CNTN1 | USP9X 4 0.853 adjusted up or down by %0th, Vioth, /šth, Wºrd, /2, etc., or by CNTN1 | PTPRJ | TRIM42 | USP9X | BTC 5 0.853 rounded-off approximations thereof, unless otherwise speci CNTN1 | PTPRJ | TRIM42 | USP9X | PPY 5 0.853 fied. Moreover, while exemplary embodiments have been CD59 || CNTN1 | FAM20C | PTPRJ | TRIM42 5 0.853 shown and described with references to particular embodi CD59 || CHKA | CNTN1 | PTPRJ | TRIM42 5 0.853 B4GALT HKA | CNTN1 || MGAT1 | USP9X 5 0.852 ments thereof, those of ordinary skill in the art will under CNTN1 | PTPRJ | TRIM42 | USP9X 4 0.852 stand that various substitutions and alterations in form and CNTN1 | FAM20C | PTPRJ | TRIM42 || USP9X 5 0.852 details may be made therein without departing from the scope CHKA | CNTN1 || MGAT1 | PTPRJ | USP9X 5 0.852 of the invention. Further still, other aspects, functions and CD59 || CNTN1 | PTPRJ | TRIM42 | PPY 5 0.852 CNTN1 | PTPRJ | TRIM42 | USP9X | CPM 5 0.852 advantages are also within the scope of the invention. B4GALT1 | CHKA | CNTN1 | FAM20C | USP9X 5 0.851 [0263] Exemplary flowcharts are provided herein for illus CHKA | CNTN1 || MGAT1 | USP9X | INS 5 0.851 trative purposes and are non-limiting examples of methods. One of ordinary skill in the art will recognize that exemplary US 2015/0330.997 A1 Nov. 19, 2015

methods may include more or fewer steps than those illus comparing the level of the one or more markers in the first trated in the exemplary flowcharts, and that the steps in the sample(s) with a level of the one or more markers in the exemplary flowcharts may be performed in a different order second sample(s), wherein a difference in the level of the than shown. one or more markers in the first sample(s) as compared to the level of the one or more markers in the second INCORPORATION BY REFERENCE sample(s) indicates that the subject will respond to the treatment. [0264] The contents of all references, including patents and 4. The method of any one of claims 1-3, wherein the level patent applications, cited throughout this application are of two, three, or four markers is determined. hereby incorporated herein by reference in their entirety. The 5. The method of any one of claims 1-3, wherein the one or appropriate components and methods of those references more markers is selected from the group consisting of may be selected for the invention and embodiments thereof. USP9X, SEPT3, DAG1, PTPRJ, CPM, MMP7, B4GALT1, Still further, the components and methods identified in the MGAT1, CD59, CASR, DMP4, and SCG5. Background section are integral to this disclosure and can be 6. The method of any one of claims 1-3, wherein the level used in conjunction with or substituted for components and in the subject sample(s) is determined by mass spectrometry methods described elsewhere in the disclosure within the or immunoassay. 7. The method of any one of claims 1-3, wherein the sample scope of the invention. (s) from the subject is a fluid sample(s) or a tissue sample(s). We claim: 8. The method of any one of claims 1-3, wherein the subject 1. A method for determining whether a subject has or will is at risk of developing type 2 diabetes. develop impaired glucose tolerance, the method comprising 9. The method of any one of claims 1-3, further comprising determining the level of one or more marker listed in any of determining one or more of the level of INS, the HBA1c level, Tables 1-3 in a sample(s) from the subject; and the fasting plasma glucose level in a sample(s) from the comparing the level of the one or more markers in the subject. subject sample(s) with a level of the one or more markers 10. A kit for determining whether a subject has or will in a control sample(s), wherein a difference in the level develop impaired glucose tolerance, the kit comprising of the one or more markers in the subject sample(s) as reagents for determining the level of one or more markers compared to the level of the one or more markers in the listed in any of Tables 1-3 in a subject sample(s) and instruc control sample(s) indicates that the subject has or will tions for use of the kit to determine whether the subject has or develop impaired glucose tolerance. will develop impaired glucose tolerance. 2. A method for determining whether a subject has or will 11. A kit for determining whether a subject has or will develop type 2 diabetes, the method comprising develop type 2 diabetes, the kit comprising reagents for deter determining the level of one or more marker listed in any of mining the level of one or more markers listed in any of Tables Tables 1-3 in a sample(s) from the subject; 1-3 in a subject sample(s) and instructions for use of the kit to comparing the level of the one or more markers in the determine whether the subject has or will develop type 2 subject sample(s) with a level of the one or more markers diabetes. in a control sample(s), wherein a difference in the level 12. A kit of monitoring the effectiveness of a treatment in a of the one or more markers in the subject sample(s) as subject having impaired glucose tolerance and/or type 2 dia compared to the level of the one or more markers in the betes the kit comprising reagents for determining the level of control sample(s) indicates that the subject has or will one or more markers listed in any of Tables 1-3 in a subject develop type 2 diabetes. sample(s) and instructions for use of the kit to monitor the 3. A method for monitoring the effectiveness of a treatment effectiveness of the treatment. in a subject having impaired glucose tolerance and/or type 2 13. The kit of any one of claims 10-12, further comprising diabetes, the method comprising reagents for obtaining a biological sample from a subject or a determining the level of one or more marker listed in any of control sample. Tables 1-3 in a first sample(s) from the subject prior to 14. The kit of any one of claims 10-12, wherein the one or the initiation of the treatment; more markers is selected from the group consisting of determining the level of one or more marker listed in any of USP9X, SEPT3, DAG1, PTPRJ, CPM, MMP7, B4GALT1, Tables 1-3 in a second sample(s) from the subject after at MGAT1, CD59, CASR, DMP4, and SCG5. least a portion of the treatment has been administered;