Patent Application
20160208329
This invention relates to diagnosis and treatment of inflammatory and immune diseases. More specifically, this invention relates to systems, devices and methods for diagnosing and treating a disease that is susceptible to an anti-TL1A therapy.
All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
TL1A activation is involved in pathogenesis of a variety of inflammatory and immune diseases. For example, genome-wide association studies (GWAS) have implicated TL1A in the pathogenesis of inflammatory bowel disease (IBD) such as Crohn's disease (CD). Evidence in preclinical mouse models also supports the role of TL1A in the pathogenesis of IBD. In addition, intestinal tissues from CD patients demonstrate increased expression of TL1A at sites of active disease. However, IBD is a heterogeneous disease, and previously, treatment of IBD patients has been by trial and error. While anti-TL1A therapy (e.g., treatment with an anti-TL1A antibody) is of help to some CD patients, not all patients will benefit from anti-TL1A therapy.
As such, there is a need of biomarkers, devices, systems, and methods for defining a biomarker signature for TL1A activation, for identifying patients who have most likely been inflicted by TL1A activation and will be most suitable for anti-TL1A therapy, and for guiding treatment options for these patients.
Various embodiments of the present invention provide a method of selecting a treatment for a subject. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and prescribing an anti-TL1A therapy to the subject if the subject has a high expression level relative to the reference value, or prescribing no anti-TL1A therapy to the subject if the subject does not have a high expression level relative to the reference value.
Various embodiments of the present invention provide a method of identifying a subject likely to be responsive to an anti-TL1A therapy. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and identifying the subject as likely to be responsive to an anti-TL1A therapy if the subject has a high expression level relative to the reference value, or identifying the subject as unlikely to be responsive to an anti-TL1A therapy if the subject does not have a high expression level relative to the reference value.
Various embodiments of the present invention provide a method of treating a subject with an anti-TL1A therapy. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and administering an anti-TL1A therapy to the subject if the subject has a high expression level relative to the reference value, or administering no anti-TL1A therapy to the subject if the subject does not have a high expression level relative to the reference value.
Various embodiments of the present invention provide a method of diagnosing a disease in a subject. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and diagnosing a TL1A-associated disease in the subject if the subject has a high expression level relative to the reference value, or diagnosing no TL1A-associated disease in the subject if the subject does not have a high expression level relative to the reference value.
Various embodiments of the present invention provide a method diagnosing susceptibility to a TL1A-associated disease in a subject. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and diagnosing susceptibility to a TL1A-associated disease in the subject if the subject has a high expression level relative to the reference value, or diagnosing no susceptibility to a TL1A-associated disease in the subject if the subject does not have a high expression level relative to the reference value.
Various embodiments of the present invention provide a method of treating a disease in a subject. The method may comprise or may consist essentially of or may consist of: administering an anti-TL1A therapy to the subject, thereby treating the disease, wherein the subject has a high expression level relative to a reference value of one or more biomarkers associated with TL1A signaling.
Various embodiments of the present invention provide a method for diagnosing a disease in a subject. The method may comprise or may consist essentially of or may consist of: obtaining a sample from a subject; assaying the expression level of one or more genes in the sample; comparing the expression level to a reference value of expression level of the one or more genes; and diagnosing a disease in the subject according to the relative difference between the expression level and the reference value. In some embodiments, the method further comprises diagnosing the disease in the subject if the subject has an expression level higher than the reference value, or not diagnosing the disease in the subject if the subject does not have an expression level higher than the reference value. In other embodiments, the method further comprises diagnosing the disease in the subject if the subject has an expression level lower than the reference value, or not diagnosing the disease in the subject if the subject does not have an expression level lower than the reference value. In various further embodiments, the method further comprises prescribing an anti-TL1A therapy to the subject if the subject is diagnosed with the disease. In various further embodiments, the method further comprises administering an anti-TL1A therapy to the subject if the subject is diagnosed with the disease. In one embodiment, the disease is an IBD subtype, for example, an IBD subtype responsive to an anti-TL1 A therapy.
Various embodiments of the present invention provide a method for diagnosing susceptibility to an IBD subtype in a subject. The method may comprise or may consist essentially of or may consist of: obtaining a sample from the subject; assaying the expression level of one or more genes in the sample; comparing the expression level to a reference value of expression level of the one or more genes; and diagnosing susceptibility to the IBD subtype in the subject if the subject has an expression level different from the reference value, or not diagnosing susceptibility to the IBD subtype in the subject if the subject does not have an expression level different from the reference value.
In various methods described herein, the one or more biomarkers or genes assayed may be those described in Table 1, Table 4, Table 5 and/or Table 6 herein. In various methods described herein, the TL1A-associated disease may include but are not limited to fibrosis, ulcerative colitis (UC), Crohn's disease (CD), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease, allergic lung inflammation, asthma, atherosclerosis, lupus, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, type 1 diabetes, lung carcinoma, colon carcinoma, leukemia, lymphoma, transplant rejection, graft versus host disease, or central nervous system injury. In various methods described herein, the IBD subtype may be characterized by being treatable with an anti-TL1A therapy, that is, an IBD subtype responsive to an anti-TL1A therapy.
Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.
All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Allen et al., Remington: The Science and Practice of Pharmacy 22nd ed., Pharmaceutical Press (Sep. 15, 2012); Hornyak et al., Introduction to Nanoscience and Nanotechnology, CRC Press (2008); Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology 3rd ed., revised ed., J. Wiley & Sons (New York, N.Y. 2006); Smith, March's Advanced Organic Chemistry Reactions, Mechanisms and Structure 7th ed., J. Wiley & Sons (New York, N.Y. 2013); Singleton, Dictionary of DNA and Genome Technology 3rd ed., Wiley-Blackwell (Nov. 28, 2012); and Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N. Y. 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see Greenfield, Antibodies A Laboratory Manual 2nd ed., Cold Spring Harbor Press (Cold Spring Harbor N.Y., 2013); Köhler and Milstein, Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion, Eur. J. Immunol. 1976 July, 6(7):511-9; Queen and Selick, Humanized immunoglobulins, U.S. Pat. No. 5,585,089 (1996 December); and Riechmann et al., Reshaping human antibodies for therapy, Nature 1988 Mar. 24, 332(6162):323-7.
One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. Indeed, the present invention is in no way limited to the methods and materials described.
“Subject” or “individual” or “patient” or “animal” or “mammal” refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, treatment or therapy is desired. Mammalian subjects include, but are not limited to, humans; domestic animals; farm animals; zoo animals; sport animals; pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; rodents such as mice, rats, hamsters and guinea pigs; and so on. In certain embodiments, the mammal is a human subject. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be included within the scope of this term.
“Biological sample” or “Sample” as used herein means any biological material from which nucleic acids and/or proteins can be obtained. As non-limiting examples, the term encompasses whole blood, plasma, saliva, cheek swab, or other bodily fluid or tissue that contains nucleic acids and/or proteins.
“Treatment” and “treating,” as used herein refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition, prevent the pathologic condition, pursue or obtain beneficial results, or lower the chances of the individual developing the condition even if the treatment is ultimately unsuccessful. Those in need of treatment include those already with the condition as well as those prone to have the condition or those in whom the condition is to be prevented.
“Beneficial results” may include, but are in no way limited to, lessening or alleviating the severity of the disease condition, preventing the disease condition from worsening, curing the disease condition, preventing the disease condition from developing, lowering the chances of a patient developing the disease condition and prolonging a patient's life or life expectancy. In some embodiments, the disease condition is a TL1A-associated disease.
“Patient outcome” refers to whether a patient's health improves or worsens as a result of treatment as well as whether a patient survives or dies as a result of treatment. As provided in this invention, prescribing and administering an appropriate treatment (e.g., an anti-TL1A therapy or not) according to the specific conditions of individual patients increases their chances of health improvement and/or survival.
“TL1A” as used herein is a TNF-like cytokine factor encoded by the gene TNFSF15. Examples of TL1A include mouse TL1A such as NCBI reference sequence NM_177371.3, rat TL1A such as NCBI reference sequence AF520787.1, and human TL1A such as NCBI reference sequence NM_005118, NM_001204344.1, among others.
“Anti-TL1A therapy”, as used herein refers to therapeutic agents and methods that suppress TL1A gene expression, DR3 gene expression, or block the signaling of TL1A and DR3 (the receptor for TL1A) proteins. Examples of anti-TL1A therapy include, but are not limited to, an agent that specifically binds TL1A or DR3 and blocks TL1A-DR3 interaction, an anti-TL1A antibody blocking TL1A-DR3 signaling, an anti-DR3 antibody blocking TL1A-DR3 signaling, a soluble decoy DR3 polypeptide (e.g., a soluble DR3-Fc fusion protein), or a nucleic acid antagonist of TL1A or DR3, such as a ribozyme, aptamer or antisense molecule targeting TL1A or DR3, or a combination thereof.
As disclosed herein, the inventors discovered a TL1A-specific biomarker signature of 22 genes. In accordance with various embodiments herein, the invention includes devices, systems, and methods for stratifying patient population based on this biomarker signature to identify individuals most likely to been exposed in vivo to the pro-inflammatory effects of TL1A activation. As this particular population of patients would potentially benefit from an anti-TL1A therapy, for example, an anti-TL1A therapy may be prescribed or administrated to them. In another embodiment, by evaluating changes in the biomarker signature, one can monitor the progress and/or assess the effectiveness of an anti-TL1A therapy in a patient.
In one embodiment, the invention provides for a method of selecting a treatment for a subject. In one embodiment, the present invention provides a method of diagnosis and/or treatment by obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and prescribing an anti-TL1A therapy to the subject if the subject has a high expression level relative to the reference value, or prescribing no anti-TL1A therapy to the subject if the subject does not have a high expression level relative to the reference value. In some embodiments, the method further comprises stimulating the sample with IL12, IL18, or TL1A, or a combination thereof, before assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample. In another embodiment, the one or more biomarkers associated with TL1A signaling is listed in Table 1, Table 4, Table 5 and/or Table 6 herein.
In one embodiment, the invention provides for a method of identifying a subject likely to be responsive to an anti-TL1A therapy. In one embodiment, the present invention provides a method of identifying a subject likely to be responsive to an anti-TL1A therapy by obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and identifying the subject as likely to be responsive to an anti-TL1A therapy if the subject has a high expression level relative to the reference value, or identifying the subject as unlikely to be responsive to an anti-TL1A therapy if the subject does not have a high expression level relative to the reference value. In some embodiments, the method further comprises stimulating the sample with IL12, IL18, or TL1A, or a combination thereof, before assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample. In another embodiment, the one or more biomarkers associated with TL1A signaling is listed in Table 1, Table 4, Table 5 and/or Table 6 herein.
In another embodiment, the invention provides for a method of treating a subject with an anti-TL1A therapy. In one embodiment, the method includes obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and administering an anti-TL1A therapy to the subject if the subject has a high expression level relative to the reference value, or administering no anti-TL1A therapy to the subject if the subject does not have a high expression level relative to the reference value. In some embodiments, the method further comprises stimulating the sample with IL12, IL18, or TL1A, or a combination thereof, before assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample. In another embodiment, the one or more biomarkers are described in Table 1, Table 4, Table 5 and/or Table 6 herein.
In another embodiment, the invention provides for a method of diagnosing a TL1A-associated disease in a subject. In another embodiment, the method comprises obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and diagnosing a TL1A-associated disease in the subject if the subject has a high expression level relative to the reference value, or diagnosing no TL1A-associated disease in the subject if the subject does not have a high expression level relative to the reference value. In another embodiment, the one or more biomarkers are described in Table 1, Table 4, Table 5 and/or Table 6 herein.
In various embodiments, the invention provides for a method diagnosing susceptibility to a TL1A-associated disease in a subject. In one embodiment, the method comprises: obtaining a sample from the subject, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample, comparing the expression level to a reference value of expression level, and diagnosing susceptibility to a TL1A-associated disease in the subject if the subject has a high expression level relative to the reference value, or diagnosing no susceptibility to a TL1A-associated disease in the subject if the subject does not have a high expression level relative to the reference value. In another embodiment, the one or more biomarkers associated with TL1A signaling is described in Table 1, Table 4, Table 5 and/or Table 6 herein. In another embodiment, the TL1A-associated disease includes fibrosis. In another embodiment, the TL1A-associated disease includes Inflammatory Bowel Disease (IBD).
In various embodiments, the invention provides a method of treating a disease in a subject. In one embodiment, the method comprises: administering an anti-TL1A therapy to the subject, thereby treating the disease, wherein the subject has a high expression level relative to a reference value of one or more biomarkers associated with TL1A signaling. In another embodiment, the one or more biomarkers are described in Table 1, Table 4, Table 5 and/or Table 6 herein.
In various embodiments, the invention provides for a method of diagnosing an IBD subtype in a subject. In one embodiment, the method comprises: obtaining a sample from the subject; assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample; comparing the expression level to a reference value of expression level; and diagnosing the IBD subtype in the subject if the subject has a high expression level relative to the reference value, or not diagnosing the IBD subtype in the subject if the subject does not have a high expression level relative to the reference value. In some embodiments, the method further comprises stimulating the sample with IL12, IL18, or TL1A, or a combination thereof, before assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample. In another embodiment, the one or more biomarkers are described in Table 1, Table 4, Table 5 and/or Table 6 herein. In one embodiment, the IBD subtype is characterized by being treatable with an anti-TL1A therapy.
In various embodiments, the invention provides for a method for diagnosing a disease in a subject. In one embodiment, the method comprises: obtaining a sample from a subject; assaying the expression level of one or more genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein in the sample; comparing the expression level to a reference value of expression level of the one or more genes; and diagnosing a disease in the subject according to the relative difference between the expression level and the reference value. In some embodiments, the method further comprises stimulating the sample with IL12, IL18, or TL1A, or a combination thereof, before assaying the expression level of one or more genes in the sample. In some embodiments, the method further comprises diagnosing the disease in the subject if the subject has an expression level higher than the reference value, or not diagnosing the disease in the subject if the subject does not have an expression level higher than the reference value. In other embodiments, the method further comprises diagnosing the disease in the subject if the subject to has an expression level lower than the reference value, or not diagnosing the disease in the subject if the subject does not have an expression level lower than the reference value. In various further embodiments, the method further comprises prescribing an anti-TL1A therapy to the subject if the subject is diagnosed with the disease. In various further embodiments, the method further comprises administering an anti-TL1A therapy to the subject if the subject is diagnosed with the disease.
In various embodiments, the disease is a TL1A-associated disease. In various embodiments, the disease is fibrosis, Crohn's disease (CD), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease, allergic lung inflammation, asthma, atherosclerosis, lupus, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, type 1 diabetes, lung carcinoma, colon carcinoma, leukemia, lymphoma, transplant rejection, graft versus host disease, or central nervous system injury. In various embodiments, the disease is a subtype of a disease, such as an IBD subtype responsive to an anti-TL1A therapy.
In various embodiments, the method comprises diagnosing the disease in the subject if the subject has an expression profile different from a reference profile, or not diagnosing the disease in the subject if the subject does not have an expression profile different from a reference profile. In accordance with the present invention, the expression profile may comprise a plurality of gene expression levels, in which some gene expression levels may be higher and other gene expression levels may be lower than the reference profile.
In various embodiments, the invention provides for a method of diagnosing susceptibility to an IBD subtype in a subject. In one embodiment, the method comprises: obtaining a sample from the subject; assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample; comparing the expression level to a reference value of expression level; and diagnosing susceptibility to the IBD subtype in the subject if the subject has a high expression level relative to the reference value, or diagnosing no susceptibility to the IBD subtype in the subject if the subject does not have a high expression level relative to the reference value. In another embodiment, the one or more biomarkers are described in Table 1, Table 4, Table 5 and/or Table 6 herein. In one embodiment, the IBD subtype is characterized by being treatable with an anti-TL1A therapy.
In various embodiments, the invention provides for a method for diagnosing susceptibility to an IBD subtype in a subject. In one embodiment, the method comprises: obtaining a sample from the subject; assaying the expression level of one or more genes in the sample; comparing the expression level to a reference value of expression level of the one or more genes; and diagnosing susceptibility to the IBD subtype in the subject if the subject has an expression level different from the reference value, or not diagnosing susceptibility to the IBD subtype in the subject if the subject does not have an expression level different from the reference value. In various embodiments, the one or more genes is listed in Table 1, Table 4, Table 5 and/or Table 6 herein. In various embodiments, the IBD subtype is a subtype responsive to an anti-TL1A therapy.
In some embodiments, assaying the expression level of one or more genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein in the sample comprise assaying at least two, three, four, or five genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein. In other embodiments, assaying the expression level of one or more genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein in the sample comprise assaying all of genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein. In another embodiment, assaying the expression level of one or more genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein in the sample comprise assaying any number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55) of genes listed in Table 1, Table 4, Table 5 and/or Table 6 herein. In various embodiments, the methods described herein comprise assaying the expression level of one or more genes listed Table 6.
In various embodiments, the methods described herein comprise assaying the expression level of one or more genes selected from the group consisting of BIRC3, C17orf49, CCL20, CSF2, CD274, CD74, EPSTI1, FAS, GBP1, GBP4, GBP5, HAPLN3, IFNG, IRF1, NFKBIA, NFKB2, RELB, RGS1, SGK1, STAT1, TAP1, and TRAFD1. In various embodiments, the methods described herein comprise assaying the expression level of one or more genes selected from the group consisting of BATF, CCL20, CD274, CD83, CDKN1A, CHAC1, CSF2, DUSP5, FEZ1, GADD45G, HMSD, IFNG, IL22, IL26, IL411, IRF8, LTA, MFSD2A, MYO1B, NFKBIA, RPL21, SGK1, TNFRSF18, TNFRSF4, TRAF4, and XIST.
In accordance with various embodiments herein, the subject can be human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse or rat. In one embodiment, the subject has a symptom of a TL1A-associated disease, is suspected of having a TL1A-associated disease, or is diagnosed with a TL1A-associated disease. In another embodiment, the subject has received, is receiving, or will receive an anti-TL1A therapy. In another embodiment, the subject has been, is being, or will be treated for a TL1A-associated disease. In another embodiment, the subject is in complete or partial remission, or has a recurrence of a TL1A-associated disease. In one embodiment, the subject has a symptom of an IBD subtype. In another embodiment, the subject is suspected of having an IBD subtype. In some embodiments, the IBD subtype is a subtype responsive to an anti-TL1A therapy.
In one embodiment, the sample comprises a T cell, CD4+ T cell, CD8+ T cell, CD56+ T cell, CD45R0+ T cell, CD45RA+ T cell, NK cell, peripheral blood mononuclear cell (PBMC), or peripheral blood lymphocyte (PBL), or a combination thereof. In various embodiments, the sample is a cell, tissue, or body fluid. In various embodiments, the sample can be serum, urine, blood, plasma, saliva, semen, lymph, or a combination thereof. In various embodiments, the sample can be obtained before, during, or after a treatment of TL1A-associated disease. In various embodiments, the sample can be obtained before, during, or after an anti-TL1A therapy.
In accordance with various embodiments herein, the TL1A-associated disease is fibrosis, Crohn's disease (CD), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease, allergic lung inflammation, asthma, atherosclerosis, lupus, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, type 1 diabetes, lung carcinoma, colon carcinoma, leukemia, lymphoma, transplant rejection, graft versus host disease, or central nervous system injury.
Examples of “TL1A-associated diseases” include, but are not limited to, fibrosis, Crohn's disease (CD), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease, allergic lung inflammation, asthma, atherosclerosis, lupus, rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, type 1 diabetes, lung carcinoma, colon carcinoma, leukemia, lymphoma, transplant rejection, graft versus host disease, or central nervous system injury.
IBD includes several forms of inflammatory diseases and conditions affecting various parts of the gastrointestinal (GI) tract, such as the colon and small intestine. Examples of IBD include, but are not limited to, Crohn's disease (CD), ulcerative colitis (UC), other forms of colitis such as collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behçet's disease, and indeterminate colitis, among others. Crohn's disease (CD) and ulcerative colitis (UC) are two main forms of IBD. Hallmarks of IBD can include inflammation of the digestive tract in compartments of the epithelial mucosa or transmural lesions in the bowel wall.
In various embodiments, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample comprises assaying an mRNA level. In various embodiments, assaying an mRNA level comprises using RNA sequencing, northern blot, in situ hybridization, hybridization array, serial analysis of gene expression (SAGE), reverse transcription PCR, real-time PCR, real-time reverse transcription PCR, or quantitative PCR, or a combination thereof.
In various embodiments, assaying an mRNA level comprises contacting the sample with a polynucleotide probe capable of specifically hybridizing to mRNA of one or more biomarkers associated with TL1A signaling and thereby forming a probe-target hybridization complex.
Hybridization-based RNA assays include, but are not limited to, traditional “direct probe” methods such as, northern blot or in situ hybridization (e.g., Angerer (1987) Meth. Enzymol 152: 649). The methods can be used in a wide variety of formats including, but not limited to, substrate (e.g. membrane or glass) bound methods or array-based approaches. In a typical in situ hybridization assay, cells are fixed to a solid support, typically a glass slide. If a nucleic acid is to be probed, the cells are typically denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to the nucleic acid sequence encoding the protein. The targets (e.g., cells) are then typically washed at a predetermined stringency or at an increasing stringency until an appropriate signal to noise ratio is obtained. The probes are typically labeled, e.g., with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long so as to specifically hybridize with the target nucleic acid(s) under stringent conditions. The preferred size range is from about 200 bases to about 1000 bases. Hybridization protocols suitable for use with the methods of the invention are described, e.g., in Albertson (1984) EMBO J. 3: 1227-1234; Pinkel (1988) Proc. Natl. Acad. Sci. USA 85: 9138-9142; EPO Pub. No. 430,402; Methods in Molecular Biology, Vol. 33: In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994), Pinkel, et al. (1998) Nature Genetics 20: 207-211, and/or Kallioniemi (1992) Proc. Natl Acad Sci USA 89:5321-5325 (1992). In some applications, it is necessary to block the hybridization capacity of repetitive sequences. Thus, in some embodiments, tRNA, human genomic DNA, or Cot-I DNA is used to block non-specific hybridization.
In various embodiments, assaying an mRNA level comprises contacting the sample with one or more polynucleotide primers capable of specifically hybridizing to mRNAs of genes listed in Table 1, Table 4, Table 5 and/or Table 6, forming a primer-template hybridization complex, and performing a PCR reaction. In some embodiments, the one or more polynucleotide primers are primers listed in Table 2. In other embodiments, the one or more polynucleotide primers comprises about 15-45, 20-40, or 25-35 bp sequences that are identical (for forward primers) or complementary (for reverse primers) to sequences of genes listed in Table 1, Table 4, Table 5 and/or Table 6. As a non-liming example, the one or more polynucleotide primers for INFG (e.g., NM_000619.2 with 1240 bp) can comprise sequences that are identical (for forward primers) or complementary (for reverse primers) to INFG's bp 1-20, 5-25, 10-30, 15-35, 20-40, 25-45, 30-50, so on and so forth, until the end of INFG, 1201-1220, 1205-25, 1210-1230, 1215-1235, 1220-1240. While not listed here exhaustively because of the space, all these polynucleotide primers for INFG and other genes listed in Table 1, Table 4, Table 5 and/or Table 6 can be used in the present invention. In various embodiments, the one or more polynucleotide primers are labeled with radioisotopes or fluorescent molecules. As the labeled primers emit radio or fluorescent signals, the PCR products containing the labeled primers can be detected and analyzed with a variety of imaging equipment.
Methods of “quantitative” amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. Detailed protocols for quantitative PCR are provided in Innis, et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.). Measurement of DNA copy number at microsatellite loci using quantitative PCR anlaysis is described in Ginzonger, et al. (2000) Cancer Research 60:5405-5409. The known nucleic acid sequence for the genes is sufficient to enable one of skill in the art to routinely select primers to amplify any portion of the gene. Fluorogenic quantitative PCR may also be used in the methods of the invention. In fluorogenic quantitative PCR, quantitation is based on amount of fluorescence signals, e.g., TaqMan and sybr green. Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren, et al. (1988) Science 241:1077, and Barringer et al. (1990) Gene 89: 117), transcription amplification (Kwoh, et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), self-sustained sequence replication (Guatelli, et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874), dot PCR, and linker adapter PCR, etc.
Expression Level Assay—Protein
In various embodiments, assaying the expression level of one or more biomarkers associated with TL1A signaling in the sample comprises assaying a protein level. In various embodiments, assaying a protein level comprises using western blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, or mass spectrometry, or a combination thereof.
In various embodiments, assaying a protein level comprises contacting the sample with antibodies capable of specifically binding to proteins of genes listed in Table 1, Table 4, Table 5 and/or Table 6 and thereby forming antigen-antibody complexes. In the methods and assays of the invention, the expression levels of proteins encoded by biomarker genes listed in Table 1, Table 4, Table 5 and/or Table 6, or fragments or variants thereof can be determined using antibodies specific for those individual proteins or fragments or variants thereof and detecting immunospecific binding of each antibody to its respective cognate biomarker protein.
Antibodies, both polyclonal and monoclonal, can be produced by a skilled artisan either by themselves using well known methods or they can be manufactured by service providers who specialize making antibodies based on known protein sequences. In the present invention, the protein sequences of biomarker gens are known and thus production of antibodies against them is a matter of routine.
For example, production of monoclonal antibodies can be performed using the traditional hybridoma method by first immunizing mice with an antigen which may be an isolated protein of choice or fragment thereof (for example, a protein encode by a biomarker gene listed in Table 1, Table 4, Table 5 and/or Table 6, or a fragment thereof or a variant thereof) and making hybridoma cell lines that each produce a specific monoclonal antibody. The antibodies secreted by the different clones are then assayed for their ability to bind to the antigen using, e.g., ELISA or Antigen Microarray Assay, or immuno-dot blot techniques. The antibodies that are most specific for the detection of the protein of interest can be selected using routine methods and using the antigen used for immunization and other antigens as controls. The antibody that most specifically detects the desired antigen and protein and no other antigens or proteins are selected for the processes, assays and methods described herein. The best clones can then be grown indefinitely in a suitable cell culture medium. They can also be injected into mice (in the peritoneal cavity, surrounding the gut) where they produce an antibody-rich ascites fluid from which the antibodies can be isolated and purified. The antibodies can be purified using techniques that are well known to one of ordinary skill in the art.
Any suitable immunoassay method may be utilized, including those which are commercially available, to determine the expression level of a biomarker protein or a variant thereof assayed according to the invention. Extensive discussion of the known immunoassay techniques is not required here since these are known to those of skill in the art. Typical suitable immunoassay techniques include sandwich enzyme-linked immunoassays (ELISA), radioimmunoassays (RIA), competitive binding assays, homogeneous assays, heterogeneous assays, etc.
For example, in the assays of the invention, “sandwich-type” assay formats can be used. An alternative technique is the “competitive-type” assay. In a competitive assay, the labeled probe is generally conjugated with a molecule that is identical to, or an analog of, the analyte. Thus, the labeled probe competes with the analyte of interest for the available receptive material. Competitive assays are typically used for detection of analytes such as haptens, each hapten being monovalent and capable of binding only one antibody molecule.
The antibodies can be labeled. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, label with a chemiluminescent compound. For example, the detection antibody can be labeled with catalase and the conversion uses a colorimetric substrate composition comprises potassium iodide, hydrogen peroxide and sodium thiosulphate; the enzyme can be alcohol dehydrogenase and the conversion uses a colorimetric substrate composition comprises an alcohol, a pH indicator and a pH buffer, wherein the pH indicator is neutral red and the pH buffer is glycine-sodium hydroxide; the enzyme can also be hypoxanthine oxidase and the conversion uses a colorimetric substrate composition comprises xanthine, a tetrazolium salt and 4,5-dihydroxy-1,3-benzene disulphonic acid. In one embodiment, the detection antibody is labeled by covalently linking to an enzyme, label with a fluorescent compound or metal, or label with a chemiluminescent compound.
Direct and indirect labels can be used in immunoassays. A direct label can be defined as an entity, which in its natural state, is visible either to the naked eye or with the aid of an optical filter and/or applied stimulation, e.g., ultraviolet light, to promote fluorescence. Examples of colored labels which can be used include metallic sol particles, gold sol particles, dye sol particles, dyed latex particles or dyes encapsulated in liposomes. Other direct labels include radionuclides and fluorescent or luminescent moieties. Indirect labels such as enzymes can also be used according to the invention. Various enzymes are known for use as labels such as, for example, alkaline phosphatase, horseradish peroxidase, lysozyme, glucose-6-phosphate dehydrogenase, lactate dehydrogenase and urease.
The antibody can be attached to a surface. Examples of useful surfaces on which the antibody can be attached for the purposes of detecting the desired antigen include nitrocellulose, PVDF, polystyrene, and nylon.
In some embodiments of the processes, assays and methods described herein, detecting the level of antibodies reactive to a biomarker protein or a variant thereof includes contacting the sample from the cancer patient with an antibody or a fragment thereof that specifically binds a biomarker protein or a variant thereof, forming an antibody-protein complex between the antibody and the biomarker protein or a variant thereof present in the sample, washing the sample to remove the unbound antibody, adding a detection antibody that is labeled and is reactive to the antibody bound to the biomarker protein or a variant thereof in the sample, washing to remove the unbound labeled detection antibody and converting the label to a detectable signal, wherein the detectable signal is indicative of the level of biomarker protein or a variant thereof in the sample from the patient. In some embodiments, the effector component is a detectable moiety selected from the group consisting of a fluorescent label, a radioactive compound, an enzyme, a substrate, an epitope tag, electron-dense reagent, biotin, digonigenin, hapten and a combination thereof. In some embodiments, the detection antibody is labeled by covalently linking to an enzyme, labeled with a fluorescent compound or metal, labeled with a chemiluminescent compound. The level of biomarker protein may be obtained by assaying a light scattering intensity resulting from the formation of an antibody-protein complex formed by a reaction of biomarker protein in the sample with the antibody, wherein the light scattering intensity of at least 10% above a control light scattering intensity indicates the likelihood of chemotherapy resistance.
In various embodiments, the reference value of expression level is the median or mean expression level from a population of subjects who have no TL1A-associated disease. In one embodiment, the reference value of expression level is the median or mean expression level from a population of subjects who have no IBD. In various embodiments, the reference value of expression level is the median or mean expression level from a population of subjects who are unlikely to be responsive to an anti-TL1A therapy. In various embodiments, the reference value of expression level is the median or mean expression level from a population of subjects who are not responsive to an anti-TL1A therapy. In additional embodiments, the reference value is the expression level of a biomarker gene or a variant thereof in a sample obtained from the subject at a different (for example, an earlier) time point, such as during diagnosis, before treatment, during treatment, after treatment or a combination thereof.
Various statistical methods, for example, a two-tailed student t-test with unequal variation, may be used to measure the differences in expression levels of a biomarker gene between the subject's sample and a control sample from a normal/healthy individual, or a reference value of expression level generate by computer algorithm pooling many control samples, as described herein. A significant difference may be achieved where the p value is equal to or less than 0.05.
In various embodiments, the expression level of a biomarker gene or a variant thereof in the subject as compared to the reference value is higher by at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%. In various embodiments, the expression level of a biomarker gene or a variant thereof in the subject as compared to the reference value is increased by at least or about 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, 50-fold, 55-fold, 60-fold, 65-fold, 70-fold, 75-fold, 80-fold, 85-fold, 90-fold, 95-fold, or 100-fold.
In various embodiments, the anti-TL1A therapy comprises an agent that specifically binds TL1A or DR3 and blocks TL1A-DR3 interaction. In one embodiment, the anti-TL1A therapy comprises an anti-TL1A antibody or a fragment thereof, antagonistic anti-TL1A antibodies, or an isolated antigen-binding polypeptide that binds specifically to TL1A, or a combination thereof. In one embodiment, the anti-TL1A therapy comprises a soluble form of TL1A that specifically binds DR3. In one embodiment, the anti-TL1A therapy comprises an anti-DR3 antibody or a fragment thereof, antagonistic anti-DR3 antibodies, an isolated antigen-binding polypeptide that binds specifically to DR3, or a combination thereof. In one embodiment, the anti-TL1A therapy comprises a soluble form of DR3 that specifically binds TL1A. In one embodiment, the anti-TL1A therapy comprises a soluble decoy DR3 polypeptide, a polypeptide comprising a DR3 extracellular domain, a DR3-Fc protein, or a polypeptide comprising a DR3 pre-ligand assembly domain (a DR3-PLAD peptide), or a combination thereof. In one embodiment, the anti-TL1A therapy comprises a dominant negative DR3. In one embodiment, the anti-TL1A therapy comprises agents targeting TL1A or DR3 expression (e.g., ribozymes, aptamers and antisense nucleic acids), a nucleic acid antagonist of TL1A, or a nucleic acid antagonist of DR3), or a combination thereof. In one embodiment, the anti-TL1A therapy comprises GEP and GEP peptides including the peptide(s) denoted Atsttrin, Atsttrin-α variants, or a combination thereof.
The duration and/or dose of treatment with anti-TL1A therapies may vary according to the particular anti-cancer agent or combination thereof. An appropriate treatment time for a particular anti-TL1A therapeutic agent will be appreciated by the skilled artisan. The invention contemplates the continued assessment of optimal treatment schedules for each anti-TL1A therapeutic agent, where the TL1A-specific biomarker signature of the subject as determined by the methods of the invention is a factor in determining optimal treatment doses and schedules.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
CD4+ T cells from normal individuals were treated with recombinant TL1A following priming with IL12 and IL18. RNA sequencing was utilized to measure TL1A mediated gene activation and to identify biomarkers responsive to TL1A signaling.
In one example, CD4+ cells were isolated from normal donors, rested overnight, and then treated for 8 hours in three groups: untreated (UT), primed (IL12+IL18), and stimulated (IL12+IL18+TL1A)). RNA were isolated from the cells and used for Fluidigm qPCR for 24 genes (22 biomarker genes and 2 housekeeping ActB and EEF1A1). Real-time PCR of 22 genes validated these genes as markers for activation by TL1A (
All primers were optimized for efficiency and absence of off-target amplification using cyber-green qPCR.
Samples were prepared with the Illumina TruSeq RNA library preparation kit and sequenced on the Illumina GA IIx.
RNA sequencing data were prescreened: all failed probe data were removed and all genes with fewer than 3 samples (out of 12) with FPKM>5 were removed. Total 8695 genes passed prescreen (out of 24789).
RNA sequencing data were analyzed using BRB Array Tools developed by Richard Simon & BRB-ArrayTools Development Team. It is available at the website of Biometric Research Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute. BRB-ArrayTools is an integrated package for the visualization and statistical analysis of DNA microarray gene expression data. It was developed by professional statisticians experienced in the analysis of microarray data and involved in the development of improved methods for the design and analysis of microarray based experiments. The array tools package utilizes an Excel front end. Scientists are familiar with Excel and utilizing Excel as the front end makes the system portable and not tied to any database. The input data is assumed to be in the form of Excel spreadsheets describing the expression values and a spreadsheet providing user-specified phenotypes for the samples arrayed. The analytic and visualization tools are integrated into Excel as an add-in. The analytic and visualization tools themselves are developed in the powerful R statistical system, in C and Fortran programs and in Java applications. Visual Basic for Applications is the glue that integrates the components and hides the complexity of the analytic methods from the user. The system incorporates a variety of powerful analytic and visualization tools developed specifically for microarray data analysis. In one example, genes with highest 20% variance were selected and genes missing >50% values were eliminated.
Fluidigm qPCR technology was used. In one example, PCR was performed in 48×48 format according to protocol with modification of primer concentrations which were adjusted to optimal concentrations as noted in Table 3.
PBMC (peripheral blood mononuclear cells) were isolated from healthy volunteers by separation on Ficoll-Hypaque gradients. CD4+ T cells were isolated according to manufacturer's recommendations using negative selection by depletion with magnetic beads (Stemcell Technologies, Vancouver, BC, Canada) and were at least 95% pure.
CD4+ T cells were cultured overnight (37° C. with 5% CO2) in RPMI 1640 with 10% fetal calf serum. For the primed group (IL12+IL18), cells were treated with IL-12 (0.5 ng/ml) and IL-18 (50 ng/ml) for 8 hours at 37° C. prior to RNA isolation. For the TL1A-stimulated group (IL12+IL18+TL1A), cells were treated with IL-12 (0.5 ng/ml), IL-18 (50 ng/ml), and recombinant TL1A (100 ng/ml) (Fitzgerald, North Acton, Mass.) for 8 hours at 37° C. prior to RNA isolation. RNA was isolated using RNeasy Plus Mini Kit (Qiagen, Germantown, Md.). Other examples are described in Papadakis et al. (TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells; J Immunol. 2004 Jun. 1; 172(11):7002-7), which is incorporated herein by reference as though fully set forth.
Other examples are described in Papadakis et al. (TL1A synergizes with IL-12 and IL-18 to enhance IFN-gamma production in human T cells and NK cells; J Immunol. 2004 Jun. 1; 172(11):7002-7), which is incorporated herein by reference as though fully set forth.
In one example, 20 normal control (NL), 20 CD, and 18 UC samples were rested overnight, activated for 8 hours with (IL12+IL18) or (IL12+IL18/TL1A), and analyzed for expression levels of 48 genes. In another example, 21 NL, 15 NL-H, 20 CD, and 18 UC samples were rested overnight, activated for 8 hours with (IL12+IL18) or (IL12+IL18+TL1A), and analyzed for expression levels of 20 genes. Results are shown in
The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described can be achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some preferred embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by inclusion of one, another, or several advantageous features.
Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.
Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.
In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.
Preferred embodiments of this application are described herein, including the best mode known to the inventors for carrying out the application. Variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.
All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of same that may have a limiting affect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.
It is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.
Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and/or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the specification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).
The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit to the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.
While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.)
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/054425 | 9/5/2014 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61874487 | Sep 2013 | US |
