Universal Detection System

Abstract

Provided herein are systems and methods for the detection and analysis of biomolecules. Specifically, provided herein are sensor systems that use enzymes containing an allosteric site that interacts with an inhibitor to determine the presence, absence, or amount of one or more analytes of interest in a sample. In some embodiments, the allosteric site comprises a grafted epitope corresponding to one or more analytes of interest.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 385,147, filed November 28, 2022, the entire disclosure of which is incorporated herein by reference. Array List The text of the computer readable sequence listing filed herewith, entitled "40764-209_SEQUENCE_LISTING", created on November 28, 2023, and having a file size of 197,680 bytes, is hereby incorporated by reference in its entirety. Provided herein are systems and methods for the detection and analysis of biomolecules. Specifically, provided herein are sensor systems that use enzymes containing an allosteric site that interacts with an inhibitor to determine the presence, absence, or amount of one or more analytes of interest in a sample. In some embodiments, the allosteric site comprises a grafted epitope corresponding to one or more analytes of interest. [Background technology]

[0002] Molecular diagnostics, a collection of technologies used to analyze biological markers, has emerged as an important component of medical and healthcare testing. In medicine, technologies are used to diagnose and monitor disease, detect risk, monitor health conditions, and determine which treatments will best benefit a patient. Traditionally, biological samples are collected from a subject and sent to a laboratory for analysis using one or more molecular diagnostic tests. Many tests are time-consuming and utilize complex, expensive equipment available in limited locations. To obtain relevant information more quickly, development of technologies that can be used at the point of care or directly by patients outside of medical facilities has begun. For example, technologies have been developed that allow diabetic patients to self-monitor their blood glucose levels anytime, anywhere. Unfortunately, such technologies are not available for the majority of the many biomarkers that provide important information about a patient's health and well-being. A new technology platform is needed that provides rapid results, offers user convenience, and offers the ability to analyze a variety of different biomarkers, including large, complex molecules such as proteins.

[0003] One previously described approach involves an enzyme that is engineered to bind to an analyte at an allosteric site, whereby the enzyme's activity decreases or increases upon binding of the analyte to the allosteric site (see WO2021 / 067608, incorporated herein by reference in its entirety). However, this approach involves redesigning the enzyme for each different analyte to be detected, often involving a molecular evolution approach. Thus, new approaches are needed. Summary of the Invention

[0004] Provided herein are compositions and methods related to universal sensing systems. The present disclosure provides enzymes suitable for use in the detection systems described herein. As described in more detail herein, the enzymes comprise an engineered allosteric site that includes a grafted epitope corresponding to an analyte of interest. The epitope is selected or designed to bind to an inhibitor that can also bind to the analyte. For example, the epitope can have an amino acid sequence that corresponds to the amino acid sequence of the inhibitor-binding site of the analyte. Upon contact with an inhibitor that can bind to the analyte, the enzymatic activity of the enzyme can be inhibited as the inhibitor, which also binds to the grafted epitope, can be inhibited. The provided enzymes are useful for detecting an analyte (e.g., its concentration) in a sample, such as a biological sample. In the absence of the analyte (or in the presence of only low concentrations of the analyte), the enzymatic activity of the enzyme is typically inhibited because an inhibitor can bind to the enzyme's epitope and thus inhibit the enzyme's activity. In the presence of the analyte (or in the presence of increased concentrations of the analyte), the enzymatic activity of the enzyme is typically restored (referred to herein as "removal of inhibition") because the inhibitor at least partially binds to the analyte. Thus, the enzymes provided herein are suitable for use in detection systems for detecting an analyte of interest.

[0005] The enzymes provided herein, and systems / uses comprising them, offer significant advantages over previously discussed approaches. Specifically, the enzymes are easily configured for use in the detection of a variety of analytes. Epitopes corresponding to a given analyte can be readily identified and grafted into allosteric sites of the enzymes described herein. Thus, the approaches described herein provide a universal detection paradigm for a variety of analytes, including macromolecular analytes such as proteins and peptides. Once an inhibitor that binds to the analyte at a graftable epitope is identified, the epitope can be grafted into the enzymes described herein, and the resulting epitope-grafted enzyme can be used to detect the analyte, for example, in the sensor systems described herein.

[0006] Enzyme-based detection systems can be applied to detect any analyte of interest in any sample type. Specifically, the detection system uses an enzyme to modify a substrate. The modification of the substrate results in a detectable event. The detectable event is detected by a sensor, processed, and reported to a user. The enzyme includes an epitope-grafted sequence. The epitope-grafted sequence corresponds to an epitope of the analyte of interest (e.g., having an amino acid sequence and / or structure that corresponds to the epitope of the analyte, although as discussed below, "corresponding" does not necessarily mean 100% identical). The detection system further uses an inhibitor that competitively binds to the analyte of interest and the epitope-grafted sequence. When the inhibitor binds to the enzyme's epitope-grafted sequence, the enzyme is inhibited, reducing or preventing the enzyme from processing the substrate and resulting in a change in the detectable signal (e.g., resulting in a low or undetectable signal). When the inhibitor does not bind to the enzyme's epitope-grafted sequence, the enzyme processes the substrate, resulting in a change in the detectable signal (e.g., producing a detectable signal or increasing the amount of a detectable signal). In the presence of the analyte near the sensor, the inhibitor binds to the analyte, deinhibiting the enzyme to the extent that the inhibitor transfers from the inhibited enzyme to the analyte, resulting in a detectable event based on a change in signal. In the absence of the analyte near the sensor, the inhibitor is more likely to bind via the enzyme's epitope-grafted sequence, maintaining the enzyme in an inhibited state, and the sensor signal will be reduced or undetectable compared to established background levels.

[0007] By utilizing different epitope sequences, sensing systems can be readily designed to detect any analyte or combination of analytes of interest. When combinations of analytes are to be detected together, two or more epitope sequences may be inserted into a single enzyme at the same or different allosteric positions, and multiple different enzymes, each with its own different epitope-grafted sequence, are used. As demonstrated in the experimental section below, many diverse analytes have been detected, including macromolecular protein analytes. The universal sensor system described herein has advantages over enzyme switch technologies, such as those described in WO2021 / 067608, which is incorporated herein by reference in its entirety. With the enzyme switch approach, the enzyme must be redesigned for each different analyte to be detected, which often involves a molecular evolution approach. The universal sensor system described herein requires far fewer manipulations to replace one epitope-grafted sequence with another.

[0008] In some embodiments, provided herein is an enzyme comprising an epitope-grafted allosteric site that is inhibited by contact with an inhibitor and deinhibited in the presence of an analyte that binds to the inhibitor. In some embodiments, the enzyme is a glucose metabolic enzyme. In some embodiments, the glucose metabolic enzyme is an FAD-dependent glucose dehydrogenase (FAD-GDH) enzyme. In some embodiments, the FAD-GDH enzyme is a fungal FAD-GDH (an FAD-GDH enzyme derived from a fungal organism). In some embodiments, the FAD-GDH enzyme is a Mucor FAD-GDH. In some embodiments, the enzyme is an FAD-GDH derived from an organism selected from the group consisting of Mucor hiemalis, Mucor circinelloides, Mucor ambiguus, Mucor lusitanicus, Mucor guilliermondii, Mucor subtilissimus, and Mucor prainii. In some embodiments, the FAD-GDH enzyme is an Aspergillus genus FAD-GDH (e.g., Aspergillus flavus). In some embodiments, the enzyme is a wild-type enzyme except for epitope grafting. In some embodiments, in addition to epitope grafting, the enzyme comprises a synthetic sequence variation. In some embodiments, the synthetic sequence variation comprises a sequence variation that increases enzyme stability compared to the non-variant enzyme. In some embodiments, the enzyme comprises a sequence selected from the group consisting of SEQ ID NOs: 1-64, 65-72, 75-127, and 132-134, or a sequence at least 70% identical thereto. In some embodiments, the enzyme is a FAD-GDH enzyme, and the allosteric site is located in a surface region corresponding to residue ranges 45-70, 335-362, and 439-457 of SEQ ID NO: 1. In some embodiments, the epitope-grafted sequence comprises an epitope sequence corresponding to an analyte. In some embodiments, the analyte is a protein.In some embodiments, the analyte is a peptide. In some embodiments, the analyte is selected from the group consisting of a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker. In some embodiments, the epitope graft sequence comprises 3 to 30 amino acids.

[0009] In some embodiments, provided herein are compositions comprising an enzyme comprising an epitope-grafted allosteric site that is inhibited by contact with an inhibitor and deinhibited in the presence of an analyte that binds to the inhibitor. In some embodiments, the enzyme is a glucose metabolic enzyme. In some embodiments, the glucose metabolic enzyme is an FAD-dependent glucose dehydrogenase (FAD-GDH) enzyme. In some embodiments, the FAD-GDH enzyme is a fungal FAD-GDH. In some embodiments, the FAD-GDH enzyme is a Mucor FAD-GDH. In some embodiments, the enzyme is a FAD-GDH from an organism selected from the group consisting of Mucor hiemalis, Mucor circinelloides, Mucor ambigus, Mucor lusitanicus, Mucor guilliermondii, Mucor subtilisimus, and Mucor prainii. In some embodiments, the FAD-GDH enzyme is an Aspergillus FAD-GDH (e.g., Aspergillus flavus). In some embodiments, the enzyme is a wild-type enzyme except for the epitope grafting. In some embodiments, in addition to the epitope grafting, the enzyme comprises a synthetic sequence variation. In some embodiments, the synthetic sequence variation comprises a sequence variation that increases enzyme stability compared to the non-variant enzyme. In some embodiments, the enzyme comprises a sequence selected from the group consisting of SEQ ID NOs: 1-64, 65-72, 75-127, and 132-134, or a sequence at least 70% identical thereto. In some embodiments, the enzyme is a FAD-GDH enzyme, and the allosteric site is located in a surface region corresponding to residue ranges 45-70, 335-362, and 439-457 of SEQ ID NO: 1. In some embodiments, the epitope grafted sequence comprises an epitope sequence corresponding to an analyte. In some embodiments, the analyte is a protein. In some embodiments, the analyte is a peptide. In some embodiments, the analyte is selected from the group consisting of a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker. In some embodiments, the epitope grafted sequence comprises between 3 and 30 amino acids.

[0010] In some embodiments, provided herein is a system comprising any of the above enzymes or compositions, an analyte, and an inhibitor that binds to the epitope-grafted sequence. In some embodiments, the inhibitor binds to the analyte with higher affinity than the inhibitor binds to the epitope-grafted sequence. In some embodiments, the inhibitor is an immunoglobulin. In some embodiments, the immunoglobulin is an antibody. In some embodiments, the immunoglobulin is an antibody fragment. In some embodiments, the system further comprises a substrate for the enzyme. In some embodiments, the substrate is glucose. In some embodiments, the system further comprises a sensor. In some embodiments, the sensor is an electrochemical sensor. In some embodiments, the sensor detects a product of the enzyme that reacts with the substrate. In some embodiments, the system further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, intestinal fluid, saliva, and urine.

[0011] In some embodiments, provided herein is a reaction mixture comprising any of the above enzymes or compositions. In some embodiments, the reaction mixture comprises an inhibitor that binds to the analyte and the epitope-grafted sequence. In some embodiments, the inhibitor is an immunoglobulin. In some embodiments, the immunoglobulin is an antibody. In some embodiments, the immunoglobulin is an antibody fragment. In some embodiments, the reaction mixture further comprises a substrate for the enzyme. In some embodiments, the substrate is glucose. In some embodiments, the reaction mixture further comprises a sample. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is selected from the group consisting of blood, serum, plasma, intestinal fluid, saliva, and urine.

[0012] In some embodiments, provided herein are kits comprising any of the above enzymes, compositions, or systems. In some embodiments, the kits comprise an inhibitor that binds to the analyte and the epitope-grafted sequence. In some embodiments, the inhibitor is an immunoglobulin. In some embodiments, the immunoglobulin is an antibody. In some embodiments, the immunoglobulin is an antibody fragment. In some embodiments, the kits comprise a substrate for the enzyme. In some embodiments, the substrate is glucose. In some embodiments, the kits comprise a sensor. In some embodiments, the sensor is an electrochemical sensor. In some embodiments, the kits comprise a control sample containing the analyte. In some embodiments, the kits comprise a control sample lacking the analyte. In some embodiments, provided herein is the use of the enzyme, composition, system, reaction mixture, or kit described above. In some embodiments, provided herein is the use of the enzyme, composition, system, reaction mixture, or kit described above for detecting the presence, absence, or amount of an analyte in a sample.

[0013] In some embodiments, provided herein are methods for detecting an analyte, comprising: a) contacting a sample suspected of containing the analyte with an enzyme as described above; and b) directly or indirectly detecting the activity of the enzyme. In some embodiments, the detecting step comprises electrochemical measurement of a by-product of the enzyme reacting with a substrate. In some embodiments, the present disclosure provides: i) an enzyme containing an engineered allosteric site containing a grafted heterologous epitope, the enzyme having enzymatic activity that is inhibited by the binding of an inhibitor to the grafted epitope; ii) an enzyme whose epitope comprises an amino acid sequence corresponding to an inhibitor binding site of the polypeptide analyte, and the inhibitor is capable of competitively binding to the polypeptide analyte and the grafted epitope; iii) any of the above enzymes, wherein the epitope comprises an amino acid sequence that has at least 70%, at least 80%, or at least 90% sequence identity with the corresponding sequence of the inhibitor binding site being analyzed; iv) Any of the above enzymes, wherein the subject of analysis is a peptide, polypeptide, or protein. v) any of the above enzymes, wherein the epitope is a linear epitope vi) any of the above enzymes, wherein the epitope is a conformational epitope vii) Any of the above enzymes, wherein the analysis target is selected from a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker. viii) any of the above enzymes, wherein the epitope comprises about 3 to about 30 amino acids; ix) Any of the above enzymes, wherein the epitope comprises about 5 to about 15 amino acids, preferably about 8 to about 10 amino acids. x) Any of the above enzymes, wherein the enzyme is a glucose metabolic enzyme. xi) Any of the above enzymes, wherein the enzyme is an FAD-dependent glucose dehydrogenase (FAD-GDH) (e.g., fungal FAD-GDH). xii) Any of the above enzymes, wherein the enzyme is derived from an FAD-GDH of the family Mucoraceae or Aspergillaceae, preferably the enzyme is derived from an FAD-GDH of the genus Mucor or Aspergillus. xiii) Any of the above enzymes, wherein the enzyme is derived from Mucor hiemalis, Mucor circinelloides, Mucor ambygus, Mucor lusitanicus, Mucor guilliermondii, Mucor prainii, Mucor subtilisimus, and Aspergillus flavus. xiv) Any of the above enzymes, wherein the enzyme is derived from FAD-GDH from Mucor hiemalis, Mucor circinelloides, Mucor ambygus, Mucor plenii, and Mucor subtilisimus. xv) any of the above enzymes, wherein the enzyme has at least 70%, at least 80%, or at least 90% identity to: a) SEQ ID NO: 1, 66-72, or 119-127; b) 1, 66-72, 119-127, or 132-134; c) 1, 66-72, 110-115, or 119-127; d) 1, 66-72, 110-112, 114, or 119-127; e) 1, 66-72, 110-112, 114, 119-127, or 132-134; or f) 1, 66-72, 110-115, 119-127, or 132-134, where the sequence identity may be assessed over the entire sequence of the enzyme excluding the epitope. xvi) Any of the above enzymes, wherein the allosteric site is located on the surface of the enzyme. xvii) any of the above enzymes, wherein the allosteric site is located on a surface corresponding to surface 2 of the enzyme of SEQ ID NO: 1, wherein surface 2 comprises residues F341, E344, E348, and K358 of SEQ ID NO: 1, and surface 2 optionally comprises residues T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, and K358 of SEQ ID NO: 1. xviii) any of the above enzymes, wherein an epitope is grafted (i) at a position corresponding to about position 330 to about position 370 of SEQ ID NO: 1; optionally at about position 335 to about position 362 of SEQ ID NO: 1; further, an epitope may be grafted at a position corresponding to positions T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, or K358 of SEQ ID NO: 1; an epitope may be grafted at position 341 or 358 of SEQ ID NO: 1, or (ii) at a position corresponding to about position 320 to about position 335 of SEQ ID NO: 131, optionally at about position 325 to about position 330, and further optionally at about position 327 to about position 329 of SEQ ID NO: 131; or an epitope may be grafted at a position corresponding to position 328 of SEQ ID NO: 131. xix) Any of the above enzymes further comprising one or more amino acid modifications that increase the stability of the enzyme. xx) any of the above enzymes containing Cys at positions corresponding to positions 153 and 192 of SEQ ID NO: 118 xxi) A system comprising any of the above enzymes, and an inhibitor capable of binding to a grafted epitope of said enzyme and an analyte comprising said epitope. xxii) A system in which the affinity of the analyte to the inhibitor is higher than the affinity of the epitope-grafted enzyme to the inhibitor. xxiii) Any of the above systems, wherein the inhibitor is an immunoglobulin, antibody, or antibody fragment. xxiv) Any of the above systems further comprising a substrate for said enzyme. xxv) Any of the above systems, wherein the substrate is glucose. xxvi) Any of the above systems, further comprising an analyte having a binding site for said inhibitor, wherein the amino acid sequence of said binding site corresponds to the amino acid sequence of a grafted epitope of said enzyme. xxvii) Any of the above systems, wherein the analyte is a peptide, polypeptide, or protein, preferably wherein the analyte is selected from a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, or a transplant rejection biomarker. xxviii) Any of the above systems, wherein the analyte is present in a biological sample, and the biological sample may be selected from blood, serum, plasma, intestinal fluid, saliva, and urine. xxix) A sensor comprising any of the above enzymes or systems. xxx) A sensor that is an electrochemical sensor xxxi) Any of the above sensors configured to directly or indirectly detect the turnover rate of a substrate by said enzyme. xxxii) Any of the above sensors configured to detect the product of turnover of a substrate by said enzyme. xxxiii) methods for detecting an analyte, e.g., determining the presence, absence, or concentration of an analyte in a sample, comprising the steps of: a) contacting a sample with any of the above enzymes in the presence of the analyte and an inhibitor capable of binding to a grafted epitope of said enzyme; and b) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme. xxxiv) A method comprising the steps of: a) contacting a sample with any of the above enzymes in the presence of an analyte and an inhibitor capable of binding to a grafted epitope of said enzyme; b) allowing the inhibitor to inhibit the enzyme; c) allowing any analyte present in the sample to bind to the inhibitor, thereby deinhibiting the enzyme; and d) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme, wherein the enzymatic activity of the enzyme may be proportional to the concentration of the analyte in the sample. xxxv) Any of the above methods, wherein the sample is a biological sample selected from blood, serum, plasma, intestinal fluid, saliva, or urine. xxxvi) A method for identifying an allosteric site of an enzyme, which allosteric site can be inhibited by an inhibitor, comprising the steps of: a) generating one or more antibodies and / or antibody mimetics that bind to the enzyme; b) screening the ability of said one or more antibodies and / or antibody mimetics to allosterically inhibit the enzymatic activity of the enzyme, thereby identifying antibodies and / or antibody mimetics that allosterically inhibit the enzymatic activity of the enzyme; and c) identifying amino acids of the enzyme in contact with said antibodies and / or antibody mimetics that allosterically inhibit the enzymatic activity of the enzyme. xxxv) the method as described above, further comprising the step of determining the retention of enzyme activity when the amino acid is modified. xxxvi) Any of the above two methods further comprising the step of grafting an epitope into the amino acid sequence of the enzyme at a position corresponding to the allosteric site, wherein the epitope comprises an amino acid sequence capable of binding to an inhibitor. xxxvii) Any of the above three methods, wherein the enzyme, allosteric site, inhibitor, and / or epitope is as defined above. xxxviii) A method of diagnosing the health of a subject, comprising: a) contacting a biological sample from said subject with any of the sensors described above; and b) determining the presence, absence, or concentration of an analyte associated with the health of the subject in the sample according to any of the methods described above. xxxix) Use of any of the above enzymes, systems, or sensors in any of the above methods to determine the presence, absence, or concentration of an analyte in a sample. to provide.

[0014] Provided herein below are exemplary compositions of matter, methods, devices (e.g., sensors), systems, reaction mixtures, kits, and apparatus for detecting and / or monitoring analytes in a sample. It is understood that the teachings of the present disclosure are not limited to the particular embodiments described, which may, of course, vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. By way of example, and not limitation, a sensor system can be designed to recognize, identify, and / or quantify one or more analytes in a sample. Sensor systems can be utilized in a variety of conditions and configurations, including sensors for measuring the presence or level of an analyte in a subject. The configuration of such sensors can depend on the analyte being measured and the type of sample the system monitors for the analyte. In some embodiments, the sensor is configured for in vivo detection and / or measurement of the analyte in a subject. The analyte can be present in any type of sample. For example, the sensor can test for the analyte in dermal fluid, interstitial fluid, subcutaneous fluid, urine, or blood (e.g., capillary blood). In some embodiments, the sensor is configured for detecting or measuring the analyte using a handheld or benchtop device. In such embodiments, the sample is transferred from its source to the device for measurement.

[0015] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present specification, including definitions, will govern. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure. All articles, patents, patents, and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0016] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or phrases that do not exclude the possibility of additional acts or structures. The singular forms "a," "and," and "the" include plural references unless the context clearly dictates otherwise. This disclosure also contemplates other aspects "comprising," "consisting of," and "consisting essentially of" the aspects or elements presented herein, whether or not expressly stated.

[0017] Recitation of numerical ranges herein expressly contemplates each intervening number to the same degree of precision. For example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated. Ranges include all intermediate ranges, e.g., 6 to 7, 6 to 8, 7 to 9, 8 to 9, and 7 to 8.

[0018] The term "sample" is used in its broadest sense. Samples include biological and environmental samples. Biological samples may be obtained from any source, including animals, plants, and microorganisms, and may encompass liquids, solids, tissues, and gases. Materials obtained from clinical or forensic situations containing the analyte of interest are also within the intended meaning of the term "sample." In some embodiments, the sample is a biological sample from an animal (e.g., a human). Biological samples include, but are not limited to, blood, serum, plasma, intestinal fluid, urine, stool, saliva, tissue, cerebrospinal fluid, semen, vaginal fluid, mucus, lymph, cellular fluid, aqueous humor, bone marrow, bronchoalveolar lavage fluid, oral swab, earwax, gastric juice, gastrointestinal fluid, milk, nasal wash, liposuction, peritoneal fluid, sebum, synovial fluid, tears, sweat, and vitreous humor. Environmental samples include, but are not limited to, water, air, snow, and soil. The sample may be in a processed form, including dry samples (e.g., dried blood spots) and solid samples (e.g., formalin-fixed, paraffin-embedded (FFPE)). In some embodiments, the sample is located in vivo in an animal.

[0019] "Enzyme" refers to a protein or fragment thereof that has activity (alternatively referred to as catalytic activity, enzymatic activity, or enzymatic activity) toward one or more reactants (e.g., enzyme substrates). Examples of one or more reactants (e.g., enzyme substrates) are glucose, lactate, glutamate, ascorbic acid, cholesterol, choline acetylcholine, hypoxanthine, norepinephrine, 5-hydroxytryptamine, phenylethylamine, and e / e-methylhistamine, polyphenols, ethanol, aldehydes, or malate.

[0020] As used herein, the term "epitope" refers to a sequence (e.g., an amino acid sequence) recognized by a binding molecule. The term epitope includes sequences recognized by antibodies, antibody fragments, and antibody mimetics, including aptamers, affimers, and DARPins. Epitopes include structural epitopes and linear epitopes. "Epitope grafting," as used herein, refers to a molecule containing a heterologous epitope sequence. For example, an epitope grafted enzyme is an enzyme that has been modified to include an epitope sequence from a different molecule (e.g., an analyte of interest). In some embodiments, an epitope grafted enzyme comprises an epitope sequence from a different molecule (e.g., an analyte of interest) that is grafted into the enzyme's sequence by insertion, e.g., by adding the epitope to the enzyme's sequence, or by replacing one or more amino acids (e.g., consecutive amino acids) of the enzyme's sequence with the epitope sequence. In some embodiments, the epitope is grafted into the enzyme's sequence by replacing one or more amino acid sequences of the enzyme's sequence with the epitope sequence, where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or at least 30 amino acids (e.g., consecutive amino acids) of the enzyme's sequence are replaced with the epitope sequence. In some embodiments, the number of replaced amino acids in the sequence of the enzyme is the same as the number of amino acids in the sequence of the epitope.

[0021] "Antigen-binding molecule" refers to a molecule that binds to a specific antigen. Examples include, but are not limited to, proteins, nucleic acids, aptamers, affimers, DARPins, synthetic molecules, etc. "Antigen binding protein" refers to a protein that binds to a specific antigen. "Antigen binding proteins" include, but are not limited to, immunoglobulins, including polyclonal, monoclonal, chimeric, single chain, camelid, VHH, and humanized antibodies, Fab fragments, F(ab')2 fragments, and Fab expression libraries. "Specific binding" or "specifically binding," when used in reference to the interaction of a binding molecule with an antigen, means that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the antigen; in other words, the antibody recognizes and binds to a specific structure rather than the antigen in general.

[0022] "Affimer," as used herein, refers to a peptide that specifically or selectively binds to a target (e.g., an analyte, an epitope-grafted sequence). Generally, affimers can be small peptides or proteins, typically having a molecular weight less than 12 kDa. Affimers may have the ability to recognize a specific epitope or antigen and have binding affinities approaching those of antibodies (e.g., in the low nanomolar to picomolar range), but the term "affimer," as used herein, does not encompass antibodies, immunoglobulins, the Fab region of an antibody, or the Fc region of an antibody. Affimers can have the same specificity advantages of antibodies, but can be smaller, chemically synthesized or chemically modified, and have the advantage of being free of cell culture contaminants.

[0023] "Aptamer," as used herein, refers to an oligonucleotide or peptide molecule that binds to a specific target molecule. Aptamers are usually created by selecting them from a large pool of random sequences, although natural aptamers also exist. "DARPin" (designed ankyrin repeat protein), as used herein, refers to genetically engineered antibody-mimetic proteins that typically exhibit high specificity and high affinity target protein binding. They are typically derived from natural ankyrin repeat proteins, which are responsible for diverse functions such as cell signaling, cellular regulation, and structural integrity, and are one of the most common classes of natural binding proteins. DARPins contain at least three repeat motifs or modules, the most N-terminal and most C-terminal modules of which are called "caps" because they hide the hydrophobic core of the protein.

[0024] "Sensor," as used herein, refers to a device or molecule configured to detect the presence and / or measure the level (e.g., presence, absence, or concentration) of one or more (e.g., multiple) analytes in a sample. Sensors can include biological, mechanical, and electrical components. An "electrochemical sensor" is a chemical sensor in which an electrode is used as the transducer element in the presence of an analyte. In some embodiments, a detectable signal is produced by electrochemical oxidation and reduction reactions on the sensor. These reactions are converted into an electrical signal that can be related to the amount, concentration, or level of the analyte or the activity of an enzyme in the sample. "Sensing layer," as used herein, refers to a component of a sensor that includes components that facilitate the electrooxidation or electroreduction of a compound, either directly at an electrode or via one or more electron transfer agents, one or more cofactors, or a combination of one or more electron transfer agents and one or more cofactors. In some sensor embodiments, the sensing layer is disposed near or on the working electrode. "Sensing region," as used herein, refers to the active chemical area of ​​a sensor.

[0025] "Identical," "identity," or "sequence identity," as used herein in the context of two or more polypeptide or polynucleotide sequences, means that the sequences have a specified percentage of residues that are the same over a particular region, as determined using standard algorithms, taking into account the extent to which the sequences are identical over a window of comparison. Typically, identity is assessed over the entire length of the sequence. In some embodiments, where the sequence is that of an epitope-transfer enzyme described herein, the sequence is typically assessed over the entire length of the sequence, excluding the epitope. Identity or sequence identity may be determined using computer algorithms such as the GAP, BESTFIT, FASTA, and BLAST family of programs disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al. (John Wiley & Sons Inc NY, 1995-1999).

[0026] "Subject" or "patient," as used herein, refer interchangeably to any vertebrate, including, but not limited to, mammals (e.g., cows, pigs, camels, llamas, horses, goats, rabbits, sheep, hamsters, guinea pigs, cats, dogs, rats, and mice, non-human primates (e.g., monkeys such as cynomolgus or rhesus monkeys, chimpanzees), and humans). In some embodiments, the subject can be human or non-human. In some embodiments, the subject is human. A subject or patient can receive one or more forms of treatment.

[0027] The terms "variant protein," "protein variant," or "variant," when used interchangeably herein, refer to a protein that differs from that of a parent protein by at least one amino acid modification. The term "protein variant" can refer to the protein itself, a composition comprising the protein, or the amino acid sequence encoding it. In some embodiments, a protein variant has at least one amino acid modification compared to a parent or reference protein, e.g., about 1 to about 100 amino acid modifications (e.g., 2 to 100, 1 to 50, 2 to 40, 5 to 30, 10 to 20, and all ranges therebetween) compared to the parent protein. In some embodiments, a protein variant has about 1 to about 40 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about 1 to about 30 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about 1 to about 20 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about 1 to about 10 amino acid modifications compared to the parent protein. In some embodiments, a protein variant has about 1 to about 5 amino acid modifications compared to the parent protein. In some embodiments, the protein variant sequences herein retain at least about 70%, or at least about 80% identity to the parent or reference protein sequence. In other embodiments, the protein variant sequences herein retain at least about 90% identity. In still other embodiments, the protein variant sequences herein retain at least about 95%, 96%, 97%, 98%, or 99% identity. As one of skill in the art will recognize, any sequence identity provided herein can apply to any protein disclosed herein.

[0028] A variant protein typically retains the properties of the unmodified parent or reference sequence, or in some cases may have improved properties. For example, a variant enzyme typically retains the enzymatic activity of the unmodified sequence. In some cases, the enzymatic activity may be comparable to that of the unmodified sequence. In some cases, the enzymatic activity may be improved. Typically, a variant sequence retains 70%, 80%, or 90% or more of the enzymatic activity of the unmodified sequence.

[0029] The term "amino acid" or "any amino acid," as used herein, refers to any and all amino acids, including naturally occurring amino acids (e.g., α-amino acids), unnatural amino acids, modified amino acids, and non-natural amino acids. It includes both D- and L-amino acids. Natural amino acids include those found in nature, e.g., the 23 amino acids that are incorporated into peptide chains to form the building blocks of a wide range of proteins. These are primarily L-stereoisomers, although a small number of D-amino acids occur in bacterial envelopes and some antibiotics. "Non-standard" natural amino acids include, for example, pyrolysine (found in methanogens and other eukaryotes), selenocysteine ​​(present in many non-eukaryotes and most eukaryotes), and N-formylmethionine (encoded by the start codon AUG in bacteria, mitochondria, and chloroplasts). "Unnatural" or "non-natural" amino acids are non-proteinogenic amino acids (e.g., not naturally encoded or found in the genetic code), either naturally occurring or chemically synthesized. Over 140 unnatural amino acids are known, with thousands of possible combinations. Examples of "unnatural" amino acids include β-amino acids (β3 and β2), homoamino acids, proline and pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, alpha-methyl amino acids, and N-methyl amino acids. Unnatural or non-natural amino acids also include modified amino acids. "Modified" amino acids include amino acids (e.g., natural amino acids) that have been chemically modified to include a group, groups, or chemical moiety not naturally occurring in amino acids.

[0030] For the most part, the names of naturally occurring and non-naturally occurring aminoacyl residues used herein follow the naming conventions set forth by the IUPAC Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB Commission on Biochemical Nomenclature, as set forth in "Nomenclature of α-Amino Acids (Recommendations, 1974)" Biochemistry, 14(2), (1975). In this disclosure, amino acids are referred to. In addition to the names of amino acids, three-letter and one-letter codes are also used herein. For clarity, the amino acids referred to in this disclosure are referred to as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Qln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0031] Throughout this specification, unless naturally occurring amino acids are referred to by their full names (e.g., alanine, arginine, etc.), they are represented by their conventional three-letter or one-letter abbreviations (e.g., Ala or A for alanine, Arg or R for arginine, etc.). The term "L-amino acid," as used herein, refers to the "L" isomeric form of a peptide; conversely, the term "D-amino acid" refers to the "D" isomeric form of a peptide (e.g., the D isomeric form of phenylalanine is DPhe, (D)Phe, D-Phe, or DF). The D isomeric form of an amino acid residue can be substituted for any L-amino acid residue so long as the desired function is retained by the peptide.

[0032] In the case of rare or unnatural amino acids, unless referred to by their full name (e.g., sarcosine, ornithine, etc.), frequently used three- or four-letter codes for those residues are used, including Sar or Sarc (sarcosine, i.e., N-methylglycine), Aib (α-aminoisobutylamine), Dab (2,4-diaminobutanoic acid), Dapa (2,3-diaminopropanoic acid), γ-Glu (γ-glutamic acid), GABA (γ-aminobutanoic acid), β-Pro (pyrrolidine-3-carboxylic acid), and 8Ado (8-amino-3,6-dioxaoctanoic acid), Abu (2-aminobutyric acid), βhPro (β-homoproline), βhPhe (β-homophenylalanine) and Bip (β,β-diphenylalanine), and Ida (iminodiacetic acid).

[0033] An amino acid "replacement" or "substitution" refers to the substitution of one amino acid at a given position or residue for another amino acid at the same position or residue in a polypeptide sequence. Amino acids are broadly grouped as "aromatic" or "aliphatic." Aromatic amino acids contain an aromatic ring. Examples of "aromatic" amino acids include histidine (H or His), phenylalanine (F or Phe), tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acids are broadly grouped as "aliphatic." Examples of "aliphatic" amino acids include glycine (G or Gly), alanine (A or Ala), valine (V or Val), leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine (S or Ser), threonine (T or Thr), cysteine ​​(C or Cys), proline (P or Pro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (N or Asn), glutamine (Q or Gin), lysine (K or Lys), and arginine (R or Arg).

[0034] Amino acid replacements or substitutions can be conservative, semi-conservative, or non-conservative. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the replacement of one amino acid with another amino acid that shares common properties. A functional method for defining common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of the same organism (Schulz and Schirmer, Principles of Protein Structure, Springer-Verlag, New York (1979)). Such analysis can define groups of amino acids in which amino acids within a group preferentially exchange with each other and, therefore, most closely resemble each other in their effect on overall protein structure (Schulz and Schirmer, supra).

[0035] Examples of conservative amino acid substitutions include substitutions of amino acids within the above subgroups, such as arginine for lysine to maintain a positive charge, and vice versa, aspartic acid for glutamic acid to maintain a negative charge, and vice versa, threonine for serine to maintain a free -OH, and asparagine for glutamine to maintain a free -NH. "Semi-conservative substitutions" include substitutions of amino acids within the same group listed above but not within the same subgroup. For example, substitutions of asparagine for aspartic acid or lysine for asparagine include amino acids within the same group but in different subgroups. "Non-conservative mutations" include amino acid substitutions between different groups, such as tryptophan for lysine or serine for phenylalanine. In some embodiments, the variant enzyme lacks one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, etc.) from the N-terminus compared to the corresponding wild-type enzyme. In some such embodiments, a methionine is added to the new N-terminus of the truncated enzyme.

[0036] The term "analyte," as used herein, refers to a substance or chemical component that is of interest in an analytical procedure, e.g., to be identified and / or measured. Analytes include, but are not limited to, proteins, nucleic acids, lipids, carbohydrates, and minerals. Analytes include "biomarkers," which are measurable indicators of some biological state or condition. As used herein, a "macromolecular" analyte refers to an analyte having a molecular weight greater than 1000 daltons. [Brief explanation of the drawings]

[0037] [Figure 1] This figure shows a table of kinetics of sera from mice inoculated with Mucor mutant FAD-GDH, measured as the slope of the linear regression of the trace from 640 to 1280 seconds (-ΔA600 / min measured by the addition of 1:50 or 1:500 diluted sera). The ranking of kinetics of 1:50 or 1:500 is listed from 1 to 27, with high-ranking sera (high inhibition) shown in dark gray and low-ranking sera (low inhibition) shown in light gray. ND, serum not tested in this experiment. [Figure 2] 1 is a graph of the calculated percent inhibition in the FAD-GDH colorimetric screening assay by selected hybridoma supernatants. Negative values ​​for percent inhibition indicate an observed stimulation of GDH activity by these samples. [Figure 3] Figure 3A is a graph of dose-dependent inhibition of the measured reaction rate of GDH activity using various concentrations of mAb 1-286 (x-axis, shown on log scale). Figure 3B is a graph of the remaining absorbance of the reaction after 45 minutes, showing endpoint inhibition by various concentrations of mAb 1-286 (x-axis, shown on log scale). Parameters from the curve fits of Figures 3A and 3B are shown in the respective insets. [Figure 4] Figure 1 shows a graph of the initial reaction rate plotted against the glucose concentration for each of the antibody dilutions shown. Vmax and Km parameters were calculated from the curve fitting and are listed in the inset table. [Figure 5]Lineweaver-Burk plot of the data presented in Figure 4. The intersection of the various lines at a common point on the x-axis left of the origin indicates the allosteric mechanism of inhibition. [Figure 6] Figures 6A-6C show zoomed-in images of the structure of non-glycosylated mucormycosis FAD-GDH in complex with rFab 286. Figure 6A shows the interface formed between rFab 286 (cartoon representation) and FAD-GDH (surface representation) in the X-ray crystal structure. Surfaces 1 (white), 2 (black), and 3 (gray) are indicated by numbers, and the substrate access hole is labeled. Figure 6B is a 45-degree rotation of the view in Figure 6A, visualizing surface 3. Figure 6C is a top-down view of the epitope of rFab 286 on FAD-GDH, with the rFab removed for clarity. Bound FAD is seen deep within the active site (arrow). [Figure 7] Figure 1 shows a graph of percent inhibition of GDH activity for select alanine-scanning mutants of nonglycosylated Mucor FAD-GDH measured in the absence or presence of mAb 1-286 (1 nM). Inhibition was calculated for each replicate, and the mean ± SD is shown. Mutations in surface 1 showed a range of impaired GDH activity and blunted inhibitory responses. Mutations F341A, E344A, and E348A in surface 2 indicate that these residues are associated with functional responses to inhibitory antibodies. The DQETAAAA mutant combines D338A, Q342A, E344A, and T345A; DQTAAA, DQAA, DTAA, and QTAA contain combinations of alanine mutations made at these four positions. [Figure 8]SDS-PAGE analysis of purified recombinant FAD-GDH mutant proteins. Lanes 1, 13, and 23: nonglycosylated, wild-type FAD-GDH (lacking alanine mutations); lane 2, Q48A; lane 3, F49A; lane 4, V50A; lane 5, M56A; lane 6, Y57A; lane 7, Q59A; lane 8, T63A; lane 9, D64A; lane 10, L65A; lane 11, C66A; lane 12, R69A; lane 14, E348A; lane 15, E349A; lane 16, Y354A; lane 17, K358A; lane 18, L65A; lane 19, L69A; lane 20, E348A; lane 21, E349A; lane 22, Y354A; lane 23, K358A; lane 24, L65A; lane 25, L65A; lane 26, L65A; lane 27, L65A; lane 28, L65A; lane 29, L66A; lane 30, L65A; lane 31, L65A; lane 32, L65A; lane 33, L65A; lane 34, L65A; lane 35, L65A; lane 36, L65A; lane 37, L65A; lane 38, L65A; lane 39, L66A; lane 40, L65A; lane 41, L65A; lane 42, L65A; lane 43, L65A; lane 44, Lane 18, Y442A; lane 19, T446A; lane 20, D447A; lane 21, L450A; lane 22, N452A; lane 24, M56A (repeat expression and purification success); lane 25, C66A (repeat success); lane 26, T337A; lane 27, V340A; lane 28, N343A; lane 29, L346A; lane 30, L65A; lane 31, D338A; lane 32, F341A; lane 33, E344A; lane 34, L450A. Purified but not analyzed by SDS-PAGE: V61A. The black arrow indicates the expected position of the approximately 70 kDa FAD-GDH band based on standard proteins run on the same gel. [Figure 9] Graph and curve fitting of GDH activity of wild-type (WT) or each of the three mutant FAD-GDH enzymes as measured by titrating the concentration of inhibitory mAb 1-286 as shown in Table 3. [Figure 10] 1 is a graph of percent inhibition of HA-grafted FAD-GDH enzyme by titration of anti-HA antibody, anti-Myc antibody, or mAb 1-286. [Figure 11] 1 is a graph of percent inhibition by VHH-1, VHH-10, VHH-859, and VHH-898 elevated against ungrafted 19031FAD-GDH by titration of enzyme concentration. [Figure 12] 1 is a graph of percent inhibition of V5 epitope-grafted FAD-GDH enzyme in the presence of anti-V5 monoclonal antibody or mAb 1-286. [Figure 13]1 is a graph of percent inhibition of TnI epitope-grafted FAD-GDH enzyme in the presence of anti-TnI monoclonal antibody or mAb 1-286. [Figure 14] Figure 14A shows plots of the rate of DCPIP reduction by FAD-GDH and the blank-subtracted reaction rate versus assay concentration of FAD-GDH. Data points correspond to final concentrations of 0, 8, 44, 80, and 116 ng / ml. Linear regression of the data points is shown as a dotted line, with the trend line and quality of fit (R2) in bold. Figure 14B shows a graph of the kinetic absorbance of blank or an exemplary single concentration of FAD-GDH assayed in duplicate. [Figure 15] Figure 15A is a graph of reaction rate per minute for serially diluted concentrations of D-glucose. Data points correspond to 6, 12.1, 24.2, 48.5, 97, and 194 mM glucose. Figure 15B is a graph of an estimate of the Km of the FAD-GDH enzyme for glucose. A double reciprocal plot of the data from Figure 15A using the four highest concentrations tested. The x-intercept was calculated from the equation and corresponds to an estimated apparent Km of 64.7 mM. [Figure 16] Figure 16A is a graph of the absorbance of DCPIP reduction reactions containing either PBS or two dilutions of normal mouse serum (NMS). NMS does not inhibit the kinetics of the glucose-driven FAD-GDH reaction. Figure 16B is a linear regression analysis of reactions containing either PBS or two dilutions of normal mouse serum (NMS). NMS does not inhibit the kinetics of the glucose-driven FAD-GDH reaction, as all three traces overlap and have similar kinetics. [Figure 17] Figure 17A is a graph of percent inhibition by the top inhibitory sera. The reaction rate for PBS + enzyme + no glucose (control) was subtracted from the reaction rate with inhibitory sera at each dilution. Percent inhibition was plotted as the difference from the NMS reading at each dilution. Figure 17B is a summary table of percent inhibition by the top inhibitory sera. [Figure 18] 10 is a graph showing the de-inhibition of WT and 358HA epitope-grafted FAD-GDH. [Figure 19] 19A-19F are graphs showing the percent inhibition of Mucor (Mucor prainii, Mucor guilliermondii, Mucor hiemalis, Mucor subtilisimus, Mucor circinelloides, and Mucor ambigus, respectively) epitope grafting by the 1-286 antibody and anti-epitope antibodies. [Figure 20] Figures 20A and 20B are graphs showing the inhibition of FAD-GDH at various VHH doses, and Figure 20C is a graph of the percent inhibition of either glycosylated or non-glycosylated FAD-GDH in the presence of IgG 103 and its fragment Fab 103. [Figure 21] Figure 21A is a graph of the percent inhibition of ungrafted or V5 epitope-grafted FAD-GDH using various α-V5 antibody concentrations, and Figures 21B and 21C are graphs of the percent inhibition of two versions of the V5 epitope-grafted FAD-GDH enzyme in the presence of the V5 peptide. [Figure 22] 1 is a graph of percent inhibition of three TnI epitope-grafted (358TN1, 358TN4, and 358TN8) FAD-GDH enzymes at various α-TnI antibody concentrations. [Figure 23] 1 is a graph of the percent inhibition of FAD-GDH grafted enzymes with various epitopes (V5 / TnI / Flag / HA / Myc) in response to the corresponding anti-epitope antibodies at 100 nM concentration. [Figure 24] Figure 24A is a graph of percent inhibition of purified 358HNL-H3 enzyme at various α-HNL antibody concentrations, and Figure 24B is a graph of deinhibition of the enzyme in the presence of HNL peptide. [Figure 25] 1 is a graph of FAD-GDH inhibition assays using epitope-grafted enzymes 341BP and 358BP and various α-NTproBNP antibodies and the reversal of inhibition by NT-ProBNP antigen. [Figure 26] 1 is a graph of FAD-GDH deinhibition using different concentrations of inhibitor and antigen. [Figure 27]1 is a graph of the stability comparison between non-grafted FAD-GDH, 358HA epitope grafted and 358HACC epitope grafted with an additional disulfide bond. [Figure 28] 1 is a graph of percent inhibition of periplasmic extracts resulting from phage display of non-glycosylated, non-grafted FAD-GDH binding proteins. [Figure 29] Graph of a competitive binding assay of four identified and reformatted anti-FAD-GDH IgG and 1-286 antibody epitopes. [Figure 30] Figure 30A is a schematic diagram showing two formats of a competitive binding assay of inhibitory IgG 10-3 (clone 3) and 1-286 on ungrafted FAD-GDH. Figure 30B is a graph of the results of the assay format shown on the right of Figure 30A, where plates were coated with 10-3 and titrated with 1-286 antibody. Figure 30B is a graph of the results of the assay format shown on the left of Figure 30A, where plates were coated with 1-286 and titrated with 10-3 antibody. [Figure 31] 1 is a graph of the calculated percent inhibition of inhibitory IgG of non-grafted FAD-GDH plotted as a function of concentration for IC50 determination, as determined in the table below. [Figure 32] Samples of purified Aspergillus flavus FAD-GDH with epitopes grafted at position 328 of the TN1, HA, or HNL epitopes were analyzed by SDS-PAGE and Coomassie Brilliant Blue staining. Arrows indicate the migration positions of the purified enzymes. The positions of molecular weight (MW) standards are marked in kilodaltons (kDa) in the four standard lanes (unlabeled). [Figure 33] FIG. 1 shows either wild-type, non-grafted Mucor FAD-GDH19-031 (negative control) or various Aspergillus flavus epitope-grafted constructs engineered at amino acid position 328 tested for inhibition by various commercially available antibodies at the concentrations indicated. [Figure 34]Figure 1 shows the epitope-grafted Aspergillus flavus FAD-GDH enzyme tested for inhibition by various antibodies at the final concentrations shown. HNL 2-6128 is a negative control for all three grafted Aspergillus flavus enzymes because this antibody recognizes a different sequence than the HNL epitope grafted onto the enzyme. [Figure 35] Figure 35A shows ungrafted, wild-type Aspergillus flavus FAD-GDH or epitope-grafted, Aspergillus flavus 328 HA constructs tested for inhibition by anti-HNL (control) or anti-HA ab182009 antibody in a dose-response experiment. Figure 35B shows ungrafted, wild-type Aspergillus flavus FAD-GDH or epitope-grafted, Aspergillus flavus 328 HA constructs tested for inhibition by anti-HNL (control) or anti-HA ab236632 antibody in a dose-response experiment. DETAILED DESCRIPTION OF THE INVENTION

[0038] As discussed above, provided herein are systems, methods, and compositions, including kits, devices, and reaction mixtures, that utilize epitope-grafting enzymes for the analysis of analytes in samples. Exemplary embodiments of the present technology are provided below. It is understood that the teachings of the present disclosure are not limited to these exemplary embodiments.

[0039] I) Enzymes A) Sensor enzyme In some embodiments, the techniques provided herein use one or more enzymes. When exposed to a substrate, the enzyme produces a reaction product. The reaction product is detected, directly or indirectly, to determine the activity of the enzyme. The enzyme is designed or configured such that its enzymatic activity changes in response to the presence, absence, or amount of an analyte in a sample. Thus, by measuring the activity of the enzyme, a determination of the presence, absence, or amount of the analyte in a sample is achieved.

[0040] In some embodiments, the enzyme is any enzyme having an enzymatic activity that is detectably altered in the presence of an analyte of interest. In some embodiments, the enzyme comprises one or more allosteric sites where, when an inhibitor or inhibitors bind, the activity of the enzyme is altered (e.g., decreased). In some embodiments, the allosteric site comprises a heterologous sequence. In some embodiments, the heterologous sequence is epitope-grafted. In some embodiments, the inhibitor or inhibitors specifically bind to the epitope-grafted sequence contained within the enzyme and, upon binding, inhibit the enzyme activity. Thus, in some embodiments, provided herein is an enzyme that comprises a modified allosteric site that comprises a grafted heterologous epitope, and has an enzymatic activity that is inhibited by the binding of an inhibitor to the grafted epitope.

[0041] In some embodiments, the enzyme is a glucose metabolic enzyme (i.e., an enzyme that utilizes glucose as a substrate). For example, in some embodiments, the enzyme is glucose dehydrogenase (GDH) (i.e., an enzyme that catalyzes the oxidation of glucose in the presence of a cofactor such as nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), or pyrroloquinoline quinone (PQQ)) or glucose oxidase (GO) (i.e., an enzyme that catalyzes the oxidation of glucose to hydrogen peroxide). Ferri et al., Diabetes Sci. Technol., Glucose Electrochemistry, 5(5), 1068-76 (2011), the entire contents of which are incorporated herein by reference, provides a summary of exemplary glucose metabolic enzymes suitable for use in sensors. In some embodiments, the enzyme is flavin-adenine-dinucleotide-dependent glucose dehydrogenase (FAD-GDH). In some embodiments, the GDH is pyrroloquinoline quinone glucose dehydrogenase (PQQ-GDH). In some embodiments, the GDH is nicotine adenine dinucleotide (phosphate)-dependent glucose dehydrogenase (NAD(P)-GDH).

[0042] In some embodiments, the enzyme is derived from a microorganism. In some embodiments, the enzyme is derived from a bacterium or a fungus. In some embodiments, the enzyme is derived from a mold. In some embodiments, the enzyme is derived from an organism of the phylum Mucoromycota or Ascomycota. In some embodiments, the enzyme is derived from an organism of the order Mucorales or Eurotiales. In some embodiments, the enzyme is derived from the family Mucoraceae or Aspergillaceae. In some embodiments, the enzyme is derived from the genus Mucor or Aspergillus (e.g., subgenus Circumdati, e.g., section Flavi). In some embodiments, the enzyme is derived from Mucor hiemalis, Mucor circinelloides, Mucor ambigus, Mucor lusitanicus, Mucor guilliermondii, Mucor subtilisimus, Mucor plenii, Aspergillus flavus, and / or Aspergillus oryzae. In some embodiments, the enzyme is a FAD-GDH derived from the genus Mucor (e.g., Mucor hiemalis, Mucor circinelloides, Mucor ambigus, Mucor lusitanicus, Mucor guilliermondii, Mucor subtilisimus, and / or Mucor plenii). In some embodiments, the enzyme is derived from FAD-GDH from Mucor hiemalis, Mucor circinelloides, Mucor ambygus, Mucor plenii, and Mucor subtilisimus.

[0043] In some embodiments, the enzyme is a wild-type enzyme. Examples of such wild-type enzymes into which epitope grafting can be inserted are set forth in SEQ ID NOs: 66-72, 119-127, and 131. In some embodiments, the enzyme is a modified enzyme (e.g., a synthetically modified enzyme) that includes one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc.) variations compared to the wild-type enzyme. In some embodiments, the enzyme is selected from the group consisting of UCW69416.1 (SEQ ID NO: 119), UCW69417.1 (SEQ ID NO: 120), UCW69418.1 (SEQ ID NO: 121), UCW69419.1 (SEQ ID NO: 122), UCW69420.1 (SEQ ID NO: 123), UCW69421.1 (SEQ ID NO: 124), UCW69422.1 (SEQ ID NO: 125), UCW69423.1 (SEQ ID NO: 126), or UCW694 24.1 (SEQ ID NO: 127), or any one of the sequences related to SEQ ID NOs: 1-118 or 132-134 (excluding any epitope-grafted sequences identified by underlining in Table 1). Sequence variations include point mutations, insertions and deletions, and chimeric enzymes (i.e., enzymes having sequences from two or more different enzymes). As noted above, amino acid modifications are typically conservative substitutions.

[0044] One or more synthetic sequences may be added to the enzyme to facilitate expression or purification of the enzyme, hi some such embodiments, the sequences used to facilitate expression or purification are removed prior to use of the enzyme in the sensor.

[0045] In some embodiments, one or more amino acids are modified to increase a desired property of the enzyme compared to the wild-type enzyme. Desired properties include, but are not limited to, enzyme activity (e.g., specific activity, turnover rate, K m, titratability), allosteric inhibition, de-inhibition, stability (e.g., thermal stability, shelf-life stability, stability when embedded or otherwise associated with the sensor surface, etc.), operability, ability to adsorb to the sensor surface, ability to create a fusion protein (e.g., fusion with an inhibitor), immobilization (e.g., compatibility by addition of a binding moiety), ability to conform onto a surface, compatibility with the sensor layer, biocompatibility with the sensing conditions (e.g., sample, pH, salt), resistance to interfering agents, avoidance of generation of interfering by-products (e.g., peroxides), affinity for the inhibitor, and substrate specificity. In some embodiments, one or more variants are made to increase the stability of the enzyme. For example, one or more cysteine ​​substitutions can be made in the enzyme to allow stabilizing disulfide bond formation (see Example 15). In some embodiments, pairs of cysteine ​​mutations are introduced that are spatially close to each other to form disulfide bonds and stabilize the enzyme structure.

[0046] Enzymes can be produced in host cells. Thus, in some embodiments, the present disclosure includes nucleic acids and expression systems for recombinant expression of enzymes in host cells or organisms. Nucleic acid sequences can be altered to generate variant enzymes as described above and to enhance protein expression compared to wild-type nucleic acid sequences. For example, nucleic acid variants can encode the same amino acids but result in different expression profiles in a given host expression system. The nucleic acid sequence encoding the enzyme can be provided in an expression vector suitable for expression in a desired host cell. Alternatively, the nucleic acid sequence can be inserted into the genome of a host cell or organism. Suitable host cells include, but are not limited to, bacterial cells (e.g., E. coli), yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris), baculovirus, plant, and animal cells. In some embodiments, the enzyme is produced in a cell-free system.

[0047] B) Substrate of the sensor enzyme In some embodiments, enzyme activity is assessed directly or indirectly by measuring the presence or amount of a substrate processed by the enzyme. Any suitable natural or synthetic substrate can be used with the selected enzyme. For example, if the enzyme is a glucose-metabolizing enzyme, glucose can be used as the substrate. In some embodiments, the substrate is modified to facilitate detection of enzymatic processing. For example, in some embodiments, a detectable label (e.g., fluorescent, luminescent, radioactive, chemical, affinity tag, etc.) is added to the substrate so that reaction products, either bearing or lacking the label, can be assessed. In some embodiments, by-products of enzymatic substrate processing are detected directly or indirectly, as a measure of enzyme activity. For example, the enzyme glucose oxidase is composed of two identical protein subunits and a cofactor (i.e., flavin adenine dinucleotide (FAD)) in its active site. With FAD, glucose oxidase catalyzes the oxidation of its reactant glucose at its first hydroxyl group using molecular oxygen as an electron acceptor to produce the products gluconolactone and hydrogen peroxide. The hydrogen peroxide product produced can be detected (e.g., electrochemical oxidation at an electrode and multiple electron transfers can be detected). Alternatively, oxygen consumption can be measured. Glucose dehydrogenase can utilize multiple different cofactors (e.g., NAD, PQQ, etc.). When FAD is used as a cofactor, glucose dehydrogenase catalyzes the oxidation of glucose to produce gluconolactone and FADH2. FADH2 is electrochemically oxidized at an electrode and multiple electron transfers can be detected.

[0048] C) Allosteric site In some embodiments, the epitope grafting sequence (e.g., amino acid sequence) is inserted into an allosteric site of an enzyme. Some substrates bind to enzymes at sites other than the active site. This other site is called the allosteric site. The allosteric site allows molecules to activate (fully or partially) or inhibit enzyme activity. Such molecules bind to the allosteric site and alter the structure or shape of the enzyme. The epitope grafting sequence provides an allosteric site that alters the activity of the enzyme when a drug (e.g., an inhibitor) binds to the epitope grafting sequence located at the allosteric site. In some embodiments, two or more allosteric sites may be available in the enzyme.

[0049] In some embodiments, the enzyme has one or more surface regions amenable to grafting of heterologous epitopes. Such surface regions may include allosteric sites. Surface regions amenable to addition of heterologous sequences are regions on the surface of the enzyme that, when modified to insert a heterologous sequence, do not eliminate measurable enzymatic activity. In some embodiments, at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%) of the enzymatic activity is maintained after addition of the heterologous sequence to the surface region compared to the enzyme without the heterologous sequence. Enzymatic activity can be assayed, for example, by measuring the amount of substrate processed by the enzyme over a given period of time. Assays for assessing enzymatic activity are provided in the Examples section below. In some embodiments, the surface region is located in an allosteric region of the enzyme.

[0050] For example, in some embodiments, the enzymes herein have surface regions comprising amino acids 45-70, 335-362, and / or 439-457 of SEQ ID NO: 1, or variants thereof, or the corresponding regions of SEQ ID NOs: 66-72 or 119-127. Epitope grafts can be inserted at any position within these surface regions.

[0051] In some embodiments, the enzyme has an allosteric site located on its surface that includes residues F341, E344, E348, and K358 of SEQ ID NO: 1 (or the corresponding residues of SEQ ID NOs: 66-72 or 119-127, or variant sequences). In some embodiments, the surface includes residues T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, and K358 of SEQ ID NO: 1 (or the corresponding residues of SEQ ID NOs: 66-72 or 119-127, or variant sequences). An epitope can also be grafted into any one of these positions (see below regarding "at").

[0052] In some embodiments, the epitope is grafted into a position corresponding to about position 330 to about position 370 of SEQ ID NO: 1, or the corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to about position 335 to about position 362 of SEQ ID NO: 1, or the corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to positions T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, and K358 of SEQ ID NO: 1, or the corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to position 341 or 358 of SEQ ID NO: 1, or a corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to position 341 of SEQ ID NO: 1, or a corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to position 358 of SEQ ID NO: 1, or a corresponding region of SEQ ID NOs: 66-72 or 119-127, or a variant sequence. In some embodiments, the epitope is grafted into a position corresponding to about position 320 to about 335 of SEQ ID NO: 131 (or a corresponding region of a variant sequence), for example, about position 325 to about 330 of SEQ ID NO: 1 (or a corresponding region of a variant sequence), for example, about position 327 to about 329 of SEQ ID NO: 131 (or a corresponding region of a variant sequence). In some embodiments, the epitope is grafted into the position corresponding to position 328 of SEQ ID NO: 131 (or the corresponding position of a variant sequence). In some embodiments, the epitope is grafted into the sequence corresponding to position 328 of SEQ ID NO: 131. For epitopes grafted at the recited positions ("at"), the added epitope sequence can be (i) substitution of the relevant amino acid with the epitope; (ii) insertion of the epitope N-terminal to the relevant amino acid; or (iii) insertion of the epitope C-terminal to the relevant amino acid.For positions 328, 341, and 358, "at" typically means that the epitope is grafted after that residue (ie, C-terminus to position 328, 341, or 358). In some embodiments, allosteric sites are identified and modified as discussed in the Examples below.

[0053] D) Epitope grafting The epitope sequence is provided in a region of the enzyme that is suitable for allosteric control of enzyme activity. The epitope sequence provides a recognition sequence for interaction with an inhibitor. When the inhibitor interacts with the epitope sequence, the activity of the enzyme is altered. For example, in some embodiments, interaction of the inhibitor with the epitope sequence located at the allosteric site of the enzyme reversibly inhibits enzyme activity. In such a state, the enzyme may be considered "inhibited." Inhibition need not eliminate all enzyme activity. A detectable decrease in enzyme activity is suitable for many sensor applications. When an analyte that is also recognized by the inhibitor is present near the enzyme, the inhibitor will associate weakly with the epitope-grafted sequence of the enzyme, resulting in increased enzyme activity. Introduction of the analyte and association of the inhibitor with the analyte rather than the epitope-grafted sequence in the enzyme "de-inhibits" the enzyme.

[0054] The epitope sequence may be selected based on one or more of several parameters. First, the epitope sequence should provide sufficient structure to allow the inhibitor to associate (e.g., bind) with the allosteric site of the enzyme containing the epitope-grafted sequence. Second, the association of the inhibitor with the allosteric site containing the epitope-grafted sequence should inhibit enzyme activity. Third, the strength of the inhibitor's association with the epitope-grafted sequence should be such that the presence of the analyte in a sample introduced to the enzyme deinhibits the enzyme. In some such embodiments, the epitope sequence and inhibitor are selected such that, when present, the inhibitor preferentially binds to the analyte across the allosteric site containing the epitope-grafted sequence. This may be achieved, for example, by using an epitope-grafted sequence that provides a sequence / structure for which the inhibitor has weaker affinity than the corresponding sequence / structure found in the analyte. One or more amino acid differences in the epitope-grafted sequence compared to the corresponding sequence in the analyte may be used to provide differential binding of the inhibitor to the epitope-grafted enzyme compared to the analyte. In some embodiments, the affinity of the analyte to the inhibitor is higher than the affinity of the epitope-grafted enzyme to the inhibitor. The affinity of the analyte to the inhibitor and / or epitope is expressed as K D The affinity of the analyte to the inhibitor may be determined as a K value and can be determined using standard methods known in the art. In some embodiments, the affinity of the analyte to the inhibitor is at least 2-fold, at least 3-fold, at least 5-fold, at least 10-fold, or at least 50-fold higher than the affinity of the epitope-grafted enzyme to the inhibitor. In some embodiments, the inhibitor has a high dissociation rate (K off ) are designed, selected, or screened for.

[0055] In some embodiments, the epitope comprises an amino acid sequence corresponding to an inhibitor binding site of an analyte, such as a peptide, polypeptide, or protein. The inhibitor binding site on a polypeptide can be identified by one of skill in the art. For example, the analyte can be contacted with an inhibitor, and the inhibitor binding site can be predicted, for example, by X-ray crystallography. This and other methods are described in the Examples. In some embodiments, the epitope comprises an amino acid sequence having at least 70%, at least 80%, or at least 90% sequence identity with the corresponding sequence of the inhibitor-binding site of the target molecule. The epitope may be designed or constructed to bind to the inhibitor less strongly than the inhibitor binds to the target molecule, e.g., less strongly than the inhibitor binds to the inhibitor-binding site of the target molecule. The strength of binding of the inhibitor to the epitope may be controlled by altering the sequence of the epitope-grafted epitope compared to the sequence of the inhibitor-binding site of the target molecule. For example, a grafted epitope having a sequence containing one, two, three, four, five, or more modifications (e.g., substitutions, e.g., conservative substitutions) compared to the sequence of the inhibitor-binding site of the target molecule may exhibit altered (e.g., decreased) binding strength to the inhibitor compared to the target molecule.

[0056] In some embodiments, the inhibitor is capable of competitively binding to the polypeptide analyte and the grafted epitope, hi some embodiments, the epitope thus comprises an amino acid sequence corresponding to the inhibitor binding site of the polypeptide analyte, and the inhibitor competitively binds to the grafted epitope and the analyte.

[0057] In some embodiments, the epitope grafting sequence comprises 3 to 30 amino acids. The lower end of the range should include sufficient structure to allow recognition by the inhibitor. In some embodiments, the epitope grafting sequence comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the epitope grafting sequence has 5 to 15 amino acids (e.g., 8 to 10). In other words, in some embodiments, the epitope grafting sequence comprises 3 to 30 amino acids, e.g., 5 to 15 amino acids, 8 to 10 amino acids, etc. In some embodiments, the epitope grafting sequence is selected to include one or more or all polar amino acids (serine (Ser), threonine (Thr), cysteine ​​(Cys), asparagine (Asn), glutamine (Gln), and tyrosine (Tyr)).

[0058] In some embodiments, the epitope-grafted sequence is a linear epitope. In some embodiments, the epitope-grafted sequence is a conformational epitope. A linear or continuous epitope is an epitope that is recognized by a binding molecule (e.g., an antibody, antibody fragment, or antibody mimic such as an aptamer, affimer, DARPin, etc.) due to its linear sequence of amino acids, or primary structure. In contrast, a conformational epitope is recognized by its three-dimensional shape. In some embodiments, the epitope-grafted sequence is a discontinuous epitope, i.e., an epitope that is composed of multiple distinct segments from the primary amino acid sequence. In some embodiments, the epitope grafted sequence is a linear epitope having a length of 3 to 30 amino acids, such as 5 to 15 amino acids, 8 to 10 amino acids, etc. In some embodiments, the epitope-grafted sequence is a discontinuous epitope comprising multiple (e.g., 2, 3, or 4) segments, each having a length of 3-15 amino acids, e.g., 5-12 amino acids, 8-10 amino acids, etc. Typically, the total length is as described above.

[0059] In some embodiments, the epitope-grafted sequence is inserted into the enzyme while retaining the original amino acids of the enzyme. In other embodiments, one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15) from the enzyme are removed and replaced with the epitope-grafted sequence. In some embodiments, prior to removal, the removed amino acids are N-terminal to a selected epitope-grafted sequence insertion site identified in the allosteric site of the enzyme. In some embodiments, the optimal location of a given epitope-grafted sequence within the allosteric site of the enzyme is determined by a screening method. In some embodiments, the screening method includes inserting the grafted sequence at alternating positions across the allosteric site or within subregions of the allosteric site to identify the optimal location (see, e.g., SEQ ID NOS: 8-13 and 14-16, which show alternating placement of the V5 epitope IPNPLLGLD at alternating positions within the enzyme). In some embodiments, the screening method includes a test linker sequence on one or both sides of the epitope-grafted sequence. In some embodiments, the screening method identifies the effect of design features on enzymatic activity, inhibition of enzymatic activity, and / or de-inhibition of enzymatic activity.

[0060] II) Inhibitors In some embodiments, an inhibitor is used that interacts with one or more epitope-grafted allosteric sites on the enzyme and inhibits enzyme activity. The inhibitor also interacts with at least a portion of the analyte of interest that corresponds to the epitope-grafted sequence, such that when the enzyme is bound to the inhibitor and in an inhibited state, the enzyme is deinhibited in the presence of an analyte that competes with the enzyme for binding to the inhibitor. Any agent that recognizes the epitope-grafted sequence that inhibits the enzyme, or recognizes the analyte or a portion thereof (e.g., recognizes the corresponding epitope present in the analyte) that deinhibits the enzyme in the presence of the analyte, can be used.

[0061] In some embodiments, the inhibitor is an antigen-binding protein. In some embodiments, the inhibitor is an antibody or antibody mimetic. In some embodiments, the inhibitor is an immunoglobulin (e.g., an antibody or antibody fragment). In some embodiments, the inhibitor is an antibody. As used herein, the term "antibody" is used broadly to refer to whole antibodies, monoclonal antibodies (including human, humanized, or chimeric antibodies), polyclonal antibodies, and antibody fragments (e.g., Fab', F(ab')2, Fv, single-chain antibodies) that are capable of binding to an antigen and contain the aforementioned complementarity-determining regions (CDRs), so long as they exhibit the desired biological activity. As used herein, an "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In some embodiments, the inhibitor is a nanobody (e.g., a VHH). In some embodiments, the inhibitor is a camelid single-domain antibody. In some embodiments, the inhibitor is a bispecific antibody configured to bind to two or more different analytes, such that the presence of either analyte results in competition with the inhibitor and partial or complete de-inhibition of the sensor enzyme.

[0062] In some embodiments, the inhibitor is an aptamer. Aptamers are oligonucleotide or peptide molecules that bind to specific target molecules. Aptamers are usually created by selecting them from a large pool of random sequences, although natural aptamers also exist. In some embodiments, the inhibitor is an affimer. Affimers are small proteins that typically bind to target proteins with nanomolar-range affinities. They are engineered non-antibody binding proteins designed to mimic the molecular recognition characteristics of monoclonal antibodies. These affinity reagents can be optimized to increase their stability, tolerate temperature and pH ranges, reduce their size, and increase their expression in host cells.

[0063] In some embodiments, the inhibitor is a DARPin. DARPins (an acronym for designed ankyrin repeat proteins) are genetically engineered antibody-mimetic proteins that typically exhibit high specificity and high affinity target protein binding. They are derived from natural ankyrin repeat proteins, one of the most common classes of natural binding proteins, and are responsible for diverse functions such as cell signaling, regulation, and cellular structural integrity. DARPins contain at least three repeat motifs or modules, the most N-terminal and most C-terminal modules of which are called "caps" because they hide the hydrophobic core of the protein.

[0064] In some embodiments, as described herein, binding of an inhibitor to an epitope-grafted enzyme reduces the enzymatic activity of the epitope-grafted enzyme by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%) compared to the enzymatic activity of the epitope-grafted enzyme in the absence of the inhibitor. In some embodiments, as described herein, unbinding of the inhibitor from the inhibitor-bound epitope-grafted enzyme (i.e., deinhibition of the enzyme) restores at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%) of the enzymatic activity of the epitope-grafted enzyme in the absence of the inhibitor. In some embodiments, the inhibition of enzyme activity resulting from binding of an inhibitor to an epitope-grafted enzyme, as described herein, is at least 70%, 80%, 90%, 95%, 97%, 98%, 99% or 100% reversible. In some embodiments, the inhibitor is used at a concentration that reduces the enzymatic activity of the epitope-grafted enzyme by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100%) compared to the enzymatic activity of the epitope-grafted enzyme in the absence of the inhibitor. It is routine for one of skill in the art to determine the appropriate inhibitor concentration based on the desired level of enzyme inhibition and the inhibitor being used. For example, in some embodiments, the inhibitor concentration is about 0.1 nM to about 10 μM, e.g., about 1 nM to about 1 μM. In some embodiments, the concentration is about 0.1 nM to about 1 μM, e.g., about 1 nM to about 100 nM.

[0065] III) Analysis target As demonstrated in the Examples section below, the universal sensor system technology provided herein is capable of detecting and analyzing a wide variety of analytes, including macromolecular proteins.

[0066] In some embodiments, the analyte is a prognostic or diagnostic analyte for the health and / or well-being of a patient. For example, the analyte can be any molecule of interest for diagnosis, screening, disease staging, forensic analysis, pregnancy tests, drug testing, and other reasons. The analyte can be a biopolymer marker of a physiological condition, including health, disease, drug response, efficacy, safety, injury, trauma, traumatic brain injury, pain, chronic pain, pregnancy, atherosclerosis, myocardial infarction, type I or type II diabetes, sepsis, cancer, Alzheimer's dementia, multiple sclerosis, etc. The analyte can include proteins, peptides, polypeptides, amino acids, hormones, steroids, vitamins, drugs, including those administered for therapeutic purposes and those administered for illicit purposes, bacteria, viruses, and metabolites or antibodies of any of the above substances.

[0067] In some embodiments, the analyte is one or more of TnI, TnT, BNP, NTproBNP, proBNP, HCG, TSH, NGAL (also known as LCN2), theophylline, digoxin, and phenytoin. In some embodiments, the analyte is acid phosphatase, alanine aminotransferase, albumin (BCG / BCP), alkaline phosphatase, alanine aminotransferase, alpha-1-acid glycoprotein, alpha-1-antitrypsin, alpha-fetoprotein, amikacin, amphetamine / methamphetamine, amylase, apolipoprotein A1, apolipoprotein B, anti-HBc (IgG and IgM) antibodies, aspartate aminotransferase, barbiturates, benzodiazepines, beta-2-microglobulin, beta-hCG, bilirubin, cancer antigen 15-3, cancer antigen 125, cancer antigen 19-9XR, carcinoembryonic antigen (CEA), cannabinoids, carbamazepine, ceruloplasmin, cholesterol, cocaine, complement C3, complement C4, cortisol, creatine kinase, creatine, CRPVario, C-peptide, cyclic citrullinated peptides, cyclosporine, dehydroepiandrosterone sulfate (DHEA-S), ecstasy, estradiol, ferritin, folic acid, follicle-stimulating hormone (FSH), free prostate-specific antigen (PSA), free triiodothyronine (T3), free thyroxine (T4), gamma-glutamyltransferase, gentamicin, haptoglobin, HCV (antibody to hepatitis C virus), HDL, hemoglobin A1c, hepatitis B surface antigen, HIV antigen and / or antibody, homocysteine, holotranscobalamin (B-12 marker), human epididymis protein 4 (HE4), immunoglobulin A, immunoglobulin G, immunoglobulin M, insulin, IgM antibody, LDL, lactate dehydrogenase , lipase, lipoprotein A, luteinizing hormone, methadone, microalbumin, myoglobin, opiates, parathyroid hormone, phencyclidine, phenobarbital, phenytoin, prealbumin, procalcitonin (PCT), protein (urine / CSF), progesterone, prolactin, prostate-specific antigen (PSA), propoxyphene, rheumatoid factor, salicylic acid, serum benzodiazepines, SHBG, sirolimus, T3, T4, tacrolimus, testosterone, theophylline, thyroglobulin antibodies, tobramycin, TPO antibodies, transferrin, tricyclic antidepressants, triglycerides, vancomycin, glial fibrillary acidic protein (GFAP), and ubiquitin carboxy-terminal hydrolase L1 (UCHL1).

[0068] In some embodiments, two or more analytes are detected. In some embodiments, two or more analytes are detected in an "and" format, where the presence or amount of each analyte is determined independently. In other embodiments, two or more analytes are detected in an "or" format, where the presence of any one of the analytes produces a detectable signal that identifies the presence of at least one analyte but does not distinguish between the analytes.

[0069] IV) Sample The enzymes and sensor systems described herein find use in the analysis of analytes in any desired sample type. Samples include both biological and environmental samples. Samples can be detected in a laboratory, in the field, or in any other suitable location. The sample can be provided to a sensor for testing, or the sensor can be applied to a source of the sample. For example, in some embodiments, the sensor is physically close to, attached to, or contained within the sample source (e.g., on or within a subject or environmental sample). In some embodiments, the sample is a biological sample. Biological samples may be obtained from any source, including animals, plants, and microorganisms, and encompass liquids, solids, tissues, and gases. Materials obtained from clinical or forensic settings containing the analyte of interest are also within the intended meaning of the term sample. Biological samples include, but are not limited to, whole blood, serum, plasma, saliva, ocular lens fluid, amniotic fluid, synovial fluid, cerebrospinal fluid, tears, lymph, interstitial fluid, peritoneal fluid, bronchial washings, ascites fluid, bone marrow aspirate, pleural effusion, urine, milk, sweat, saliva, semen, mucus, stool, tissue (such as skeletal muscle tissue, liver tissue, lung tissue, kidney tissue, cardiac muscle tissue, brain tissue, bone marrow, cervical tissue, skin), organs (e.g., biopsy samples), vaginal fluid, aqueous humor, earwax, gastric juice, gastrointestinal fluid, nasal washings, liposuction, sebum, tears, breath, and vitreous humor. Such samples may be evaluated in vitro, ex vivo, or in vivo.

[0070] In some embodiments, the sample is an environmental sample, including but not limited to water, air, snow, and soil. The sample may be in a processed form, including dried (e.g., dried blood spot) and fixed (e.g., formalin-fixed paraffin-embedded (FFPE)) samples. In some embodiments, the sample is located in vivo in an animal.

[0071] When a sensor is used to measure one or more analytes in vivo, it may be placed on or within a subject such that a desired sample within the subject comes into contact with the sensor chemistry. For example, the sensor may be placed in a wearable device that facilitates contact between the sensor chemistry and the subject's interstitial fluid or blood. Such systems, and technology used therein, are described in U.S. Patent Nos. 6,932,894; 7,620,438; 7,670,470; 7,826,382; 7,920,907; 8,106,780; 8,115,635; 8,147,666; 8,223,021; and 8,280,470, each of which is incorporated herein by reference in its entirety. No. 4; U.S. Patent No. 8,358,210; U.S. Patent No. 8,377,271; U.S. Patent No. 8,380,274; U.S. Patent No. 8,390,455; U.S. Patent No. 8,409,093; U.S. Patent No. 8,410,939; U.S. Patent No. 8,437,829; U.S. Patent No. 8,542,122; U.S. Patent No. 8,617,069; U.S. Patent No. 8,688,188; U.S. Patent No. 8,737,259; U.S. Patent No. 8,760,297; U.S. Patent No. 8,816,862; U.S. Patent No. Nos. 8,915,850; U.S. Patent No. 9,000,929; U.S. Patent No. 9,007,781; U.S. Patent No. 9,008,743; U.S. Patent No. 9,014,774; U.S. Patent No. 9,042,955; U.S. Patent No. 9,060,805; U.S. Patent No. 9,184,875; U.S. Patent No. 9,186,098; U.S. Patent No. 9,186,113; U.S. Patent No. 9,215,992; U.S. Patent No. 9,226,714; U.S. Patent No. 9,232,916; U.S. Patent No. 9,262,264 No. 5,453; U.S. Patent No. 9,271,670; U.S. Patent No. 9,314,198; U.S. Patent No. 9,336,423; U.S. Patent No. 9,351,669; U.S. Patent No. 9,402,544; U.S. Patent No. 9,402,570; U.S. Patent No. 9,414,778; U.S. Patent No. 9,474,475; U.S. Patent No. 9,532,737; U.S. Patent No. 9,549,694; U.S. Patent No. 9,636,068; U.S. Patent No. 9,687,183; U.S. Patent No. 9,693,713;U.S. Patent No. 9,713,443; U.S. Patent No. 9,750,444; U.S. Patent No. 9,808,186; U.S. Patent No. 9,831,985; U.S. Patent No. 9,895,091; U.S. Patent No. 9,907,470; U.S. Patent No. 9,931,066; U.S. Patent No. 9,980,669; U.S. Patent No. 9,993,188; U.S. Patent No. 10,010,280; U.S. Patent No. 10,028,680; U.S. Patent No. 10,136,816; U.S. Patent No. 10,136,845; U.S. Patent No. 10,178,954; U.S. Patent No. 10,201, 301; U.S. Patent No. 10,213,139; U.S. Patent No. 10,349,877; U.S. Patent No. 10,492,685; U.S. Patent No. 10,653,344; U.S. Patent No. 10,736,547; U.S. Patent No. 10,765,351; U.S. Patent No. 10,820,842; U.S. Patent No. 10,923,218; U.S. Patent No. 10,952,611; U.S. Patent No. 11,051,724; U.S. Patent No. 11,119,090; U.S. Patent No. 11,179,068; U.S. Patent No. 11,202,591; and U.S. Patent No. 11,213,229. In some embodiments, the sensor is placed in a wearable mouthpiece that facilitates contact between the sensor chemistry and saliva. In some embodiments, the sensor is placed along a device that collects biological fluids, such as a syringe, dialysis tubing, breathing tube, catheter channel, etc. In some embodiments, the sensor is contained within an implant (e.g., a stent, a transplant, an artificial joint or prosthetic limb, etc.); In some embodiments, the sensor is directly exposed to the sample without any modification or alteration of the sample, hi some embodiments, the sample is pre-processed to remove one or more components prior to exposing the sample to the sensor chemistry.

[0072] V) Detection / Sensor Systems In some embodiments, provided herein is a system comprising an epitope-grafted enzyme and an inhibitor capable of binding thereto, wherein the enzyme and inhibitor are typically as described herein. In some embodiments, the system further comprises a substrate for the epitope-grafted enzyme, hi some embodiments, the enzyme is epitope-grafted FAD-GDH and the substrate is glucose. In some embodiments, the system further includes an analyte having a binding site for the inhibitor. The analyte may be an analyte described in more detail herein, for example, the analyte may be a peptide, polypeptide, or protein described herein. In some embodiments, the analyte is present in a biological sample as described herein.

[0073] Also provided is a sensor comprising the enzyme described herein. The sensor may comprise the system described herein. The sensor may be an electrochemical sensor. In some embodiments, enzymes are integrated into electrochemical sensors. A summary of suitable sensor structures and sensor systems using these sensors that utilize the enzymes of the present disclosure is provided. However, this summary should be understood as non-limiting to the aspects disclosed herein, and alternative sensors and systems are intended to be within the scope of the present disclosure.

[0074] Typically, multiple copies of the enzyme are used on the sensor surface. The concentration and spacing of the enzyme may be selected based on the desired sensor performance. For example, in some embodiments, a low concentration of enzyme allows for the detection of low amounts of analyte. In some embodiments, when maximum sensitivity is desired, a more diluted and spread enzyme is used on the sensor surface. In some embodiments, two or more different enzymes that detect different analytes are used in a single sensor system. In some embodiments, a monolayer of enzymes is used. In some embodiments, the sensor contains or includes one or more (e.g., multiple) enzymes on the active area of ​​a single working electrode, or on two or more separate working electrodes. The single working electrode configuration of the sensor may employ two-electrode or three-electrode detection motifs.

[0075] In some embodiments, an electron transfer agent may be present in one or more sensing regions (e.g., active regions) of any sensor or sensor structure. A suitable electron transfer agent / mediator compound may facilitate the transfer of electrons to the working electrode when a reactant undergoes a redox reaction. The choice of electron transfer agent within each active region may affect the observed redox activity of each. When multiple active regions are present, the electron transfer agents within each active region may be the same or different.

[0076] Suitable electron transfer agents may include electroreducible and electrooxidizable ions, complexes, or molecules (e.g., quinones) with redox potentials several hundred millivolts higher or lower than the redox potential of the electrode. In some embodiments, suitable electron transfer agents may include low-potential osmium complexes, such as those described in U.S. Pat. Nos. 6,134,461 and 6,605,200, the entire disclosures of which are incorporated herein by reference. Further examples include those described in U.S. Pat. Nos. 6,736,957, 7,501,053, and 7,754,093, the disclosures of each of which are incorporated herein by reference in their entireties. Other suitable electron transfer agents may include metal compounds or complexes of ruthenium, osmium, iron (e.g., polyvinylferrocene or hexacyanoferrate), or cobalt, including, for example, metallocene compounds thereof. Suitable examples of electron transfer mediators and polymer-bound electron transfer mediators may include those described in U.S. Patent Nos. 8,444,834, 8,268,143, and 6,605,201, the disclosures of which are incorporated herein by reference in their entireties. Suitable ligands for metal complexes may also include, for example, bidentate or high-dentate ligands, such as bipyridine, biimidazole, phenanthroline, or pyridyl (imidazole). Other suitable bidentate ligands may include, for example, amino acids, oxalic acid, acetylacetone, diaminoalkanes, or o-diaminoarenes. Any combination of monodentate, bidentate, tridentate, tetradentate, or high-dentate ligands may be present in the metal complex to achieve a full coordination sphere.

[0077] In some embodiments, the active or sensing region may also include a cofactor capable of catalyzing a reaction of a reactant associated with at least one oxidase or dehydrogenase domain portion of the enzyme. In some aspects, the cofactor is a non-protein organic molecule, such as pyrroloquinoline quinone (PQQ), flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), flavin mononucleotide (FMN), or the like. In certain embodiments, the cofactor may be attached to a polymer, cross-linking the cofactor to the electron transfer agent. A second cofactor may also be used in certain embodiments. In some embodiments, diaphorase is included when the enzyme turns over NAD or NADP. In some embodiments, the sensor is provided as a component of a benchtop device, in some embodiments, the sensor is provided as part of a handheld device, in some embodiments, the sensor is provided as part of a wearable device, in some embodiments, the sensor is integrated into or connected to a medical device, such as a catheter (e.g., an indwelling catheter), endoscope, etc.

[0078] VI) Data analysis / software In some embodiments, a system is provided that includes a computer processor that contains or executes software that controls one or more or all of: sensor control, sensor monitoring, data collection from the sensor, data analysis, data reporting (e.g., display), data storage, data migration (e.g., cloud or network), and generation of an alert or other signal that notifies a user (e.g., a user, patient, healthcare professional, etc.) of an event of interest (e.g., presence of an analyte, a change in analyte concentration, a threshold analyte concentration responsive to the needs of the present invention, etc.). These processes may be embodied in software, firmware, hardware, or any combination thereof.

[0079] Certain steps, operations, or processes described herein may be implemented or performed by one or more hardware or software modules, alone or in combination with other devices. In one embodiment, the software modules are implemented by a computer program product that includes a computer-readable medium containing computer program code that can be executed by a computer processor to perform any or all of the described steps, operations, or processes. Embodiments of the technology may also relate to apparatus for performing the operations herein. This apparatus may be specially constructed for the required purposes and / or may include a general-purpose computing device selectively activated or reconfigured by a computer program stored in the computer. Such a computer program may be stored on a non-transitory, tangible computer-readable storage medium, or any type of medium suitable for storing electronic instructions that can be coupled to a computer system bus. Furthermore, any computer system referred to in the specification may include a single processor or may be configured using a multiple-processor design to increase computational complexity.

[0080] In some embodiments, the system tracks, analyzes, and / or reports one or more of each of the following: a) sensor operating status (power status, battery status, etc.); b) raw signal from the sensor (e.g., electrochemical signal, fluorescent signal, etc.); c) presence or absence of a detected analyte; d) analyte concentration or concentration change; e) indicators of a change in health status. In some embodiments, the processor and / or software is located on a personal computing device (e.g., a handheld or wearable computing device, a tablet, a laptop computer, a desktop computer) associated with a user of the sensor (e.g., a patient, a caregiver, a healthcare professional, a family member). In some embodiments, the processor and / or software is located on a computing device remote from the user (e.g., a remote server) and is in electronic communication with the sensor or an intermediate device that receives information from the sensor.

[0081] VII) Further Methods In some embodiments, provided herein are methods for determining the presence, absence, or concentration of an analyte in a sample, such as a sample (e.g., a biological sample) described herein. In some embodiments, the method comprises: a) contacting the sample with an enzyme described herein in the presence of an analyte and an inhibitor capable of binding to a grafted epitope of the enzyme; and b) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme Includes. In some embodiments, the method comprises: a) contacting the sample with an enzyme described herein in the presence of an analyte and an inhibitor capable of binding to a grafted epitope of said enzyme; b) allowing the inhibitor to inhibit the enzyme; c) binding the analyte present in the sample to the inhibitor, thereby deinhibiting the enzyme; and d) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme. may include: In some embodiments, the measurement is an electronic measurement, which can be performed in some embodiments when an enzyme is included in a system or sensor described herein.

[0082] In some embodiments, the enzymatic activity of the enzyme is proportional to the concentration of the analyte in the sample. For example, the presence, absence, or concentration of the analyte in the sample can be associated with a health condition as described herein. The health condition can be, for example, a pathological condition or a lifestyle condition. For example, the presence of a disease biomarker can be associated with the presence of a disease. By monitoring the presence, absence, or concentration of a given analyte over time, the associated health condition can be monitored. This can be useful, for example, to inform a physician about appropriate medication prescribing or to inform a subject about appropriate lifestyle choices. Thus, in some embodiments, provided herein are methods of diagnosing the health of a subject, comprising: (a) contacting a biological sample from the subject with an enzyme or sensor described herein; and (b) determining the presence, absence, or concentration of an analyte associated with the subject's health in the sample according to the provided methods. In some embodiments, provided herein are epitope-grafted enzymes described herein for use in methods of diagnosing the health of a subject, such use comprising contacting a biological sample from the subject with the enzyme; and (b) determining the presence, absence, or concentration of an analyte associated with the subject's health in the sample as described herein.

[0083] In some further embodiments, provided herein are methods for identifying an allosteric site on an enzyme that can be inhibited by an inhibitor, comprising: a) generating one or more antibodies and / or antibody mimetics that bind to the enzyme; b) screening the ability of said one or more antibodies and / or antibody mimetics to allosterically inhibit the enzymatic activity of the enzyme, thereby identifying antibodies and / or antibody mimetics that allosterically inhibit the enzymatic activity of the enzyme; and c) identifying amino acids of the enzyme contacted by said antibody and / or antibody mimetic that allosterically inhibit the enzymatic activity of the enzyme; The present invention provides a method comprising: In some embodiments, the method further comprises determining the retention of enzyme activity when the amino acid is modified.

[0084] In some embodiments, the method further comprises grafting an epitope into the amino acid sequence of the enzyme at a position corresponding to the allosteric site, wherein the epitope comprises an amino acid sequence capable of binding to an inhibitor. In some embodiments, the epitope is an epitope described in more detail herein. In some embodiments, these methods may be used to identify, design, or improve the enzymes described herein. Methods for identifying allosteric sites on enzymes are described in more detail in the Examples. Also provided are epitope-modifying enzymes obtainable by such methods.

[0085] array The following sequences are referenced throughout the examples. In addition to the sequences shown below, the sequences may contain a C-terminal G4S linker followed by a His8 tag. The sequences may further include a secretion signal (e.g., LFSLAFLSALSLATASPAGRAK (SEQ ID NO: 130), which is listed below in certain sequences for illustrative purposes (indicated by double underlining), but in some embodiments, the listed sequence omits the secretion signal peptide sequence). For glycosylated, secreted expression in Pichia culture, each protein sequence is added to its N-terminus with an AKS signal sequence preceding the listed sequence. The sequences also include an N-terminal methionine residue.

[0086] [Table 1] JPEG2025540754000002.jpg237170 JPEG2025540754000003.jpg238170 JPEG2025540754000004.jpg237170 JPEG2025540754000005.jpg237170 JPEG2025540754000006.jpg237170 JPEG2025540754000007.jpg236170 JPEG2025540754000008.jpg240170 JPEG2025540754000009.jpg246170 JPEG2025540754000010.jpg238170 JPEG2025540754000011.jpg245170 JPEG2025540754000012.jpg236170 JPEG2025540754000013.jpg236170 JPEG2025540754000014.jpg187170 JPEG2025540754000015.jpg208170 JPEG2025540754000016.jpg216170 JPEG2025540754000017.jpg208170 JPEG2025540754000018.jpg210170 JPEG2025540754000019.jpg210170 JPEG2025540754000020.jpg209170 JPEG2025540754000021.jpg208170 JPEG2025540754000022.jpg209170 JPEG2025540754000023.jpg208170 JPEG2025540754000024.jpg208170 JPEG2025540754000025.jpg209170 JPEG2025540754000026.jpg209170 JPEG2025540754000027.jpg208170 JPEG2025540754000028.jpg231170 JPEG2025540754000029.jpg192170 JPEG2025540754000030.jpg207170 JPEG2025540754000031.jpg207170 JPEG2025540754000032.jpg206170 JPEG2025540754000033.jpg208170 JPEG2025540754000034.jpg186170 [Example]

[0087] Example 1 A general method for the discovery of novel allosteric sites. Allostery means that an effector binds to an enzyme at a site distal to the active site and transmits a signal that alters enzyme activity. Effectors can be small molecules, peptides, or antibodies. Antibodies are significantly larger than small molecules and peptides and are therefore more suitable for discovering allosteric effector sites of enzymes that use small molecule substrates.

[0088] Pools of potential inhibitory antibodies can be generated in several ways, including immunizing animals or screening synthetic antibody libraries using phage display. Once a pool of antibodies specific to the enzyme of interest is found, the pool can be screened for its ability to inhibit the target. The antibody pool can take the form of serum from immunized animals or a pool of phage enriched for the target of interest. If appropriate controls (i.e., pools of antibodies generated against different targets) are used, the antibody pool can be used in an enzyme assay of choice to determine whether the pool contains sufficient amounts of inhibitory antibodies. If the antibody pool shows inhibition beyond the control, it can be concluded that the pool contains antibodies that inhibit the enzyme of interest. If the enzyme of interest uses a small molecule as a substrate, some antibodies may inhibit the enzyme in an allosteric manner. The antibody pool can then be divided into individual clones and screened using the enzyme assay of choice in a high-throughput manner to find individual clones that inhibit the enzyme. Once clones are identified, they can be screened for mode of inhibition using Lineweaver-Burk analysis, described below. Any antibodies determined to inhibit in a non-competitive or non-competitive manner can be considered allosteric inhibitors. Confirmation of the binding site can be facilitated by a crystal structure or similarly definitive structural analysis of the epitope-paratope interaction.

[0089] General FAD-GDH Assay Protocol The FAD-GDH (flavin adenine dinucleotide-glucose dehydrogenase) activity assay measures enzyme activity by monitoring the change in optical absorbance of the reaction mixture at 600 nm. The reaction mixture contains the enzyme (FAD-GDH), substrate (glucose), electron carrier (phenazine ethosulfate, PES), and color reporting reagent (2,6-dichlorophenolindophenol, DCPIP). FAD-GDH converts one molecule of glucose to gluconolactone, while PES transfers two electrons to DCPIP, which is reduced to colorless DCPIPH2 as the final electron acceptor. In the assay design, the assay mixture contains three components: 10 μL of 10× enzyme solution (purified FAD-GDH or Pichia expression supernatant solution), 10 μL of 10× substrate (D-glucose) solution, and 80 μL of 1.25× reaction master mix (electron carrier PES and color reporting reagent DCPIP). The total assay volume is 100 μL with final concentrations of 1× enzyme (final concentrations vary depending on the experimental design), 1× substrate (100 mM), and 1× reaction master mix (2 mM PES and 0.5 mM DCPIP).

[0090] material Phenazine ethosulfate (PES), Sigma (P4544-5G) 2,6-Dichlorophenol-indophenol (DCPIP), Sigma (D1878-5G) Triton X-100, Sigma(T9284-100ML) PIPES, Sigma (P6757-25G) Coster assay plate, 96-well, clear black, flat bottom, nonsterile, Corning (Cat. No. 3631) Millex-GV Sterile 33mm Low Protein Binding Durapore PVDF Membrane 0.22μm, Millipore (SLGV133RS) BMG Plate Reader, AP20-506

[0091] method Reagent preparation Preparation of enzyme dilution buffer (50 mM potassium phosphate buffer pH 6.5): Bring potassium phosphate to pH 6.5 with monobasic and dibasic potassium phosphate. Add approximately 35 mL of 1 M monobasic potassium phosphate to 15 mL of 1 M dibasic potassium phosphate. Titrate to pH 6.5 with monobasic or dibasic potassium phosphate. Dilute this stock to 1 L and bring to 50 mM potassium phosphate pH 6.5. Filter through a 0.22 μM filter. Preparation of assay buffer (50 mM PIPES-NaOH buffer pH 6.5 with 0.1% Triton X-100 solution): Add 1.51 g of PIPES to 60 mL of water and stir. Add 1.0 mL of 10% Triton solution and adjust the pH to 6.5 ± 0.05 with 6 N NaHO. Transfer to a 100 mL graduated cylinder and fill to the 100 mL mark with distilled water. Filter-sterilize the solution using a 0.22 μm filter.

[0092] Preparation of 1 M D-glucose solution: Add 9.0 g of D-glucose to a 50 mL conical. Add distilled water until the solution reaches the 40 mL mark. Mix gently by swirling until completely dissolved. Stir the solution at room temperature for at least 16 hours (allowing time for the glucose to mutarotate). Transfer the solution to a 50 mL graduated cylinder and add distilled water to the 50 mL mark. Filter sterilize the solution using a 0.22 μm filter.

[0093] Assay plate preparation and reading Preparation of 10x enzyme dilution: Dilute the enzyme stock solution or Pichia expression supernatant to a 10x concentration with enzyme dilution buffer. Preparation of reaction mixture: Add 357 μL of 80 mM PES solution and 839 μL of 8 mM DCPIP solution to 8.6 mL of assay buffer. Add 179 μL of DI water to bring the total volume to 10 mL. Vortex to mix thoroughly. Transfer 10 μL of 10x enzyme solution to a 96-well assay plate. Add 80 μL of reaction master mix using a multichannel pipette. Incubate the plate at room temperature for 5-10 minutes. Load the plate into a BMG plate reader. Add 10 μL of 1M D-glucose solution to each well and shake at 500 rpm for 30 seconds. Read the plate for 15 reading cycles. The time interval between each reading cycle is 87 seconds. The total reading time is 20 minutes. Data Processing Data is analyzed with MARS software. Linear regression from GraphPad Prism 8 is used to calculate the slope and R-squared value for each reaction well. The slopes from each individual sample well and control well (wild-type FAD-GDH enzyme) are used to calculate the following equation: Activity % = Slope 試料 / Tilt 対照 ×100% Relative activity is calculated using

[0094] Example 2 Discovery of the first allosteric site of Mucor FAD-GDH and identification of the inhibitory antibody 1-286 Mouse Immunization. Five CAF1 / J, SJL / J, and RBF / DnJ female mice were inoculated with the Mucor mutant FAD-GDH. 35 μg of FAD-GDH (untransplanted: 19031 FAD-GDH) was diluted in potassium phosphate pH 5.5, 0.1% (v / v) Triton X-100, 0.1 ml of Adjulite complete Freund's adjuvant, and sterile 0.9% NaCl to a final volume of 0.2 ml per animal. Five additional animals were inoculated similarly, except that 0.2 ml of AddaVax adjuvant was used, and 0.005 mg of human / mouse CpG DNA was substituted for Adjulite, resulting in a 0.4 ml inoculum per mouse. The inoculum was administered in two axillary and two inguinal sites on the ventral side of the animals. Six weeks later, a second immunization was performed using the same protocol. Another immunization was administered four weeks later. The animals were allowed an additional 5 months to develop an immune response, at which point serum was collected and screened for inhibition of FAD-GDH enzyme activity in vitro.

[0095] Screening of mouse sera for inhibitory antibodies. Sera were labeled #71 to #99 in tubes corresponding to different mice. Normal mouse serum (NMS) was used as a control. Serial dilutions of each serum were prepared in phosphate-buffered saline, pH 7.2 (PBS). GDH colorimetric assays were performed in 96-well plates according to the general FAD-GDH assay protocol described in Example 1. Each reaction was initiated by adding 12.5 μl of diluted serum in 50 mM PIPES / Triton buffer, 2 mM phenazine methosulfate (PMS), and 0.17 mM dichlorophenol indophenol (DCPIP), 0.04 μg FAD-GDH, and 12.5 μl of 400 mM D-glucose solution. The final volume of the reaction well was 125 μl. Absorbance at 600 nm was read continuously over 30 min at 37°C in a spectrophotometer.

[0096] The resulting data were plotted, and the slope of the linear portion of the curve was used to calculate the reaction rate (-ΔA600 / min). Comparisons were made between different serum dilutions (1:50 or 1:500) and ranked by the degree of inhibition (Figure 1). Control reaction results included: PBS + FAD-GDH + 40 mM glucose: 0.0053; normal mouse serum (NMS) + enzyme + glucose: 0.0052 or 0.0051 at 1:50 and 1:500, respectively; and PBS + enzyme + water (substitute for glucose): 0.0016. Using this screening method, several sera were found to inhibit the FAD-GDH enzyme reaction rate. None of the sera demonstrated a GDH stimulatory effect in these assays. Mice #80, #81, and #90 were selected to generate hybridoma fusions; their spleens were harvested and perfused; splenocytes were harvested, frozen, and stored in liquid nitrogen until use.

[0097] Fusion of human myeloma with mouse B cells. Splenocyte harvest samples were thawed, mixed with HSFM culture medium (Gibco; catalog number ME130092L1) supplemented with 10% (v / v) FBS, 10 ml / L L-glutamine, and 24 μg / ml CpG DNA, rinsed thoroughly, and transferred to a culture flask. Cells were allowed to recover overnight in a 37°C incubator with 5% CO2. B cells were enriched using the EasySep Mouse B Cell Isolation Kit according to the manufacturer's instructions, yielding 1.4 × 10 6 This resulted in 10 B cells. Early passage human myeloma cells were thawed from cryopreservation to 37°C and recovered in supplemented HSFM culture medium. After several days of expansion in growth flasks, cells were counted and yielded 1.47 x 10 7 1.47 x 10 mouse B cells and 1.47 x 10 7Myeloma cells were fused in a microslide electrofusion chamber using standard methods using Cytofusion Medium C (BTX; Catalog No. 47-0001). The fused bulk culture was cultured in HSFM medium supplemented with HAT (Sigma; Catalog No. H0262) for selection. Cells were then pelleted, resuspended, and plated in a mixture of HSFM and CloneMatrix semi-solid medium containing goat anti-mouse DyLight488. Individual hybridoma clones showing enriched fluorescent signals were picked using Clonepix2 and arrayed into 96-well culture plates. Ten plates of anti-FAD-GDH hybridoma clones were picked and grown to confluence.

[0098] ELISA screening of hybridoma supernatants. A 1 μg / ml dilution of FAD-GDH was prepared in PBS buffer and passively coated onto a 96-well ELISA plate (BrandTech; catalog no. 781722). After washing the plates with water, they were then blocked with blocking buffer (PBS supplemented with 5% (w / v) BSA and 0.1% (v / v) Tween-20), washed again, and then incubated with hybridoma supernatants (one clone per well). The plates were washed again and then incubated with Affipure sheep anti-mouse peroxidase-conjugated antibody (Jackson ImmunoResearch; catalog no. 515-035-062) for detection. After washing, the plates were reacted with a colorimetric peroxidase developing solution. The reaction was terminated by adding 1N sulfuric acid, and the absorbance of each well was read at 492 nm. Any well with an absorbance value above a nominal cutoff of 0.2 was considered positive for binding. A total of 89 clones were identified as binding to FAD-GDH in the ELISA assay and were carried forward.

[0099] FAD-GDH Inhibition Assay with Selected Hybridoma Clones Colorimetric assay of FAD-GDH was performed in the presence of 89 selected hybridoma clone supernatants (60 μl / well) with reagents and methods consistent with the general FAD-GDH assay protocol in Example 1. Absorbance was read at 600 nm for 30 minutes. Percent inhibition was calculated using the following equation: (slope 培地のみ -Tilt 抗体 ) / tilt 培地のみ ) × 100 was calculated for each clone by comparing it with the medium-only control (Figure 2).

[0100] Isotype determination of selected clones. A panel of clones showing the highest inhibition or activation of FAD-GDH was selected for isotype testing. ELISA plates were passively coated with sheep anti-mouse IgG antibody and washed. Antibody-containing hybridoma supernatants were screened using the SBA-Clonetyping System-HRP kit (Southern Biotech; catalog number: 5300-05). Table 1 lists the identified isotypes detected in each clone along with their percent inhibition measured in the FAD-GDH assay.

[0101] [Table 2] The data was evaluated and clones meeting the following criteria were selected for scaling to a 500 ml expansion and purification experiment: greater than 30% change in enzyme activity in the colorimetric screening assay, IgG isotype, and clone. Clones meeting these criteria were #134, 236, 228, 275, 286, and 618.

[0102] Hybridoma clone cultivation and anti-FAD-GDH antibody purification. FAD-GDH clones 1-134, 1-228, 1-236, 1-275, 1-286, and 1-618 were inoculated into 500 ml of supplemented HSFM and cultured for 2 weeks. The culture supernatant was filtered through a 0.45 μm filter and then purified using a HiPrep Protein A column (Cytiva; catalog number: 28-4082-61) and subsequently desalted into PBS using a HiPrep 26 / 10 Desalting column (Cytiva; catalog number: 17-5087-01). The absorbance of the purified protein was measured at 280 nm, and the protein concentration was determined using 1.38 AU of a 1 mg / ml solution measured with a 1 cm path length. Concentrations and yields are provided in Table 2.

[0103] [Table 3] Enzyme Assay Screening of Purified Anti-FAD-GDH IgG Clones. Purified antibodies from clones 1-134, 1-286, 1-228, 1-275, 1-236, and 1-618 were tested for inhibition or stimulation of FAD-GDH enzyme activity. Only 1-286 showed inhibition in the screening assay; the other clones showed no inhibition. An eight-point, two-fold dilution series of 1-286 was prepared and tested for dose-dependent inhibition of FAD-GDH using the reagents and methods described in the general FAD-GDH assay protocol in Example 1. Absorbance at 600 nm was measured for 30 minutes, and the reaction rate (slope of the line) was determined using the linear portion of each curve and linear regression and plotted against antibody concentration. Figure 3A shows the dose-dependent relationship between the measured slope and antibody concentration. The IC50 of mAb 1-286 was measured to be 3.7 μg / ml under these experimental conditions. After a total reaction time of 45 min, absorbance at 600 nm was read again and the remaining absorbance was plotted against antibody concentration, and high absorbance values ​​were associated with extensive inhibition at high concentrations of mAb 1-286, indicating a saturable and dose-dependent inhibitory response (Figure 3B).

[0104] Enzyme assay to determine the allosteric mechanism of mAb 1-286 inhibition Using the reagents and methods described in the general FAD-GDH assay protocol in Example 1, the initial rates of the FAD-GDH reaction were measured under serial dilutions of D-glucose from 100 mM to 0 mM and mAb 1-286 from 5 nM to 0 nM. The reaction was allowed to proceed for 30 min, and absorbance was read continuously at 600 nm. The initial rates were calculated as μM / min and plotted against the concentration of glucose in mM (Figure 4).

[0105] A double reciprocal (Lineweaver-Burk) plot was constructed from the data in Figure 4 to determine the mechanism of enzyme inhibition by mAb 1-286. The data, shown in Figure 5, show the intersection of the various lines at a common tangent point on the x-axis to the left of the origin with the various y-intercepts measured. The data indicate an allosteric mechanism of inhibition and exclude a competitive inhibition mechanism.

[0106] Example 3 Characterization of the allosteric site by crystal structure analysis To reduce the heterogeneity of the recombinant FAD-GDH protein for crystallization, DNA encoding non-glycosylated FAD-GDH was designed to remove the amino-terminal signal sequence that directs the nascent folded protein into the secretory pathway. The protein is therefore recombinantly expressed without glycan conjugation by expression in the host organism. Following standard expression conditions for glycosylated FAD-GDH, expression of the non-glycosylated FAD-GDH protein was induced by methanol in a Pichia pastoris clonal transformant. Cell pellets resulting from 2 L expression cultures were collected by centrifugation and stored at -20°C until purification.

[0107] Cytoplasmic proteins were released by resuspending cells in 200 ml of Yeastbuster reagent (EMD / Millipore) supplemented with 1x THP (Millipore), 1 mM MgCl2 (Sigma), and 400 U / ml OmniCleave endonuclease (Lucigen) and incubating at 22°C with constant agitation for 1-2 hours. The lysate was then centrifuged at 18,000 rpm for 30 minutes at 8°C in a JA-20 rotor (Beckman) to pellet insoluble material. The supernatants were pooled, filtered using a 0.22 μm cellulose acetate vacuum filtration unit (Corning), diluted with 800 ml of buffer A (20 mM potassium phosphate, pH 7.0), and mixed with constant agitation for 15 minutes. The mixture was filtered using a 0.45 μm cellulose acetate vacuum filtration unit (Corning) and loaded onto a 5 ml HiTrap SP HP cation exchange column (GE / Cytiva) using an AKTA Pure FPLC (GE / Cytiva). After loading, the column was washed with 50 ml of buffer A, and the protein was then eluted with buffer B (20 mM potassium phosphate, pH 7.0, 1 M NaCl) using a gradient of 0 to 700 mM NaCl. The protein elutes between 200 and 300 mM NaCl as a sharp peak with characteristic absorbance at both 280 nm and 450 nm.

[0108] Peak elution fractions containing nonglycosylated FAD-GDH were pooled and concentrated to ≤5 ml using an Amicon-15 concentrator with a 30 kDa MWCO membrane (Millipore). The sample was then filtered using a MILLEX GV syringe-driven filter unit (Millipore) and injected onto a HiLoad 26 / 600 Superdex 200 pg column equilibrated with Buffer C (20 mM sodium phosphate, pH 7.2, 150 mM NaCl) using 10 ml of Superloop (GE / Cytiva). FAD-GDH typically eluted as a single, symmetrical peak observed between 180 and 220 ml. The peak eluate was again pooled, concentrated to ≥20 mg / ml, frozen, and stored at -80°C. By SDS-PAGE and Coomassie Brilliant Blue staining analysis, the purified, nonglycosylated FAD-GDH protein appeared as a distinct band of approximately 70 kDa.

[0109] The crystal structure of FAD-GDH was solved either alone or in a 1:1 complex with rFab 286. For the enzyme-only structure, a complex of unglycosylated FAD-GDH and rFab 286 was subjected to a sitting-drop sparse matrix screen in JCSG Core Suites I-IV (Nextal Biotech). After 3 days, crystals were observed in Suite I condition F10 (0.1 M phosphate citrate, pH 4.2, 5% PEG 1000, 40% ethanol). Grid screening of the F10 hit condition was performed in 0.1 M sodium acetate, pH 4.2, by varying the PEG 1000 concentration from 3 to 8% and the ethanol concentration from 30 to 45%. Crystals grew to their maximum size after 1 week of incubation at 20°C. Crystals were collected by looping, transferred to a fresh drop of mother liquor containing 20% ​​glycerol for cryoprotection, and flash-frozen in liquid nitrogen. The loop-containing crystals were transferred to a UniPuck and X-ray diffraction experiments were performed. The crystal structure of FAD-GDH was solved from diffraction data extending to 1.94 Å using molecular replacement with Aspergillus flavus FAD-GDH (Protein Data Bank ID: 4YNT) as a search model. The structure revealed the presence of the FAD-GDH enzyme in a packed crystal lattice with no density for rFab 286. It was believed that the ethanol present in the mother liquor disrupted the interaction of FAD-GDH with rFab 286, resulting in crystals of FAD-GDH alone.

[0110] The 1:1 complex structure of FAD-GDH and rFab 286 was formed by mixing the enzyme and Fab at a molar ratio of 1:1.3 and loading onto a HiLoad 16 / 600 Superdex 200 pg column in buffer D (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM TCEP). The peak fractions corresponding to the complex were collected, concentrated to approximately 30 mg / ml, and screened using the sitting drop vapor diffusion method at JCSG Core Suites I-IV (Nextal Biotech). The plates were incubated at 20°C, and initial hits were observed after two days. Four initial hits were identified in buffers ranging from pH 6.0 to 7.5, containing 10% PEG (6K or 8K) as a precipitant and 8% ethylene glycol or 5% MPD as an additive. The best crystals were collected by loop, transferred to a fresh drop of mother liquor containing 35% PEG600 as a cryoprotectant, and flash-frozen in liquid nitrogen. The loop containing the crystal was transferred to a Uni Puck and x-ray diffraction experiments were performed at beamline BL13-XALOC, ALBA, Barcelona, ​​Spain.

[0111] The interface formed between the enzyme and rFab spanned three noncontiguous segments of the enzyme sequence that are proximal to each other in the folded enzyme (Figures 6A–6C). These regions, designated Surface 1, Surface 2, and Surface 3, together form the structural epitope of mAb 1-286 (rFab 286). The rFab was observed to bind adjacent to the putative substrate entry channel of the enzyme but did not appear to sterically obstruct it to any appreciable extent. Direct contacts were formed between the complementarity-determining regions (CDRs) of the rFab heavy chain and residues of FAD-GDH on both Surface 2 (predominantly) and Surface 1. Although direct interactions between rFab 286 and Surface 3 are not supported by the structure, a wall-like structure formed by Surface 3 residues may aid in directionality and / or facilitate rFab interaction with the other two surfaces. Forty-six FAD-GDH residues within a 4 Å distance of rFab 286 were selected for detailed epitope characterization using alanine-scanning mutagenesis, as described in the subsequent Examples. When the apo structure of FAD-GDH was compared with that in the complex with rFab 286, no significant changes in the FAD-GDH structure were detected.

[0112] Example 4 Characterization of allosteric sites by surface mapping Alanine-scanning mutagenesis was used to assess the specific importance of amino acid residues within the three surfaces for binding to rFab 286 and functional response. Amino acid residues within surfaces 1, 2, or 3 of nonglycosylated FAD-GDH (SEQ ID NO: 1) were mutated from their native residue to alanine, except where the native amino acid was either alanine or glycine. A panel of 46 mutant enzymes was expressed as nonglycosylated, His-tagged recombinant proteins in Pichia pastoris cultured in 24-well plates. Protein expression was induced by the addition of 0.5% methanol, and cells were pelleted and lysed with YeastBuster™ Master Mix (Novagen / EMD Millipore). Soluble cytosolic proteins were isolated by centrifugation according to the manufacturer's instructions. FAD-GDH variant activity in the soluble lysates was measured in the absence or presence of mAb 1-286. Non-glycosylated FAD-GDH lacking any alanine mutations consistently showed greater than 80% inhibition by 1 nM mAb 1-286.

[0113] Comparison of activity of alanine mutants Purified, non-glycosylated FAD-GDH and alanine mutant FAD-GDH enzymes were diluted to a final concentration of 100 nM and then serially diluted across the wells of a 96-well plate using enzyme dilution buffer (50 mM potassium phosphate buffer pH 6.5). Enzyme solution (10 μL) was transferred from the enzyme dilution plate to a 96-well assay plate. A reaction master mix (80 μL; 0.6 mM DCPIP and 2.5 mM PES in 50 mM PIPES buffer, pH 6.5, 0.1% v / v Triton) was added before incubation at ambient temperature for 10 minutes. After loading into the plate reader, 10 μL of 1 M D-glucose solution was injected into each reaction well with shaking (500 rpm for 30 seconds). The plate was read for 15 read cycles, with an 87-second time interval between each read cycle. Data from wells that showed reaction kinetics that were too fast or too slow were excluded from activity calculations. Data were trimmed to include only the linear portion of the reaction, and the slope and R-squared value for each reaction were calculated using linear regression from GraphPad Prism 8. Relative activity was calculated using the following equation: Activity % = Slope アラニン変異体 / Tilt 野生型 ×100% was calculated using

[0114] IC50 measurement of antibody 1-286 Preparation of 10x enzyme solution. The 100 nM enzyme solution prepared above was diluted to a final concentration of 6.25 nM. The 1-286 antibody stock solution was diluted to a final concentration of 10 μM and then serially diluted (4-fold serial dilutions) in a 96-well plate using assay buffer. Enzyme solution (10 μL) (unmutated FAD-GDH or alanine mutant) was transferred to a column of the assay plate along with 10 μL of 1-286 antibody dilution. Reaction mix (70 μL) was added to the enzyme and incubated for 10 minutes at ambient temperature. After loading into the plate reader, 10 μL of 1 M D-glucose solution was injected into each well with shaking (500 rpm for 30 seconds). The plate was read for 30 read cycles with an 87-second time interval between each read cycle. Data sets were trimmed to include only the linear portion of each reaction, and linear regression was used to calculate the slope and R-squared value for each reaction well. Percent inhibition was calculated using the slope of each well and the slope of the negative control well (buffer only, no antibody) using the following equation: Inhibition % = (slope 抗体 -TiltNeg ) / tilt Neg ×100% A four-parameter nonlinear regression analysis of log antibody concentration versus percentage inhibition was performed to calculate IC50 values.

[0115] Evaluation of alanine-scanning mutants identified F341, E344, and E348 within surface 2 as the residues most critical for the inhibitory response of FAD-GDH to mAb 1-286. Each of these residues forms contacts with the CDRs of rFab 286. Several mutations in the first half of surface 1 revealed altered GDH activity and reduced the extent of inhibition by 1-286 (Figure 7). Consistent with the lack of polar contacts observed between surface 3 of FAD-GDH and rFab 286 in the structure, alanine mutations introduced into surface 3 were not observed to alter the ability of the mutant enzyme to respond to mAb 1-286.

[0116] Selected C-terminal His-tagged, non-glycosylated FAD-GDH mutants were expanded and purified from Pichia expression shake-flask culture pellets by IMAC and size-exclusion chromatography. Purified proteins were visualized by SDS-PAGE and Coomassie Brilliant Blue staining (Figure 8). The mutant enzymes were then tested for in vitro GDH activity. To determine the IC50 value of mAb 1-286 compared to wild-type, the antibody was titrated against a defined amount of non-glycosylated FAD-GDH, and the resulting GDH activity was measured (Table 3). In particular, the titration curves of inhibition of mAb 1-286 and wild-type (non-glycosylated, no mutations) FAD-GDH, F341A, E344A, and E348A, are shown in Figure 9. IC50 values ​​were calculated from sigmoidal curve fitting: wild-type (WT), 0.1 nM; F341A, 2.0 nM; E344A, 71.4 nM; E348A, 20.2 nM. Higher IC50 values ​​correlate with poorer responses, so the order of stringency is: E344A > E348A >> F341A. The difference between the inhibition of the DQETAAAA and DQTAAA combination mutants in Figure 7 also highlights the importance of E344 of FAD-GDH in response to mAb 1-286. Based on these data, it was concluded that surface 2 is the major surface contributing to the enzyme-antibody interaction that results in inhibitory function.

[0117] [Table 4]

[0118] Example 5 Characterization of the inhibition of epitope-grafted FAD-GDH Epitope-grafted FAD-GDH was used to assess inhibition of enzyme activity in the presence of antibodies or antibody fragments specific for particular epitopes. HA epitope ( YPYDVPDYA) were inserted into positions 341 (341HA) and 358 (358HA) of FAD-GDH(19031) (SEQ ID NOs: 55 and 56), and the constructs were purified as described elsewhere herein. Following the general FAD-GDH assay protocol of Example 1, 1.5 nM of enzyme was treated with antibodies at concentrations ranging from 320 nM to 0 nM. Neither 341HA nor 358HA showed a response to an irrelevant control antibody (α-Myc Ab). However, both 341HA and 358HA showed dose-dependent inhibition by α-HA Ab. 341HA was not inhibited by 1-286 Ab, whereas 358HA showed dose-dependent inhibition by 1-286 Ab (Figure 10). Because the 341 site of FAD-GDH is central to the binding site for 1-286 and 358 is peripheral to the interaction, an epitope inserted at 341 but not at 358 is likely to disrupt 1-286 binding.

[0119] The VHH for FAD-GDH (19031) was identified by phage display. Following the general FAD-GDH assay protocol in Example 1, 1.5 nM of enzyme was treated with VHH at concentrations ranging from 40 μM to 0 nM. Both VHH-1 and the epitope-grafted VHH-859 showed dose-dependent inhibition of FAD-GDH, similar to the 1-286 Ab (Figure 11). Based on these results, small VHH format-specific binding proteins could bind and inhibit enzyme activity as effectively as large format IgG and Fab. V5( IPNPLLGLD ) and TnI( ISASRKLQS The epitopes were inserted at various positions into FAD-GDH(19031) (see SEQ ID NOS: 5-31 and 43 and Table 4) and expressed in Pichia pastoris as described herein. Following the general FAD-GDH assay protocol in Example 1, 10x enzyme dilutions were prepared by two-fold serial dilutions of Pichia expression supernatants of each epitope-grafted construct. The supernatant dilution concentration with optimal reaction rate was identified, showing a linear absorbance decrease of approximately 1.0 AU over 15 reading cycles (or a 20-minute reading window). The identified supernatant dilution (50 μL) was transferred to a new 96-well plate and used in the general FAD-GDH assay protocol described in Example 1. The final antibody concentration (1×) was 50 nM for both the α-V5 and 1-286 antibodies.

[0120] Various TnI epitope-grafted constructs (see Table 4) were screened using both 1-286 mAb and α-TnI mAb at a final concentration of 50 nM. The calculated percent inhibition is summarized in the graph below. FAD-GDH (19031) does not respond to α-TnI antibody at a concentration of 50 nM, but the TnI epitope-grafted constructs show varying degrees of inhibition by α-TnI antibody (Figure 13). Inhibitory responses to anti-TnI Abs were observed in 339TN, 340TN, 341TN, 342TN, 343TN, 344TN, 356TN, 357TN, and 358TN; the rest of the constructs, 356TN, 357TN, and 358TN, showed weak responses to 1-286 mAb because the epitope-grafted site is located within the binding site of 1-286. In constructs 356TN, 357TN, and 358TN, epitopes were inserted towards the ends of the 1-286 binding site, thus largely preserving the response to 1-286.

[0121] Various V5 epitope grafts (see Table 4) were screened using both 1-286 mAb and α-V5 mAb at a final concentration of 50 nM. The calculated percent inhibition is summarized in the graph below. FAD-GDH (19031) does not respond to α-V5 antibody at 50 nM concentration, but V5 epitope grafts show varying degrees of inhibition by α-V5 antibody (Figure 12).

[0122] [Table 5]

[0123] FAD-GDH assay using mouse serum To evaluate the inhibitory activity of antibodies raised against 19031 FAD-GDH, FAD-GDH activity is measured spectrophotometrically (2,6-dichloroindophenol sodium salt hydrate (DCPIP)) in the absence or presence of immunized animal serum. Samples are prepared in 1 ml of assay reaction mixture (ARM) (0.1 M D-glucose, 34.9 mM PIPES / Triton buffer, 0.14 mM phenazine methosulfate (PMS), 0.68 mM DCPIP) in a quartz glass cuvette containing a stir bar, pre-warmed to 37 °C for 35-45 seconds. The reaction is initiated by the addition of 0.25 μg / mL enzyme (33.3 μl) in ED buffer. The amount of enzyme required for a linear response was titrated with saturating glucose to a final concentration of 194 mM.

[0124] Figure 14A shows the kinetics of DCPIP reduction by FAD-GDH in initial studies. These were calculated using linear regression. The three columns highlighted in gray are shown as filled gray circles in the graph. The DCPIP assay shows a linear response across the amount of FAD-GDH added to the cuvette. There is a good linear fit up to and including a final concentration of 116 ng / ml in the cuvette. An enzyme concentration midway through the linear portion of the curve is chosen and the glucose concentration is titrated. Followed by 1.25 μg / ml FAD-GDH (40 ng / ml in the cuvette). The mean specific activity was determined as 99% of the specific activity of the indicated amount from the blank difference rate per minute as shown below: [[(average enzyme ΔA600 / min)-(blank ΔA600 / min)]×3.1×1000] / [(16.8×1×0.1)×0.25mg / ml]= 382.33U / mg

[0125] To determine the robust output range with subsaturating substrate concentrations, FAD-GDH assays were performed with a glucose titration. Following the general FAD-GDH assay protocol, each reaction contained 12.5 μl of diluted serum in 50 mM PIPES / Triton buffer, 2 mM phenazine methosulfate (PMS), and 0.17 mM dichlorophenol indophenol (DCPIP), and 0.04 μg FAD-GDH. Various concentrations of D-glucose solution (12.5–100 mM) were added last to initiate each reaction. Absorbance at 600 nm was measured for 30 minutes at 37°C. Enzyme rates were calculated using linear regression. The DCPIP assay shows a nearly linear response of 1.25 μg / ml FAD-GDH (40 ng / ml in the cuvette) between 12.1 and 48.5 mM glucose. The apparent Km for glucose is 64.7 mM under these conditions.

[0126] GDH assays were performed with polyclonal sera from normal mice to calibrate FAD-GDH activity in 96-well plates according to a general FAD-GDH assay protocol. Each reaction contained 12.5 μl of diluted serum in 50 mM PIPES / Triton buffer, 2 mM phenazine methosulfate (PMS), and 0.17 mM dichlorophenol indophenol (DCPIP), 0.04 μg FAD-GDH, and the equivalent of 20 mM D-glucose solution was added to initiate each reaction. Absorbance at 600 nm was measured for 30 minutes at 37°C. Enzyme rates were calculated using linear regression (Figures 16A and 16B). The DCPIP assay was used to determine the degree of inhibition of activity by normal mouse sera. Negligible interference was observed with 1:25 or 1:50 dilutions of normal mouse sera, far exceeding the range of serum in diagnostic assays. To increase the dynamic range for detecting inhibition, the amount of glucose in the assay was increased to 40 mM.

[0127] GDH assays were performed with polyclonal serum from mice immunized with FAD-GDH to calibrate FAD-GDH activity in 96-well plates according to a general FAD-GDH assay protocol. Each reaction contained 12.5 μl of diluted serum in 50 mM PIPES / Triton buffer, 2 mM phenazine methosulfate (PMS), and 0.17 mM dichlorophenol indophenol (DCPIP), and 0.04 μg FAD-GDH. 12.5 μl of 40 mM D-glucose solution was added to initiate each reaction. Absorbance at 600 nm was measured at 37°C for 30 minutes. Enzyme rates were calculated using linear regression (Figures 17A, 17B, and 1). The DCPIP assay was used to determine the degree of inhibition of FAD-GDH activity by various samples of FAD-GDH-immunized mouse serum. Of these, the most inhibitory sera consistently included Abs 77, 90, 81, 92, and 78.

[0128] Example 6 Inhibition release assay using the 358HA construct This example assays de-inhibition of the enzyme by the 358HA epitope-grafted construct and HA peptide. The general FAD-GDH assay protocol of Example 1 was performed using 1.5 nM of both the control enzyme (ungrafted FAD-GDH) and 358HA, final antibody concentrations from 5 nM to 0 nM, 2-fold serum dilutions, and final HA peptide concentrations from 1 μM to 0 nM, 4-fold serial dilutions. The percentage of inhibition decreased as the antigen (HA) peptide concentration increased, indicating successful competition of the antigen with the enzyme-bound antibody, resulting in the release of the antibody-bound enzyme and reactivation of the enzyme's catalytic function. Deinhibition was observed in a dose-dependent manner at various antibody concentrations (5 nM, 2.5 nM, 1.3 nM, and 0.6 nM) (Figure 18). No inhibition or deinhibition was observed with non-transplanted FAD-GDH.

[0129] Example 7 Epitope grafting into six Mucor FAD-GDH sequences (Mucor prainii, Mucor guilliermondii, Mucor hiemalis, Mucor subtilisimus, Mucor circinelloides, and Mucor ambygus) Six Mucor epitope-grafted constructs were expressed in Pichia pastoris. Enzyme dilutions were prepared by serial two-fold dilutions of Pichia expression supernatants of each epitope-grafted construct. The general FAD-GDH assay protocol from Example 1 was used as described in Example 5. Antibody inhibition studies were also completed as in Example 5. The final antibody concentration was 50 nM for all four antibodies tested (α-HA, α-HNL, α-TnI, and 1-286). The percentage of inhibition of Mucor (Mucor prainii, Mucor guilliermondii, Mucor hiemalis, Mucor subtilisimus, and Mucor ambigus) epitope grafting by the 1-286 antibody and anti-epitope antibodies (α-HA / α-HNL / α-TNI) is shown in Figures 19A-F. * Epitope grafts indicated by " did not show viable enzymatic activity, and therefore percent inhibition was not measured. Both the in-house, non-grafted FAD-GDH (19031) and wild-type Mucor FAD-GDH showed no response to anti-epitope antibodies (α-HA / α-HNL / α-TNI). Six Mucor graft panels had at least one epitope graft that responded to anti-epitope antibodies. These epitope grafts are indicated by " above the percent inhibition bar. * " is shown.

[0130] Example 8 VHH and Fab inhibitors Inhibition by VHH-1 and VHH-859 material 19031 FAD-GDH Purified VHH-1, VHH-10, VHH-859, and VHH-898 method 10x substrate dilution made by serially diluting (2 / 3 fold) 1M glucose with DI water Final dilution concentrations (1x) ranging from 100 mM to 0 mM, 2 / 3-fold serial dilutions. Final VHH-1 concentrations (1x) ranging from 2 μM to 0 μM, 2-fold serial dilutions. Final VHH-859 concentrations (1x) ranging from 1 μM to 0 μM, 2-fold serial dilutions. The slope and R-squared value for each reaction well was calculated using linear regression analysis from GraphPad Prism 8. The slope and substrate concentration were plotted for each reaction well, and the Michaelis-Menten equation was applied to fit the data.

number

[0131] Inhibition test of Fab IO3 material 19031 FAD-GDH Purified IgG IO3 (4.7 mg / mL) and purified Fab IO3 (enzymatically digested from IgG IO3, 5.4 mg / mL) method Final glycosylated FAD-GDH concentration (1x) 1.0 nM. Final non-glycosylated FAD-GDH concentration (1x) 0.7 nM. Final antibody 1-286, IgG 103, and Fab 103 concentrations (1x) are 100 nM, 100 nM, and 1.5 μM, respectively. result Both IgG 103 and its digested antibody fragment Fab 103 inhibit FAD-GDH (FIG. 20C).

[0132] Example 9 V5 epitope-grafted antibody inhibition titration and V5 antigen detection Step 1: Determine the range material Non-grafted FAD-GDH (19031) and V5 epitope-grafted (339V5 (SEQ ID NO: 4) / 340V5 (SEQ ID NO: 5) / 341V5 (SEQ ID NO: 6)) FAD-GDH constructs Anti-V5 antibody, mouse monoclonal 1 mg / mL, Sigma (Cat. No. V8012-50UG) method Final enzyme concentrations (1x, non-grafted FAD-GDH (19031) and three V5 epitope-grafted FAD-GDH) were 0.625 nM. Final antibody (α-V5 mAb) concentrations (1x) ranged from 650 nM to 0 nM, with two-fold serial dilutions. result The calculated percent inhibition at various α-V5 antibody concentrations is plotted in Figure 21A. All V5 epitope grafts showed dose-dependent inhibition by α-V5 antibodies. Ungrafted FAD-GDH (19031) showed no inhibition at any given α-V5 antibody concentration.

[0133] Step 2. Relief of V5 epitope-grafted enzyme inhibition by a single dose of V5 peptide material α-V5 antibody, mouse monoclonal 1 mg / mL, Sigma (catalog no. V8012-50UG) Non-grafted and 341V5 epitope-grafted FAD-GDH V5 peptide (CGKPIPDPLLGLDST), 10 mg / mL, Sigma (catalog no. V7754-4MG); resuspended in water at 10 mg / mL method Final enzyme concentrations (1x) were 3 nM for V5 epitope grafted (341V5) and 1.5 nM for control enzyme (ungrafted FAD-GDH). Final α-V5 antibody concentration (1x) was 300 nM. Final V5 peptide concentration (1x) was 3 μM. Percent inhibition was calculated using the following formula:

[0134] Inhibition % = (slope 試料 -Tilt Neg ) / tilt Neg ×100% The slope of each sample well and the slope of the negative control wells (buffer only, no antibody or V5 peptide) were calculated using the slope of each sample well and the slope of the negative control wells (buffer only, no antibody or V5 peptide) using the slope of each sample well. result The α-V5 antibody (enzyme + Ab + buffer) showed approximately 50% inhibition of 341V5 FAD-GDH but no inhibition of non-grafted FAD-GDH. The percent of inhibition was reduced by approximately 9% with the 341V5 epitope graft in the presence of the V5 peptide (enzyme + Ab + Ag), whereas the V5 peptide had no effect on the non-grafted enzyme (indicated by the arrow in Figure 21B).

[0135] Step 3. V5 peptide titration to release V5 epitope-grafted enzyme inhibition Ingredients (same as above) method The final enzyme concentration (1x) was 3 nM for the V5 epitope-grafted (341V5) and 1.5 nM for the control enzyme (ungrafted FAD-GDH). The final α-V5 antibody concentration (1x) was 300 nM. The final V5 peptide concentration (1x) was 3 μM–0 μM, in a 2-fold serial dilution. Percent inhibition was calculated using the following formula: Inhibition % = (slope 試料 -Tilt Neg ) / tilt Neg ×100% The slope of each sample well and the slope of the negative control wells (buffer only, no antibody or V5 peptide) were calculated using the slope of each sample well and the slope of the negative control wells (buffer only, no antibody or V5 peptide) using the slope of each sample well. Log V5 peptide concentration and percentage inhibition are plotted. result The V5-peptide dose-dependently reversed the inhibition of the α-V5 antibody against the V5 epitope-grafted FAD-GDH (341V5) (FIG. 21C).

[0136] Example 10 TnI epitope-grafted antibody inhibition titration material Purified TnI epitope-grafted enzymes (358TN1 (SEQ ID NO: 28), 358TN4 (SEQ ID NO: 29), and 358TN8 (SEQ ID NO: 30)) α-TnI 19C7 Mouse IgG, 2mg / mL, Abcam, Mouse mAb against cardiac troponin 19C7 method Final enzyme concentrations (1x) for 358TN1, 358TN4, and 358TN8 enzymes were 1.5 nM, 3.5 nM, and 3.5 nM, respectively. Final antibody (α-TnI mAb) concentrations (1x) were 1.0 μM to 0 nM, in 4-fold serial dilutions. result The calculated percent inhibition at various α-TnI antibody concentrations is plotted in Figure 22. All three TnI epitope grafts (358TN1, 358TN4, and 358TN8) showed dose-dependent inhibition by α-TnI antibodies.

[0137] Example 11 Cardiac troponin I (TNI), V5, HA, c-Myc, and FLAG antibody inhibition material Epitope-grafted constructs from Pichia expression supernatants included six TnI (19C7) epitope-grafted (341TN1 (SEQ ID NO: 25), 341TN4 (SEQ ID NO: 26), 341TN8 (SEQ ID NO: 27), 358TN1 (SEQ ID NO: 28), 358TN4 (SEQ ID NO: 29), and 358TN8 (SEQ ID NO: 30)), 24 V5 epitope-grafted (341VL1-341VL11, 341VLFL, 358VL1-358 VL11, and 358VLFL (SEQ ID NOs: 31-54), two FLAG epitope grafts (341FLAG (SEQ ID NO: 59) and 358FLAG (SEQ ID NO: 60)), two c-Myc epitope grafts (341Myc (SEQ ID NO: 57) and 358Myc (SEQ ID NO: 58)), and two hemagglutinin (HA) epitope grafts (341HA (SEQ ID NO: 55) and 358HA (SEQ ID NO: 56)).

[0138] α-c-Myc 9E10 mouse mAb, 1 mg / mL, Millipore (Cat. No. MABE282) α-HA mouse mAb, 0.5 mg / mL, Sigma (Cat. No.: SAB1305536-400UL) α-Flag M2 9E10 mouse mAb, 1 mg / mL, Millipore (Cat. No. F1804-200UG) α-V5 antibody, mouse mAb 1mg / mL, Sigma (Cat. No.: V8012-50UG) α-Tn1 19C7 Mouse IgG, 2mg / mL, Abcam, Mouse mAb against cardiac troponin 19C7 The final antibody concentration (1x) was 100 nM for all antibodies. result Non-transplanted FAD-GDH did not show an inhibitory response to either anti-epitope antibody. All of the FAD-GDH grafted enzymes bearing various epitopes (V5 / TnI / Flag / HA / Myc) showed inhibitory responses to the corresponding anti-epitope antibodies at a concentration of 100 nM, as shown in FIG.

[0139] Example 12 HNL analysis target detection material Purified 358HNL-H3 enzyme (SEQ ID NO: 116) Rabbit α-HNL mAb, 0.75 mg / mL, Abcam (catalog no. ab206427) method The final enzyme concentration (1x) was 1.25 nM for 358HNL-H3. The final antibody (α-HNL) concentration (1x) was 450 nM to 0 nM, diluted 4-fold. result The percent of inhibition was calculated at various α-HNL antibody concentrations and is shown in Figure 24 A. HNL epitope-grafted 358HNL-H3 showed dose-dependent inhibition by α-HNL antibody.

[0140] Abolition of HNL epitope-grafted antibody inhibition by a single dose of HNL peptide material Purified 358HNL-H1 (SEQ ID NO: 117) and 358HNL-H3 (SEQ ID NO: 116) Rabbit α-HNL mAb, 0.75 mg / mL, Abcam (catalog no. ab206427) 340V5 peptide SGSGPGSQPGEFTLGNIKS (SEQ ID NO: 128) (reconstituted with DI water to 20 mg / mL) 341V5 peptide SGSGQPGEFTLGNIKSYPG (SEQ ID NO: 129) (reconstituted with DI water to 20 mg / mL) method Final enzyme concentrations (1x) were 0.5 nM for 358HNL-H1 and 1.5 nM for 358HNL-H3. Final α-HNL antibody concentrations (1x) were 25 nM. Final 340V5 and 341V5 peptide concentrations (1x) were 4 μM. Reactions were performed in wells of a multiwell plate.

[0141] Percent inhibition was calculated using the following formula: Inhibition % = (slope 試料 -Tilt Neg ) / tilt Neg ×100% The slope of each sample well and the slope of the negative control wells (buffer only, no antibody or HNL peptide) were calculated using the slope of each sample well and the slope of the negative control wells (buffer only, no antibody or HNL peptide) using the slope of each sample well. result The α-HNL antibody showed approximately 20% inhibition in 358HNL-H1 and 60% inhibition in 358HNL-H3 (antibody + Ab + buffer). The percent inhibition was reduced to approximately 0% in 358HNL-H1 in the presence of the 340V5 and 341V5 peptides. Similarly, the percent inhibition was reduced to approximately 30% in 358HNL-H3 in the presence of the 340V5 and 341V5 peptides (enzyme + Ab + Ag). See Figure 24B.

[0142] Example 13 NTproBNP antigen detection material Purified NTproBNP epitope-grafted 341BP (SEQ ID NO: 63) and 358BP (SEQ ID NO: 64) Mouse α-NTproBNP antibody, 1 mg / mL, Biorad (Cat. No. MCA2641) Mouse α-NTproBNP antibody, 3.5 mg / mL, Novus (Cat. No. NB200-439) Purified NTproBNP antigen 4.5mg / mL (E248770251-22-005) method FAD-GDH inhibition assays were performed using the epitope-grafted enzymes 341BP and 358BP. The final enzyme concentration (1×) was 1 nM for both 341BP and 358BP. The final α-NTproBNP antibody concentration (1×) was 500 nM. The final NTproBNP antigen concentration (1×) was 5 μM. Reactions were performed in wells of a multiwell plate. Percent inhibition was calculated using the following formula: Inhibition % = (slope 試料 -Tilt Neg ) / tilt Neg ×100% The slope of each sample well and the slope of the negative control wells (buffer only, no antibody or NTproBNP antigen) were calculated using the slope of each sample well and the slope of the negative control wells (buffer only, no antibody or NTproBNP antigen) using the slope of each sample well. result The Biorad α-NTproBNP antibody showed approximately 25% inhibition of 341BP and 7% inhibition of 358BP (antibody + Ab + buffer). The percent of inhibition was reduced to approximately 0% in the presence of 5 μM NTproBNP antigen. Similarly, the Novus α-NTproBNP antibody showed approximately 7% inhibition of 358BP (enzyme + Ab + buffer). The percent of inhibition was reduced to approximately 0% in the presence of 5 μM NTproBNP antigen (Figure 25). The Novus antibody showed very little inhibition of 358BP, therefore, de-inhibition was not confirmed.

[0143] Example 14 Deinhibition titration In this example, enzyme deinhibition was evaluated using different concentrations of inhibitor and antigen. In these examples, non-transplanted FAD-GDH (purified 19031 FAD-GDH 38 mg / mL (E239543171-22-011)) was used as the enzyme, and inactive FAD-GDH was used as the analyte (purified 19031HHAA FAD-GDH 63 mg / mL (E247909068-22-015)). Inhibition and deinhibition were evaluated using mouse 1-286 mAb (mouse 1-286 mAb, 8 mg / mL (E241086302-18-013)) as the inhibitor, which binds to the allosteric site on non-transplanted FAD-GDH and inhibits FAD-GDH activity.

[0144] Non-grafted antibodies were used at a final enzyme concentration (1x) of 0.5 nM. 1-286 Ab was used at concentrations titrated from 4 nM to 0 nM by two-fold serial dilutions. Reactions were performed in wells of a multi-well plate. Antigen was used at concentrations titrated from 25 nM to 0 nM by two-fold serial dilutions. Percent inhibition was calculated using the following formula: Inhibition % = (slope 試料 -Tilt Neg ) / tilt Neg ×100% The slope of each sample well and the slope of the negative control well (buffer only, no antibody antigen) were calculated using the formula: result An antigen titration curve at 0 nM antibody showed no increase or decrease in percent inhibition, confirming that the deactivated FAD-GDH used as antigen showed no detectable residual enzyme activity within the concentrations tested. Increasing the antigen concentration reduced the percent inhibition. This dose-dependent decrease in inhibition was repeatedly observed at various concentrations of antibody. This demonstrated successful competition of the antigen with the enzyme-bound antibody, releasing the antibody-bound enzyme and reactivating the enzyme catalytic function (Figure 26).

[0145] Example 15 Improved stability by introducing disulfide bonds This example demonstrated that the introduction of a disulfide bond into FAD-GDH resulted in a significant improvement in stability in both time and temperature stress tests. material Purified WT 19031 (SEQ ID NO: 1), 358HA (SEQ ID NO: 56) and 358HACC (SEQ ID NO: 118) method The enzyme was diluted to 1 mg / mL with PBS buffer. The diluted enzyme samples were divided into aliquots and initially frozen at -80°C. At each time point, one aliquot of each sample was thawed and stored in a 37°C incubator. Sample activity was measured at a final concentration of 0.5 nM by FAD-GDH activity assay. result Figure 27 shows the enzyme sample activity measured at each time point. Compared to the ungrafted 19031 enzyme, 358HA shows a significant loss of activity over extended time points, indicating suboptimal stability. 358HACC, a disulfide bond-containing construct using 358HA as the parent sequence, shows a significant improvement in stability.

[0146] Example 16 Discovery of a second allosteric site on FAD-GDH Phage display of nonglycosylated FAD-GDH was used to identify the second allosteric site of FAD-GDH. An aliquot of the Superhuman 2.0 library from Distributed Bio was panned against nonglycosylated FAD-GDH 19031. Blocked, neutravidin-coated beads were used for deselection. Selection was performed in solution using biotinylated nonglycosylated FAD-GDH, which was then captured on magnetic neutravidin-coated particles. The nonglycosylated FAD-GDH bait was decreased in concentration from a maximum of 100 nM to 5 nM over rounds 1 through 4. Wash intensity was increased from 6 × 30 s in round 1 to 2 × 30 min washes and 4 × 30 s in round 4. After each round, phage were harvested and collected at least 1 × 10 11 Phage were amplified to a titer. Output titers are shown in Table 5 for rounds 1, 2, and 4.

[0147] [Table 6] To confirm the enrichment of scFvs against the non-glycosylated FAD-GDH target, an ELISA assay was completed. A 96-well streptavidin-coated plate was blocked and coated with non-glycosylated FAD-GDH. Phage output from rounds 2–4 was diluted 1:1 in a 12-point dilution series in PBST. Phage was added to the FAD-GDH-coated wells, followed by the addition of an anti-M13 phage HRP conjugate. HRP activity was measured by reading the absorbance at 492 nm. The absorbance was then plotted against the phage dilution. An increasing signal over the rounds indicated that the phage output contained scFvs specific for the FAD-GDH target.

[0148] ER2738 cells containing phagemids selected from rounds 3 and 4 of the anti-FAD-GDH SuperHuman 2.0 campaign were streaked onto 225mm x 225mm 2XYT agar plates containing 2% glucose, Carb-100, and Tet-20 at 1:100,000x and 1:1,000,000x dilutions of the original glycerol stock and incubated overnight at 30°C. Individual clones representing single antibody clones on the phagemid were aliquoted into individual wells of a 96-deep-well plate prepared with 500µl of 2XYT medium, 2% glucose, and Carb-100 per well. Plates were covered with breathable lids and grown overnight at 37°C in a short-throw shaking incubator at 900 RPM. After overnight growth, wells were allowed to confluent with cells. A new set of 96-deep-well plates was prepared with 1 ml of 2XYT medium and Carb-100. The confluent growth (20 μL) was transferred to the new 96-deep-well plates and returned to the shaking incubator at 37°C and 900 RPM for 2.5 hours. Storage medium (20% glycerol + 2XYT) was added in a volume of 500 μL to the remaining medium in the confluent 96-well plates to create temporary glycerol stocks for storage at -80°C.

[0149] Activity assay screening of scFVs for FAD-GDH inhibition An activity assay mixture was prepared with final concentrations of 80 mM PIPES + 0.2% Triton; 5.36 mM PES; 0.68 mM DCPIP; and 57.14 pM enzyme, non-glycosylated. Nunc clear-bottom, black-walled plates were filled with 25 μl of prepared periplasmic extract (PPE), 85 μl of potassium dihydrogen phosphate, pH 6.5, 0.1% Triton X-100, and 75 μl of the reaction mixture prepared above. The final concentration of enzyme in the reaction was 42 pM. The reaction was initiated with 20 μl of 1 M glucose, and the plate was read every 5 minutes at 600 nm for 2 hours. Percent inhibition was calculated for each clone.

[0150] The streptavidin-coated plate was blocked with 200 μl of blocking solution. Then, 2 μg / ml of biotinylated, non-glycosylated FAD-GDH was diluted in PBS, and 100 μl was added to each well. After coating, the plate was washed, and then 50 μl of PPE and 50 μl of PBS were added to each well and incubated for 1 hour. After incubation, the plate was washed, and anti-V5-HRP antibody was diluted 1:5000 in the blocker, and 100 μl was added to each well. After 1 hour of incubation, the plate was washed, and OPD substrate was prepared. 100 μl of substrate was added to each well, and the plate was developed for 4 minutes. After 4 minutes, 100 μl of 1N sulfonic acid was added to each well, and the absorbance was then read at 492 nm (Figure 28).

[0151] Clones that showed binding and inhibition of FAD-GDH were selected and grown in 3 ml cultures containing LB plus Carb-100. The cultures were spun down, plasmids were prepared, and phagemids were extracted. The phagemids were then used as templates for Sanger sequencing. Thirteen unique clones showed binding and inhibition of FAD-GDH.

[0152] A competitive ELISA was used to assess whether the 13 identified anti-FAD-GDH scFV inhibitors bind to epitopes different from the 1-286 antibody epitope. ELISA plates were coated with the 1-286 anti-FAD-GDH antibody by diluting the 1-286 antibody to a concentration of 2 μg / ml in PBS, and 100 μl of diluted antibody was dispensed into each well. The plates were incubated for 2 hours, washed, and blocked with blocking buffer by adding 200 μl of blocking buffer to each well, which were then allowed to incubate for 1 hour. The plates were washed, and 2 μg / ml of nonglycosylated FAD-GDH diluted in the block was added to each well in a volume of 100 μl and allowed to incubate for 1 hour. After incubation, the plates were washed, and scFV PPE from each of the 13 identified FAD-GDH inhibitors was added to the wells. The plates were incubated for 1 hour, washed, and then 100 μl of 0.1 μg / ml anti-V5-HRP conjugate was added. The plates were incubated for 1 hour and then read by adding 100 μl of prepared OPD substrate to each well and allowing them to develop for 3 minutes. Subsequently, 100 μl of 1N sulfonic acid was added to each well, and the plates were then read at 492 nm. The absence of a signal indicated that the antibodies competed for the same site on FAD-GDH and that they bound to the same or similar epitopes. The presence of a signal indicated that the scFvs were able to bind to FAD-GDH even in the presence of the 1-286 antibody, and therefore, that their binding site was different from the 1-286 epitope (Table 6). scFVs #3, 6, 7, and 13 appeared to bind to epitopes different from 1-286.

[0153] [Table 7]

[0154] scFv#3, 6, 7, and 13 were reformatted for expression as IgG in CHO. Abbott pHybe vectors were used for expression, DNA was synthesized, and sequence verified by a third party. Expi-CHO cells were cultured at approximately 6.0 x 10 per ml. 6 The cells were cultured to a cell density of 100 cells and a total volume of 1 L per construct. Using the ThermoFisher Expi-CHO transfection kit and protocol, 1 μg / ml DNA of both the heavy and light chain plasmids expressing inhibitory anti-FAD-GDH IgG3, 6, 7, and 13 was transfected into each liter of Expi-CHO cells. The cells were returned to the incubator and shaken at 140 RPM, 8% CO2, 80% humidity, and 37°C. Cell viability was monitored over a 10-day period. When viability fell below 80%, all cultures were harvested by spinning in a floor centrifuge and the supernatant was retained. The supernatant was filtered through a 0.4 μm filter and stored at 4°C.

[0155] The supernatants were purified using a HiTrap 5ml MAbSelect Xtra column on an AKTA Pure. Each supernatant was loaded onto the MAbSelect column at a flow rate of 5ml / min. After the supernatant was fully loaded, the column was washed with PBS, and the protein was then eluted with a citrate pH gradient ranging from 0.1M citrate at pH 4.0, 3.6, 3.3, and 2.8. Fractions were eluted into 1M Tris Tris-HCl pH 9.0 for neutralization. Peak fractions were pooled and loaded onto a HiLoad 26 / 600 Superdex 200pg gel filtration column. Peak fractions were collected and pooled.

[0156] A competitive ELISA was used to assess whether the four identified and reformatted anti-FAD-GDH IgG inhibitors bound to sites distinct from the 1-286 antibody epitope. BRAND plastic ELISA plates were coated with the 1-286 anti-FAD-GDH antibody by diluting the 1-286 antibody to 2 μg / ml in PBS, and 100 μl of diluted antibody was dispensed into each well. The plates were incubated for 2 hours, washed, and then blocked with blocking buffer by adding 200 μl of blocking buffer to each well and allowing them to incubate for 1 hour. The plates were washed, and 2 μg / ml nonglycosylated FAD-GDH diluted in block was added to each well in a volume of 100 μl and allowed to incubate for 1 hour. After incubation, the plates were washed, and serial dilutions of inhibitory IgG (IO-3, IO-6, IO-7, and IO-13) prepared in block were added to the wells. The plates were incubated for 1 hour, washed, and then 100 μl of 0.1 μg / ml donkey anti-human (H+L)-HRP conjugate was added. The plates were incubated for 1 hour and then read by adding 100 μl of prepared OPD substrate to each well and allowing them to develop for 3 minutes. Subsequently, 100 μl of 1N sulfonic acid was added to each well, and the plates were then read at 492 nm. The absence of a signal indicated that the antibodies competed for the same site on FAD-GDH and that they bound to the same or similar epitope. The presence of a signal indicated that the antibody was able to bind to FAD-GDH even in the presence of the 1-286 antibody. IO-3 was the only full-length IgG that did not compete with 1-286, indicating that it bound to a site different from that of 1-286 (Figure 29).

[0157] To further confirm the finding that IO-3 did not compete with 1-286 and to test whether IO-3 binds to nonglycosylated 19031 FAD-GDH or glycosylated 19031 FAD-GDH (WT-FAD-GDH), two different ELISA formats were designed. A diagram summarizing the different assay formats is shown in Figure 30A. In the first ELISA, BRAND plastic plates were coated with 1-286 anti-FAD-GDH antibody by diluting the 1-286 antibody to a concentration of 2 μg / ml in PBS and dispensing 100 μl of diluted antibody into each well (Figure 30A, left). In the second ELISA, BRAND plastic plates were coated with IO-3 anti-FAD-GDH antibody by diluting the IO-3 antibody to a concentration of 2 μg / ml in PBS and dispensing 100 μl of diluted antibody into each well (Figure 30A, right). The plates were incubated for 2 hours, washed, and blocked with blocking buffer by adding 200 μl of blocking buffer to each well and incubating them for 1 hour. The plates were washed, and 2 μg / ml of non-glycosylated FAD-GDH or glycosylated FAD-GDH (WT FAD-GDH) was diluted and added to each well in a volume of 100 μl and incubated for 1 hour. After incubation, the plates were washed, and serial dilutions of either IO-3 (on 1-286-coated plates) or 1-286 (on IO-3-coated plates) were prepared in block, and 100 μl was added to each well. The plates were incubated for 1 hour, washed, and then 100 μl of 0.1 μg / ml donkey anti-human (H+L)-HRP conjugate was added to ELISA format 1, and 100 μl of goat anti-mouse (H+L)-HRP conjugate was added to ELISA format 2. The plates were incubated for 1 hour and then read by adding 100 μl of prepared substrate to each well and allowing them to develop for 3 minutes, after which 100 μl of 1N sulfonic acid was added to each well and the plates were then read at 492 nm.

[0158] IO-3 did not compete with 1-286 using non-glycosylated FAD-GDH, but glycosylated WT FAD-GDH showed no binding of IO-3 (Figures 30B and 30C), indicating that glycosylation somehow interferes with the binding of IO-3 to FAD-GDH.

[0159] Colorimetric FAD-GDH activity assay with inhibitory IgG3, 6, 7, and 13. A dilution series was prepared for each of inhibitor IgGs 3, 6, 7, and 13 in assay buffer (50 mM PIPES-NaOH and 0.1 mM Triton X-100). The dilution series was prepared so that the highest final concentration of IgG in the assay was 400 nM, and four-fold serial dilutions were made down to a final IgG concentration of 0.024 nM in the assay. Assays were assembled in black-walled 96-well plates with clear bottoms and contained diluted antibody and final concentrations of 30 mM PIPES, 2 mM PES, 0.5 mM DCPIP, and 1 μM FAD-GDH. The reaction was initiated with 10 μl of 1 M glucose, and absorbance was measured at 600 nm for 40 minutes. Each of the inhibitory IgGs showed at least some inhibition, with IgG3 and IgG6 showing the highest percentage of inhibition overall (Figure 31).

[0160] Example 17 Demonstration of anti-epitope antibody inhibition of Aspergillus flavus FAD-GDH Aspergillus flavus FAD-GDH (SEQ ID NO: 131) was modified with epitopes in a region of the enzyme corresponding to the region successfully modified in the Mucor FAD-GDH enzyme described above. The Mucor FAD-GDH insert, comprising surface 2, is a large protruding structure on the surface of the enzyme, containing both undefined and helical secondary structure segments. Aspergillus flavus FAD-GDH naturally lacks the insert sequence and instead folds as a short connector without defined secondary structure. The N-terminal (N'-) distal end of the short connector in Aspergillus flavus FAD-GDH was selected for epitope grafting with several epitopes. Specifically, position 328 of SEQ ID NO: 131 was selected for insertion of cardiac troponin I (TNI), hemagglutinin (HA), or human neutrophil lipocalin (HNL) epitopes, resulting in the proteins SEQ ID NOs: 132-134.

[0161] A panel of three grafted proteins and wild-type Aspergillus flavus FAD-GDH were expressed as secreted proteins in Pichia pastoris and purified from their supernatants using IMAC and a preparative sizing chromatography step. All three purified proteins showed activity in the DCPIP assay, indicating that they were likely well-folded and functional enzymes in these preparations. A sample gel of purified Aspergillus flavus FAD-GDH proteins with grafting at the 328 amino acid residue position is shown in Figure 32.

[0162] Using anti-epitope antibodies, we observed inhibition of various Aspergillus flavus FAD-GDH328 grafts. As shown in Figure 33, the antibodies were ineffective in inhibiting wild-type Mucor 19-031, which served as a negative control for the experiment. At the antibody concentrations listed, inhibition was observed, most notably with the anti-HNL antibody ab206427 and the three anti-HA antibodies tested. The inhibition observed with the matched epitope-antibody pairs exceeded nonspecific inhibition of wild-type Aspergillus flavus FAD-GDH in each case, indicating that inhibition of each graft was specific. The three Aspergillus flavus FAD-GDH epitope grafts at position 328 were further tested using a negative control anti-HNL antibody, 2-6128, which does not recognize the HNL sequence used for epitope grafting. In Figure 34, only the matched anti-HNL antibody, ab206427, inhibited the Aspergillus flavus grafted enzyme 328HNL. Similar degrees of inhibition of the 328HA graft were achieved using different concentrations of the two different antibodies tested.

[0163] Dose-response inhibition experiments were performed for each of the two antibodies, ab182009 and ab236632 (Figures 35A and 35B). In Figure 35A, the anti-HA antibody ab182009 demonstrated dose-dependent inhibition of the grafted enzyme (open squares) that consistently exceeded background, nonspecific inhibition of the ungrafted enzyme (open circles). An unrelated antibody (anti-HNL 2-6128) was used as a negative control for inhibition of either ungrafted or grafted Aspergillus flavus FAD-GDH (filled triangles and filled circles). In Figure 35B, samples were compared in a dose-response inhibition assay as in Figure 35A, except the anti-HA antibody was ab236632. The measured inhibition was dose-responsive and consistently higher for the grafted enzyme (filled squares) than for the ungrafted enzyme control (open circles).

Claims

1. An enzyme comprising an epitope-grafted allosteric site that is inhibited by contact with an inhibitor and that is deinhibited in the presence of an analyte that binds to the inhibitor.

2. The enzyme of claim 1, which is a glucose metabolic enzyme.

3. The enzyme according to claim 2, wherein the glucose metabolic enzyme is an FAD-dependent glucose dehydrogenase (FAD-GDH) enzyme.

4. The enzyme according to claim 3, wherein the FAD-GDH enzyme is a Mucor FAD-GDH or an Aspergillus FAD-GDH.

5. Mucor hiemalis (M. hiemalis), Mucor circinelloides (M. circinelloides), Mucor ambigus (M. ambiguus), Mucor lusitanicus (M. lusitanicus), Mucor guilliermondii (M. guilliermondii), Mucor subtilissimus (M. subtilissimus), Mucor prainii (M. prainii), and Aspergillus flavus The enzyme according to claim 4, which is an FAD-GDH derived from an organism selected from the group consisting of A. flavus.

6. The enzyme according to any one of claims 1 to 5, which is a wild-type enzyme except for the epitope grafting.

7. The enzyme of any one of claims 1 to 5, which comprises synthetic sequence variations in addition to epitope grafting.

8. 8. The enzyme of claim 7, wherein the synthetic sequence variation comprises a sequence variation that increases the enzyme stability compared to the non-variant enzyme.

9. 2. The enzyme of claim 1, comprising a sequence selected from the group consisting of SEQ ID NOs: 1-64, 65-72, 75-127, and 132-134, or a sequence at least 70% identical thereto.

10. 2. The enzyme of claim 1, which is a FAD-GDH enzyme, and the allosteric sites are located in surface regions corresponding to residue ranges 45-70, 335-362, and 439-457 of SEQ ID NO:

1.

11. The enzyme of claim 1 , wherein the epitope-grafted sequence comprises an epitope sequence corresponding to an analyte.

12. The enzyme according to claim 11 , wherein the analyte is a protein.

13. The enzyme according to claim 11 , wherein the analyte is a peptide.

14. The enzyme of claim 11, wherein the analyte is selected from the group consisting of a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker.

15. The enzyme of claim 11, wherein the epitope grafted sequence comprises 3 to 30 amino acids.

16. A composition comprising an enzyme comprising an epitope-grafted allosteric site that is inhibited by contact with an inhibitor and that is deinhibited in the presence of an analyte that binds to the inhibitor.

17. The composition of claim 16 , wherein the enzyme is a glucose metabolic enzyme.

18. 18. The composition of claim 17, wherein the glucose metabolic enzyme is an FAD-dependent glucose dehydrogenase (FAD-GDH) enzyme.

19. 19. The composition according to claim 18, wherein the FAD-GDH enzyme is a Mucor FAD-GDH or an Aspergillus FAD-GDH.

20. 20. The composition of claim 19, wherein the enzyme is a FAD-GDH derived from an organism selected from the group consisting of Mucor hiemalis, Mucor circinelloides, Mucor ambigus, Mucor lusitanicus, Mucor guilliermondii, Mucor subtilisimus, Mucor plenii, and Aspergillus flavus.

21. 21. The composition of any one of claims 16 to 20, wherein the enzyme is a wild-type enzyme except for the epitope grafting.

22. The composition of any one of claims 16 to 20, wherein the enzyme comprises synthetic sequence variations in addition to epitope grafting.

23. 23. The composition of claim 22, wherein the synthetic sequence variation comprises a sequence variation that increases enzyme stability compared to a non-variant enzyme.

24. 17. The composition of claim 16, wherein the enzyme comprises a sequence selected from the group consisting of SEQ ID NOs: 1-64, 65-72, 75-127, and 132-134, or a sequence at least 70% identical thereto.

25. The composition of any one of claims 16 to 24, wherein the enzyme is in contact with a sensor surface.

26. 26. The composition of claim 25, wherein the sensor is an electrochemical sensor.

27. 17. The composition of claim 16, wherein the enzyme is a FAD-GDH enzyme and the allosteric sites are located in surface regions corresponding to residue ranges 45-70, 335-362, and 439-457 of SEQ ID NO:

1.

28. 27. The composition of claim 26, wherein the epitope grafted sequence comprises an epitope sequence corresponding to an analyte.

29. 29. The composition of claim 28, wherein the analyte is a protein.

30. 29. The composition of claim 28, wherein the analyte is a peptide.

31. 29. The composition of claim 28, wherein the analyte is selected from the group consisting of a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker.

32. 17. The composition of claim 16, wherein the epitope grafted sequence comprises 3 to 30 amino acids.

33. 33. A system comprising the composition of any one of claims 16 to 32 and an inhibitor that binds to the analyte and the epitope-grafted sequence.

34. 34. The system of claim 33, wherein the inhibitor binds to the analyte with a higher affinity than the inhibitor binds to the epitope-grafted sequence.

35. The system of claim 33 , wherein the inhibitor is an immunoglobulin.

36. 35. The system of claim 34, wherein the immunoglobulin is an antibody.

37. 37. The system of claim 36, wherein the immunoglobulin is an antibody fragment.

38. 34. The system of claim 33, further comprising a substrate for the enzyme.

39. 39. The system of claim 38, wherein the substrate is glucose.

40. 34. The system of claim 33, further comprising a sensor.

41. 41. The system of claim 40, wherein the sensor is an electrochemical sensor.

42. 41. The system of claim 40, wherein the sensor detects a product of the enzyme reacting with a substrate.

43. 34. The system of claim 33, further comprising a sample.

44. 44. The system of claim 43, wherein the sample is a biological sample.

45. 45. The system of claim 44, wherein the biological sample is selected from the group consisting of blood, serum, plasma, intestinal fluid, saliva, and urine.

46. A reaction mixture comprising the composition of any one of claims 16 to 32.

47. 47. The reaction mixture of claim 46, further comprising an inhibitor that binds to the analyte and the epitope-grafted sequence.

48. 48. The reaction mixture of claim 47, wherein the inhibitor is an immunoglobulin.

49. 49. The reaction mixture of claim 48, wherein the immunoglobulin is an antibody.

50. 49. The reaction mixture of claim 48, wherein the immunoglobulin is an antibody fragment.

51. 47. The reaction mixture of claim 46, further comprising a substrate for the enzyme.

52. 52. The reaction mixture of claim 51 , wherein the substrate is glucose.

53. 47. The reaction mixture of claim 46, further comprising a sample.

54. 54. The reaction mixture of claim 53, wherein the sample is a biological sample.

55. 55. The reaction mixture of claim 54, wherein the biological sample is selected from the group consisting of blood, serum, plasma, intestinal fluid, saliva, and urine.

56. A kit comprising the enzyme or composition according to any one of claims 1 to 32.

57. 57. The kit of claim 56, further comprising an inhibitor that binds to the analyte and the epitope-grafted sequence.

58. 58. The kit of claim 57, wherein the inhibitor is an immunoglobulin.

59. 59. The kit of claim 58, wherein the immunoglobulin is an antibody.

60. 58. The kit of claim 57, wherein the immunoglobulin is an antibody fragment.

61. 57. The kit of claim 56, further comprising a substrate for the enzyme.

62. 62. The kit of claim 61, wherein the substrate is glucose.

63. 57. The kit of claim 56, further comprising a sensor.

64. 64. The kit of claim 63, wherein the sensor is an electrochemical sensor.

65. 57. The kit of claim 56, further comprising a control sample comprising the analyte.

66. 57. The kit of claim 56, further comprising a control sample lacking said analyte.

67. Use of an enzyme or composition according to any one of claims 1 to 32.

68. Use of an enzyme or composition according to any one of claims 1 to 32 for detecting the presence, absence or amount of an analyte in a sample.

69. Use of a system according to any one of claims 33 to 45.

70. Use of the system of any one of claims 33 to 45 for detecting the presence, absence or amount of an analyte in a sample.

71. Use of a reaction mixture according to any one of claims 46 to 55.

72. Use of the reaction mixture of any one of claims 46 to 55 for detecting the presence, absence or amount of an analyte in a sample.

73. Use of a kit according to any one of claims 56 to 66.

74. Use of a kit according to any one of claims 56 to 66 for detecting the presence, absence or amount of an analyte in a sample.

75. 16. A method for detecting an analyte, comprising the steps of: a) contacting a sample suspected of containing the analyte with an enzyme according to any one of claims 1 to 15; and b) directly or indirectly detecting the activity of the enzyme.

76. 76. The method of claim 75, wherein the detecting step comprises electrochemical measurement of a by-product of the enzyme reacting with a substrate.

77. An enzyme comprising an engineered allosteric site, wherein the engineered allosteric site comprises a grafted heterologous epitope, and wherein the enzyme has an enzymatic activity that is inhibited by binding of an inhibitor to the grafted epitope.

78. 78. The enzyme of claim 77, wherein the epitope comprises an amino acid sequence corresponding to an inhibitor binding site of the polypeptide analyte, and the inhibitor is capable of competitively binding to the polypeptide analyte and the grafted epitope.

79. 79. The enzyme of claim 77 or 78, wherein the epitope comprises an amino acid sequence having at least 70%, at least 80%, or at least 90% sequence identity with the corresponding sequence of the inhibitor binding site being analyzed.

80. The enzyme according to any one of claims 77 to 79, wherein the subject of analysis is a peptide, polypeptide, or protein.

81. The enzyme according to any one of claims 77 to 80, wherein the epitope is a linear epitope.

82. The enzyme according to any one of claims 77 to 80, wherein the epitope is a structural epitope.

83. The enzyme according to any one of claims 77 to 82, wherein the subject to be analyzed is selected from a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, and a transplant rejection biomarker.

84. The enzyme of any one of claims 77 to 83, wherein the epitope comprises from about 3 to about 30 amino acids.

85. The enzyme according to any one of claims 77 to 84, wherein the epitope comprises from about 5 to about 15 amino acids, preferably from about 8 to about 10 amino acids.

86. The enzyme according to any one of claims 77 to 85, which is a glucose metabolic enzyme.

87. The enzyme according to any one of claims 77 to 86, which is FAD-dependent glucose dehydrogenase (FAD-GDH).

88. The enzyme according to any one of claims 77 to 87, which is derived from a FAD-GDH of the family Mucoraceae or Aspergillaceae, preferably from a FAD-GDH of the genus Mucor or Aspergillus.

89. The enzyme according to any one of claims 77 to 88, which is derived from FAD-GDH from Mucor hiemalis, Mucor circinelloides, Mucor amvigus, Mucor lusitanicus, Mucor guilliermondii, Mucor plenii, Mucor subtilisimus, and Aspergillus flavus.

90. The enzyme according to any one of claims 77 to 89, which is derived from FAD-GDH from Mucor hiemalis, Mucor circinelloides, Mucor amvigus, Mucor plenii, and Mucor subtilisimus.

91. 91. The enzyme of any one of claims 77 to 90, having at least 70%, at least 80%, or at least 90% identity to SEQ ID NOs: 1, 66-72, 119-127, or 131.

92. 92. The enzyme of claim 91, wherein sequence identity is assessed across the entire sequence of the enzyme excluding the epitope.

93. 93. The enzyme of any one of claims 77 to 92, wherein the allosteric site is located on the surface of the enzyme.

94. 94. The enzyme of any one of claims 77 to 93, wherein the allosteric site is located on a surface corresponding to surface 2 of the enzyme of SEQ ID NO: 1, wherein surface 2 comprises residues F341, E344, E348, and K358 of SEQ ID NO:

1.

95. 95. The enzyme of claim 94, wherein surface 2 comprises residues T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, and K358 of SEQ ID NO:

1.

96. 96. The enzyme of any one of claims 77 to 95, wherein the epitope is grafted at a position corresponding to (i) about position 330 to about 370, optionally about position 335 to about 362 of SEQ ID NO:1; or (ii) about position 320 to about 335, optionally about position 325 to about 330 of SEQ ID NO:131, further optionally about position 328 of SEQ ID NO:

131.

97. 97. The enzyme of claim 96, wherein the epitope is transplanted into a position corresponding to T337, D338, V340, F341, N434, E344, L346, E348, E349, Y354, or K358 of SEQ ID NO:

1.

98. 98. The enzyme of claim 96 or 97, wherein the epitope is grafted into a position corresponding to position 341 or 358 of SEQ ID NO:

1.

99. 99. The enzyme of any one of claims 77 to 98, further comprising one or more amino acid modifications that increase the stability of the enzyme.

100. The enzyme according to any one of claims 77 to 99, comprising Cys at positions corresponding to positions 153 and 192 of SEQ ID NO:

118.

101. 101. A system comprising an enzyme according to any one of claims 77 to 100 and an inhibitor, wherein the inhibitor is capable of binding to a grafted epitope of the enzyme and to an analyte comprising the epitope.

102. 102. The system of claim 101, wherein the affinity of the analyte for the inhibitor is higher than the affinity of the epitope-grafted enzyme for the inhibitor.

103. 103. The system of claim 101 or 102, wherein the inhibitor is an immunoglobulin, an antibody, or an antibody fragment.

104. The system of any one of claims 101 to 103, further comprising a substrate for the enzyme.

105. The system of any one of claims 101 to 104, wherein the substrate is glucose.

106. 106. The system of any one of claims 101 to 105, further comprising an analyte having a binding site for the inhibitor, wherein the amino acid sequence of the binding site corresponds to the amino acid sequence of a grafted epitope of the enzyme.

107. The system of any one of claims 101 to 106, wherein the analyte is a peptide, polypeptide or protein, and preferably the analyte is selected from a cardiovascular disease biomarker, a cancer biomarker, an infectious disease biomarker, an inflammatory biomarker, a metabolic biomarker, or a transplant rejection biomarker.

108. The system of any one of claims 101 to 107, wherein the analyte is present in a biological sample.

109. 109. The system of claim 108, wherein the biological sample is selected from blood, serum, plasma, intestinal fluid, saliva, and urine.

110. A sensor comprising an enzyme according to any one of claims 77 to 100 or a system according to any one of claims 101 to 109.

111. 111. The sensor of claim 110, which is an electrochemical sensor.

112. 112. The sensor of claim 110 or 111, configured to directly or indirectly sense the turnover rate of a substrate by the enzyme.

113. 113. A sensor according to any one of claims 110 to 112, configured to detect a product of turnover of a substrate by the enzyme.

114. 1. A method for determining the presence, absence, or concentration of an analyte in a sample, comprising: a) contacting a sample with an enzyme according to any one of claims 77 to 100 in the presence of an analyte and an inhibitor capable of binding to a grafted epitope of said enzyme; and b) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme A method comprising:

115. a) contacting a sample with an enzyme according to any one of claims 77 to 100 in the presence of an analyte and an inhibitor capable of binding to a grafted epitope of said enzyme; b) allowing the inhibitor to inhibit the enzyme; c) binding the analyte present in the sample to the inhibitor, thereby deinhibiting the enzyme; and d) obtaining one or more measurements characteristic of the enzymatic activity of the enzyme; 115. The method of claim 114, comprising:

116. 116. The method of claim 115, wherein the enzymatic activity of the enzyme is proportional to the concentration of the analyte in the sample.

117. 117. The method of any one of claims 115 or 116, wherein the sample is a biological sample selected from blood, serum, plasma, intestinal fluid, saliva, or urine.

118. 1. A method for identifying an allosteric site of an enzyme, wherein the allosteric site can be inhibited by an inhibitor, comprising: a) generating one or more antibodies and / or antibody mimetics that bind to the enzyme; b) screening the one or more antibodies and / or antibody mimetics for their ability to allosterically inhibit the enzymatic activity of the enzyme, thereby identifying antibodies and / or antibody mimetics that allosterically inhibit the enzymatic activity of the enzyme; and c) identifying amino acids of the enzyme that contact said antibodies and / or antibody mimetics that allosterically inhibit the enzymatic activity of the enzyme; A method comprising:

119. 119. The method of claim 118, further comprising determining the retention of enzyme activity when the amino acid is modified.

120. The method of claim 118 or 119, further comprising the step of grafting an epitope into the amino acid sequence of the enzyme at a position corresponding to the allosteric site, wherein the epitope comprises an amino acid sequence capable of binding to an inhibitor.

121. 121. The method of any one of claims 118 to 120, wherein the enzyme, allosteric site, inhibitor and / or epitope is as defined in any one of claims 77 to 100.

122. 1. A method for diagnosing the health of a subject, comprising: a) contacting a biological sample from the subject with a sensor according to any one of claims 110 to 113; and b) determining the presence, absence or concentration of an analyte associated with the subject's health in the sample according to the method of any one of claims 114 to 117. A method comprising:

123. 114. Use of an enzyme according to any one of claims 77 to 100, a system according to any one of claims 101 to 109, or a sensor according to any one of claims 110 to 113 for determining the presence, absence or concentration of an analyte in a sample.

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