Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56983—Viruses
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]

Definitions

• the described invention relates in general to systems, devices, reagents, and methods for detecting various analytes of interest in biological samples or other sample types, and more specifically to a biosensor-based system for detecting and identifying analytes of interest in real-time based on the emission of a detectable signal when the biosensor reacts with an analyte of interest in a sample being tested.
• the following patents provide additional background information regarding the technology of the present invention and are incorporated by reference herein, in their entirety, for all purposes: U.S. Patent Nos. 9,023,640; 9,752,199; 9,850,546; 9,850,547; and 9,850,548.
• a biosensor is a system or device for the detection of an analyte that combines a sensitive biological component with a physicochemical detector component.
• the components of a typical biosensor system include a biological element, a transducer or detector element, and associated electronics or signal processors that display test results in a meaningful and useful manner.
• the biological element typically includes biological material such as tissue, microorganisms, organelles, cell receptors, enzymes, antibodies, nucleic acids, and the like that may be created by known biological engineering processes.
• the transducer or detector element works in a physicochemical manner (e.g., optical, piezoelectric, and/or electrochemical) that transforms the signal resulting from the interaction of the analyte with the biological element into another signal that can be more easily measured and quantified.
• Biosensors originated from the integration of molecular biology and information technology (e.g., microcircuits, optical fibers, etc.) to qualify or quantify biomolecule- analyte interactions such as antibody- antigen interactions.
• a first biosensor system for the detection of target analytes.
• This system includes a living biological cell of a predetermined type; a signal-generating reporter associated with the living biological cell; a signal transduction pathway or activator mechanism associated with the signal-generating reporter; a universal detector element associated with the activator mechanism; and an analyte binding element associated with the universal detector element, wherein the analyte binding element is specific to both the universal detector element and a target analyte.
• a second biosensor system for the detection of target analytes.
• This system includes a living biological cell of a predetermined type; a signal-generating reporter within the living biological cell, wherein the signal-generating reporter is responsive to predetermined changes occurring within the living biological cell; a signal transduction pathway or activator mechanism associated with the signal-generating reporter, wherein the activator mechanism is operative to induce the predetermined changes within the living biological cell; a universal detector element associated with the activator mechanism, wherein the universal detector element is operative to trigger the activator mechanism; an analyte binding element associated with the universal detector element, wherein the analyte binding element is specific to both the universal detector element and a target analyte; and wherein upon the binding of an analyte binding element to which a target analyte is also bound to the universal detector element, the universal detector element triggers the activator mechanism causing the predetermined changes to occur within the living biological cell, thereby causing the signal-
• a third biosensor system for the detection of target analytes.
• This system includes a living biological cell of a predetermined type; a signal-generating reporter within the living biological cell, wherein the signal-generating reporter is responsive to predetermined changes occurring within the living biological cell; a signal transduction pathway or an activator mechanism associated with the signal-generating reporter, wherein the activator mechanism is operative to induce the predetermined changes within the living biological cell; a universal detector element associated the activator mechanism, wherein the universal detector element is operative to trigger the activator mechanism; an analyte binding element associated with the universal detector element, wherein the analyte binding element is specific to both the universal detector element and a target analyte; and wherein upon the binding of an analyte binding element to which a target analyte is also bound to the universal detector element, the universal detector inhibits the activator mechanism causing the predetermined changes to be reduced within the living biological cell, thereby causing the signal- generating reporter
• FIGS, la-b are illustrations of a first biosensor in accordance with an exemplary embodiment of the present invention, wherein Jurkat T cells have been engineered to produce aequorin and to express the transmembrane non-antibody signal transducing element IgGbp- CD3Q
• FIGS. 2a-b are illustrations of a second biosensor in accordance with an exemplary embodiment of the present invention, wherein MC/9 mast cells have been engineered to produce aequorin, and wherein the MC/9 cells express the native receptor FcsRI, which binds to the soluble non-antibody signal transducing element IgGbp-IgE;
• FIGS. 3a-b are illustrations of a third biosensor in accordance with an exemplary embodiment of the present invention, wherein MC/9 mast cells have been engineered to produce aequorin, and wherein the MC/9 cells express the native receptor FcsRI, which binds to the soluble non-antibody signal transducing element IgGbp-IgE, which has been excreted by the MC/9 mast cells;
• FIGS. 4 is an illustration of a fourth biosensor in accordance with an exemplary embodiment of the present invention, wherein biosensor cells have been engineered to produce aequorin and to express the transmembrane non-antibody signal transducing element mSA- CD3 ⁇ , which binds to a biotinylated detector element; and
• FIGS. 5 is an illustration of a fifth biosensor in accordance with an exemplary embodiment of the present invention, wherein biosensor cells have been engineered to produce aequorin and to express the transmembrane non-antibody signal transducing element mSA- CD3 ⁇ , which binds to a biotinylated detector element.
• the present invention relates in general to systems, devices, reagents, and methods for detecting various analytes and/or other targets of interest in biological samples or other sample types, and more specifically to a biosensor-based system for detecting and identifying analytes of interest in real time based on the emission of a detectable signal when the biosensor reacts with an analyte of interest in a sample being tested.
• the engineered cells of the present invention are extremely sensitive and effective biosensors and because these biosensor cells have an intrinsic detection capacity, they provide a versatile system that can be readily adapted to detect a wide variety of different infectious agents or other targets by simply selecting alternative soluble detector (e.g., antibody) molecules with specificity for a particular pathogen or other target of interest.
• the system of this invention can be readily configured for multiplex detection of several infectious agents or other analytes in a single assay, providing for great flexibility and utility.
• the versatility of the present invention is derived from a unique combination of elements and in particular from the combination of a universal biosensor cell with a specific soluble detector (e.g., antibody).
• the universal biosensor cell has the capacity to respond to the presence of essentially any target molecule that can be recognized by the detector molecule. Because, in some embodiments, the detector or detector antibody is added to the system as a soluble factor, the system may be configured to detect an alternative target by simply selecting an appropriate alternate detector or detector antibody.
• the specificity of the disclosed system is determined by the detector molecule, which is selected based on its specificity and affinity for a target molecule that is characteristic of an infectious agent or other target analyte.
• the combination of this universal biosensor cell and soluble detector also enables the construction of multiplex assays by simply including a plurality of detector molecules (e.g., antibodies) within the test system, wherein the target molecules are selected based on their specificity for alternative infectious agents or other analytes.
• Genetic manipulation and modification of the biosensor cell types used with this invention typically involve the use of appropriately selected gene delivery vehicles that contain genetic elements that function efficiently in the cell type of choice.
• a promoter element that directs high level expression of introduced transgenes in the specific biosensor cell of choice.
• such a promoter element may be derived directly from the biosensor cell itself and then used to express a transgene of interest.
• an appropriate element may be determined empirically by comparing the function of alternative promoter elements in the context of alternative gene delivery vehicles in order to identify effective promoter, transgene, vector combinations for the cell type of choice.
• Transgenes such as the gene encoding a luminescent reporter protein may be introduced into the biosensor cell using standard techniques such as electroporation or chemical transfection reagents such as, for example, lipofectamine. Other genetic engineering methods known to those of ordinary skill in the art are also compatible with the present invention.
• An exemplary embodiment of this invention includes a living, engineered biosensor cell, wherein the living engineered biosensor cell is typically a component of the mammalian immune system; a reporter protein, wherein the reporter protein is expressed by and present within the living, engineered cell, and wherein the reporter protein emits a detectable signal in response to certain predetermined changes in the cytosol of the living, engineered cell; a signal transduction pathway expressed by the living, engineered cell, wherein the signal transduction pathway controls a biological or biochemical process within the cytosol of the living, engineered cell, and wherein the at least one biological or biochemical process, when it occurs, causes the reporter protein to emit a detectable signal; at least one type of detector molecule, wherein each detector molecule is adapted to bind to a specific analyte; at least one analyte, wherein the at least one analyte binds to the detector molecule that is specific to that analyte; and a plurality of transmembrane non
• This system may also include a device for mixing the living cells together with soluble components and a sample containing an analyte or infectious agent of interest while maintaining the viability and functionality of the living biosensor cell, and a detector for detecting the signal emitted by the biosensor cell.
• Another exemplary embodiment of this invention includes a living, engineered cell, wherein the living engineered cell is a component of the mammalian immune system, wherein the living engineered cell is a mast cell, and wherein the mast cell expresses at least one predetermined receptor; a reporter protein, wherein the reporter protein is aequorin that is expressed by the living, engineered cell, and wherein the aequorin emits a detectable signal of light in response to certain predetermined changes in the cytosol of the living, engineered cell; a signal transduction pathway expressed by the living, engineered cell, wherein the signal transduction pathway controls a biochemical process within the cytosol of the living, engineered cell, wherein the biochemical process controlled by the signal transduction pathway further includes an increase in intracellular calcium, and wherein the increase in intracellular calcium, when it occurs, causes the aequorin to emit detectable light; at least one type of detector molecule, wherein each detector molecule is adapted to bind to a specific analy
• Each signal transducing element is adapted to bind to the at least one predetermined receptor and to receive a detector molecule.
• an aggregation of the receptors occurs on the cell surface, the signal transduction pathway is activated, the increase in intracellular calcium occurs, and detectable light is emitted by the aequorin.
• This system may also include a device for mixing the living cells together with soluble components and a sample containing an analyte or infectious agent of interest while maintaining the viability and functionality of the living biosensor cell, and a detector for detecting the signal emitted by the biosensor cell.
• Still another exemplary embodiment of this invention includes a biosensor for the rapid detection, wherein the biosensor futher includes a living, engineered cell, wherein the living, engineered cell is derived from a cellular component of the mammalian immune system (i.e., an immunocyte); a reporter protein, wherein the reporter protein is engineered into and expressed by the living, engineered cell, and wherein the reporter protein emits a detectable signal in response to certain predetermined changes in the cytosol of the living, engineered cell; a signal transduction pathway engineered into or occurring naturally within the living, engineered cell, wherein the signal transduction pathway controls a biological process within the cytosol of the living, engineered biosensor cell, and wherein the biological process, when it occurs, causes the reporter protein to emit a detectable signal; and a plurality of non-antibody signal transducing elements that directly or indirectly bind to an analyte in a sample to be analyzed, wherein the bound non-antibody signal transducing elements then cooperate with a
• Exemplary embodiments of this invention include a living, engineered biosensor cell that is typically a component of the mammalian immune system, e.g., an immunocyte.
• the biosensor cell is a human or mouse B cell.
• B cells or B lymphocytes are a type of white blood cell of the lymphocyte subtype that function in the humoral immunity component of the adaptive immune system by secreting antibodies.
• the biosensor cell is a human or mouse T cell.
• T cells or T lymphocytes are another type of lymphocyte that play a central role in cell-mediated immunity as part of the adaptive immune system. T cells are distinguishable from other lymphocytes due to the presence of a T-cell receptor on the cell surface.
• the biosensor cell is a mast cell.
• a mast cell is also a type of white blood cell known as a granulocyte that is derived from the myeloid stem cell that is a part of the immune and neuroimmune systems.
• Other types of cells are compatible with this invention, including basophils, which are another type of white blood cell, and which are similar in both appearance and function to mast cells.
• the living biological cell can be a prokaryotic cell or a eukaryotic cell such as a eukaryotic cell that includes a Ca 2+ signaling system.
• the living cell can be a yeast cell or an insect cell such as an insect cell that is a Drosophila Schneider 2 (S2) cell, an sf9 cell, or an insect cell that has been engineered to use aequorin as the reporter.
• the living biological cell can be a mammalian cell such as an HEK cell, a CHO cell, a COS cell, or a 3T3 cell.
• the living biological cell can be an engineered cell.
• the engineered cell can be derived from a native, passaged, or cultured mammalian cell.
• the engineered cell can be derived from a non-reproducing cell, a fixed cell, a drug- or chemically- treated cell, an osmotically-treated cell, a radiated cell, an artificial or synthetic cell, or a nonliving cell, provided that the engineered cell comprises a functional ligand, signal-transduction pathway and reporter.
• the cell can be an artificial or synthetic cell.
• the engineered cell can be derived from a plant cell, an animal cell, an insect cell or other non-mammalian animal cell, a component of a mammalian immune system, a follicular dendritic cell, natural killer cell, macrophage, monocyte, mononuclear phagocyte, neutrophil, eosinophil, or basophil.
• the engineered cell can also be a cell that expresses Fc receptor types, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils and mast cells.
• the cell can be any prokaryotic or eukaryotic cell that has a suitable receptor, signaling pathway, and signal output method, either naturally, through genetic engineering, or through chemical addition.
• the cell can be an artificial or nonliving unit provided that it has a functional receptor, signaling pathway, and signal output method.
• An example of a cell useful in this system is a macrophage cell, such as the human cell line U937, which expresses an Fc receptor on the cell surface.
• An antigen can be bound to an antibody by addition of the antibody to the target and this antigen- antibody complex will bind to the Fc receptor on the cell and stimulate signaling which results in an increase in intracellular calcium.
• the cells can be fixed, frozen, desiccated, or freeze-dried.
• Exemplary embodiments of this invention include a reporter element, such as a reporter protein or enzyme that is produced or expressed by the living, engineered biosensor cell.
• the reporter protein emits a detectable signal in response to certain predetermined changes in the cytosol of the living, engineered biosensor cell.
• the reporter protein is a bioluminescent photoprotein such as aequorin, which is derived from the hydrozoan Aequorea Victoria.
• Aequorin has been previously used for engineering living biosensor cells to produce light signals in response to activation of a wide variety of signal transduction pathways; thus, various methods for manipulating the production of aequorin in living cells are well known to the skilled artisan.
• a skilled artisan may select and employ any appropriate gene delivery vehicle such as, for example, bacterial plasmid vectors or viral vectors, for introducing the appropriate genetic material into the biosensor cells. Production of the reporter protein within the biosensor cell will then be controlled by expression of the introduced genetic material.
• gene delivery vehicle such as, for example, bacterial plasmid vectors or viral vectors.
• Photoproteins or other types of reporter proteins, enzymes, and molecules may be incorporated into and utilized with various alternate embodiments of the present invention.
• the reporter can be a protein that has fluorescent properties that undergo a detectable change in response to the activation of the at least one biochemical pathway and a resultant change in the living biological cell.
• the reporter or reporter protein can be other calcium- sensitive luminescent or fluorescent molecules, such as obelin, thalassicolin, mitrocomin (halistaurin), clytin (phialidin), mnemopsin, berovin, Indo-1, Fura-2, Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, cameleons (A. Miyawaki et al., (1999) Proc. Natl. Acad. Sci. 96, 213540), or similar molecules.
• the reporter protein can be a chimeric protein that includes a Ca 2+ binding domain and an associated fluorescent protein.
• the associated fluorescent protein can be a green fluorescent protein (GFP).
• GFP green fluorescent protein
• the protein can bind other components of the phosphatidyl inositol pathway (i.e., the pathway used in the embodiments described herein) and change its fluorescence.
• An example is a fluorescent protein that has been engineered to bind diacylglycerol.
• the reporter can be an enzyme that is adapted to produce a luminescent or fluorescent signal.
• the reporter protein can be an enzyme such as luciferase or alkaline phosphatase that yields a luminescent or fluorescent signal respectively.
• the reporter can be a dye that has fluorescent, ultraviolet, or visible properties, wherein the fluorescent, ultraviolet, or visible properties undergo a detectable change in response to the activation of the at least one biochemical pathway and the resultant change in the living biological cell.
• Exemplary embodiments of this invention include an activator mechanism in the form of a signal transduction pathway expressed by the living, engineered biosensor cell.
• the signal transduction pathway controls at least one biological process within the cytosol of the living, engineered cell, and the at least one biological process, when it occurs, causes the reporter protein to emit a detectable signal.
• the signal transduction pathway is any biochemical pathway in which an increase in intracellular Ca2+ concentration is induced in response to activation of a cell surface signal transducing molecule, such as a receptor protein.
• the biosensor cells used with this invention may be selected from a set of living cells that are capable of producing an increase in cytoplasmic Ca2+ in response to activation of a cell surface signal transduction molecule.
• B cells, T cells, and mast cells have the capacity to induce an increase in Ca2+ concentration in response to activation of cell surface signal transducing molecules such as the B cell receptor, the T cell receptor, and the Fc epsilon receptor (mast cells), respectively.
• cell surface signal transducing molecules such as the B cell receptor, the T cell receptor, and the Fc epsilon receptor (mast cells), respectively.
• mammalian cells growing in culture typically generate populations of cells in which specific individual cells may have differing capacities to induce an increase in Ca2+ concentration
• the transfectants created by the introduction of transgenes into a cell are a mixed population of cells derived from a large number of independent gene insertion events.
• FACS fluorescence-activated cell sorting
• aequorin has been used previously for engineering living biosensor cells to produce light signals in response to activation of a wide variety of signal transduction pathways, particularly wherein such signal transduction pathways lead to an increase in cytoplasmic Ca2+ ions within a living cell.
• biosensor cells that produce aequorin as the reporter protein are charged with coelenterazine (CTZ) prior to their use in a detection assay.
• CTZ coelenterazine
• This charging step covalently links the aequorin to a hydrophobic prosthetic group (e.g., CTZ) and upon calcium (Ca2+) binding, the CTZ undergoes an irreversible reaction that includes a conformation change, and emits blue light (at 469 nm).
• a hydrophobic prosthetic group e.g., CTZ
• Ca2+ calcium
• the signal transduction pathway can transmit a first signal by way of release of calcium ions from the endoplasmic reticulum into the cytosol and a second signal may be released by the reporter in response to the calcium ions.
• This signaling pathway is the second-messenger cascade found in B cells, T cells, mast cells, macrophages, and other immune cells, wherein crosslinking of the cell surface receptors activates a tyrosine kinase, which then phosphorylates phospholipase C, which then cleaves phosphatidylinositol 4,5- bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol; IP3 then opens calcium channels to release calcium from intracellular stores such as the endoplasmic reticulum or to let in extracellular calcium, thereby elevating the calcium concentration in the cell's cytosol.
• PIP3 phosphatidylinositol 4,5- bisphosphate
• IP3
• second- messenger cascades could be employed, such as a G-protein-adenylyl cyclic -cAMP-protein kinase A cascade.
• the signal transduction pathway can also transmit a signal by way of release of diacylglycerol, ceramide, or other lipophilic second messenger molecule, wherein the reporter emits a second signal in response to release of diacylglycerol, ceramide, or other lipophilic second messenger molecule.
• the signal transduction pathway can also transmit a signal by way of release or production of nitric oxide ("NO"), cAMP, cGMP, or other cyclic nucleotide, wherein the reporter emits the second signal in response to this release or production.
• the signal transduction pathway can also transmit a signal by way of release or production of superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox messenger, wherein the reporter emits the second signal in response to release or production of the superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox messenger molecule.
• the activator mechansim includes a change in cell pH or temperature or a change in cell electrical or magnetic properties.
• Exemplary embodiments of the present invention include various non-antibody signal transducing elements that function as universal detector elements for recogrnizing target analytes.
• Each signal transducing element is typically adapted to receive, i.e., bind, am analyte binding element (also referred to herein as a "detector molecule") that is itself adapted to receive, i.e., bind, a specific analyte of interest.
• the signal transducing element is a transmembrane chimeric fusion protein that is engineered into and expressed on the surface of the biosensor cell, and that is adapted to activate the signal transduction pathway that ultimately results in the reporter protein emitting a detectable signal.
• the signal transducing element is a soluble chimeric fusion protein that is adapted to bind to a cell surface signal transducer, such as a native receptor or receptor protein that is adapted to activate the signal transduction pathway that ultimately results in the reporter protein emitting a detectable signal.
• the signal transducing element is a soluble chimeric fusion protein that is engineered into and expressed by the biosensor cell. The soluble chimeric fusion protein is then secreted/excreted into the extracellular space where it binds to a cell surface signal transducer, such as a native receptor or receptor protein that is adapted to activate the signal transduction pathway that ultimately results in the reporter protein emitting a detectable signal.
• the chimeric fusion proteins of this invention may include: (i) a component of a protein that is adapted to bind to the at least one type of detector molecule (e.g., a soluble antibody); and (ii) a component of a receptor complex normally expressed by the living, engineered biosensor cell.
• the component of the protein that is adapted to bind to the at least one type of detector molecule may be derived from a bacterial binding protein (i.e., an antibody binding protein derived from a bacteria) such as, for example, the IgG binding domain of a strep G protein (referred to herein as IgGbp or Igbp in the Figures).
• Tandem repeats of this IgG binding domain may be included to increase the affinity of the binding protein for the soluble antibody.
• the component of the chimeric fusion protein that is adapted to bind to the at least one type of detector molecule is an antibody binding domain derived from a receptor protein such as, for example, the murine Fc gamma RI (FcyRI) receptor.
• FcyRI murine Fc gamma RI
• the component of the receptor complex normally expressed by the living, engineered biosensor cell is IgM (for B cell biosensors); Iga/ ⁇ (for B cell biosensors); IgE (for mast cell biosensors); CD 19 (for B cell biosensors), CD3zeta (for T cell biosensors), or FcsRI (for mast cell biosensors).
• the non-antibody signal transducing elements of this invention may include either complete protein sequences or engineered protein fragments such as selected protein domains derived from larger protein molecules.
• fragments of larger molecules may be created using standard genetic engineering techniques such as synthetic gene technology.
• fragments of larger proteins are used to engineer antibody binding motifs as aspects of chimeric fusion proteins it is important to design the engineered proteins to ensure proper conformational folding of the selected protein fragments. Therefore, it is useful to include (in the fusion proteins) short spacer or linker elements that do not readily form protein secondary structures. Short combinations of amino acids such as glycine, serine and alanine, for example, may be used for these spacer or linker elements.
• the amino acid sequence glycine (G), serine (S), alanine (A), serine (S), glycine (G), serine (S), glycine (G) is used to separate a binding domain from a component of a receptor complex in an engineered protein molecule (see SEQ ID NO: 19).
• the linker typically joins the carboxyl terminus of one element to the amino terminus of another.
• Peptide linkers may vary from 0 to 25 amino acids in length or any intermediate integer value and typically, but not always, comprise hydrophilic amino acids such as glycine (G) and serine (S).
• each signal transducing element binds to a detector molecule that binds to a specific analyte of interest.
• a detector molecule that has bound to an analyte will either (i) bind to a transmembrane signal transducing element; or (ii) to a signal transducing element that will itself bind to a cell surface signal transducer (e.g., native receptor).
• a first non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes a bacterial binding protein (IgGbp) fused to the IgM heavy chain constant domain (B cell) with a GSASGSG linker.
• SEQ ID NO: 1 provides the DNA sequence for signal transducing element IgGbp-IgM and SEQ ID NO: 2 provides the protein sequence for signal transducing element IgGbp-IgM.
• a second non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes a bacterial binding protein (IgGbp) fused to the Iga/ ⁇ component of the B cell receptor with a GSASGSG linker.
• SEQ ID NO: 3 provides the DNA sequence for signal transducing element IgGbp-Iga/ ⁇ and SEQ ID NO: 4 provides the protein sequence for signal transducing element IgGbp-Iga/ ⁇ .
• a third non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes a bacterial binding protein (IgGbp) fused to the CD3 zeta chain of the T-cell receptor with a GSASGSG linker.
• SEQ ID NO: 5 provides the DNA sequence for signal transducing element IgGbp-CD3 ⁇
• SEQ ID NO: 6 provides the protein sequence for signal transducing element IgGbp-CD3 ⁇ .
• a fourth non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes the FcyRI antibody binding domain fused to the IgM heavy chain constant domain (B cell) with a GSASGSG linker.
• SEQ ID NO: 7 provides the DNA sequence for signal transducing element FcyRI-IgM and
• SEQ ID NO: 8 provides the protein sequence for signal transducing element FcyRI-IgM.
• a fifth non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes the FcyRI antibody binding domain fused to the Iga/ ⁇ component of the B-cell receptor with a GSASGSG linker.
• SEQ ID NO: 9 provides the DNA sequence for signal transducing element FcyRI-Iga/p and SEQ ID NO: 10 provides the protein sequence for signal transducing element FcyRI-Iga/p.
• a sixth non-antibody signal transducing element in accordance with an exemplary embodiment of the present invention includes the FcyRI antibody binding domain fused to the CD3 zeta chain of the T-cell receptor with a GSASGSG linker.
• SEQ ID NO: 11 provides the DNA sequence for signal transducing element Fc ⁇ RI-CD3 ⁇ and
• SEQ ID NO: 12 provides the protein sequence for signal transducing element Fc ⁇ RI-CD3 ⁇ .
• a seventh exemplary non-antibody signal transducing element in accordance with the presentinvention includes a bacterial binding protein (IgGbp) fused to the IgE constant domain (B cell) with a GSASGSG linker.
• SEQ ID NO: 13 provides the DNA sequence for signal transducing element IgGbp-IgE and SEQ ID NO: 14 provides the protein sequence for signal transducing element IgGbp-IgE.
• An eighth exemplary non-antibody signal transducing element in accordance with the present invention includes the FcyRI antibody binding domain fused to the IgE constant domain (B cell) with a GSASGSG linker.
• SEQ ID NO: 15 provides the DNA sequence for signal transducing element FcyRI-IgE and
• SEQ ID NO: 16 provides the protein sequence for signal transducing element FcyRI-IgE.
• a ninth exemplary non-antibody signal transducing element in accordance with the present invention includes monomeric streptavidin fused to the CD3 zeta chain of the T-cell receptor with a GSASGSG linker.
• SEQ ID NO: 17 provides the DNA sequence for signal transducing element and SEQ ID NO: 18 provides the protein sequence for signal transducing element
• Monomeric streptavidin is a recombinant form of streptavidin that includes mutations that break the streptavidin tetramer into a monomer and to enhance the solubility of the resultant isolated subunit.
• the universal detector element includes an antibody VDJ region, Fab fragment or other antibody determinant.
• the universal detector element can include a T cell VJ region, VDJ region, or other T cell receptor determinant.
• the universal detector element can include a synthetic peptide; a small organic determinant, which is not a peptide; a protein or peptide determinant; a lectin determinant, a carbohydrate-binding module, or other carbohydrate-binding determinant; a lipid-binding determinant; or a metallothione determinant that binds a metal or other metal-binding determinant.
• the universal detector element can be covalently bound to a signal transduction pathway expressed by the living biological cell.
• a signal transduction pathway expressed by the living biological cell.
• An example is a membrane- anchored antibody where the anchored portion is part of the signal transduction pathway, i.e., transmits a signal from outside the cell into the cell.
• the universal detector element is not modular, but is an integral part of the signal transduction pathway, e.g., part of a chimeric protein forming that pathway.
• the universal detector element can be non-covalently bound to the signal transduction pathway.
• An example is an antibody externally bound to an Fc receptor on a signal transduction molecule where the molecule bearing the Fc receptor transmits the signal through the membrane.
• the universal detector element is modular and the cell containing the signal transduction pathway can be loaded with a universal detector element of choice.
• the universal detector element can include a determinant that non-covalently binds to a portion of the signal transduction pathway or the universal detector element can include an Fc determinant that non-covalently binds it to an Fc binding portion of the signal transduction pathway.
• the universal detector element can include a biotin or (strep)avidin determinant that non-covalently binds it to a biotin- or (strep)avidin-binding portion of the signal transduction pathway.
• Exemplary embodiments of this invention include at least one type of analyte binding element, also referered to herein as a "detector molecule", wherein each analyte binding element is adapted to bind to a specific target analyte.
• the analyte binding element may a soluble antibody that is not in any way expressed by the biosensor cells.
• the particular analyte binding element used with the present invention is selected based on its ability to unambiguously identify the target analyte of interest.
• the analyte binding element is a soluble antibody such as a commercially available IgG that is specific for a particular analyte, such as an infectious agent.
• the analyte binding element is a biotinylated molecule (or streptavidin-based molecule) that is specific for a predetermined analyte such as, for example, a biotinylated autoantigen molecule that is specific for an anti- autoantigen antibody.
• a detector or target molecule according to this invention may include an autoantigen or an autoantibody associated with an autoimmune disease.
• autoimmune diseases or disorders include rheumatoid arthritis (RA), juvenile RA (JRA), diabetes mellitus type 1, systemic lupus erythematosus, Hashimoto's thyroiditis, Graves' disease, scleroderma, celiac disease, Crohn's disease, ulcerative colitis, Sjogren's syndrome, multiple sclerosis, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatica, temporal arteritis/giant cell arteritis, and Guillain-Barre syndrome.
• RA rheumatoid arthritis
• JRA juvenile RA
• diabetes mellitus type 1 systemic lupus erythematosus
• Hashimoto's thyroiditis Graves' disease
• Detector or target molecules may also comprise tumor- specific or tumor-associated antigens or antibodies to such antigens; or biologically active molecules, such as EGF, peptide hormones, including insulin and growth hormone, cytokines, interleukins, interferons, TNF, etc. or antibodies to such biologically active molecules.
• biologically active molecules such as EGF, peptide hormones, including insulin and growth hormone, cytokines, interleukins, interferons, TNF, etc. or antibodies to such biologically active molecules.
• the system of this invention can further include an analyte binding element that includes an IgG fragment and the IgG fragment can be a single chain antibody or a single chain diabody.
• the detector can also be an affibody (i.e., engineered binding protein), an aptamer (e.g., DNA or RNA molecule that has been engineered to bind ligands), or a soluble receptor such as a soluble receptor for an infectious virus.
• An intended use of the present invention is the detection of various analytes that are or might be present within samples to be tested.
• an analyte that is to be detected will bind to a detector molecule, such as a soluble antibody, that is specific to that analyte.
• a sample to be tested may be taken from a large number of food sources, including: (i) meats such as beef, pork, lamb, bison, poultry, and seafood; and (ii) plants and vegetables.
• a sample to be tested may also be taken from many other sources such as water, consumable fluids, preservative fluids, and bodily fluids such as blood.
• Analytes that may be detected include virtually anything that will bind with specificity to the detector or detector molecule such as chemicals, toxins, and infectious agents such as viruses, bacteria, and other biological materials or agents.
• the specific infectious agent is Escherichia coli, although other infectious agents (such as Salmonella, Listeria, and Campylobacter) and contaminants may be detected with the present invention.
• Escherichia coli 0157 H7, 026, 045, O103, 0111, 0121, and 0145 in either separate assays or multiplexed assays, may all potentially be detected using this invention.
• the present invention is capable of detecting many different analytes including meat pathogens, and those found on spinach, lettuce, and other vegetables and foods.
• An analyte may contain one or more epitopes of an antigen or allergen, including both linear or conformation epitopes; it may also contain one or more ligands or receptors recognized by reciprocal receptors or ligands.
• Exemplary analytes include a bacterium, such as Bacillus (e.g., B. anthracis), Enterobacteriaceae (e.g., Salmonella, Escherichia coli, Yersinia pestis, Klebsiella, and Shigella), Yersinia (e.g., Y.
• Staphylococcus e.g., S. aureus
• Streptococcus Gonorrheae
• Enterococcus e.g., E. faecalis
• Listeria e.g., L. monocytogenes
• Brucella e.g., B. abortus, B. melitensis, or B. suis
• Vibrio e.g., V. cholerae
• Pseudomonas e.g., P. pseudomallei or P. aeruginosa
• Burkholderia e.g., B. mallei or B.
• pseudomallei Shigella (e.g., S. dysenteriae), Rickettsia (e.g., R. rickettsii, R. prowazekii, or R. typhi), Francisella tularensis, Chlamydia psittaci, Coxiella burnetii, Mycoplasma (e.g., M. mycoides), etc.; allergens, such as peanut dust, mycotoxins, mold spores, or bacterial spores such as Clostridium botulinum and C.
• Rickettsia e.g., R. rickettsii, R. prowazekii, or R. typhi
• Francisella tularensis Chlamydia psittaci, Coxiella burnetii, Mycoplasma (e.g., M. mycoides), etc.
• allergens such as peanut dust, mycotoxins, mold spor
• toxins such as ricin, mycotoxin, tetrodotoxin, anthrax toxin, botulinum toxin, staphylococcal entertoxin B, or saxitoxin
• a virus such as Adenoviridae (e.g., adenovirus), Arenaviridae (e.g., Machupo virus), Bunyaviridae (e.g., Hantavirus or Rift Valley fever virus), Coronaviridae, Orthomyxoviridae (e.g., influenza viruses), Filoviridae (e.g., Ebola virus and Marburg virus), Flaviviridae (e.g., Japanese encephalitis virus and Yellow fever virus), Hepadnaviridae (e.g., hepatitis B virus), Herpesviridae (e.g., herpes simplex viruses), Papovaviridae (e.g., papilloma viruses), Paramy
• a helminth such as cestodes (tapeworms), trematodes (flukes), or nematodes (roundworms, e.g., Ascaris lumbricoides, Trichuris trichiura, Necator americanus, or Ancylostoma duodenale); a parasite (e.g., any protozoa or helminths described herein); a fungus, such as Aspergilli, Candidae, Coccidioides immitis, and Cryptococci; an environmental contaminant; a water additive; an agricultural marker; a nucleic acid (e.g., oligonucleotides, polynucleotides, nucleotides, nucleosides, molecules of DNA, or molecules of RNA, including a chromosome, a plasmid, a viral genome, a primer, or a gene); a protein (e.g., a glycoprotein,
• Targets also include food-borne pathogens, such as Salmonella (e.g., Salmonella Typhimurium), pathogenic E. coli (e.g., 0157:H7), Bacillus (e.g., B. cereus), Clostridium botulinum, Listeria monocytogenes, Yersinia (e.g., Y. enterocolitica), Norovirus (e.g., Norwalk virus), Shigella, Staphylococcus aureus, Toxoplasma gondii, Vibrio (e.g., V. vulnificus, V. cholera, V.
• Salmonella e.g., Salmonella Typhimurium
• E. coli e.g., 0157:H7
• Bacillus e.g., B. cereus
• Clostridium botulinum Listeria monocytogenes
• Yersinia e.g., Y. enterocolitica
• Norovirus e.g
• weaponized pathogens such as Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella (e.g., B. suis), Burkholderia mallei, Burkholderia pseudomallei, Shigella, Clostridium botulinum, Variola (e.g., V.
• Filoviridae e.g., Ebola virus and Marburg virus
• Arenaviridae e.g., Lassa virus and Machupo virus
• Clostridium perfringens any food- borne pathogen (e.g., Salmonella species, Escherichia coli 0157:H7, or Shigella), Chlamydia psittaci, Coxiella burnetii, Staphylococcal aureus, Rickettsia (e.g., R. prowazekii or R.
• Alphavirus e.g., Venezuelan equine encephalitis virus, eastern equine encephalitis virus, or western equine encephalitis virus
• Vibrio cholerae Cryptosporidium parvum
• Henipavirus e.g., Nipah virus
• Bunyaviridae e.g., Hantavirus or Rift Valley fever virus
• Flaviviridae e.g., Japanese encephalitis virus and Yellow fever virus
• Coccidioides spp e.g., Venezuelan equine encephalitis virus, eastern equine encephalitis virus, or western equine encephalitis virus
• Vibrio cholerae e.g., Cryptosporidium parvum
• Henipavirus e.g., Nipah virus
• Bunyaviridae e.g., Hantavirus or Rift Valley fever virus
• Flaviviridae e.g.
• Epitopes that can be detected as analytes or portions of an analyte are typically antigenic determinant sites on an antigen to which an immunogolublin (or antigen binding fragment thereof) can specifically bind.
• Epitopes can be formed both from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of a protein.
• Epitopes can be found on the Fab (variable) region of immunoglobulins (referred to as "idiotypic determinants”) and comprise the immunoglobulin's "idiotype".
• the epitope and antigen can be naturally occurring or artificially produced.
• the epitope or antigen can be isolated or purified from a matrix or substance of origin, synthesized, or recombinantly produced, for example.
• Epitopes and antigens useful as analytes can be from a human or non-human animal, plant, bacteria, protozoan, parasite, virus, etc.
• the analyte is a polypeptide, nucleic acid molecule, carbohydrate, glycoprotein, lipid, lipoprotein, glycolipid, or small molecule.
• the analyte is selected from among a cancer antigen, autoantigen, allergen, endogenous antigen, infectious agent antigen, drug (small molecule) antigen, toxin, venom, biologic antigen, environmental antigen, transplant antigen, and implant antigen.
• An analyte may comprise an epitope of a cancer antigen.
• the analyte is a tumor-associated antigen.
• the analyte is a tumor-specific antigen.
• the analyte is a tumor-associated antigen (TAA)
• TAA is a carbohydrate antigen having one or more post-translational modifications that differ from the wild-type protein, comprises a fusion region of a protein resulting from a gene fusion that is present in malignant cells but not present in non-malignant cells, and/or wherein the TAA comprises a receptor tyrosine kinase (RTK) that is deregulated and/or dysfunctional in tumor cells due to autocrine activation, chromosomal translocations, RTK overexpression, or gain-of-function mutations in the RTK gene or protein.
• RTK receptor tyrosine kinase
• the analyte is an immunoglobulin expressed by a B-cell malignancy.
• B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma and multiple myeloma. Additional B-cell malignancies include, for example.
• B-cell prolymphocytic leukemia lymphoplasmocytic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extra-nodal and nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition diseases, heavy chain diseases), and follicular lymphoma (e.g., Grades I, II, III, or IV).
• the analyte is a tumor-associated antigen derived from tumor cells obtained from the subject.
• the tumor- associated antigen is one or more antigens selected from among 17-1 A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE-1, .beta.-catenin, BCG, bcr/abl, Bcr/abl el4a2 fusion junction, bcr-abl (b3a2), bcr-abl (b3a2), bcr-abl pl90 (ela2), bcr-abl p210 (b2a2), bcr-abl p210 (b3a2), bcr-abl p210 (b3a2), bullous pemphigoid antigen-1, CA19-9, CA125, CA215, CAG-3, CAMEL, Cancer-testis antigen, Caspase-8, CCL3, CCL4, CD16, CD20
• the tumor associated antigen is identified by the SEREX (serological analysis of recombinant cDNA expression library) approach or based on the serological screening of cDNA expression library generated from tumor tissues of various origin or cancer cell lines, and identifying immunogenic tumor proteins based on their reactivity with autologous patient sera.
• the analyte is a tumor-associated antigen that is acarbohydrate antigen having one or more post-translational modifications that differ from the wild-type protein.
• the tumor-associated antigen comprises a fusion region of a protein resulting from a gene fusion that is resent in malignant cells but not present in non- malignant cells.
• the tumor-associated antigen comprises a receptor tyrosine kinase that is deregulated and/or dysfunctional in tumor cells due to autocrine activation, chromosomal translocations, RTK overexpression, or gain-of-function mutations in the RTK gene or protein.
• the analyte may comprise an epitope of an antigen of an infectious or noninfectious agent that can be either pathogenic or non-pathogenic to the subject.
• the analyte can be derived from a mutualistic, parasitic, or commensal microorganism, including any microorganism in a animal or plant biome, such as probiotic or commensal microorganisms in the human digestive tract, mucosal surfaces, or epithelium.
• the bacterial pathogen is selected from among Acinetobacter baumannii (formerly Acinetobacter calcoaceticus), Actinobacillus, Actinomyces pyogenes (formerly Corynebacterium pyogenes), Actinomyces israelii, nocardia asteroids, N. brasiliensis, Aeromonas hydrophila, Amycolata autotrophica, Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum), Arizona hinshawii— all serotypes, Bacillus anthracis, Bacteroides fragilis, Bartonella henselae, B. quintana, B.
• pseudomallei Coxiella burnetii, Francisella tularensis, Mycobacterium bovis (except BCG strain, BSL II— Bacterial Agents Including Chlamydia), M. tuberculosis, Mycobacteria other than tuberculosis (MOTT), Pasteurella multocida type B— "buffalo” and other virulent strains.
• the analyte can be derived from a viral pathogen.
• the analyte is derived from a viral pathogen selected from among Adenoviruses, human— all types, Alphaviruses (Togaviruses), Eastern equine encephalitis virus, Eastern equine encephalomyelitis virus, Venezuelan equine encephalomyelitis vaccine strain TC-83, Western equine encephalomyelitis virus, Arenaviruses, Lymphocytic choriomeningitis virus (non- neurotropic strains), Tacaribe virus complex, Bunyaviruses, Bunyamwera virus, Rift Valley fever virus vaccine strain MP-12, Calciviruses, Coronaviruses.
• Flaviviruses Togaviruses— Group B Arboviruses, Dengue virus serotypes 1, 2, 3, and 4, Yellow fever virus vaccine strain 17D, Hepatitis A, B, C, D, and E viruses, the Cytomegalovirus, Epstein Barr virus, Herpes simplex types 1 and 2, Herpes zoster, Human herpesvirus types 6 and 7, Influenza viruses types A, B, and C, Papovaviruses, Papilloma viruses, Newcastle disease virus, Measles virus, Mumps virus, Parainfluenza viruses types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), Respiratory syncytial virus, Human parvovirus (B 19), Coxsackie viruses types A and B, Echoviruses, Polioviruses, Rhinoviruses, Alastrim (Variola minor virus), Smallpox (Variola major virus), Whitepox Reoviruses, Coltivirus, human Rotavirus, and Orb
• the analyte can be derived from a parasite.
• the analyte is derived from a parasite selected from among Ancylostoma human hookworms including A. duodenale, A. ceylanicum, Ascaris including Ascaris lumbricoides suum, Babesia including B. divergens, B. microti, Brugia filaria worms including B. malayi, B. timori, Coccidia, Cryptosporidium including C. parvum, Cysticercus cellulosae (hydatid cyst, larva of T. solium), Echinococcus including E. granulosis, E.
• E. vogeli Entamoeba histolytica, Enterobius, Fasciola including F. gigantica, F. hepatica, Giardia including G. lamblia, Heterophyes, Hymenolepis including H. diminuta, H. nana, Isospora, Leishmania including L. braziliensis, L. donovani, L. ethiopia, L. major, L. mexicana, L. peruvania, L. tropica, Loa loa filaria worms, Micro sporidium, Naegleria fowleri, Necator human hookworms including N. americanus, Onchocerca filaria worms including, O.
• the analyte can be a fungal pathogen.
• the analyte is derived from a fungal pathogen selected from among Aspergillus fumigates, Blastomyces dermatitidis, Cladosporium bantianum, Candida albicans, C.
• the analyte can be a toxin.
• the analyte is a toxin selected from among Abrin, Botulinum neurotoxins, Clostridium perfringens epsilon toxin, Conotoxins, Diacetoxyscirpenol, Ricin, Saxitoxin, Shiga-like ribosome inactivating proteins, Shigatoxin, Staphylococcal enterotoxins, T-2 toxin, and tetrodotoxin.
• the analyte is selected from among Hepatitis B surface antigen (HBsAg), B. burgdorferi OspA, HPV LI, RSV F protein, Influenza hamagglutanin, Influenza stem-loop region, Influenza M2, P. falciparum merozoite surface protein 1-10, GLURP, SERA, S-antigen, 6-cys family, AMA1, EBA175, 140, 181, MTRAP, PTRAMP, ASP, Rhl, 2a, 2b, 4, 5, RAPl, 2, 3, RAMA, RHOPHl, 2, 3, P.
• HBAg Hepatitis B surface antigen
• B. burgdorferi OspA HPV LI
• RSV F protein Influenza hamagglutanin
• Influenza stem-loop region Influenza M2
• P. falciparum merozoite surface protein 1-10 P. falciparum merozoite surface protein 1-10
• GLURP GLURP
• SERA S
• vivax circumsporozoite protein sporozoite surface proetin2, SSP2/TRAP, CSP-N, CSP-R, CSP-C, MSP-1, MSP-9, DBPRIII, AMA-1, Pvs25, Pvs28, S. aureus capsular polysaccharide, poly-N-acetyl glucosamine, HIV gpl20, gp41, and Dengue virus conserved regions.
• analyte comprises at least one epitope of an allergen.
• allergens can be naturally occurring, or artificial such as allergens contained in allergy vaccines.
• allergens include, but are not limited to, animal products (for example, Fel d 1, fur dander, cockroach calyx, wool, dust mite excretion), drugs (for example, penicillin, sulfonamides, salicylates, local anaesthetic), food (for example, celery and celeriac, corn, eggs (e.g., albumin), fruit, legumes (for example, beans, peas, peanuts, soybeans), milk, seafood (e.g., shellfish), sesame, soy, tree nuts (for example, pecans, almonds), wheat, insect venom (for example, fire ants, bee sting venom, wasp sting venom), latex, metal, plant pollen (for example, grass (e.g., ryegrass, timothy-grass, weeds (e.g., ragweed, plantago, nettle, Art
• the analyte is an allergen derived from a latex protein, for example, unprocessed latex sap, raw latex containing ammonia, or finished latex product in which the proteins have been exposed to chemicals and high temperatures.
• the allergen is the allergen of a mite, for example, Dermatophagoides farinae, Dermatophagoides pteronyssinus, Acarus siro, Blomia tropicalis, Chortoglyphus arcuatas, Euroglyphus cannei, Lepidoglyphus destructor, Tyrophagus putrescentiae, or Glyphagus demesticus.
• the allergen is from venom, for example, Bombus spp., Vespa crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Vespula spp., Dolichovespula maculata, or Dolichovespula arenaria.
• venom for example, Bombus spp., Vespa crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Vespula spp., Dolichovespula maculata, or Dolichovespula arenaria.
• the analyte is an allergen from an insect, for example, Camponotus pennsylvanicus, Solenopsis invicta, Solenopsis richteri, Periplaneta americana, Blattella germanica, Blatta orientails, Tebanus spp., Musca domestica, Ephemeroptera spp., Culicidae sp., or Heterocera spp.
• an insect for example, Camponotus pennsylvanicus, Solenopsis invicta, Solenopsis richteri, Periplaneta americana, Blattella germanica, Blatta orientails, Tebanus spp., Musca domestica, Ephemeroptera spp., Culicidae sp., or Heterocera spp.
• the allergen analyte is epithelia, dander, or hair from an organism, for example, Serinus canaria, Felis catus (domesticus), Bos taurus, Gallus gallus (domesticus), Canis familiaris, Arias platyrhynchos, Meriones unguiculatus, Capra hircus, Anser domesticus, Cavia porcellus (cobaya), Mesocrietus auratus, Sus scrofa, Equus caballus, Mus musculus, Psittacidae, Columba fasciata, Oryctolagus cuniculus, Rattus norvegicus, or Ovis aries.
• the allergen analyteis from fungi for example,
• Cephalosporium acremonium Alternaria tenuis, Aspergillus glaucus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor, Aureobasidium pullulan (Pullularia pullulans), Drechslera sorokiniana, Helminthosporium sativum, Botrytis cinerea, Candida albicans, Chaetomium globosum, Cladosporium herbarum, Cladosporium sphaerospennum (Homodendrum hordei), Drechslera spicifera (Curvularia spicifera), Epicoccum nigrum (Epicoccum purpurascens), Epidermophyton floccosum, Fusarium moniliforme, Fusarium solani, Geotrichum candidum, Gl
• the allergen is from a smut, for example, Ustilago nuda, Ustilago cynodontis, Ustilago candis, Sporisorium cruentum, Ustilago avenae, or Ustilago tritici.
• a smut for example, Ustilago nuda, Ustilago cynodontis, Ustilago candis, Sporisorium cruentum, Ustilago avenae, or Ustilago tritici.
• the allergen analyte is from a grass, for example, Paspalum notatum, Cynodon dactylon, Poa compressa, Bromus inennis, Phalaris arundinacea, Zea cans, Elytrigia repens (Agropyron repens), Sorghum haelpense, Poa pratensis, Festuca pratensis (elatior), Avena sativa, Dactylis glomerata, Agrostis gigantea (alba), Secale cereale, Leymus (Elymus) condensatus, Lolium perenne ssp. multiflorum, Lolium perenne, Anthoxanthum odoratum, Phleum pratense, Holcus lanatus, Triticum aestivum, or Elymus (Agropyron) smithii.
• a grass for example, Paspalum notatum, Cynodon dactylon, Po
• the allergen analyte is from a weed, for example, Atriplex polycarpa, Baccharis halimifolia, Baccharis sarothroides, Hymenoclea salsola, Amaranthus hybridus, Xanthium strumarium (commune), Rumex crispus, Eupathium capiUifolium, Solidago spp., Amaranthus tuberculatus (Acnida tamariscina), Allenrolfea occidentalis, Chenopodium botrys, Kochia scoparia, Chenopodium album, Iva xanthifolia, Iva angustifolia, Chenopodium ambrosioides, Artemisia vulgaris, Artemisia ludoviciana, Urtica dioica, Amaranthus spinosus, Plantago lanceolata, Iva axillaris, Atriplex lentiformis, Ambrosia dumosa, Ambrosia dumosa,
• the allergen analyte is from a tree, for example, Acasia spp., Alnus glutinosa, Alnus rubra, Alnus incana ssp. rugosa, Alnus rhombifolia, Fraxinus velutina, Fraxinus pennsylvanica, Fraxinus latifolia, Fraxinus americana, Populus tremuloides, Myrica cerifera, Fagus grandifolia (americana), Casuarina equisetifolia, Betula lenta, Betula pendula, Betula nigra, Betula occudentalis (fontinalis), Betula populifolia, Acer negundo, Cryptomeria japonica, Juniperus ashei (sabinoides), Juniperus virginiana, Tamarix gallica, Populus balsamifera ssp.
• a tree for example, Acasia
• the allergen is from a flower, for example, Chrysanthemum leucanthemum, Taraxacum officinale, or Helianthus annuus.
• the allergen is from a farm plant, for example, Medicago sativa, Ricinus communis, Trifolium pratense, Brassica spp., or Beta vulgaris.
• the allergen analyte is from plant food (an edible plant), for example, Prunus dulcis, Malus pumila, Prunus armeniaca, Musa paradisiaca (sapientum), Hordeum vulgare, Phaseolus lanatus, Phaseolus vulgaris, Phaseolus sp., Phaseolus sp., Phaseolus vulgaris, Rubus allegheniensis, Vaccinium sp., Brassica oleracea var. botrytis, Fagopyrum esculentum, Brassica oleracea var. capitata, Theobroma cacao, Cucumis melo, Daucus carota, Brassica oleracea var.
• Botrytis Apium graveolens var. dulce, Prunus sp., Cinnamomum verum, Coffea arabic, Zea cans, Vaccinium macrocarpon, Cucumis sativus, Allium sativum, Zingiber officinale, Vitis sp., Citrus paradisi, Humulus lupulus, Citrus limon, Lactuca sativa, Agaricus campestris, Brassica sp., Myristica fragrans, Avena sativa, Olea europaea, Allium cepa var.
• the allergen analyte is from fish or shellfish, for example,
• the allergen is an animal food product, for example, from Bos taurus, Ovis aries, or Sus scrofa.
• the allergen is a poultry product, for example, chicken (Gallus gallus) products or turkey (Meleagris gallopavo) products.
• the allergen is from a dairy product, for example, bovine casein or bovine milk.
• the allergen is a nut, for example, Bertholletia excelsa, Anacardium oceidentale, Cocos nucifera, Corylus americana, Arachis hypogaea, Carya illinoensis, Juglans nigra, or Juglans regia.
• the allergen is dust, for example, barley grain dust, corn grain dust, house dust, mattress dust, oat grain dust, wheat grain dust, upholstery dust, or latex dust.
• the antigen analyte is an autoantigen associated with an autoimmune disorder.
• the autoimmune disorder is a cell or organ- specific autoimmune disorder
• the autoantigen analyte is selected from among: acetylcholine receptor (myasthenia gravis), actin (chronic active hepatitis, primary biliary cirrhosis), adenine nucleotide translocator (ANT) (dilated cardiomyoapthy, myocarditis), beta- adrenorecep tor (dilated cardiomyopathy), aromatic L-amino acid decarboxylase (autoimmune polyendocrine syndrome type I (APS-1)), asialoglycoprotein receptor (autoimmune hepatitis), bactericidal/permeability- increasing protein (Bpi) (cystic fibrosis vasculitides), calcium-sensing receptor (acquired hypoparathyroidism), cholesterol side-chain clea
• the autoimmune disorder is a systemic autoimmune disorder
• the autoantigen analyte is selected from among: ACTH (ACTH deficiency), aminoacyl-tRNA histidyl synthetase (myositis, dermatomyositis), aminoacyl-tRNA synthetase (polymyositis, dermatomyositis), cardiolipin (SLE), carbonic anhydrase II (SLE, Sjogren syndrome, systemic sclerosis), collagen (rheumatoid arthritis (RA), SLE, progressive systemic sclerosis), centromere- associated protein (systemic sclerosis), DNA-dependent nucleosome- stimulated ATPase (dermatomyositis), fibrillarin (scleroderma), fibronectin (SLE, RA, morphea), glucose-6-phosphate isomerase (RA), Beta2-glycoprotein I (Beta2-GPI) (ACTH (ACTH defici
• the autoimmune disorder is a plasma protein autoimmune disorder or cytokine autoimmune disorder
• the autoantigen analyte is selected from among: CI inhibitor (autoimmune CI deficiency), Clq (SLE, membrane proliferative glomerulonephritis (MPGN)), cytokine (e.g., IL-1 alpha, IL-lbeta, IL-, IL-10, LIF) (RA, systemic sclerosis), factor II (prolonged coagulation time), factor V (prolonged coagulation time), factor VII (prolonged coagulation time), factor VIII (prolonged coagulation time), factor IX (prolonged coagulation time), factor X (prolonged coagulation time), factor XI (prolonged coagulation time), factor XII (prolonged coagulation time), thrombin (prolonged coagulation time), vWF (prolonged coagulation time), glycoprotein Ilb/IIIg and Ib/IX (autoimmune thrombocyto
• the autoimmune disorder is a cancer or paraneoplastic autoimmune disorder
• the autoantigen analyte is selected from among: amphiphysin (neuropathy, small lung cell cancer), cyclin B 1 (hepatocellular carcinoma), DNA topoisomerase II (liver cancer), desmoplakin (paraneoplastic pemphigus), gephyrin (paraneoplastic stiff man syndrome), Hu protein (paraneoplastic encephalomyelitis), neuronal nicotinic acetylcholine receptor (subacute autonomic neuropathy, cancer), p53 (cancer, SLE), p62 (IGF-II mRNA- binding protein) (hepatocellular carcinoma), recoverin (cancer-associated retinopathy), Rl protein (paraneoplastic opsoclonus myoclonus ataxia), beta IV spectrin (lower motor neuron syndrome), synaptotagmin (Lambert-Eaton mya
• the antigen analyte is an endogenous antigen that is an aberrantly expressed polypeptide.
• endogenous antigens include, but are not limited to, amyloid beta (A-beta), alpha synuclein, cystatin C, tau, ABri, ADan, superoxide dismutase (SOD), mutant Huntington, PrP sc or a fragment of any of the foregoing.
• the analyte comprises at least one epitope of an implant to be introduced into a subject, metabolic or degradation products of an implant material, or substances that specifically bind to an epitope of an implant material, such as antibodies developed to an implant material or its degradation products
• implants can include, for example, electrically powered implants (for example, artificial pacemakers), bioimplants (biomaterial surgically implanted in a subject's body to replace damaged tissue (for example, orthopedic reconstructive prosthesis), cardiac prostheses (artificial valves), skin, and cornea), contraceptive implants, dental implants, orthopedic implants, and adhesion prevention devices.
• metals such as cobalt chrome (Co— Cr) alloys, titanium, and titanium alloys
• polymers such as ultrahigh molecular weight polyethylene (UHMWPE) and polymethyl methacrylate cement (PMMA); and bioceramics, such as hydroxyapatite and Bioglass.
• UHMWPE ultrahigh molecular weight polyethylene
• PMMA polymethyl methacrylate cement
• bioceramics such as hydroxyapatite and Bioglass.
• the non-antibody binding element can be a bacterial binding protein, or an antibody binding domain.
• the predetermined analyte can be selected from beneficial gut bacteria, pathogenic bacteria, protein toxins, protein biomarkers, small molecule toxins, metabolites, or chemical warfare agents.
• the assay is modified into a competitive format, wherein the small molecule analyte is linked to a protein or other macromolecular carrier in such a way that antibodies to the free analyte also recognize the immobilized analyte (if no antibodies are available they can be generated by immunizing animals with the immobilized analyte).
• the immobilized analyte will aggregate cell receptors that are bound to the anti-analyte antibody and give a luminescent signal. If the immobilized analyte is mixed with the free analyte to be measured, which cannot lead to aggregation, the luminescent signal will be reduced.
• the activator can be a receptor and the non- antibody binding element can be a ligand that is specific for the receptor and causes a conformational change (rather than aggregation) in the receptor when bound thereto, wherein the ligand is adapted to bind to the receptor only after it has bound to the predetermined analyte.
• the ligand can be fused to a detector, wherein the detector is operative to prevent the ligand from binding to the receptor unless the ligand has first bound to the predetermined analyte.
• the activator can also be a receptor that has been engineered to bind a predetermined analyte, wherein the receptor undergoes a conformational change upon binding the predetermined analyte. Again, this variant does not rely on an aggregation effect.
• an aggregation event can be mediated by a carrier molecule such as serum albumin that binds multiple copies of a target such as physiological or drug metabolites.
• a first biosensor 100 in accordance with an exemplary embodiment of the present invention includes Jurkat T cells 102 that have been engineered to produce aequorin 104 and that have been charged with CTZ 106 to form an aequorin/CTZ complex, as previously described.
• This particular biosensor has also been engineered to express the transmembrane non-antibody signal transducing element 108, which is IgGbp-CD3 ⁇ (SEQ ID NOS: 5-6), although the transmembrane non-antibody signal transducing element ⁇ ⁇ - ⁇ 3 ⁇ (SEQ ID NO: 11-12) may also be used with biosensor 100.
• Biosensor cell 102 also includes at least one signal transduction pathway 110, the activation of which results in an increase of intracellular Ca2+ 112. As shown in FIG. lb, when a sufficient number of detector molecules 114 (e.g., soluble antibodies) to which target analyte 116 (e.g., E.
• coli 0157 is bound bind to transmembrane non-antibody signal transducing elements 108, signal transduction pathway 110 is activated, intracellular Ca2+ 112 increases, the aequorin/CTZ complex undergoes a conformational change and emits a signal (photon) of light 118 which is detected by photo multiplier tube 120, and spike 122 is graphically displayed on a testing device (see description below), indicating the presence of target analyte 116 within a sample being tested.
• the display may be both qualitative and quantitative with regard to target analyte 116.
• a second biosensor 200 in accordance with an exemplary embodiment of the present invention includes MC/9 (ATCC ® CRL-8306 TM ) mast cells 202 that have been engineered to produce aequorin 204 and that have been charged with CTZ 206 to form an aequorin/CTZ complex, as previously described.
• This particular biosensor expresses the native Fc epsilon receptor (i.e., FcsRI) 207, which binds to soluble non-antibody signal transducing element 208, which is IgGbp-IgE (SEQ ID NOS: 13-14), although the non- antibody signal transducing element FcyRI-IgE.
• FcsRI native Fc epsilon receptor
• SEQ ID NO: 15-16 may also be used with biosensor 200.
• detector molecules 214 e.g., soluble antibodies
• target analyte 216 e.g., E.
• coli 0157 is bound bind to non-antibody signal transducing elements 208 that have previously bound to native Fc epsilon receptors 207, signal transduction pathway 210 is activated, intracellular Ca2+ 212 increases, the aequorin/CTZ complex undergoes a conformational change and emits a signal (photon) of light 218 which is detected by photo multiplier tube 220, and spike 222 is graphically displayed on a testing device (see description below), indicating the presence of target analyte 216 within a sample being tested.
• the display may be both qualitative and quantitative with regard to target analyte 216.
• a third biosensor 300 in accordance with an exemplary embodiment of the present invention includes MC/9 (ATCC ® CRL-8306 TM ) mast cells 302 that have been engineered to produce aequorin 304 and that have been charged with CTZ 306 to form an aequorin/CTZ complex, as previously described.
• This particular biosensor expresses the native Fc epsilon receptor (i.e., FcsRI) 307, which binds to non-antibody signal transducing element 308, which is IgGbp-IgE (SEQ ID NOS: 13-14), although the non-antibody signal transducing element FcyRI-IgE.
• FcsRI native Fc epsilon receptor
• biosensor cells 302 may also be used with biosensor 300.
• biosensor cells 302 have been further engineered to express IgGbp-IgE and excrete this non-antibody signal transducing element into the extracellular space, wherein it binds to the native FcsRI expressed on the cell surface.
• detector molecules 314 e.g., soluble antibodies
• target analyte 316 e.g., E.
• coli 0157 is bound bind to non-antibody signal transducing elements 308 that have previously bound to native Fc epsilon receptors 307, signal transduction pathway 310 is activated, intracellular Ca2+ 312 increases, the aequorin/CTZ complex undergoes a conformational change and emits a signal (photon) of light 318 which is detected by photo multiplier tube 320, and spike 322 is graphically displayed on a testing device (see description below), indicating the presence of target analyte 316 within a sample being tested.
• the display may be both qualitative and quantitative with regard to target analyte 316.
• a fourth biosensor 400 in accordance with an exemplary embodiment of the present invention includes biosensor cells 402 that have been engineered to produce aequorin and to express transmembrane non-antibody signal transducing element 408, which is mSA-CD3 (SEQ ID NO: 17-18).
• Non-antibody signal transducing element (monomeric streptavidin-CD3 ⁇ ) binds to a biotinylated detector molecule 414, which specifically binds to a target molecule 416 such as, for example, epidermal growth factor (EGF).
• EGF epidermal growth factor
• An anti-target molecule antibody 417 such as, for example, anti-EGF, creates target multimers that cluster multiple signal transducing elements and induce signal transduction as previously described.
• the monomeric streptavidin component is replaced with a biotinylated component and alternate linkage means may be employed.
• a fifth biosensor 500 in accordance with an exemplary embodiment of the present invention includes biosensor cells 502 that have been engineered to produce aequorin and to express transmembrane non-antibody signal transducing element 508, which is mSA-CD3C (SEQ ID NO: 17-18).
• Non-antibody signal transducing element mSA-CD3C (monomeric streptavidin-CD3 ⁇ ) binds to a biotinylated detector molecule 514, which in some embodiments is an autoantigen molecule.
• Biotinylated detector molecule 514 specifically binds to a target molecule 516, which in some embodiments is an anti-autoantigen molecule.
• Autoantibodies in a serum sample create target multimers that cluster multiple signal transducing elements and induce signal transduction as previously described.
• the monomeric streptavidin component is replaced with a biotinylated component and alternate linkage means may be employed.
• the amino acid sequences of the signal-transducing polypeptide used to produce the chimeric proteins of the invention may have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to the proteins or domains identified by or in the following accession numbers: IgM heavy chain (GenBank: CAC20458.1), Ig-alpha (P11912.2, GL547896), Ig-beta (P40259.1 GL728994), CD19 (AAA69966.1 GL901823), CD3zeta (P20963.2, GI: 23830999), IgE alpha (1F2Q_A, GL9257150) and Fc- epsilonRl subunit alpha (P12319.1, GI: 119865).
• Staphylococcus aureus Protein A (P02976.3, GI: 110283003) is encoded by the spa gene of Staphylococcus aureus and its structure, including its Ig-binding segments, and immunoglobulin-binding properties are well-known and are incorporated by reference to Graille, et al, Proc Natl Acad Sci U S A. 2000 May 9;97(10):5399-404; and Roben, et al. J Immunol. 1995 Jun 15;154(12):6437-45.
• Variants of Protein A or its immunoglobulin-binding segments having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to known Protein A amino acid sequences and the capacity to bind to an immunoglobulin or other analyte, such as those described by Graille, et al. and Roben, et al., may be produced by molecular biological techniques well-known in the art including by direct synthesis of a nucleic acid encoding an immunoglobulin-binding amino acid sequence.
• Variants of Protein G or its immunoglobulin- binding segments having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to known Protein G amino acid sequences and the capacity to bind to an immunoglobulin or other analyte, such as those described by Bailey, et al. and Watanabe, et al. may be produced by molecular biological techniques well-known in the art including by direct synthesis of a nucleic acid encoding an immunoglobulin-binding amino acid sequence.
• Fc receptors bind to the Fc portion of an immunoglobulin and many types such Fc receptors are known, including FcyRI and FcsRI. The structural and functional binding characteristics of these FcRs are incorporated by reference to Fridman, FASEB J. 1991 Sep; 5(12):2684-90.
• Variants of FcRs or their immunoglobulin-binding segments having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to a known FcR amino acid sequence, such as a sequence described by Fridman, may be produced by molecular biological techniques well-known in the art including by direct synthesis of a nucleic acid encoding an immunoglobulin-binding amino acid sequence.
• a signal transducing protein according to the invention may have at least 70%,
• variants may be constructed by methods well known in the molecular biological arts or by chemical synthesis of polynucleotides encoding the variant chimeric reporter proteins, insertion of the encoding sequences into a vector, and transformation or transfection of a competent cell with the vector.
• biosensors of this invention resulted in a mixed population of biosensor cells when cultured. Some cells did not express the engineered factors while others expressed the factors at varying levels. Following successful electroporation and gene insertion, biosensor cells were cultured and tested for biological response (flash signal) as mixed populations. Single cell sorting was performed using a Flow Cytometer. Cells were isolated and then expanded for analysis to select those that expressed high levels of the desired proteins. For this process, fluorescently labeled antibodies were used to target different receptors on the biosensor cells, thereby enabling the sorting process. Individual clones were screened for signaling and the best clones were selected. Through this process, the most suitable clones were identified and isolated.
• FACS Fluorescence-Activated Cell Sorting
• a secondary FITC-labeled antibody was added (0.5-1 ⁇ g) to the cells and mixed before incubating on ice (or at 4°C) for 20-40 minutes. Cells were protected from light during the entire process. A 2 mL volume of cold wash buffer was added then cells centrifuged and supernatant discarded. The wash step was repeated and cells re-suspend in 0.5-1 mL of wash buffer. Cells were incubated on ice until the time for sorting. Sorting was done as soon as possible (at least on the same day). Cloning and culturing cells after single cell sorting was conducted as described below.
• Biosensor cells were sorted into 96 well plates with each well containing one cell and 100-200 ⁇ ⁇ cell growth media. Plates were scanned/monitored for the next 10 to 14 days to determine the rate of growth and to judge when to transfer to a 24 well plate. During scanning, different markings were used for different conditions. Some wells were marked if they contained live cells, but were not ready for transfer and contaminated wells were also marked. Cells were transferred to a 24 well plate containing 1.0 mL of the appropriate media in each well. In cases of contaminated cells, washing was done by adding all of the cell suspension from a well into 5ml of sterile lx PBS in a 15mL conical tube.
• clones were screened using anti-CD3s antibodies (positive control) and monoclonal antibodies against bacteria with the respective bacteria while Digitonin was used for chemical response test.
• MC/9-Aeq clones were screened using anti-FcsRI antibodies (biological response) and Digitonin (chemical response).
• FACS fluorescence-activated cell sorting
• MC/9 mast cells were cultured in Complete Mast Cell Media (DMEM - Sigma, Cat. No. D5796; lx Pen/Strep; 10% FBS; 10% T-Stim Supplement; 50 ⁇ ⁇ - mercaptoethanol).
• Jurkat T-cells were cultured in complete RPMI media (RPMI-ThermoFisher; 10% FBS; lx Pen/Strep).
• RPMI-ThermoFisher 10% FBS; lx Pen/Strep
• Appropriate antibiotics were added to the growth media 2-3 days after electroporation to select for cells that had successfully integrated the linearized DNA constructs.
• Cell concentration was kept between 4.0 x 10 5 and 1.0 x 10 6 cells/mL for optimal cell growth.
• Different cell lines and clones were processed for long term storage and stocks were frozen in liquid nitrogen as follows: (i) cells were centrifuged at 150 RCF for 10 minutes and the supernatant was discarded; (ii) the cell pellet was re-suspended in freezing media (RPMI; 50% FBS; 10% DMSO) at a concentration of 5.0 x 10 5 cells/mL; and (iii) volumes of 1 mL were aliquoted into 2 mL Nunc Cryo-vials and frozen at -80°C for 24 hours before being transferred to liquid nitrogen for long term storage.
• RPMI 50% FBS
• DMSO re-suspended in freezing media
• volumes of 1 mL were aliquoted into 2 mL Nunc Cryo-vials and frozen at -80°C for 24 hours before being transferred to liquid nitrogen for long term storage.
• biosensor cells of this invention were centrifuged in 50 mL conical tubes at
• the biosensor cells of this invention were demonstrated to be more effectively charged at lower concentrations rather than higher concentrations. For example, charging cells at a density of 25,000 cells/180 ⁇ ⁇ versus 400,000 cells/180 ⁇ ⁇ was shown to result in a two-fold increase in detectable signal.
• Jurkat-Fc ⁇ RI-CD3 ⁇ clone P5G7 cells were charged at both 400,000 cells/180 and 25,000 cells/180 then tested at 400,000 cells/180 in each reaction. Overnight E. coli Ol l l bacteria culture was used with 23 nM anti-E. coli Ol l l mAb. Biosensor cells charged at a lower concentration gave a higher signal for the same number of bacteria cells tested.
• Biosensor cell density is mostly changed by concentrating the cells after charging to allow for optimal pathogen detection. Different concentrations were used for pathogen detection depending on the target pathogen and quality of the antibody, when the detector molecule is a soluble antibody. Biosensor cells are concentrated by centrifugation at 150 RCF for 10 minutes and the cell pellet is re-suspended in the desired volume of the testing medium. The charging medium may also serve as the testing medium. In certain instances, addition of normal FBS to the media triggered a biological response resulting in a biosensor signal (flash) due to high concentration of antibodies in normal FBS. Therefore, commercially available antibody-depleted FBS was used in the charging process, which reduced the antibody triggered signal without totally eliminating it. Additional methods were used to further deplete traces of antibodies in the commercially available antibody-depleted FBS.
• the analyte bioassay is formatted with the biosensor cell and a soluble monoclonal antibody (mAb) that is specific for that analyte (e.g., pathogen).
• mAb monoclonal antibody
• the specificity of the bioassay is directly related to the selective binding of the soluble antibody to the target analyte and the specificity and sensitivity of the biosensor is determined by detection and measurement of bioluminescence.
• biosensor cells are initially charged using the light-emitting molecule, coelenterazine (CTZ). The soluble antibody of choice and the sample being analyzed are then added.
• CTZ coelenterazine
• a target pathogen If a target pathogen is present in the sample, it interacts with the soluble antibody, which binds to a fusion protein expressed by the biosensor cell, ultimately triggering a signal cascade that results in light emission from the biosensor cell.
• the emitted light is detected by a photo multiplier tube (PMT) in the testing device and the signal emitted by the biosensor cell is displayed as photon counts per second.
• PMT photo multiplier tube
• detector molecules e.g., antibodies
• biosensor cells e.g., antibodies
• analytes e.g., bacteria
• the bioassay aspect of the present invention herein may be carried out in a testing subunit or test cartridge designed for use with a bench-top or portable testing system and device such as that disclosed in U.S. Patent No. 9,023,640), which is incorporated by reference herein, in its entirety.
• the test cartridge which may be a single-use, disposable item, receives both the sample and the biosensor and introducing the biosensor into the test cartridge mixes the sample and the biosensor in a predictable and controlled manner.
• the test cartridge further includes a reaction chamber for receiving the test sample and the biosensor, wherein the reaction chamber has a predetermined internal geometry and has been further adapted to minimize or eliminate background noise for the purpose of improving the overall signal to noise ratio. At least one stabilizer may be located in the reaction chamber for minimizing shear force damage to the test sample and biosensor during the mixing process.
• reaction chamber and fluid channels that lead to the reaction chamber within the test cartridge are designed to achieve several objectives.
• An inlet channel for fluid entering the reaction chamber includes a tubular shape and the diameter of the tube is relatively small and tapers to become smaller at the inlet to the reaction chamber. This increases the velocity of fluid entering the reaction chamber and promotes more vigorous and homogenous mixing due to the bulk motion of the reagents within the reaction chamber. It is desirable to mix the reagents and sample in a way to promote mixing beyond molecular diffusion, in order to minimize the duration of the test by ensuring that any infectious agent present in the sample rapidly encounters the biosensor.
• the inlet channel may be offset from the central axis of the reaction chamber to promote a clockwise or counterclockwise rotational motion of the reagents around the central axis of the test chamber as the fluids are mixed in order to increase homogeneity of the mixture.
• the inlet channel is also approximately tangent to the interior surface of the reaction chamber for allowing incoming fluid to travel from the inlet channel to the reaction chamber while remaining in contact with the side surface of the reaction chamber, which allows for a minimally turbulent flow and minimal introduction of air bubbles into the mixed fluids. Bubbles are undesirable due to the unpredictable refraction of light they cause as light emitted by the reagents travels through bubbles within the mixed reagents or on the surface of the mixed reagents.
• the axis of the inlet channel may be angled above horizontal (e.g., about 30 degrees) to provide a partially downward direction to the incoming fluid flow to ensure that the reagent is mixed with the fluid residing at the bottom of the reaction chamber.
• the reagents may be introduced to the test chamber using alternative fluid delivery means such as a vertical channel to deliver the reagents to the bottom of the reaction chamber, or delivering the fluid directly on the central axis of the test chamber in order to create a column of reagent flowing into the test chamber thereby promoting mixing through entrainment.
• the shape (i.e., predetermined geometry) of the reaction chamber may be a revolved section facilitating clockwise or counterclockwise motion of the mixing fluids around the central axis of the reaction chamber.
• a reaction chamber shape other than a revolved section such as a rectangular or irregular shape may be utilized.
• the revolved section used to form the reaction chamber is a portion of an ellipse for facilitating the collection of stray light emitted by the reagents and reflecting this light toward the surface of the detector, which may be a photomultiplier tube (PMT) (Hamamatsu).
• the surface of the reaction chamber may be reflective, in order to enhance the light collection properties of the elliptical shape.
• the maximum diameter of the surface of the PMT is limited to achieve a maximum signal to noise ratio of the output of the system.
• the diameter of the reaction chamber may be designed to approximately match the diameter of the PMT, which influences the elliptical shape that can be achieved in a reaction chamber designed to hold a specific volume of fluids. Due to the constrained elliptical shape, the reaction chamber surface color may be a partially diffusing white due to the additional light collection that occurs when light that would not otherwise be reflected directly to the PMT surface is partially diffused by the white surface and a fraction of this is directed toward the PMT surface. Alternatively, other surface finishes and materials such as a near-mirror finish aluminum, or a transparent material could be used if desired.
• the reaction chamber material prefferably be minimally phosphorescent, in order to prevent light emitted from the reaction chamber itself from eclipsing any emitted light from the reagents and preventing detection.
• white polymeric materials such as acrylonitrile butadiene styrene or other such polymeric materials have been found to exhibit a low level of phosphorescence, the additional light collection provided by the combination of light reflection and diffusion has been found to be a benefit to the signal to noise ratio of the light sensing circuit output.
• the testing subunit provides a system for use in sample analysis.
• the system includes a biosensor reagent, wherein the biosensor reagent includes living biological cells; a reservoir card, having a long loop portion and a short loop portion, wherein the reservoir card stores the biosensor reagent; and a test cartridge base, wherein the test cartridge base is configured to accept the reservoir card.
• the test cartridge base further includes: (i) a reaction chamber having a central axis, wherein the reaction chamber has the shape of a revolved half ellipse; and (ii) an inlet channel connected to the reaction chamber, wherein the inlet channel is positioned above the reaction chamber at an angle of 15-60 degrees above the horizontal, wherein the inlet channel is offset from the central axis of the reaction chamber, and wherein upon introducing a sample to be analyzed into the test cartridge base through the inlet channel, the sample is homogeneously mixed with the biosensor reagent while minimizing damage to the living biological cells.
• the testing subunit provides a system for rapidly detecting the presence of an analyte in a biological sample.
• This system includes a biosensor reagent including at least one antibody specific for a predetermined analyte and a bioluminescent agent, wherein the at least one antibody is expressed on the surface of living, engineered lymphocytes and wherein the bioluminescent agent is expressed by the living, engineered lymphocytes, the biosensor reagent being operative to: (i) detect the presence of a specific analyte in a sample to be tested, and (ii) emit a detectable light signal when the biosensor reagent reacts with the sample and detects the presence of the specific analyte in the sample.
• test cartridge further includes: (i) a reservoir card, wherein the reservoir card further includes the biosensor reagent; and (ii) a test cartridge base, wherein the test cartridge base is configured to accept the reservoir card.
• the test cartridge base further includes: a) a reaction chamber having a central axis, wherein the reaction chamber has the shape of a revolved half ellipse; b) an inlet channel connected to the reaction chamber, wherein the inlet channel is positioned above the reaction chamber at an angle of 15-60 degrees above the horizontal, and wherein the inlet channel is offset from the central axis of the reaction chamber; and c) wherein upon introducing the sample into the test cartridge base through the inlet channel, the sample is homogeneously mixed with the biosensor reagent while minimizing damage to the living, engineered lymphocytes and minimizing any bubbling of the mixed biosensor reagent and sample in the reaction chamber.
• a testing unit adapted to receive the test cartridge is also included.
• the testing unit including a sensor for detecting the detectable light signal emitted by the biosensor reagent upon reacting with the sample, the detection of the emitted detectable light signal being indicative of the presence of the analyte in the sample and, wherein detection of the specific analyte in the sample occurs in real time.
• the soluble antibody and sample to be tested are mixed together immediately prior introduction of the biosensor cells to the test sample.
• charged biosensor cells are centrifuged and concentrated to about 400,000 cells/180 ⁇ ⁇ (adequate for a single reaction) in the charging medium.
• a 180 ⁇ ⁇ (about 400,000 cells) aliquot of the charged biosensor cells is then loaded into the long loop portion of the reservoir card.
• 30 ⁇ ⁇ of anti-CD3s antibody in RPMI media is loaded into the short loop portion of the reservoir card. The reservoir card is then locked into the test cartridge base.
• the test cartridge base is inserted into the testing device and the charged biosensor cells are injected into the reaction chamber to initiate the reaction.
• the resulting signal is recorded for 4 to 8 minutes and at the end of the test period, the 30 ⁇ ⁇ of anti-CD3s antibody is injected from the short loop of the reservoir into reaction chamber as a positive control reaction that is recorded for 2 minutes.
• 30 ⁇ ⁇ of 0.61 mM Digitonin can be used rather than anti-CD3s antibody.
• a negative control test can be performed using a predetermined pathogen that is not specific for the antibody being used.
• the biosensor cells are coated with the soluble antibody for a period of time prior to mixing the sample to be tested with the biosensor cells.
• charged biosensor cells are centrifuged and concentrated to about 400,000 cells/180 ⁇ ⁇ (adequate for one reaction) in the charging medium.
• a 180 ⁇ ⁇ (about 400,000 cells) aliquot of the biosensor cells is then mixed with a 2 ⁇ . volume of an antibody (at 0.5 mg/mL) against the target pathogen, such as anti-E. coli Ol l l (wherein the target pathogen is E. coli Ol l l), in an Eppendorf tube.
• the biosensor cells mixed with the antibody are incubated at room temperature for 10 minutes and then loaded into the long loop portion of the reservoir card.
• 30 ⁇ ⁇ of anti-CD3s antibody in RPMI media is loaded into the short loop portion of the reservoir card.
• the reservoir card is then locked into the test cartridge base.
• a 30 ⁇ ⁇ volume of the sample to be tested is added into the reaction chamber.
• the test cartridge base is inserted into the testing device and the biosensor cells are injected into the mixing chamber to initiate the reaction.
• the resulting signal is recorded for 4 to 8 minutes and at the end of the test period, the 30 ⁇ ⁇ of anti-CD3s antibody is injected from the short loop of the reservoir into reaction chamber as a positive control reaction that is recorded for 2 minutes.
• 30 ⁇ ⁇ of 0.61 mM Digitonin can be used rather than anti-CD3s antibody.
• a negative control test can be performed using a predetermined pathogen that is not specific for the antibody being used.
• the analyte e.g., pathogenic bacteria
• the analyte is coated with the soluble antibody for a period of time prior to mixing the sample to be tested with the biosensor.
• charged biosensor cells are centrifuged and concentrated to about 400,000 cells/180 ⁇ ⁇ (adequate for one reaction) in the charging medium.
• a 180 ⁇ ⁇ (about 400,000 cells) aliquot of the biosensor cells is loaded into the long loop portion of the reservoir card.
• 30 ⁇ ⁇ of anti-CD3s antibody in RPMI media is loaded into the short loop portion of the reservoir card. The reservoir card is then locked into the cartridge base.
• the sample is incubated at room temperature for 10 minutes then added into the cartridge mixing chamber.
• the cartridge is inserted into the PMT and the biosensor cells are injected into the mixing chamber to initiate the reaction.
• the resulting signal is recorded for 4 to 8 minutes and at the end of the test period, the 30 ⁇ ⁇ of anti-CD3s antibody is injected from the short loop of the reservoir into reaction chamber as a positive control reaction that is recorded for 2 minutes.
• the exemplary bioassays described herein may include other additives that reduce background noise and enhance signal.
• anti-CD3s antibody as a positive control, the system has been demonstrated to detect fewer than 10 charged biosensor cells in a mixture of 50,000 uncharged biosensor cells.
• the biosensor itself has been demonstrated to detect 230 CFU of bacteria in a sample of 30 ⁇ .
• a proprietary monoclonal antibody (1F11) against E. coli Ol l l bacteria was used to detect E.
• E. coli 0157 was demonstrated to give negative results, thereby proving the specificity of the system.
• Numerous commercially available antibodies may also be used with the described bioassay. With regard to the proprietary monoclonal antibody (1F11), antibody analysis and selection of the monoclonal antibody was accomplished as described below.
• Antibody production was determined by an ELISA performed in 96-well multiwell plates. Each well was coated with different LPS (E. coli 0157, E. coli 0127, E. coli Ol l l, E. coli 026, Klebsiella pnuemoniae, Salmonella enterioa and naive sera) or bacteria cells (E. coli 0157, E. coli Ol l l, E. coli 26 and E. coli DH5a). Hybridoma supernatant from different clones of mAb 0157 or mAb Ol l l were added to the wells.
• LPS E. coli 0157, E. coli 0127, E. coli Ol l l, E. coli 026, Klebsiella pnuemoniae, Salmonella enterioa and naive sera
• bacteria cells E. coli 0157, E. coli Ol l l, E. coli 26 and E. coli DH5a.
• Horseradish peroxidase-conjugated (HRP) goat anti-mouse IgG was used for detection (Appendix III.A.3).
• the two hybridoma clones (IB 10 and 6G1) of E. coli 0157 exclusively recognize LPS of E. coli 0157 and E. coli 0157.
• the nine hybridoma clones of E. coli Ol l l specifically recognize LPS of E. coli Ol l l and E. coli Ol l l.
• the clones from the highest optical density (OD) reading were chosen for validation, which was accomplished as described below.
• the hybridoma cell pellets were collected and stored at -80°C before RNA extraction.
• the extracted RNA was used as a template for reverse transcription to cDNA, followed by nested PCR amplification. All positive PCR products were cloned into TA cloning vectors and sent for sequencing. The variable regions of the light chain and the heavy chain were determined after analysis of sequences.
• Four single chain antibodies (scFv) of 0157 (IB 10) customized mAb produced by FSC) and ATCC HB 10452, as well as two single chain antibodies of Ol l l produced by FSC (1F11 and 1F2) were recombinantly expressed and purified by Immobilized metal ion affinity chromatography (IMAC).
• IMAC Immobilized metal ion affinity chromatography
• sequence of scFv was constructed as the following order: pel B secretion signal + amino acid Alanine + Histidine tag + amino acids Glycine-Serine-Serine-Glycine + TEV cleavage site + amino acids Glycine-Serine- Serine-Glycine + heavy chain variable region + Linker region Serine-Alanine-Aspartic Acid- Aspartic Acid-Alanine-lysine-lysine-Aspartic Acid- Alanine- Alanine- Lysine-Lysine- Aspartic Acid- Aspardc Acid -Alanine-Lysine-Lysine-Aspartic Acid- Aspartic Acid + light chain variable region.
• the purified scFvs were tested using multi-well plates coated with LPS of 0157 or 0111.
• one polyclonal antibody (against a select bacteria) was immobilized onto a CM 5 sensor chip. The select bacteria was then bound followed by injection of a monoclonal antibody against the same bacteria in a continuous buffer flow. The interaction was monitored in real time. The relative binding of the antibody to each bacterium was recorded in resonance units (RUs). Results of the BIAcore analysis of binding an E. coli 0157 specific antibody (mAb FF754) to E. coli 0157 and E. coli 0111 indicated that 0157 mAb was specific for its target antigen.
• the receptors utilized by the present invention can include alternative Fc -bearing chimeric receptors.
• the chimeric receptors described herein comprise an extracellular domain with binding affinity and specificity for the Fc portion of an immunoglobulin ("Fc binder"), a transmembrane domain, at least one co- stimulatory signaling domain, and a cytoplasmic signaling domain comprising an ITAM.
• the chimeric receptors are configured such that, when expressed on a host cell, the extracellular ligand-binding domain is located extracellularly for binding to a target molecule (e.g., an antibody or a Fc-fusion protein) and the co-stimulatory signaling domain and the ITAM- containing cytoplasmic signaling domain are located in the cytoplasm for triggering activation and/or effector signaling.
• a chimeric receptor construct as described herein comprises, from N-terminus to C-terminus, the Fc binder, the transmembrane domain, the at least one co- stimulatory signaling domain, and the ITAM-containing cytoplasmic signaling domain.
• a chimeric receptor construct as described herein comprises, from N-terminus to C-terminus, the Fc binder, the transmembrane domain, the ITAM-containing cytoplasmic signaling domains, and the at least one co-stimulatory signaling domain.
• any of the chimeric receptors described herein can further comprise a hinge domain, which can be located at the C-terminus of the Fc binder and the N-terminus of the transmembrane domain.
• the chimeric receptor constructs described herein can contain two or more co-stimulatory signaling domains, which can link to each other or be separated by the ITAM-containing cytoplasmic signaling domain.
• the extracellular Fc binder, transmembrane domain, co-stimulatory signaling domain(s), and ITAM-containing cytoplasmic signaling domain in a chimeric receptor construct can be linked to each other directly, or via a peptide linker.
• the chimeric receptor constructs described herein comprise an extracellular domain that is an Fc binder, i.e., capable of binding to the Fc portion of an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, or monkey).
• Suitable Fc binders can be derived from naturally occurring proteins such as mammalian Fc receptors or certain bacterial proteins (e.g., protein A, protein G).
• Fc binders can be synthetic polypeptides engineered specifically to bind the Fc portion of any of the Ig molecules described herein with high affinity and specificity.
• an Fc binder can be an antibody or an antigen-binding fragment thereof that specifically binds the Fc portion of an immunoglobulin.
• examples include, but are not limited to, a single-chain variable fragment (scFv), a domain antibody, or a nanobody.
• an Fc binder can be a synthetic peptide that specifically binds the Fc portion, such as a Kunitz domain, a small modular immunopharmaceutical (SMIP), an adnectin, an avimer, an affibody, a DARPin, or an anticalin, which can be identified by screening a peptide combinatory library for binding activities to Fc.
• SMIP small modular immunopharmaceutical
• the Fc binder is an extracellular ligand-binding domain of a mammalian Fc receptor.
• an "Fc receptor” is a cell surface bound receptor that is expressed on the surface of many immune cells (including B cells, dendritic cells, natural killer (NK) cells, macrophage, neutorphils, mast cells, and eosinophils) and exhibits binding specificity to the Fc domain of an antibody.
• Fc receptors are typically comprised of at least 2 immunoglobulin (Ig)-like domains with binding specificity to an Fc (fragment crystallizable) portion of an antibody.
• binding of an Fc receptor to an Fc portion of the antibody can trigger antibody dependent cell-mediated cytotoxicity (ADCC) effects.
• the Fc receptor used for constructing a chimeric receptor as described herein can be a naturally- occurring polymorphism variant (e.g., the CD16 V158 variant), which can have increased or decreased affinity to Fc as compared to a wild-type counterpart.
• the Fc receptor can be a functional variant of a wild-type counterpart, which carry one or more mutations (e.g., up to 10 amino acid residue substitutions) that alter the binding affinity to the Fc portion of an Ig molecule.
• the mutation can alter the glycosylation pattern of the Fc receptor and thus the binding affinity to Fc.
• Fc receptors are classified based on the isotype of the antibody to which it is able to bind.
• Fc-gamma receptors FcyR
• Fc-alpha receptors FcaR
• Fc-epsilon receptors FccR
• the Fc receptor is an Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor.
• Fc-gamma receptors examples include, without limitation, CD64A, CD64B, CD64C, CD32A, CD32B, CD 16 A, and CD16B.
• An example of an Fc-alpha receptor is FcaRl/CD89.
• Fc-epsilon receptors include, without limitation, FcsRI and Fc.epsilon.RII/CD23. The table below lists exemplary Fc receptors for use in constructing the chimeric receptors described herein and their binding activity to corresponding Fc domains:
• (a) is the extracellular ligand- binding domain of CD 16 incorporating a naturally occurring polymorphism that can modulate affinity for Fc.
• (a) is the extracellular ligand-binding domain of CD 16 incorporating a polymorphism at position 158 (e.g., valine or phenylalanine).
• (a) is produced under conditions that alter its glycosylation state and its affinity for Fc.
• (a) is the extracellular ligand-binding domain of CD 16 incorporating modifications that render the chimeric receptor incorporating it specific for a subset of IgG antibodies. For example, mutations that increase or decrease the affinity for an IgG subtype (e.g., IgGl) can be incorporated.
• IgG subtype e.g., IgGl
• the Fc binder is derived from a naturally occurring bacterial protein that is capable of binding to the Fc portion of an IgG molecule.
• a Fc binder for use in constructing a chimeric receptor as described herein can be a full-length protein or a functional fragment thereof.
• Protein A is a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It is composed of five domains that each fold into a three-helix bundle and are able to bind IgG through interactions with the Fc region of most antibodies as well as the Fab region of human VH3 family antibodies.
• Protein G is an approximately 60-kDa protein expressed in group C and G Streptococcal bacteria that binds to both the Fab and Fc region of mammalian IgGs. While native protein G also binds albumin, recombinant variants have been engineered that eliminate albumin binding.
• Fc binders for use in chimeric receptors can also be created de novo using combinatorial biology or directed evolution methods.
• a protein scaffold e.g., an scFv derived from IgG, a Kunitz domain derived from a Kunitz-type protease inhibitor, an ankyrin repeat, the Z domain from protein A, a lipocalin, a fibronectin type III domain, an SH3 domain from Fyn, or others
• amino acid side chains for a set of residues on the surface can be randomly substituted in order to create a large library of variant scaffolds.
• any of the Fc binders described herein can have a suitable binding affinity for the
• binding affinity refers to the apparent association constant or KA.
• the KA is the reciprocal of the dissociation constant, K D .
• the extracellular ligand-binding domain of an Fc receptor domain of the chimeric receptors described herein can have a binding affinity K D of at least 10 "5 , 10 "6 , 10 "7 , 10 "8 , 10 "9 , 10 "10M or lower for the Fc portion of antibody.
• the Fc binder has a high binding affinity for antibody, isotype of antibodies, or subtype(s) thereof, as compared to the binding affinity of the Fc binder to another antibody, isotype of antibodies or subtypes thereof.
• the extracellular ligand-binding domain of an Fc receptor has specificity for an antibody, isotype of antibodies, or subtype(s) thereof, as compared to binding of the extracellular ligand-binding domain of an Fc receptor to another antibody, isotype of antibodies, or subtypes thereof.
• Fc- gamma receptors with high affinity binding include CD64A, CD64B, and CD64C.
• Fc-gamma receptors with low affinity binding include CD32A, CD32B, CD 16 A, and CD16B.
• the binding affinity or binding specificity for an Fc receptor or a chimeric receptor comprising an Fc binder can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy.
• the extracellular ligand-binding domain of an Fc receptor comprises an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99%) identical to the amino acid sequence of the extracellular ligand-binding domain of a naturally- occurring Fc-gamma receptor, an Fc-alpha receptor, or an Fc-epsilon receptor.
• the "percent identity" of two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci.
• Still another embodiment of this invention provides a system that includes a space or compartment for contacting an engineered cell with an analyte; an engineered cell that comprises a ligand, a signal transduction pathway, and a reporter; wherein the universal detector element binds to a predetermined analyte, the signal-transduction pathway receives a first signal induced by binding of the analyte to the ligand, transmits the first signal to the reporter, and the reporter emits a second detectable signal upon receipt of the first signal from the signal transduction pathway; and a detector.
• Still another embodiment of this invention provides a system that includes a space or compartment for contacting an engineered cell with a pre-determined analyte; an engineered cell that comprises an aggregation of ligands and signal transduction elements that constitutively transmit a signal to a detector that emits light or another detectable signal; and a detector; wherein binding of the pre-determined analyte to the aggregation of ligands and signal transduction elements attenuates signal transduction and attenuates the emission of light or other detectable signal by the reporter.
• the aggregation of ligands and signal transduction elements is maintained by a cohesive adaptor and when the cohesive adaptor is bound by the predetermined analyte, its ability to maintain the aggregation of ligands and signal transduction elements is attenuated.
• Still another embodiment of this invention provides a system that includes a space or compartment for contacting an engineered cell with a pre-determined analyte; an engineered cell that comprises a ligand, a signal transduction element that transmits an inhibitory signal when bound to the pre-determined analyte, and a reporter that constitutively emits light or another detectable signal; a detector; wherein binding of the pre-determined analyte to the universal detector elementinduces an inhibitory signal that attenuates the emission of light or other detectable signal by the reporter.
• the universal detector element can include an immunoreceptor tyrosine-based inhibition motif (ITIM). ITIMs are further described and incorporated by reference to Staub E, Rosenthal A, Hinzmann B (2004). "Systematic identification of immunoreceptor tyrosine-based inhibitory motifs in the human proteome". Cell Signal 16 (4): 435-456.

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