Patent Application
20190178889
The present invention relates to methods and materials for development of high-throughput screening assays for measuring cyclic GMP-AMP synthase (cGAS) activity and/or detecting G(2′-5′)pA(3′-5′)p (cGAMP).
Cyclic GMP-AMP synthase (cGAS) (UniProtKB—Q8N884) is a recently discovered enzyme that acts as a DNA sensor to elicit an immune response to pathogens via activation of the stimulator of interferon genes (STING) receptor. Shortly after its discovery in 2013, aberrant activation of cGAS by self-DNA was shown to underlie debilitating and sometimes fatal autoimmune diseases, such as systemic lupus erythematosus (SLE), scleroderma, and Aicardi-Goutieres Syndrome (AGS). Knockout studies in animal models have clearly indicated that inhibiting cGAS is a promising approach for therapeutic intervention. Additionally, recent studies have shown that the cGAS-STING pathway plays a key role in the innate immune response to tumors, and stimulation of the pathway is a promising strategy being tested clinically for cancer immunotherapy.
However, there are no high-throughput screening (HTS)-compatible assay methods for measuring cGAS enzyme activity, and development of a homogenous assay presents a considerable challenge, as it requires selective detection of the cyclic dinucleotide product, cGAMP (
It is against the above background that the present invention provides certain advantages and advancements over the prior art.
Although this invention disclosed herein is not limited to specific advantages or functionalities, the invention provides an antibody that specifically binds G(2′-5′)pA(3′-5′)p (cGAMP).
In one aspect of the antibodies disclosed herein, the antibody specifically binds cGAMP in the presence of excess ATP, GTP or both.
In one aspect of the antibodies disclosed herein, the cGAMP is produced in an enzymatically catalyzed reaction.
In one aspect of the antibodies disclosed herein, the antibody binds cGAMP with a Kd of less than 100 nM, less than 5 nM or less than 100 pM.
In one aspect of the antibodies disclosed herein, the binding is in a biological sample.
In one aspect of the antibodies disclosed herein, the biological sample is a cell extract or a tissue extract.
In one aspect of the antibodies disclosed herein, the antibody is a mouse monoclonal antibody.
In one aspect of the antibodies disclosed herein, the antibody is a single-chain variable fragment (scFv).
In one aspect of the antibodies disclosed herein, the antibody is conjugated to a Tb-chelate or an Eu-chelate.
The invention also provides an assay method for measuring cGAMP produced in an enzymatically catalyzed reaction, comprising:
In one aspect of the assay methods disclosed herein, the reaction is catalyzed by cyclic GMP-AMP synthase (cGAS).
The invention also provides an assay method for measuring cyclic GMP-AMP synthase (cGAS) activity, comprising:
In one aspect of the assay methods disclosed herein, the fluorescently labeled cGAMP tracer is displaced by unlabeled cGAMP in the sample.
In one aspect of the assay methods disclosed herein, the cGAMP tracer is labeled with a fluorescein, Alexa Fluor, Dylight, and/or Atto dye.
In one aspect of the assay methods disclosed herein, the signal is a time resolved Förster resonance energy transfer (TR-FRET) signal or a fluorescence polarization (FP) signal.
In one aspect of the assay methods disclosed herein, the assay method is:
In one aspect of the assay methods disclosed herein, the assay method is a high-throughput screening (HTS) assay method.
The invention also provides an assay kit for detecting and measuring cGAMP produced in an enzymatically catalyzed reaction, comprising the antibodies disclosed herein, ATP, GTP or both, and a fluorescently labeled cGAMP tracer.
The invention also provides an antibody pair comprising a first antibody and a second antibody, wherein:
In one aspect of antibody pairs disclosed herein, the first antibody binds cGAMP with a Kd of less than 100 nM, less than 5 nM, or less than 100 pM; and the second antibody binds cGAMP or a complex of the first antibody and cGAMP with a Kd of less than 100 nM, less than 5 nM, or less than 100 pM.
In one aspect of antibody pairs disclosed herein, the first antibody and/or the second antibody comprise a single-chain variable fragment (scFv).
In one aspect of antibody pairs disclosed herein, the binding is in a biological sample.
In one aspect of antibody pairs disclosed herein, the biological sample is a cell extract or a tissue extract.
In one aspect of antibody pairs disclosed herein, the biological sample is a cell lysate.
In one aspect of antibody pairs disclosed herein, the first antibody is conjugated to a Tb-chelate or an Eu-chelate and the second antibody is conjugated to a fluorescent label.
In one aspect of antibody pairs disclosed herein, the first antibody is conjugated to a fluorescent label, and the second antibody is conjugated to a Tb-chelate or an Eu-chelate.
In one aspect of antibody pairs disclosed herein, the fluorescent label comprises a fluorescein, Alexa Fluor, Dylight, and/or Atto dye.
The invention also provides an assay method for measuring cGAMP produced in an enzymatically catalyzed reaction, comprising:
In one aspect of the assay method disclosed herein, the reaction is catalyzed by cyclic GMP-AMP synthase (cGAS).
The invention also provides an assay method for measuring cyclic GMP-AMP synthase (cGAS) activity comprising:
In one aspect of the assay method disclosed herein, the signal is a time-resolved Förster resonance energy transfer (TR-FRET) signal or a fluorescence polarization (FP) signal.
In one aspect of the assay method disclosed herein, the assay method is a high-throughput screening (HTS) assay method.
The invention also provides an assay kit for detecting and measuring cGAMP produced in an enzymatically catalyzed reaction, comprising the antibody pairs disclosed herein, and ATP, GTP, or both.
These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description.
The following detailed description of the embodiments of the present invention can be best understood when read in conjunction with the following drawings, where like structure is indicated with like reference numerals and in which:
Skilled artisans will appreciate that elements in the figures are illustrated for simplicity and clarity and have not necessarily been drawn to scale. For example, the dimensions of some of the elements in the figures can be exaggerated relative to other elements to help improve understanding of the embodiment(s) of the present invention.
All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.
Before describing the present invention in detail, a number of terms will be defined. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, reference to a “nucleic acid” means one or more nucleic acids.
It is noted that terms like “preferably,” “commonly,” and “typically” are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present invention.
For the purposes of describing and defining the present invention it is noted that the term “substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The term “substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
As used herein, the terms “polynucleotide,” “nucleotide,” “oligonucleotide,” and “nucleic acid” can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.
As used herein, the terms “homogenous assay,” “homogenous format,” and “homogenous detection” can be used to refer to detection of an analyte by a simple mix and read procedure. A homogenous assay does not require steps such as sample washing or sample separation steps. Examples of homogenous assays include time-resolved Förster resonance energy transfer (TR-FRET), fluorescence polarization (FP), fluorescence intensity (FI), and luminescence-based assays.
As used herein, the term “and/or” is utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and/or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x and (y or z),” or “x or y or z.”
cGAS-cGAMP-STING Pathway
The cGAS-cGAMP-STING pathway activates the immune system in response to foreign DNA. The presence of DNA in the cytosol of eukaryotic cells is an indicator of infection or cellular damage, and it elicits a strong immune response, including type I interferon (IFN-I) induction (see
Blocking cGAS activity prevents aberrant activation of inflammatory pathways in monogenic autoimmune diseases. Inappropriate activation of the immune system by nucleic acids contributes to the pathology of a number of autoimmune diseases (
Mouse models have provided compelling evidence for the involvement of the cGAS-cGAMP-STING pathway—and specifically cGAS—in the pathogenesis of monogenic autoimmune diseases and, by extension, at least some types of SLE. 85% of AGS patients carry mutations in the DNA exonuclease Trex1 or RNase H2, which degrade dsDNA and DNA:RNA hybrids, respectively; knocking out and/or introducing AGS mutations to these nucleases causes lethal autoimmune disease in mice (Mackenzie, et al., 2016, EMBO J 35(8):831-44; Rice, et al., 2015, J Clin. Immunol. 35(3):235-43). Genetic ablation of cGAS or STING in nuclease-deficient mice protects against lethality and eliminates key autoimmune phenotypes, including ISG induction, autoantibody production, and T-cell activation (Gao, et al., 2015, Proc Natl Acad Sci USA 112(42):E5699-705; Gray, et al., 2015, J Immunol 195(5):1939-43; Mackenzie, et al., 2016, EMBO J 35(8):831-44; Yang, et al., 2007, Cell 131(5):873-86; Pokatayev, et al., 2016, J Exp Med 213(3):329-36). Similar results were observed when cGAS was eliminated in mice lacking DNase II, a lysosomal endonuclease that clears DNA from dead cells (Gao, et al., 2015, Proc Natl Acad Sci USA 112(42):E5699-705). Mutations that impair the function of RNase H2, Trex1, and other nucleic acid modifying enzymes are found less frequently in SLE (Mackenzie, et al., 2016, EMBO J 35(8):831-44) and in familiar chilblain lupus (Lee-Kirsch, et al., 2007, J Mol Med 85(5):531-7).
Furthermore, elevated levels of cGAMP were detected in Trex1 deficient mice, and knocking out cGAS prevented its accumulation (Gao, et al., 2016, Proc Nat Acad Sci USA 112(42):E5699-705). Similar results were observed when cGAS was eliminated in mice lacking DNasell, a lysosomal endonuclease that clears DNA from dead cells (Gao, et al., 2016, Proc Nat Acad Sci USA 112(42):E5699-705). RNA:DNA hybrids, which can be generated during aberrant DNA replication, can also induce a cGAS-cGAMP-STING dependent IFN-I response in cells, and activation of cGAS by RNA/DNA hybrids was demonstrated in in vitro biochemical assays (Mankan, et al., 2014, The EMBO journal, 33(24):2937-46). Mutations that impair the function of RNase H2, the major enzyme responsible for clearing DNA:RNA hybrids, are the predominant cause of AGS and are found less frequently in SLE (Mackenzie, et al., 2016, The EMBO journal, 35(8):831-44). Very recently, two groups showed that mice lacking functional RNaseH2 show strong ISG transcript upregulation, and elimination of cGAS—or STING—in the RNaseH2 deficient mice rescued the inflammatory phenotypes (Mackenzie, et al., 2016, EMBO J, 35(8):831-44; Yang, et al., 2007, Cell, 131(5):873-86; Pokatayev, et al., 2016, J Exp Med, 213(3): 329-36).
The cGAS/STING pathway drives IFN production in pDCs, and it is activated in SLE patients. The case for targeting cGAS in idiopathic SLE is rapidly building. IFN-I are strongly implicated in the pathogenesis of SLE (Elkon and Wedeman, 2012, Curr Opin Rheumatol 24(5):499-505), and approximately two thirds of SLE patients have a blood interferon (IFN) signature (Baechler, et al., 2003, Proc Nat Acad Sci USA 100(5):2610-5). Plasmacytoid dendritic cells (pDCs) are the most prolific producers of IFN-I, and their continuous stimulation is a major driver of SLE progression (Ronnblom, et al., 2003, Autoimmunity 36(8):463-72) (
Stimulation of the cGAS-cGAMP-STING pathway is also a promising approach for cancer immunotherapy (Lemos, et al., 2014, Expert Rev Clin Immunol 11(1):155-65). Immunotherapy approaches such as immune checkpoint blockade are transforming oncology, however many patients do not respond to existing agents, and alternatives are needed. In vitro and in vivo studies have shown that activation of the cGAS-STING pathway in pDCs by DNA from tumor cells and/or dead host cells is an important mechanism for initiating an antitumor T cell response in the tumor microenvironment (Ohkuri, et al., 2014, Cancer Immunol Res 2(12):1199-208; Woo, et al., 2014, Immunity 41(5):830-42) (
cGAMP Detection Methods
Simple, economical methods for detecting cGAMP will have a profound impact on efforts to elucidate the roles of the cGAS/STING pathway in immunity and to target it therapeutically. Despite rapid progress since their discovery in 2013, there are major gaps in the understanding of how cGAS and cGAMP function, both at the cellular and biochemical level. Moreover, there are very few tools for discovering and developing drugs that target the pathway, especially methods that are compatible with an automated HTS approach. Currently, detection of cGAMP in cell or tissue samples requires extraction and analysis by LC/MS (An, et al., 2017, Arthritis Rheumatol 69(4):800-7; Gray, et al., 2015, J Immunol 195(5):1939-43), and there are no methods for direct detection of cGAMP in cell lysates. The only reported method for measuring cGAS enzyme activity in vitro is the semi-quantitative determination of radioactive cGAMP formation from radiolabeled ATP and GTP precursors (Ablasser A, et al., 2013, Nature 498(7454):380-4; Diner, et al., 2013, Cell Rep 3(5):1355-61; Kranzusch, et al., 2013, Cell Rep 3(5):1362-8). Aside from the undesirability of radioassays, this method requires separation by thin layer chromatography (TLC), and thus is not amenable to HTS.
The ability to readily detect cGAMP without chromatographic separation and/or radioactive labeling would greatly accelerate basic research and drug discovery targeting the pathway. Development of a selective cGAMP antibody is an important milestone. The homogenous, fluorescent assays described herein for detecting cGAMP can have a profound impact on basic research, drug discovery, and translational studies targeting STING/cGAS for autoimmune diseases and cancer immunotherapy.
cGAS activity assays will enable basic enzymological research and screening for small molecule modulators. The cumbersome and semi-quantitative nature of the radioassays and LC/MS methods used to measure cGAS activity have hindered meaningful enzymological studies. Much of the critical information needed for understanding the physiological roles of cGAS and pursuing a targeted discovery program, such as the kinetics of activation by nucleic acids of Km values for ATP and GTP, is either non-quantitative or completely lacking. For example, the kcat value measured and provided herein by the instant inventors was the first quantitative determination of cGAS. The biochemical HTS assay for cGAS disclosed herein enables quantitative cGAS enzymological studies as well as HTS campaigns to discover first-in-class immunotherapy drugs for devastating autoimmune diseases and cancer.
Assays for detection of cGAMP in extracted cell and tissue samples will allow investigation of cGAMP as a biomarker for disease status and drug action. There is compelling evidence that pharmacological modulation of cGAMP could be used to treat serious autoimmune diseases and to invoke an anti-tumor T cell response. Assays for detecting cGAMP in cell and tissue samples from animals and humans would provide a simple, direct way to monitor the action of lead molecules and/or experimental drugs that target cGAS, and eventually for identification of responders in clinical studies, e.g., SLE patients with high levels of cGAMP in PBMCs as candidates for cGAS inhibitors (An, et al., 2017, Arthritis Rheumatol 69(4):800-7).
Methods for direct, in-well detection of cGAMP in cell lysates will enable basic cellular studies and cellular HTS assays. As the primary signaling molecule that pDC cells use to initiate responses to viral and tumor cell DNA (Chen, et al., 2016, Nat Immunol 17(10):1142-9), upstream of both T and B cell activation, cGAMP plays a fundamental roles in human immunity. Yet very little is known about the dynamics of cGAMP levels inside and outside the cell and how they impact STING activation. cGAS is cytosolic, and it is known that cGAMP is transferred between cells through gap junctions (Chen, et al., 2016, Nat Immunol 17(10):1142-9). However, exogenously added cGAMP activates the STING pathway, both in cultured cells and in animals, and the only known cGAMP phosphodiesterase, ENPP1, functions extracellularly (Chen, et al., 2016, Nat Immunol 17(10):1142-9), suggesting that there may be specific transporters. Development of simple assays for cGAMP detection directly in culture media and cell lysates will enable detailed, quantitative studies of how the unique immunomodulator controls STING pathway activation. From a drug discovery perspective, cellular cGAS assays disclosed herein will be used to test the cellular activity of cGAS modulators identified in biochemical screens and to allow cellular screening for compounds that activate or inhibit cGAS indirectly, e.g., by modulating the uptake or intracellular production of stimulatory DNA.
Assays for the cyclic nucleotides that serve as secondary messengers for G protein-coupled receptors (GPCRs), cAMP and cGMP, are a useful comparison. Since their introduction in the early 2000's, fluorescence-based assays for cAMP have become an invaluable tool for elucidating GPCR function, and they are still the predominant HTS method used for discovery of new GPCR modulating drugs (Norskov-Lauritsen, et al., 2014, Int J Mol Sci 15(2):2554-72). Development of cGAMP assays could make a similar impact, but development is more challenging: whereas cAMP acts at micromolar concentrations, cGAMP binds STING with low nanomolar affinity, thus its detection in cells requires greater sensitivity.
The disclosure provided herein includes the development and validation of cGAS enzymatic assays, establishing key feasibility for the development of highly specific cGAMP antibodies and fluorescent tracers. The disclosure further includes the optimization of assay reagents and detection formats to detect cGAMP in cell lysates and tissue samples.
Development of cGAMP immunoassays with FP and TR-FRET readouts enables mix-and-read cGAS enzyme assays with the sensitivity and robustness required for automated HTS platforms. Applicants have previously developed the use of homogenous immunodetection of nucleotides for HTS enzyme assays (Lowery, et al., 2006, Expert Opin Ther Targets, 10(1):179-90). Applicants' Transcreener assays for ADP, GDP, UDP, and AMP/GMP have been broadly used in tens of millions of wells of pharma, biotech, and academic HTS for diverse enzyme targets including kinases, ATPases, GTPases, and glyscosyltransferases (Huss, et al., 2007, J. Biomol Screen, 12(4):578-84; Reichman, et al., 2015, J. Biomol Screen, 20(10):1294-9; Liu, et al., 2007, Assay Drug Dev Techol., 5(2):225-35). See, also, U.S. Pat. Nos. 7,332,278, 7,355,010, 7,847,066, 7,378,505, and 8,088,897, which have been incorporated herein by reference in their entirety. Antibodies that selectively recognize nucleotides that differ by as little as a single phosphate (Lowery, et al., 2006, Expert Opin Ther Targets, 10(1):179-90; Klenman-Leyer, et al., 2009, Assay Drug Dev Technol., 7(1):56-67) are the core of the technology disclosed herein.
To enable HTS efforts targeting cGAS, competitive immunoassays for cGAMP have been developed with fluorescence polarization (FP) and TR-FRET signals (
In some embodiments, monoclonal antibodies to cGAMP were generated in mice. See Example 1. However, in other embodiments, polyclonal antibodies to cGAMP are generated in rabbits. The polyclonal antibodies were purified to obtain cGAMP-specific antibodies prior to labeling with lanthanides. In some embodiments, polyclonal antibodies were used for FP assays.
In some embodiments, human cGAS was expressed in E. coli to produce a functionally pure, active enzyme. In some embodiments, solubility of cGAS was optimized and/or cGAS was crystallized. Crystallization parameters such as culture temperature, inducer concentration, and E. coli host strain were modified for crystallization. In some embodiments, detection of cGAMP produced by cGAS was optimized using competitive immunoassays. Next, pilot screens were performed with a 1280 compound drug library and a 20K diversity library. See Example 2. In some embodiments, agents were tested to reduce non-specific binding to an antibody or tracer; e.g., non-ionic detergents, carrier proteins (bovine serum albumin (BSA) and bovine gamma globulin (BGG)). In some embodiments, cGAS was further purified using gradient elution from a cation exchange resin such as SP-Sepharose.
In further embodiments, cGAMP detection methods for cellular cGAS HTS assays and for translational studies with animal models were developed. Assay methods were optimized for detection of cGAMP in cell lysates and tissue samples as a marker for activation of the cGAS-cGAMP-STING pathway. Detection of cGAMP in biological samples is currently dependent on LC-MS. Development of a simple, homogenous assay can have very broad impact, not only on HTS efforts targeting cGAS or upstream targets (e.g., DNA uptake machinery), but also for monitoring cGAS activation status in tissue samples from animal models or patients. The lysate/tissue assays require a cGAMP antibody with negligible cross-reactivity to any other cellular nucleotides. Profiling the selectivity of the monoclonal antibodies identified in Example 1 against diverse nucleotides provide information needed to design alternative immunogens (e.g., conjugation to different sites on cGAMP), if necessary, to eliminate off-target binding (Staeben, et al., 2010, Assay Drug Dev Techol., 8(3):344-55; Klenman-Leyer, et al., 2009, Assay Drug Dev Technol., 7(1):56-67). Furthermore, FP and TR-FRET based competitive immunoassays for cyclic AMP (the mononucleotide) are widely used as cellular HTS assays for GPCR activation (Degorce, et al., 2009, Curr Chem Genomics, 3:22-32; Staeben, et al., 2010, Assay Drug Dev Technol., 8(3):344-55).
In some embodiments, the potential for compound interference with the cGAMP FP and TR-FRET assays was tested using the 1280 compound LOPAC library of pharmacologically active compounds (Sigma), which includes many scaffolds found in larger screening libraries. Assay robustness was assessed using a larger 20K set of compounds in an orthogonally pooled library from Lankenau Institute of Medical Research (LIMR) (Donover, et al., 2013, Comb Chem High Throughput Screen, 16(3):180-8). LIMR's library has been filtered for adherence to Lipinski's rule of five and lack of reactive groups. Principal component analysis indicated that ˜30%-50% of the collection represents unique chemical assemblies that are not present in the NIH-MLCPN library or commercial collections; it has been used to generate tractable scaffold series for diverse target types (Cheng, et al., 2013, J. Lab Autom., 19(3):297-303; Malecka, et al., 2014, ACS Chem Biol., 9(7):1603-12; Thompson, et al., 2010, J. Biomol Screen, 15(9):1107-15; Fera, et al., 2012, Chem Biol., 19(4):518-28).
In some aspects, the interference pre-screen was performed for both the FP and TR-FRET assays. These reactions mimic completed cGAS reactions; i.e., 10% conversion of ATP/GTP to cGAMP, but lack enzyme. Thus, they allow identification of compounds that cause an increase or decrease in the expected signal because of interference with the detection reagents. In some embodiments, following the interference pre-screen, live screens were performed with the LOPAC library or a larger diversity set using a cGAMP assay that provides the best performance, including resistance to interference. In some embodiments, all compounds were provided pre-dispensed in assay-ready plates at 10 μM assay concentration, n=1. In some embodiments, hits were confirmed with dose response measurements using the orthogonal Transcreener cGAMP assay method.
HTS enzyme assays were generally run under initial velocity conditions; i.e., less than 20% conversion of substrates to products, therefore the cGAS assay method disclosed herein requires an antibody that specifically binds cGAMP in the presence of an excess of the substrates, ATP and GTP. In some embodiments, an antibody with 100-fold selectivity fulfills this requirement and produces a very good signal (Staeben, et al., 2010, Assay Drug Dev Technol., 8(3):344-55; Klenman-Leyer, et al., 2009, Assay Drug Dev Technol., 7(1):56-67).
Sensitivity requirements are determined largely by the kinetic properties of the target enzyme. Most biochemical screens are performed with substrates at their Km concentrations to insure detection of competitive inhibitors. So, for measuring enzyme initial velocity, an assay must be capable of robust detection of reaction products at concentrations several-fold below the substrate Km. Though the kinetic parameters of the cGAS enzyme have not yet been reported, the target disclosed herein was a robust detection of 500 nM cGAMP. This sensitivity can allow for the use of ATP and GTP concentrations as low as 5 μM, which is likely to be well below their Km values given their high micromolar concentrations in the cell.
In some embodiments, mAb/tracer pair(s) that enable detection of 500 nM cGAMP in the presence of 5 μM ATP and GTP with a Z′ greater than 0.6 and with signal stability of at least 6 hours were used. In some embodiments, mAb/tracer pair(s) that enable detection of cGAMP over a range of 0.1 μM to 50 μM were used. In some embodiments, antibodies with at least 100-fold selectivity for cGAMP vs. ATP and GTP were used in the assays disclosed herein. In some embodiments, demonstration of a linear response in cGAMP formation to cGAS concentration, time, and ATP and GTP (at concentrations below Km) was achieved. In some embodiments, initial velocity cGAS activity (≤10% consumption of substrates) was detected with a Z′ value greater than 0.6 using ATP and GTP at their Km concentrations. In some aspects, less than 0.5% interference in the pre-screen and Z′ values greater than 0.5 in live pilot screens were observed. In some embodiments, mAb/tracer pair(s) that produce a signal of more than 100 mP using less than 10 nM cGAS under initial velocity conditions were used. Such a signal enables screening of 1,000,000 wells with 12 mg of enzyme. In some aspects, Z′ values of more than 0.7 and/or Z values of more than 0.6, and interference levels of less than 0.4% were observed.
The assays described herein surprisingly and unexpectedly comprise the following advantages: (1) far red FP and TR-FRET signals—sensitive and resistant to compound interference, which are widely used in HTS assays; (2) homogenous assays—mix and read format is highly preferred for HTS because it simplifies automation; (3) low nanomolar sensitivity—enables cost effective screening of cGAS under initial velocity conditions; (4) direct detection—assay does not rely on coupling enzymes, which are prone to interference; and (5) usable in endpoint or continuous mode—provides flexibility for experimental protocols and applications. The novel assay disclosed herein eliminates the technical hurdle preventing screening for cGAS modulators, thereby opens up investigation of promising new therapeutic approaches for debilitating and fatal autoimmune diseases and for cancer immunotherapy. Development of cGAMP antibodies is novel and could have broad utility for drug discovery and diagnostic applications targeting the cGAS/STING pathway.
Detection of endogenous cGAMP in biological samples requires a higher sensitivity than does detection of purified cGAS (see, e.g., Table 3, below). In some embodiments, biomarker samples for LC/MS were prepared by isolating large numbers of cells (106-107) and resuspending in volumes of a few microliters, resulting in minimal dilution of cellular metabolites. Based on the limited information available in published studies (An, et al., 2017, Arthritis Rheumatol 69(4):800-7; Gao et al., 2015, Proc Natl Acad Sci USA 112(42):E5699-705), cGAMP concentrations in samples prepared this way are in the 1-100 nM nanomolar range.
Detection of cGAMP for cellular HTS assays requires even greater sensitivity, as they are performed by lysing cells directly in the wells where cells are cultured, and thus rely on far fewer cells (104-105 depending on cell type and plate density), with a dilution factor of approximately 100-fold (Fujioka, et al. Dynamics of the Ras/ERK MAPK cascade as monitored by fluorescent probes. J Biol Chem. 2006; 281(13):8917-26).
In some aspects, cGAMP detection in cell lysates requires an assay with a useful range of 20 pM to 2 nM. In some embodiments, achieving these levels of sensitivity will require antibodies with cGAMP affinities in the lower end of the detection range, e.g., a Kd of 5 nM for the biomarker assay and 100 pM for the cellular HTS assay. Though antibodies against small haptens tend to have lower affinities compared with antibodies for proteins, obtaining a cGAMP antibody with low nanomolar affinity is quite reasonable: for example, Applicants' Transcreener ADP antibody—a native mouse monoclonal—binds with an affinity of approximately 12 nM (Kleman-Leyer, et al., 2009, Assay Drug Dev Technol 7(1):56-67). Developing a cGAMP antibody with affinity in the picomolar range is likely beyond the capacity in vivo affinity maturation (Boder, et al., 2009, Proc Nat Acad Sci USA 97(20):10701-5). In some embodiments, in vitro evolution was used to achieve a necessary binding affinity. In some embodiments, successive rounds of random mutagenesis of VH and VL domains was performed, and FACS was used to enrich for desirable properties of scFvs displayed on yeast (see
Additional key factors impacting sensitivity include the assay configuration and signaling mechanism used for detection. Competitive displacement assays (see Examples 1-3) generally have a lower limit of detection of approximately 0.5-1 nM, because they rely on a negative signal and cannot be configured for signal amplification. In contrast, dual antibody assays, whether solid phase (e.g., ELISA), or proximity based (e.g., TR-FRET) provide greater sensitivity, dynamic range and signal: background, and are often used to detect analytes in the low picomolar range (Arola, et al., 2016, Anal Chem 88(4):2446-52; Arola, et al., 2017, Toxins 9(4):145; Enomoto, et al., 2002, J Pharm Biomed Anal 28(1):73-9). While it has been assumed for many years that the simultaneous binding to two antibodies to a small molecule such as cGAMP is sterically prohibited, recent studies have challenged this limitation with the finding that separating small haptens (histamine, MW=111 and homovanillic acid, MW=182) by as few as five carbons can be sufficient to allow simultaneous binding of two antibodies (Quinton, et al., 2010, Anal Chem 82(6):2536-40). Moreover, there are examples of ELISAs developed for analytes of similar size as cGAMP. Native polyclonal antibodies against two fragments of imantinib (MW=493.6) were used to develop a sandwich ELISA with a working range of 130 pM to 16 nM (Saita, et al., 2017, Anal Chim Acta 969:72-8). A sandwich ELISA was developed for tacrolimus (MW=804) by using mAbs raised against the intact molecule linked to carrier protein via two different positions (Wei, et al., 2014, Clin Chem 60(4):621-30). The specificity of both these ELISAs was significantly better than competitive assays with single antibodies. In some aspects, cGAMP, with a molecular weight of 718 and the equivalent of more than 10 carbons between the adenine and guanine moieties, is a good candidate for development of a sandwich ELISA. In some embodiments, in vitro evolution is utilized to enhance the epitope recognition properties of the candidate mAbs (as scFvs), rather than relying on native antibodies.
In some embodiments, mAbs to cGAMP are generated using structurally distinct antigens. In some embodiments, at least one pair of antibodies, each with Kd≤100 nM, are generated. In some embodiments, at least one pair of antibodies, each exhibiting some differences in epitope recognition properties, are generated. In certain embodiments, mAbs are produced using antigens that completely lack adenine or guanine rings.
In some embodiments, cGAMP mAbs are generated using affinity maturation. In some embodiments, the mAb is an scFv. In some embodiments, scFvs to cGAMP having Kd of about 5 nM, or a Kd within the range of about 1 nM to about 5 nM, or a Kd of about 1 nM, are generated for a biomarker assay. In some embodiments, two scFvs to cGAMP having a simultaneous Kd of less than about 100 pM are generated for a cellular HTS assay. In some embodiments, a first scFv to cGAMP is generated, and a second scFv to the complex of the first scGv and cGAMP is generated (see, e.g.,
In some embodiments, assays for detection of cGAMP as a biomarker in cell and tissue extracts are developed. In some embodiments, assays for detection of cGAMP directly in cell lysates, e.g., for cellular HTS assays, are developed. In some embodiments, completive FP and/or TR-FRET immunoassays are developed for detection of cGAMP as a biomarker. In some embodiments, assays capable of detecting cGAMP in concentrations within the range of about 1 nM to about 100 nM are developed. In certain such embodiments, assays capable of detecting cGAMP in concentrations within the range of about 1 nM to about 100 nM, with a Z′ of at least 0.5 and/or a lower limit of detection (LLD) of less than about 0.5 nM are developed. In some embodiments, assays having less than +/−50% correlation between LC/MS and cGAS immunoassay results are generated. In some embodiments, competitive ELISA immunoassays are developed for detection of cGAMP as a biomarker.
In some embodiments, a cGAMP cellular HTS assay is validated using human cells. In some embodiments, cGAMP expression in cells from cGAMP+ and cGAMP− patients is evaluated by LC-MS. The cells from these patients are further analyzed via HTS assay in a blind fashion. In some embodiments, clinical information regarding the patient's medical history, number of classification criteria fulfilled, laboratory findings (including autoantibody specificities), and damage accrual data is obtained and measured using the Systemic Lupus International Collaborating Clinics/ACR Damage Index (SDI) (Gladman, et al., 1997, Arthritis Rheum 40(5):809-13).
In some embodiments, an S-TR-FRET detection method is utilized in a cGAMP cellular HTS assay. S-TR-FRET is a commonly used approach for homogenous HTS assays, e.g., assays for detection of phospho-proteins in cell extracts (Ayoub, et al., 2014, Front Endocrinol 5:94). Though it has not yet been used for small molecules, the recent examples of ELISAs for small molecules suggest that simultaneous binding of two antibodies to cGAMP is feasible. In some aspects, the potential gains in sensitivity and specificity over competitive FP or TR-FRET assay formats (Arola, et al., 2016, Anal Chem 88(4):2446-52; Arola, et al., 2017, Toxins 9(4):145), described above, combined with the advantages of a mix-and-read format make S-TR-FRET a highly desirable detection method for a cGAMP cellular HTS assay (
In some embodiments, assays capable of detecting endogenously produced cGAMP in cell extracts are produced. In some embodiments, assays capable of detecting endogenously produced cGAMP in cell extracts, with a Z′ of at least 0.5 and/or an LLD of less than about 10 pM are developed. In some embodiments, an ELISA, e.g., a sandwich ELISA, capable of detecting cGAMP at sub-picomolar sensitivity is produced (see, e.g.,
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
The Examples that follow are illustrative of specific embodiments of the invention, and various uses thereof. They are set forth for explanatory purposes only, and are not to be taken as limiting the invention.
The development of immunogens and tracers (see
The commercially available tracer used has a fluorescein tag, which emits at 515 nM. Fluors that emit in the far red are much preferred for HTS, as background fluorescence from screening compounds is largely in the blue-to-green region of the spectrum (Vedvik, et al., 2004, Assay Drug Dev Technol. 2(2):193-203). Three cGAMP analogs with amino linkers to different positions (BioLog) were used to synthesize a collection of 15-20 tracers using amine reactive (NHS esters), far red fluors (e.g., Alexa Fluor, Dylight, and Atto dyes with emission above 600 nM), and purified by thin layer chromatography. Tracers were tested in a matrix fashion with mAbs using FP-based equilibrium binding and competition analysis to identify the pairs that yield the most sensitive and selective immunoassay; cGAMP mAb6 and the cGAMP-Atto 633 tracer (cGAMP linked to an Atto 633 fluor) were chosen for further assay development. Representative binding curves for the 10 cGAMP mAbs with cGAMP-Atto 633 and for cGAMP mAb6 with four different tracers are shown in
Though acceptable Z′ values can often be attained with much lower signals, a screening window of at least 80 mP is desirable for unambiguous identification of hits, and enzyme reactions should generally be adjusted to produce 80% of the maximum polarization shift; therefore the minimum goal is generally 100 mP. The maximum shift for the cGAMP assay was greater than 200 mP (
Enzymes are usually screened using the Km concentrations of substrates to allow detection of competitive inhibitors, and assays are generally run under initial velocity conditions, i.e., less than 20% conversion of substrates to products. Therefore, enzyme assays need to be capable of generating a good signal at products levels of 5-10% of the substrate Km. The Km values for ATP and GTP were presumed to be at least 5 μM, given their millimolar concentrations in the cell. Accordingly, the target for practical cGAMP detection (i.e., a signal of at least 100 mP), was 500 nM. The sensitivity and dynamic range of the cGAMP assay could be tuned by changing the antibody concentration, which was present in excess over the tracer (
Use of the assay to measure cGAS enzyme activity requires an antibody that specifically binds cGAMP in the presence of an excess of substrates (ATP and GTP). As shown in
cGAS is one of four oligoadenylate synthases, nucleic acid sensors that activate innate immunity via production of short, 2′-5′ oligoadenylate secondary messengers (35); it catalyzes the formation of 2′,3′ cGAMP from ATP and GTP, with pyrophosphate as a byproduct. Binding of dsDNA and DNA:RNA hybrids to cGAS induces a conformational transition in an activation loop, not unlike the displacement of inhibitory domains by autophosphorylation in protein kinases (Zhang, et al., 2014, Cell Rep 6(3):421-30; Fabbro, et al., 2012, Methods Mol Biol 795:1-34). The full-length enzyme was used to insure that all of the potential allosteric sites were present and that the enzyme was able to sample its full conformational repertoire. Prior to running cGAS enzyme reactions, the effect of cGAS reaction components, including ATP and GTP Brij 35, NaCl, Brij 35, and dsDNA on the detection reagents was examined; no significant effects were observed. Assays using highly purified, full length human cGAS were performed. The full-length human cGAS was prepared according to Example 4, below. Additionally, third-party-provided cGAS (Sun, et al., 2013, Science 339(6121):786-91) was used for comparative purposes. Some enzyme assays were run in kinetic mode; i.e., the detection reagents were present during the enzyme reactions, and the plates were read periodically, and others were run in endpoint mode, using EDTA to quench the reaction.
Titration of the highly purified enzyme produced a sigmoidal dose-dependent polarization shift, as expected for a competitive binding assay, with a half-maximal change (EC50) of 6-12 nM cGAS, depending on the preparation (
The capability of the assay to detect activation of cGAS by dsDNA, a property critical to the physiological role of cGAS (Chen, et al., 2016, Nat Immunol 17(10):1142-9), was confirmed with a third-party-prepared enzyme, and with cGAS produced according to Example 4. Surprisingly and unexpectedly, the enzyme was shown to be highly sensitive to dsDNA, with a half maximal responses of 2.5 and 5.9 nM for herpes virus and Listeria sequences, respectively (
Having confirmed that cGAMP mAb6 has the required affinity and selectivity properties for a cGAS enzymatic assay in an FP format, development of the TR-FRET assay was relatively straightforward. Amine-reactive lanthanide chelates, including terbium, europium, and samarium, were conjugated to mAb6, and binding analysis was performed with a series of cGAMP-fluor tracers with overlapping excitation spectra. Notably, the TR-FRET assay differs from the FP assay in that the tracer, rather than the Ab, is present in excess; this minimizes consumption of expensive reagents. Additionally, the tracer fluors used for TR-FRET are generally more red-shifted than those of the FP assay in order to match the emission of lanthanides. The terbium-conjugated mAb 6 with the cGAMP-Atto 650 tracer resulted in the highest affinity of all the combinations tested, as exemplified in representative binding curves (
Though recombinant cGAS is commercially available, no associated activity data is available, and it is prohibitively expensive for HTS. Given the critical importance of a reliable source of high purity, functional cGAS for biochemical screening efforts, full-length human cGAS having a 6×His at either the N- or C-terminus were synthesized using a T7 expression vector and BL21 DE3 cells with eukaryotic tRNAs for rare codons (Rosetta DE3, Novagen). Following lysis, the soluble 6×His-cGAS and cGAS-6×His proteins were purified using immobilized metal ion affinity chromatography (IMAC); the 6×His-cGAS was further purified using cation exchange chromatography (HiTrap SP) on an Akta Start automated chromatograph system (GE Healthcare) with a 0.1-1M NaCl gradient. The two purified His-tagged cGAS constructs are shown in
Most cGAS structure/function studies have been performed with truncated constructs that include the primary DNA binding site and the catalytic domain, but lack the N-terminal 150-160 aa (Kato, et al., 2013, PloS One 8(10):e76983; Kranzusch, et al., 2013, Cell Rep 3(5):1362-8; Zhang, et al., Cell Rep 6(3):421-30). Using a cGAS produced according to this Example insures that all of the potential allosteric sites of cGAS were present, and that the enzyme was able to sample its full conformational repertoire. Notably, in this regard, a recent study showed that the N-terminal 160 aa domain makes an important contribution to DNA-dependent activation, both in vitro and in vivo (Tao, et al., 2017, J Immunol 198(9):3627-36).
As described above, detection of endogenous cGAMP in biological samples requires greater sensitivity than that of the cGAS enzymatic assays of Examples 1-3 (see Table 3). To improve sensitivity, three antigens that are structurally distinct from the guanine C-8-linked immunogen of Examples 1-3 are be produced (
Generation of Additional mAbs to cGAMP Using Structurally Distinct Antigens
cGAMP is conjugated to KLH via 6-aminohexyl carbamoyl linkers to the ribose 3′-hydroxy group of the guanosine (Biolog C191), to the ribose 3′-hydroxy group of adenosine (Biolog C192), and via a 2-4 carbon linker to the N6 of the adenine ring using EDC chemistry to generate antigens with three epitope presentations distinct from the guanine C8-linked cGAMP antigen of Examples 1-3. The ribose-linked cGAMP analogs are available from Biolog, and the adenine N6-linked analog is provided as a custom synthesis. The latter analog is important because it will present cGAMP in the opposite orientation relative the antigen of Examples 1-3, i.e., with the guanine ring fully exposed and the adenine ring less accessible, because there is precedent for such antibodies that can discriminate with more than 100-fold selectivity between cGMP and cAMP (Wescott, et al., 1985, J Cyclic Nucleotide Protein Phosphor Res 10(2):189-96). Moreover, the site attachment and conjugation chemistry used for antigen synthesis can have a profound effect on affinity and selectivity, as observed through efforts to develop anti-nucleotide antibodies for ADP, GDP, AMP, etc. (Kleman-Leyer, et al., 2009, Assay Drug Dev Technol 7(1):56-67).
As in Example 1, monoclonal antibody production is performed by Envigo (formerly Harlan; Madison, Wis.) using ten mice for each antigen. Mice are chosen for hybridoma development based on analysis of antiserum (tail bleeds) using competitive ELISAs and FP-based competition assays. Hybridomas are screened using the cognate antigen, as well as the two non-cognate antigens to identify mAbs that may exhibit some differences in epitope recognition. The most promising mAbs are tested using FP-based equilibrium binding and competition assays with a panel of tracers made by conjugating to the four different positions on cGAMP (
Affinity Maturation of cGAMP mAbs
Affinity maturation is performed using PCR-based mutagenesis of scFvs combined with FACS-based enrichment of yeast-displayed clones, an approach that can yield gains in affinity of more than 1000-fold (including scFvs for fluorescein and, more recently, the lanthanide chelate DOTA, with respective Kds of 0.27 and 8.2 pM; Table 3; Boder, et al., 2009, Proc Nat Acad Sci USA 97(20):10701-5; Orcutt, et al., 2011, Nucl Med Biol 38(2):223-33). Unlike ribosome or bacterial display, yeast display allows the use of fluorescence-activated cell sorting (FACS) for quantitative and exhaustive screening of large populations to optimize antigen binding affinity and kinetics (Boder, et al., 2012, Arch Biochem Biophys 526(2):99-106).
Generally, established FACS methods are performed, with minor modifications to reduce the mutational bias of error-prone PCR (Orcutt, et al., 2011, Nucl Med Biol 38(2):223-33), and to increase selection of stable scFvs (Julian, et al., 2017, Sci Rep 7:45259). scFvs are cloned using RT-PCR of RNA prepared from selected cGAMP-mAb hybridoma cells (3-4 clones) using standard methodology to link the VH and VL domains with a Gly-Ser linker and inserted into the pCTCON-T yeast shuttle vector in fusion with the adhesion subunit of the yeast agglutinin protein Aga2p for surface display. pCTCON-T includes a Gall promoter for inducible expression in yeast and a C-terminal 6×His tag for affinity purification (
The mutant scFv libraries are amplified to produce a quantity sufficient for yeast transformation and are cloned into pCTCON-T by homologous recombination in yeast (Oldenburg, et al., 1997, Nucleic Acids Res. 25(2):451-2). As the PCR insertion products are also homologous to each other, additional recombination events occur between inserts and lead to greater library diversity. Each of the cGAMP scFv-expressing yeast libraries is sorted by FACS for improved binders (2-3 times at least selection round), using at least 5 times the estimated library diversity. cGAMP-Atto 633 tracers (cGAMP linked to an Atto 633 fluor) with the cognate linker attachment site (
When an affinity of 1-5 nM is attained with any of the libraries, individual scFvs are cloned and characterized for affinity and selectivity for development as a biomarker assay (see Example 6, below). Further rounds are directed toward evolving scFv pairs with picomolar affinity that can bind cGAMP simultaneously for an S-TY-FRET assay (see Example 7, below). Tracers with the non-cognate structure and a non-overlapping fluor (e.g., Alexa Fluor 405) are used for counter-screening in latter rounds to remove scFvs that recognize the same epitope as another pair member, e.g., adenine specific vs. guanine specific binders.
A competitive immunoassay format with FP and/or TR-FRET signals is used for biomarker assays, because such formats are the simplest configurations for the desired detection range, and because these formats are widely used. For example, widely used assays for cyclic mononucleotides with practical detection of less than 5 nM use competitive FP and TR-FRET formats (e.g., Lance Ultra cAMP, Perkin-Elmer (Norskov-Lauritsen, et al., 2014, Int J Mol Sci 15(2):2554-72)). Assay development is similar to that described in Example 1 (see
The CGAMP biomarker assay is validated by direct comparison with LC/MS detection using a variety of therapeutically relevant cell and tissue samples from animals and humans. cGAMP levels in cell and tissue extractions are quantified by LC/MS as described below, and compared to FP and/or TR-FRET assay measurements.
cGAMP from Monocytes
THP-1 cells (0.2, 1, 5, 25M) are transfected with herring testis DNA (0.1, 0.5, 2.5, 12.5 μg) with Lipofectamine 2000. After 4 hours, cGAMP is isolated from THP-1 cells alone or THP-1 cells transfected with double-stranded DNA (dsDNA) by a methanol extraction procedure.
THP1 cells are lysed with 1 ml of 80% methanol spiked with 5 nM heavy isotope-labeled cGAMP (cGAMP*) containing 13C-, 15N-labeled AMP as an internal standard. Following sonication and harvesting, cGAMP is further purified with a solid-phase extraction column (Oasis WAX column; Waters) and resuspended in 50 μl of Optima LC-MS water (Thermo Scientific) for mass spectrometry. The cGAMP concentration (estimated 1˜100 nM) is measured by LC-MS, FP, and/or TR-FRET immunoassays for comparison. The mass spectrum peak area of the endogenous cGAMP and internal standard (100 nM final concentration) is quantified by QuanLynx software (Waters). The ratio of the peak area from endogenous cGAMP and internal standard is used to determine the concentration of endogenous cGAMP
cGAMP from Diseases Cells and Tissue
The dominant clinical manifestation in Trex1−/− mice is an autoimmune myocarditis (Morita, et al., 2004, Mol Cell Biol 24(15):6719-27). The profound reduction or loss of disease manifestations and extended survival observed in Trex1−/− mice deficient in either STING or cGAS (Gao, et al., 2015, Proc Natl Acad Sci USA 112(42):E5699-705; Gray, et al., 2015, J Immunol 195(5):1939-43), indicate that IFN-I is induced through the cGAS-STING pathway. cGAMP is detectable in the high-pM or low-nM range in the hearts of Trex1−/−, but not Trex1−/−x cGAs−/− mice (Gao, et al., 2015, Proc Natl Acad Sci USA 112(42):E5699-705). This observation was confirmed by LC-MS. At 3 months of age, Trex1−/− and Trex1+/− littermate controls are scarified (n=10 mice each) to obtain the hearts. After taking small slivers for RNA and histology, the hearts are bisected; one half is tested for cGAMP by LC-MS, and the other by competitive FP and/or TR-FRET immunoassays. Quantification by mass spectrometry is achieved using heavy isotope-labeled cGAMP (cGAMP*) containing 13C-, 15N-labeled AMP as internal standard, as in the cGAMP measurement in THP1 cells, described above.
As described above, the potential gains in sensitivity and specificity over competitive FP or TR-FRET assay formats (Arola, et al., 2016, Anal Chem 88(4):2446-52; Arola, et al., 2017, Toxins 9(4):145), as well as the advantages of a mix-and-read format, make S-TR-FERT (
While most cGAS/STING pathway studies have utilized the human monocyte cell lines THP-1, pDCs are clearly the primary cell type responsible for tumor surveillance and initiation of an IFN-driven innate immune response (Bode, et al., 2016, Eur J Immunol 46(7):1615-21; Corrales, et al., 2017, Cell Res 27(1):96-108). Therefore, in addition to THP-1 cells, human pDC line Cal-1 cells (Maeda, et al., 2005, Int J Hematol 81(2):148-54), recently shown to have a functional cGAS/STING pathway, including a robust cGAS-dependent production of IFNβ in response to cytosolic DNA (Bode, et al., 2016, Eur J Immunol 46(7):1615-21), are used for validation of the S-TR-FRET assay. Cells are grown to high density in 384-well plates and transfected with herring testis DNA (0.1, 0.5, 2.5, 12.5 μg) with Lipofectamine 2000. Following media removal, cells are lysed using buffer with a non-ionic detergent and other components that are compatible with S-TR-FRET reagents. S-TR-FRET reagents will be added, and plates are read as in the TR-FRET assay of Example 3. Unstimulated controls and spiked samples are used for comparison. A standard curve in lysis buffer is used to convert TR-FRET signals to quantitative cGAMP measurements.
Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/050060 | 9/5/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62383566 | Sep 2016 | US |
