WATERTOWN, MA, United States
WATERTOWN, MA, United States
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Methods for proteomic screening on random protein-bead arrays by mass spec is described. Photocleavable mass tags are utilized to code a protein library (bait molecules) displayed on beads randomly arrayed in an array substrate. A library of probes (prey) can be mixed with the protein-bead array to query the array. Because mass spec can detect multiple mass tags, it is possible to rapidly identify all of the interactions resulting from this mixing.


Patent
Ambergen, Inc and Massachusetts General Hospital | Date: 2014-06-27

Methods and compositions are described for the diagnosis of primary biliary cirrhosis. Novel autoantigens are described for use in assays which employ test samples from individuals.


Patent
Ambergen, Inc and Massachusetts General Hospital | Date: 2016-04-27

Methods and compositions are described for the diagnosis of primary biliary cirrhosis. Novel autoantigens are described for use in assays which employ test samples from individuals.


Patent
Ambergen, Inc | Date: 2014-10-21

Methods are described for phototransferring a compound from a first surface to a second surface. Compounds are described with photocleavable linkers. Compounds attached to a first surface through a photocleavable linker are put in proximity (or contact) with a second surface, and then phototransferred to the second surface upon exposure to electromagnetic radiation. Illuminating the compound with radiation photocleaves the compound from the first surface and transfers the compound to the second surface.


Patent
Ambergen, Inc | Date: 2015-07-23

Methods are described for phototransferring a compound from a first surface to a second surface. Compounds are described with photocleavable linkers. Compounds attached to a first surface through a photocleavable linker are put in proximity (or contact) with a second surface, and then phototransferred to the second surface upon exposure to electromagnetic radiation. Illuminating the compound with radiation photocleaves the compound from the first surface and transfers the compound to the second surface.


This invention relates to non-radioactive markers that facilitate the detection and analysis of nascent proteins translated within cellular or cell-free translation systems. Nascent proteins containing these markers can be rapidly and efficiently detected, isolated and analyzed without the handling and disposal problems associated with radioactive reagents. Preferred markers are dipyrrometheneboron difluoride (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) dyes.


Patent
Ambergen, Inc | Date: 2015-10-06

Methods are described for phototransferring a compound from a first surface to a second surface. Compounds are described with photocleavable linkers. Compounds attached to a first surface through a photocleavable linker are put in proximity (or contact) with a second surface, and then phototransferred to the second surface upon exposure to electromagnetic radiation. Illuminating the compound with radiation photocleaves the compound from the first surface and transfers the compound to the second surface.


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase II | Award Amount: 753.50K | Year: 2016

DESCRIPTION provided by applicant A fundamental challenge in the drug discovery process is to develop drugs with high efficacy and minimal side effects As an initial step this depends on identifying lead compounds which interact with high affinity and regulate the activity of specific biological drug targets such as a protein involved in a disease pathway However a second equally important step is to verify that these leads do not interact and interfere with the functioning of the vast number of other proteins which comprise the human proteome A variety of effective methods exist to perform label free high throughput screening HTS of compound libraries against single drug targets However the ability to perform HTS on compound libraries or even a small set of lead compounds against a large library of protein targets has not yet emerged Such high throughput multidimensional drug screening HT MDDS would have a significant impact of the pharmaceutical and health care industry Most importantly it would facilitate proteome wide detection of off target drug protein interactions Such interactions often cause serious adverse drug reactions SADRs responsible for the over deaths per year in the U S exceeding the number of annual deaths due to cancer In addition HT MDDS would provide a powerful method to discover new targets for existing FDA approved drugs by identifying off target interactions as well as to elucidate the molecular basis of side effects fro on label use In order to meet this challenge AmberGen has explored during Phase I the use of its proprietary Bead GPS tm technology as a means to perform HT MDDS during lead optimization Bead GPS tm is based on MALDI mass spectrometric imaging MALDI MSI of high density random bead arrays where each bead in the array carries a unique protein and corresponding photocleavable mass tag for coding PC mass tag Binding of specific compounds to a specific protein is detected by measuring simultaneously the mass of the binders and the photoreleased PC mass tag s residing on each bead Our initial results demonstrate the feasibility of using Bead GPSTM to screen a drug compound library against thousands of proteins in order to rapidly identify both on and off target interactions on a global proteome wide basis Progress during Phase I included i Development of new photocleavable linkers for facile attachment of mass tags to beads ii Demonstration of high capacity coding of protein bead libraries with single and double PC mass tags iii Construction of a model member PC mass tag coded kinase library iv Screening the kinase bead library with two known kinase inhibitors demonstrating specificity and sensitivity During Phase II we will further optimize Bead GPS tm for HT MDDS with the goal of establishing it as a standard tool for pre clinical lead optimization For this purpose we will construct a member PC mass tagged kinase protein bead library This library will be screened by Bead GPS tm against compounds from a model member kinase inhibitor library On target and off target interactions from positive hits detected by Bead GPS tm will be further characterized for binding affinity and inhibitor activity using homogenous kinase assays as well as by synchronization of fluorescent FRET and MALDI MS images of the random bead array Improvements in several aspects of the Bead GPS tm process including advanced MALDI MSI instrumentation and software will allow to fold improvements in sensitivity and scan speed In order to accelerate the development and commercialization of Bead GPS tm AmberGen will work closely with Dr Cathy Costello Director Center for Biomedical Mass Spectrometry at the Boston University Medical School and a leading expert in the field of MALDI and its biomedical applications Dr Marvin Vestal pioneer in MS MALDI instrumentation development and President of SimulTOF Systems Dr Michael Pavia a leader in the pharmaceutical industry and ex CTO of Millennium Pharmaceuticals Dr Yiwu He former Global Head and Senior Director of GlaxoSmithKline and currently Senior Program Officer at the Bill andamp Melinda Gates Foundation and Dr Gheorghe Doros Associate Professor of Biostatistics at the Boston University School of Public Health AmberGen will also explore the use of improved MALDI MSI instrumentation in collaboration with SimulTOF Systems and Bruker Daltonics PUBLIC HEALTH RELEVANCE Serious adverse drug reactions often due to unwanted off target drug interactions are responsible for over deaths per year in the U S exceeding the number of annual deaths due to cancer In order to reduce the occurrence of such adverse drug reactions AmberGen has developed during Phase I a new technology based on mass spectrometric imaging of random bead arrays that allows high throughput screening of libraries of drugs against libraries of proteins in order to detect off target interactions During Phase II we will optimize this new technology with the goal of establishing it as a standard tool for pre clinical lead identification and optimization


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2016

SUMMARY ABSTRACT Blood based multi biomarker panels hold great promise for effective early cancer detection and population wide screening For example panels consisting of three or more biomarkers which exhibit high sensitivity and specificity have been reported for several different cancers including ovarian lung breast and colorectal cancer A variety of miniaturized solid phase immunoassay platforms have also been developed to perform high throughput and low cost multiplex biomarker detection and quantification These include instruments based on microarrays microfluidics and micro bead technology A high priority is now to transition promising blood based multi biomarker cancer panels to multiplex platforms for large scale biomarker validation and ultimate use in the clinic Multiplex assay technologies are especially important to provide the cost effectiveness and high throughput capacity necessary for population wide screening However a major problem with multiplex immunoassays is the so called matrix effect Compared to most conventional single plex assays such as ELISA miniaturized multiplex assays are highly susceptible to interference caused by the presence of the more abundant non target agents in blood Such interference can originate from a variety of mechanisms including i low specificity heterophile antibodies ii matrix induced bead aggregation e g in Luminex assays and iii specific or non specific binding of non target matrix components to any component of the biomarker assay In addition high viscosity of the sample matrix e g from high total protein concentration can interfere with the microfluidics commonly used for multiplex assays Importantly the matrix effect not only limits assay sensitivity but reduces linearity and quantitative accuracy During Phase I we will evaluate a new approach to multiplex serological cancer assays termed PC PURE which is designed to eliminate the matrix effect This technology is based on the use of novel photocleavable PC linkers developed by AmberGen which are incorporated into affinity capture agents such as aptamers or antibodies The photocleavable capture agents are then tethered to micro beads affinity resins or other surfaces and used to isolate the target biomarker The biomarker capture agent complexes are gently and rapidly photo released in minutes under non denaturing conditions by low intensity near UV light into a well defined buffer enabling simultaneous pre purification and concentration of the target biomarkers prior to multiplex immunoassay Unlike conventional approaches using blocking buffers diluents and selected depletion specific to particular matrix components PC PURE eliminates all matrix effects by rapidly pre purifying the biomarkers of interest In Phase I we will evaluate the application of PC PURE to improve the multiplex detection of blood based panels of tumor shed protein biomarkers Tumor shed biomarkers have great potential for high cancer specificity but are found at extremely low abundance in the blood and hence suffer most from the matrix effect A model biomarker protein panel for ovarian cancer diagnosis will be tested Both spike in samples with known concentrations of the biomarkers and ovarian cancer patient blood samples will be analyzed on a multiplex Luminex MagPix platform and in Phase II on a Bio Plex platform designed for high throughput clinical testing This research will be conducted in collaboration with Dr Gheorghe Doros Associate Professor of Biostatistics and Director of the Biostatistics Consulting Group at the Boston University School of Public Health who will provide expert guidance for statistical analysis of the data We will also work closely with Dr Bill Jackson Founder and CSO of Base Pair Biotechnologies a leading expert on aptamers To accelerate commercialization of PC PURE we will work closely with Luminex one of the leading manufacturers of multiplex assay platforms see letters of support PROJECT NARRATIVE Multiplex serological cancer immunoassays which detect panels of biomarkers hold great promise for non invasive cancer screening However a major limitation of such assays is the matrix effect which severely degrades sensitivity dynamic range quantitative accuracy and reproducibility A new photo affinity biomarker purification approach will be developed and evaluated during Phase I which effectively eliminates the matrix effect and dramatically improves the detection of low abundance tumor shed biomarkers in the blood


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.46M | Year: 2012

DESCRIPTION (provided by applicant): There is an urgent need for early-detection cancer biomarkers which can significantly lower the high mortality and cost of treating cancer. One of the most promising candidates for such biomarkers is tumor-associated autoantigens (TAA), autoantibodies to which have been detected in patient's blood even in the early stages of cancer. In addition to use in early diagnosis, such TAAs have great value in developing anti-cancer vaccines and potentially guiding individualizedpatient therapy. One general approach to TAA discovery is screening the sera of patients having particular cancers against proteome-scale libraries of human proteins, to detect autoantibody binding. However, large-scale proteomic screens are difficult to perform using conventional technology, such as protein microarrays, due to limited sensitivity, reproducibility, scalability and expense of manufacture. An additional problem is the reliance on attachment and drying of the bait protein onto planar microarray substrates, which damages proteins, impairs the subsequent bio-assay kinetics and reduces the amount of accessible bait protein. During Phase I, we have made significant progress towards overcoming these limitations through the development of a next generation approach to proteomics, termed Bead-based Global Proteomic Screening (Bead-GPSTM). Bead-Protein libraries are produced inexpensively and rapidly using cell-free protein expression and uniformly attached to beads. Bio-assays are performed with the beads in suspension for improved kinetics. The bead library is then randomly arrayed into Pico-well slides with a capacity of up to 1-million beads. The strength of positive bait-prey interactions on the bead is quantitatively measured using fluorescence imaging and then the bead identity decoded using photocleavable mass tags combined with MALDI mass spectrometry imaging (MALDI-MSI), which provides the ability to decode tens of thousands of different bead species. During Phase I we have achieved all key milestones including: i) full production and validation of a prototype 100- member protein library based on cell-free expression methods, and, formatting the library on beads; ii) development and evaluation of an ultra-high density bead-array platform including decoding using MALDI-MSI; iii) successful screening of the prototype bead-protein library with several model autoantigens; iv) discovery and validation of a new TAA for CRC on the Bead-GPSTM platform by using candidate pre-selection with ultra-low noisegene expression profiling; v) demonstration of a MALDI-MSI bead barcoding system capable of generating tens of thousands of unique codes using lt 50 distinguishable mass tags; vi) successful screening of 94 CRC sera and controls using a model bead-proteinlibrary on Illumina's mid-plex automated VeraCodeTM platform. During the Phase II project we will continue to develop and evaluate Bead-GPSTM with special focus on the discovery of TAA biomarkers for colorectal cancer (CRC). For this purpose we will initially fabricate an intermediate 500-member library including known CRC TAAs from the literature, high probability TAAs and those discovered in Phase I. The library will be screened using Bead-GPSTM against CRC patient sera and matched controls including individuals with benign polyps and benign inflammatory diseases. Rigorous SOPs for serum collection/handling developed by an NCI (EDRN) consensus working group for biomarker discovery will be implemented by both our commercial (ABS) and institutional (Boston Medical Center) suppliers (see letters), and assisted by consultant M. Tuck, who helped spearhead the EDRN effort (see letter). For comparison, similar measurements will be run using the VeraCodeTM digital holographic bead technology in collaboration with Illumina Inc., who has agreed to provide the instrumentation (BeadXpress ) and reagents (see letter; note that AmberGen has won the Illumina Assay Design Challenge for Proteomics on VeraCode beads). The 500-member protein library will be followed by a 5,000-member library later in Phase II. To accelerate commercialization, we will work closely with Illumina in order to implement clinical multipanel assays on its VeraCodeTM system, as well as with INOVA and Bio-Rad Diagnostics, two leaders in development of clinical immunoassays (see letters). Finally, we will work closely during Phase II with Bruker Daltonics Inc., which has expressed interest in developing an integrated instrument for Bead-GPSTM. PUBLIC HEALTH RELEVANCE: The discovery of blood based early-detection cancer biomarkers can significantly reduce the high mortality rate of cancer. However, current techniques for discovery of such biomarkers, such as tumor associated antigens, are slow and unreliable. During Phase I we have developed a novel, low-cost, high-sensitivity and high-throughput proteomic approach for cancer biomarker discovery termed Bead-based Global Proteomic Screening (Bead-GPSTM). During Phase II we will evaluate the ability of this new approach to discover novel biomarkersfor the early, non-invasive, blood based diagnosis of colorectal cancer, the second leading cause of cancer deaths in the U.S.

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