Antibodies and antigen-binding fragments thereof

Abstract

Novel antibodies, or antigen-binding fragments thereof, are provided that can be used in immunoassays to aid in determining or distinguishing the smoking status of a subject. The present invention discloses an antibody or antigen-binding fragment thereof that is capable of binding to (i) N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA) and (ii) a conjugate comprising a compound of formula [II]: TIFF2024532139000029.tif5379, wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4); and each R is independently H or C. 1 ~C 6 alkyl, preferably a compound of formula [I]: TIFF2024532139000030.tif5579 The compound of formula [II] or formula [I] is bound to the immunogenic carrier via a linker, preferably the linker is bound to the compound of formula [I] via an amine group.

Description

[Technical field]

[0001] The present invention relates generally to novel antibodies, or antigen-binding fragments thereof, that can be used in immunoassays to aid in determining or distinguishing the smoking status of a subject. [Background technology]

[0002] Aerosol-generating articles in which tobacco is heated rather than burned have been proposed in the art. In heated aerosol-generating articles, the aerosol is produced by heating a substrate (such as tobacco). Research has shown that heating tobacco to temperatures below pyrolysis and combustion temperatures may reduce or eliminate some of the toxicants found in cigarette smoke. Instead of burning tobacco, heating it to temperatures typically below 300° C. is sufficient to release nicotine, but not high enough to cause significant pyrolysis. At these temperatures, the aerosol composition is simpler than that found in cigarette smoke. Tobacco combustion forms many harmful and potentially harmful components (HPHCs) in cigarette smoke. Therefore, instead of burning tobacco, heating it to lower temperatures can reduce or eliminate HPHCs.

[0003] Known heated aerosol generating articles include electrically heated aerosol generating articles and aerosol generating articles in which the aerosol is generated by the transfer of heat from a combustible fuel element or heat source to a physically separated aerosol-forming material. These so-called heat-not-burn products offer smokers an alternative to traditional cigarettes and may reduce the harmful chemicals released from tobacco while still delivering nicotine. One such tobacco heating system is IQOS (THS), which includes sophisticated electronics for heating a specially designed heated tobacco unit. THS heats tobacco sufficiently to release nicotine-containing tobacco vapor without burning the tobacco. Tobacco in a cigarette burns at temperatures in excess of 600°C, producing smoke with high levels of harmful chemicals. However, THS heats tobacco to a much lower temperature, up to 350°C, without combustion, fire, ash, or smoke. Because the tobacco is heated and not burned, the levels of harmful chemicals are significantly reduced compared to cigarette smoke.

[0004] A diagnostic test for determining or differentiating the smoking status of a subject is described in WO2018 / 211126. As an example, the test can distinguish between current smokers of traditional cigarettes (smokers), those who have switched to heat-not-burn products, also known as Reduced Risk Products or RRPs (switchers), or those who have abstained from smoking (non-smokers). The test can have a variety of applications. For example, the test can be used in clinical trials to identify and screen subjects based on smoking status. As a further example, the test can be used for insurance purposes as a compliance test to monitor switching to RRPs and compliance with switching.

[0005] Various metabolites that can be used as biomarkers of tobacco smoke exposure are described in WO2018 / 211126, including cotinine and CEMA.

[0006] There is a need in the art for agents capable of detecting CEMA with high specificity and sensitivity, particularly for use in immunoassay formats. Such agents should be easy to manufacture, have good solubility and diffusion, and be capable of detecting CEMA specifically and sensitively in complex biological samples such as urine. The present invention seeks to address this need. Summary of the Invention

[0007] The present invention relates, at least in part, to a compound of formula [II]: [ka] The present invention is based on the surprising discovery that a novel conjugate comprising: R, R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R)-R(R) is a superior conjugate that induces a robust immune response to obtain anti-CEMA antibodies or antigen-binding fragments thereof. The conjugation of CEMA to an immunogenic carrier via a linker improves recognition by the immune system and production of antibodies. In many cases, so-called anti-hapten antibody production can result in reagents with limited target affinity. Advantageously, however, the antibodies or antigen-binding fragments of the present disclosure, especially in scFv format, are highly sensitive and specific for CEMA, even in complex biological samples including urine. Of the 96 reactive clones obtained during phage display screening, 28 positive clones were identified. From these 28 positive clones, 8 sequences were taken to produce 8 scFvs, which were tested in immunoassays. Of these eight scFvs, three were selected based on their superior sensitivity and specificity for CEMA and are referred to herein as "scFv G4," "scFv B11," and "scFv E6," or "G4," "B11," and "E6."

[0008] Aspects and embodiments of the invention In one aspect, an antibody or antigen-binding fragment thereof is disclosed that is capable of binding (i) to N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA) and (ii) to a conjugate comprising a compound of formula [II]. [ka] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C1-C6 alkyl, preferably a compound of formula [I]: [ka] The compound of formula [II] or formula [I] is bound to the immunogenic carrier via a linker, preferably the linker is bound to the compound of formula [I] via an amine group.

[0009] Suitably, the immunogenic carrier is a protein, preferably bovine serum albumin or bovine thyroglobulin.

[0010] Preferably, the linker is glycol bis(succinimidyl succinate) (EGS) or disuccinimidyl suberate (DSS).

[0011] Suitably the antibody is a monoclonal antibody, preferably the antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, Fv, rIgG, or a diabody, more preferably an scFv.

[0012] Preferably, the antibody or antigen-binding fragment thereof does not bind to cotinine or 2-hydroxyethyl methacrylate (HEMA) or monohydroxybutenyl-mercapturic acid (MHBMA) or 3-hydroxypropyl mercapturic acid (3-HPMA) or dihydroxybutyl mercapturic acid (DHBMA).

[0013] Preferably, the rate of inhibition by urine is less than 20%, less than 10%, less than 5%, or no inhibition.

[0014] Suitably, the antibody or antigen-binding fragment thereof has a detection limit in an immunoassay of 160 ng / mL of CEMA in urine.

[0015] In another aspect, an antibody or antigen-binding fragment thereof is disclosed, comprising VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; or VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively.

[0016] Suitably, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2, 11 or 20.

[0017] Suitably, the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 6 or 15 or 24.

[0018] Suitably, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO:2 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:6.

[0019] Preferably, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11, and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 15, or the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 20, and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 24.

[0020] Suitably, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, an Fv, a rIgG, and a diabody, preferably an scFv.

[0021] Suitably, the antigen-binding fragment is an scFv, said scFv comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:10 or SEQ ID NO:19.

[0022] In another aspect, a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein, or a polynucleotide complementary thereto, is disclosed.

[0023] Suitably, the polynucleotide comprises one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:28, SEQ ID NO:31, and SEQ ID NO:34, or a polynucleotide complementary thereto.

[0024] In a further aspect, a vector comprising the polynucleotide sequence is provided.

[0025] Suitably, the vector further comprises an expression control sequence operably linked to the nucleic acid encoding the variable heavy chain domain and / or the variable light chain domain.

[0026] In a further aspect, a host cell containing the vector is provided.

[0027] Suitably, the host cell is a eukaryotic or prokaryotic cell.

[0028] Preferably, the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell.

[0029] Preferably, the prokaryotic cell is an E. coli cell.

[0030] In a further aspect, there is provided a method of producing an antibody or antigen-binding fragment thereof comprising incubating a host cell such that the encoded variable heavy chain domain and / or variable light chain domain is expressed by the cell, and recovering the expressed antibody or antigen-binding fragment thereof.

[0031] Suitably, the method further comprises isolating and / or purifying the recovered antibody or antigen-binding fragment thereof.

[0032] In another aspect, a method of producing the antibodies described herein is disclosed, comprising immunizing a non-human animal with a conjugate comprising a compound of formula [II]: [ka] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C1-C6 alkyl, preferably a compound of formula [I]: [ka] The aforementioned compound of formula [II] or formula [I] is bound to the immunogenic carrier via a linker, preferably the linker is bound to the compound of formula [I] via an amine group.

[0033] In another aspect, a device for determining the presence or absence of CEMA in a sample is disclosed, said device comprising an antibody or antigen-binding fragment thereof described herein immobilized on a solid phase of the device.

[0034] In another aspect, a device for determining the presence or absence of CEMA in a sample is disclosed, said device comprising a conjugate described herein immobilized on a solid phase of the device.

[0035] Suitably the device is a portable lateral flow immunoassay device, preferably a urine test strip.

[0036] Preferably, the device comprises (i) a sample pad for receiving a sample, (ii) a conjugate pad in fluid communication with the sample pad, (iii) at least one detection zone in fluid communication with a distal end of the conjugate pad, and (iv) an adsorbent pad in fluid communication with a distal end of the detection zone.

[0037] Suitably, the conjugate pad comprises an antibody or antigen-binding fragment thereof according to the present invention and as described above, said antibody or antigen-binding fragment thereof being labelled, optionally comprising a labelled antibody or antigen-binding fragment thereof capable of binding to cotinine.

[0038] Preferably, the antibody or antigen-binding fragment thereof according to the invention and described above is contained separately in an intermediate pad of the device located adjacent to the conjugate pad, and optionally, the labelled antibody or antigen-binding fragment thereof capable of binding to cotinine is contained separately in the conjugate pad. Preferably, the detection zone comprises CEMA and optionally cotinine immobilised thereon, preferably CEMA in the form of a conjugate according to the invention and described above.

[0039] In another aspect, a method for detecting CEMA in a sample comprising the use of a device described herein, or a method for detecting CEMA and cotinine in a sample comprising the use of a device described herein, is disclosed.

[0040] Suitably, the method comprises (i) applying an aliquot of a liquid biological sample, preferably urine, to the sample pad, whereby the liquid biological sample is moved by capillary action along a flow path defined by the sample pad, the conjugate pad, the detection zone, and the sorbent pad; and (ii) determining the presence or absence of CEMA in the detection zone, and optionally determining the presence or absence of cotinine in the detection zone.

[0041] In another aspect, the use of an antibody or antigen-binding fragment thereof described herein, or a device described herein, for detecting CEMA in a sample is disclosed. [Brief description of the drawings]

[0042] [Figure 1-1] Figure 1 shows the ELISA assay results in the form of a graph showing the reactivity of different scFv clones on different immunogens and proteins, including scFv G4, scFv B11, and scFv E6. The y-axis shows the optical density at 450 nm and the x-axis shows the dilution of CEMA. Optical density is measured on a BioteK EL808 reader as described in the accompanying examples. [Figure 1-2] Figure 1 shows the ELISA assay results in the form of a graph showing the reactivity of different scFv clones on different immunogens and proteins, including scFv G4, scFv B11, and scFv E6. The y-axis shows the optical density at 450 nm and the x-axis shows the dilution of CEMA. Optical density is measured on a BioteK EL808 reader as described in the accompanying examples. [Figure 1-3] Figure 1 shows the ELISA assay results in the form of a graph showing the reactivity of different scFv clones on different immunogens and proteins, including scFv G4, scFv B11, and scFv E6. The y-axis shows the optical density at 450 nm and the x-axis shows the dilution of CEMA. Optical density is measured on a BioteK EL808 reader as described in the accompanying examples. [Diagram 2] Figure 2 shows a table and graph of scFv competition ELISA results using BTG-EGS-CEMA immunogen and free CEMA. For each data set, the columns represent results from left to right at 10, 1, 0.1, 0.01, 0.001, and 0.0001 μg / ml of free CEMA. The y-axis shows optical density at 450 nm and the x-axis shows scFv conjugates. Optical density is measured on a BioteK EL808 reader as described in the accompanying examples. [Diagram 3]Figure 3 shows a table and graph of scFv competition ELISA results using BTG-DSS-CEMA immunogen and free CEMA. For each data set, the columns represent results from left to right at 10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml, 0.001 μg / ml, and 0.0001 μg / ml of free CEMA. The y-axis shows optical density at 450 nm and the x-axis shows scFv conjugates. Optical density is measured on a BioteK EL808 reader as described in the accompanying examples. [Figure 4] Figure 4 shows a graph depicting the results of a lateral flow immunoassay reactivity study in which the reactivity of selected scFvs was tested against BTG-EGS-CEMA and BTG-DSS-CEMA immunogens. For each data set, the columns represent the results from left to right with either the BTG-EGS-CEMA or BTG-DSS-CEMA immunogens. The y-axis shows the signal intensity measured in the lateral flow immunoassay and the x-axis shows the scFv conjugate under test. [Diagram 5] Figure 5 shows a graph depicting lateral flow immunoassay test results investigating scFv inhibition with free CEMA and BTG-EGS-CEMA immunogens. For each data set, columns represent results from left to right at 0 μg / ml (1x PBS), negative urine, CEMA 500 ng / ml (urine), CEMA 100 ng / ml (urine), CEMA 50 ng / ml (urine), CEMA 20 ng / ml (urine), and CEMA 10 ng / ml (urine). The y-axis shows signal intensity measured in the lateral flow immunoassay and the x-axis shows the scFv conjugate under test. [Figure 6]Figure 6 shows a graph depicting lateral flow immunoassay test results investigating scFv inhibition with free CEMA and BTG-DSS-CEMA immunogens. For each data set, columns represent results from left to right at 0 μg / ml (1x PBS), negative urine, CEMA 500 ng / ml (urine), CEMA 100 ng / ml (urine), CEMA 50 ng / ml (urine), CEMA 20 ng / ml (urine), and CEMA 10 ng / ml (urine). The y-axis shows the signal intensity measured in the lateral flow immunoassay and the x-axis shows the scFv conjugate under test. [Figure 7] Figure 7 shows a graph depicting lateral flow immunoassay testing results of the inhibition profile of selected scFvs in urine samples. For each data set, the columns represent results from left to right using G4, B11 or E6 scFv. The y-axis shows the signal intensity measured in the lateral flow immunoassay and the x-axis shows the amount of CEMA under test. [Figure 8] Figure 8 is a graph showing the specificity of scFV G4, scFV B11 and scFV E6 for cotinine and molecules with structures close to CEMA, namely 2-hydroxyethyl mercapturic acid (HEMA), monohydroxybutenyl mercapturic acid (MHBMA), 3-hydroxypropyl mercapturic acid (3-HPMA) and dihydroxybutyl mercapturic acid (DHBMA), as measured by lateral flow immunoassay. For each data set, the columns represent the results from left to right using G4, B11 or E6 scFv. The y-axis shows the signal intensity measured by lateral flow immunoassay and the x-axis shows the molecule under test for interference. [Figure 9] FIG. 9 illustrates a portable lateral flow immunoassay device format according to an embodiment of the present disclosure. [Figure 10] Figure 10 illustrates a multiplex test protocol and its interpretation that can be obtained on a portable lateral flow immunoassay device. C=control, CEM=CEMA test line, COT=cotinine test line.

[0043] definition The headings of the paragraphs used in this disclosure are for organizational purposes and are not intended to be limiting. 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 document, including definitions, governs. Preferred methods and materials are described below, however, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. The materials, methods, and examples disclosed herein are merely illustrative and are not intended to be limiting. As used herein, the singular forms ("a," "an," and "the") include both singular and plural referents unless the context clearly indicates otherwise.

[0044] The term "and / or" means either (a) or (b), or both (a) and (b).

[0045] As used herein, the terms "comprising" and "comprised of" are synonymous with "including" or "containing" and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps.

[0046] The term "consisting of" means having only the recited elements, to the exclusion of additional components, and no more.

[0047] The term "about" as used herein when referring to measurable values ​​such as parameters, amounts, lengths of time, and the like, is meant to encompass variations in and from the particular value, specifically within + / - 10% or less, preferably within + / - 5% or less, more preferably within + / - 1% or less, and even more preferably within + / - 0.1% or less of the particular value, as long as such variations are appropriate for practice in this disclosure. It should be understood that the values ​​to which the "about" modifier refers are themselves specifically and preferably disclosed.

[0048] As used herein, the term "antibody" encompasses intact monoclonal antibodies, polyclonal antibodies, multivalent (e.g., bivalent, trivalent or higher) and / or multispecific (e.g., bi- or higher specific) antibodies formed from at least two intact antibodies, as well as antigenic fragments when they exhibit the desired biological activity (particularly the ability to specifically bind to a metabolite such as CEMA), as well as multivalent and / or multispecific complexes of such fragments. The term not only includes antibodies generated by methods including immunization, but also includes any polypeptide, e.g., recombinantly expressed or synthetic polypeptide, that is engineered to encompass at least one complementarity determining region (CDR) capable of specifically binding to an epitope. Thus, the term applies to such molecules whether generated in vitro or in vivo.

[0049] The term "isolated" refers to the removal of a molecule from its natural environment.

[0050] The term "one or more," such as one or more members of a group of members, is clear in itself, but by way of further illustration, the term specifically includes a reference to any one of the aforementioned members, or to any two or more of the aforementioned members, such as, for example, any three, four, five, six, or seven of the aforementioned members, and up to all of the aforementioned members.

[0051] The term "label" refers to any atom, molecule, moiety, or biomolecule that can be used to provide a detectable, and preferably quantifiable, readout or characteristic, and that may be attached to or made part of an entity of interest, such as a metabolite or an antibody or antigen-binding fragment thereof.

[0052] The term "metabolite" is widely known in the art and may broadly refer to any substance produced by metabolism or metabolic processes. In other words, metabolites are end products resulting from metabolism. The term also encompasses detectable moieties of metabolites whose qualitative and / or quantitative assessment in a subject, alone or in combination with other data, carries information about the subject's status as a function of switching adherence. Metabolites are typically small molecules derived from combustible tobacco products. Monitoring metabolites over time may allow the progress of a subject's switching adherence over time to be determined.

[0053] A molecule is "measured" in a sample when the presence, absence, and / or quantity of said molecule or said molecules is detected or determined in the sample, preferably substantially to the exclusion of other molecules. For example, the molecule may be measured by a laboratory test as described herein.

[0054] The term "non-smoker" refers to a subject who was a former smoker but has not smoked a tobacco product (e.g., cigarettes) in the past three months. A non-smoker would be considered to have quit smoking. Preferably, the tobacco product or cigarette is a non-menthol tobacco product or cigarette.

[0055] The term "purified" does not require absolute purity. Instead, the term describes a separate environment in which the abundance (conveniently expressed in mass, or weight, or concentration) relative to other molecules is greater than in a biological sample. The separate environment describes a single medium, e.g., a single solution, a gel, a precipitate, a lyophilizate, etc. Purified molecules may be obtained by known methods, including, e.g., chromatography, preparative electrophoresis, centrifugation, precipitation, affinity purification, etc.

[0056] The terms "quantity", "amount" and "level" are synonymous and generally well understood in the art. With respect to metabolites, the terms may refer in particular to the absolute quantification of a metabolite in a sample, or the relative quantification of a metabolite in a sample, i.e., quantification relative to another value, such as relative to a baseline or reference value as taught herein, or relative to a range of values ​​representing the baseline expression of the metabolite. These values ​​or ranges may be obtained from a single subject, or a group of subjects. The absolute quantity of a metabolite in a sample may advantageously be expressed as a weight or molar amount, or more commonly as a concentration, e.g., weight per volume, or moles per volume.

[0057] As used herein, the term "sample" or "biological sample" includes any biological specimen obtained from a subject. Samples may include, but are not limited to, whole blood, plasma, serum, red blood cells, white blood cells (e.g., peripheral blood mononuclear cells), saliva, urine, stool (i.e., feces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudate, synovial fluid, cerebrospinal fluid, lymphatic fluid, fine needle aspirate, amniotic fluid, any other bodily fluid, nail clippings, cell lysates, cell secretions, inflammation fluid, vaginal secretions, or preferably biopsies such as a placental biopsy. Preferred samples may include samples that contain detectable quantities of any one or more metabolites taught herein. In one embodiment, the sample may be whole blood or a fractional component thereof, such as, for example, plasma, serum, or cell pellet. Preferably, the sample is easily obtained by a minimally invasive method that allows for the detection, removal, or separation of said sample from the subject. Samples may also include tissue samples and biopsies, tissue homogenates, and the like. The term "plasma" generally refers to the substantially colorless aqueous liquid of blood that does not contain cells, but in which blood cells (red blood cells, white blood cells, platelets, etc.) are usually suspended, containing nutrients, sugars, proteins, minerals, enzymes, etc. In the most suitable embodiment, the sample is urine (such as 24-hour urine) because it is easy and non-invasive to obtain. 24-hour urine collection is performed by collecting the subject's urine in a container for the entire 24-hour period.

[0058] The term "smoker" refers to a subject who has smoked an average of 10 or more tobacco products (e.g., cigarettes) per day in the past year. A smoker is generally a current smoker. Preferably, the tobacco product or cigarette is a non-menthol tobacco product or cigarette.

[0059] The term "switcher" refers to subjects who switched from smoking combustible tobacco products (e.g., cigarettes) to heat-not-burn (smokeless) products such as iQOS in the past three months.

[0060] The term "subject" as used herein typically refers to a human, but may also encompass reference to non-human animals, preferably warm-blooded animals, more preferably fetal animals, and even more preferably mammals such as, for example, non-human primates, rodents, dogs, cats, horses, sheep, pigs, and the like.

[0061] The term "threshold" in the context of detection means the point at which a particular or defined amount, quantity, or concentration is reached or exceeded. For example, a test line on an immunoassay device may be configured to produce a visual change when a threshold amount, quantity, or concentration is reached or exceeded. The visual change may be the appearance or disappearance of a test line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] The antibodies or antigen-binding fragments thereof described herein can be conjugated to N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA). The antibodies or antigen-binding fragments thereof described herein can be conjugated to a conjugate comprising a compound of formula [I] or [II] or [III], wherein the compound is conjugated to an immunogenic carrier via a linker.

[0063] The antibodies or antigen-binding fragments thereof described herein can specifically bind to N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA). The antibodies or antigen-binding fragments thereof described herein can specifically bind to a conjugate comprising a compound of formula [I] or [II] or [III], wherein the compound is attached to an immunogenic carrier via a linker.

[0064] Thus, the antibody or antigen-binding fragment should bind with high affinity, e.g., with a dissociation constant (K) of less than 1 μM, preferably less than 1 nM. d ) Suitably, the antibody specifically binds to CEMA and CEMA conjugates and does not significantly bind to cotinine or molecules with structures similar to CEMA, such as 2-hydroxyethyl mercapturic acid (HEMA), monohydroxybutenyl mercapturic acid (MHBMA), 3-hydroxypropyl mercapturic acid (3-HPMA), or dihydroxybutyl mercapturic acid (DHBMA).

[0065] The respective quantities or measurements for the metabolites used as tobacco smoke exposure biomarkers described herein can be evaluated simultaneously, separately, or individually. Suitably, the metabolites described herein are evaluated substantially simultaneously or simultaneously. Suitably, the metabolites described herein are evaluated simultaneously at the same time point. The amount of metabolites can be used to establish whether the subject is a current smoker of traditional cigarettes. The amount of metabolites can be used to establish whether the subject has quit smoking. The amount of metabolites can be used to establish whether the subject is a switcher from a current smoker of traditional cigarettes to a consumer of RRPs. A switching profile can be established for the subject, and switching compliance behavior can be evaluated over time to monitor the progress of switching behavior. Conveniently, the analysis can be performed in a single test.

[0066] The metabolites can comprise, consist of, or consist essentially of CEMA or CEMA and cotinine. In certain embodiments, isomers (such as stereoisomers, and / or geometric isomers, and / or optical isomers, and mixtures thereof), chemical derivatives, mimetics, variants, solvates, and salts of these metabolites are intended to be encompassed.

[0067] In certain embodiments, one or more additional metabolites, such as one or more other tobacco smoke exposure biomarkers, can also be tested simultaneously, separately, and / or individually, as desired. Additionally, more general characteristics of the sample can be assessed simultaneously, separately, and / or individually. For example, specific features can be detected to test for sample adulteration or dilution. In the case of a urine sample, features such as one or more of pH, specific gravity, oxidants, nitrites, glutaraldehyde, and creatinine levels may be measured in the urine sample.

[0068] CEMA is a specific urinary biomarker of acrolein exposure. Urinary excretion of CEMA has been shown to be consistently higher in smokers than in nonsmokers. The range in nonsmokers is mostly below 2 ng / mL and increases to higher levels of 20 ng to 205 ng / mL in smokers. In one example, smokers excreted 187 ± 181 μg / L (mean ± SD) or 184 μg / L of CEMA, while nonsmokers excreted only 4.6 ± 35 μg / L or 1.9 μg / L. Smokers consuming 20 or more cigarettes per day (CPD), who were required to smoke exactly 20, 15, 5, or 0 CPD during a 5-day restriction period, show a dose relationship between the number of cigarettes and the level of urinary CEMA (218.0, 168.0, 93.2, and 38.3 μg / 24h, respectively). Smoking cessation studies from 5 to 8 days show significantly lower urinary CEMA levels, about 7 to 10 times lower. CEMA can be detected using the antibodies of the present invention or antigen-binding fragments thereof, such as via immunoassays. Various types of immunoassay techniques are known in the art, as described herein. Various methods for measuring CEMA are known in the art, such as ultra-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. See also Anal. Bioanal. Chem. (2009) 393:969-981 and Anal Biochem. (2012) 430(1):75-82 for the determination of urinary CEMA.

[0069] Another metabolite is cotinine, the main nicotine metabolite detected in the urine of smokers. Cotinine levels in various biological fluids are widely used to estimate nicotine intake in tobacco users. Cotinine has an in vivo half-life of about 20 hours and can be detected for several days after cigarette use. Cotinine levels in blood, saliva, and urine are proportional to the amount of exposure to tobacco smoke. Cotinine levels below 10 ng / mL indicate no active smoking. Values ​​between 10 ng / mL and 100 ng / mL indicate light smoking or moderate exposure. Levels above 300 ng / mL indicate heavy smoker status, consuming more than 20 CPDs per day. In urine, values ​​between 11 ng / mL and 30 ng / mL indicate light or moderate smoking, while active smokers have levels of about 500 ng / mL or higher. In saliva, values ​​between 1 ng / mL and 30 ng / mL indicate light or moderate smoking, with levels in active smokers at about 100 ng / mL or higher. Cotinine can be detected using anti-cotinine antibodies or antigen-binding fragments thereof, such as via immunoassay. Other methods for measuring cotinine include colorimetry, gas chromatography (GC), gas chromatography mass spectrometry (GC-MS), high performance liquid chromatography, and radioimmunoassay (RIA). Methods for measuring cotinine are well documented in BMB Rep. (2014) Mar; 47(3): 130-134, J Clin Diagn Res. (2016) Mar; 10(3): ZE04-ZE06, and Ther Drug Monit. (2009) Feb; 31(1): 14-30.

[0070] Antibodies are natural immunoglobulin molecules with various structures, all based on the immunoglobulin fold. For example, IgG antibodies have two heavy chains and two light chains that are disulfide-bonded to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or an antigen-binding fragment of an antibody is the ability of the antibody or its antigen-binding fragment to specifically bind to a particular antigen.

[0071] The antigen-binding specificity of an antibody is determined by the structural features of the V-region. Variability is not evenly distributed across the 110 amino acid span of the variable domain. Instead, the V-region consists of relatively invariant stretches of 15-30 amino acids, called framework regions (FRs), separated by shorter regions of extreme variability, called "hypervariable regions", each of which is 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs, adopting a predominantly β-sheet configuration, connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the FRs, and the hypervariable regions from the other chain contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0072] Each V region typically contains three CDRs, each of which contains a "hypervariable loop" and four framework regions. Thus, an antibody binding site, which is the minimum structural unit required to bind with substantial affinity to a particular desired antigen, typically contains three CDRs and at least three, preferably four, framework regions interspersed therebetween, which hold and present the CDRs in the proper configuration. Classical four-chain antibodies have an antigen-binding site defined by the VH and VL domains in cooperation. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on a binding site formed only by the heavy chain. Single-domain engineered immunoglobulins can be prepared in which the binding site is formed by the heavy or light chain alone, in the absence of cooperation between VH and VL.

[0073] The antibodies or antigen-binding fragments thereof described herein may be isolated or purified to any degree. In some embodiments, contaminant components of its natural environment are substances that would interfere with the (diagnostic) use of the antibody and may include enzymes and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody or antigen-binding fragment is purified to (1) greater than 95% by weight of the antibody as determined by the Lowry method, most preferably greater than 99% by weight, (2) sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or preferably silver stain. Typically, an isolated antibody is prepared by at least one purification step.

[0074] The antibody may be any of the IgA, IgD, IgE, IgG, and IgM classes, preferably an IgG class antibody. The antibody may be a polyclonal antibody, such as an antiserum or immunoglobulin purified therefrom (e.g., affinity purified). The antibody may be a monoclonal antibody or a mixture of monoclonal antibodies. Monoclonal antibodies can target a particular antigen or a particular epitope within an antigen with greater selectivity and reproducibility. By way of example, and not limitation, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or trioma technology, or using human B-cell hybridoma technology (Kozbor (1983) Immunology Today 4:72) and EBV-hybridoma technology (Cole et al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Lis, Inc. 77-96).

[0075] The DNA encoding the antibody or antigen-binding fragment thereof can be sequenced to provide information for small- or large-scale recombinant production of the antibody or antigen-binding fragment thereof. Also disclosed are methods of generating antibodies, including immunizing a non-human animal with the CEMA conjugates described herein. Methods for producing antibodies and antigen-binding fragments thereof are well known in the art, as are methods for producing recombinant antibodies or antigen-binding fragments thereof (see, for example, Harlow and Lane, "Antibodies: A Laboratory Manual" (Cold Spring Harbour Laboratory, New York, 1988); Harlow and Lane, "Using Antibodies: A Laboratory Manual" (Cold Spring Harbour Laboratory, New York, 1999, ISBN 0879695447); "Monoclonal Antibodies: A Manual of Technique" (ed. Zola, CRC Press 1987, ISBN 0849364760); "Monoclonal Antibodies: A Practical Approach" (ed. Dean & Shepherd, Oxford University Press 2000, ISBN 0199637229); Methods in Molecular Biology, Vol. 248, "Antibody Engineering: Methods and Protocols" (ed. Lo, Humana Press 2004, ISBN (See, for example, US Pat. No. 1588290921). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) (Nature 352:624-628) and Marks et al. (1991) J. Mol. Biol. 222:581-597.

[0076] The antibody may be an antigen-binding fragment thereof. Such fragments include portions of an intact antibody that comprise the antigen-binding or variable regions thereof. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, and scFv fragments, diabodies, linear antibodies, single-chain antibody molecules, and multivalent and / or multispecific antibodies formed from antigen-binding fragments, e.g., dibodies, tribodies, and multibodies.

[0077] Those skilled in the art will understand that an antibody may contain one or more amino acid deletions, additions, and / or substitutions (e.g., conservative substitutions), provided that such alterations retain binding of CEMA or a CEMA conjugate. To retain binding, such alterations may be made in portions of the antibody's amino acid sequence that are not involved in binding of CEMA or a CEMA conjugate. An antibody may also contain natural or artificial modifications (e.g., glycosylation) of one or more of its constituent amino acid residues, so long as such alterations retain binding of CEMA or a CEMA conjugate.

[0078] The antibody or antigen-binding fragment thereof may be associated with or attached to a detection agent, such as a label, to facilitate detection. Examples of such detection agents include, but are not limited to, luminescent labels, colorimetric labels such as dyes, fluorescent labels, or chemical labels such as electroactive agents, enzymes, radioactive labels, or radio frequency labels. Examples of detection agents include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. The detection agent may be a particle, including, but not limited to, colloidal gold particles, colloidal sulfur particles, colloidal selenium particles, colloidal barium sulfate particles, colloidal iron sulfate particles, metal iodate particles, silver halide particles, silica particles, colloidal metal (hydrous) oxide particles, colloidal metal sulfide particles, colloidal lead selenide particles, colloidal cadmium selenide particles, colloidal metal phosphate particles, colloidal metal ferrite particles, any of the above colloidal particles coated with an organic or inorganic layer, protein or peptide molecules, liposomes, or organic polymer latex particles such as polystyrene latex beads. A more suitable and preferred particle is colloidal gold particle, which is one of the most commonly used labels. Colloidal gold may be made by any conventional means, such as the method of G. Frens, 1973 Nature Physical Science, 241:20 (1973). Alternative methods are described in US 5,578,577, US 5,141,850, US 5,079,172, US 5,202,267, US 5,514,602, US 5,616,467, and US 5,681,775.

[0079] The detection agent may be a tag that allows detection with another agent, such as a binding partner. Such tags may be, for example, FLAG tags, biotin, streptavidin, his-tags, myc-tags, maltose, maltose binding protein, or any other type of tag known in the art that has a binding partner. Examples of associations that may be utilized may include biotin:streptavidin, his-tags / metal ions, or maltose / maltose binding protein. In certain embodiments, the use of FLAG tags is preferred.

[0080] In some embodiments, the antibody is directly or indirectly labeled to allow detection of the metabolite in a sample. For example, a labeled antibody can be combined with a sample and the labeled antibody-metabolite complex is detected.

[0081] According to the present invention, CEMA can be detected using the antibodies or antigen-binding fragments thereof described herein. In particular, the antibodies or antigen-binding fragments thereof can bind to CEMA and can bind to a conjugate comprising a compound of formula [II]: [ka] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C1-C6 alkyl, preferably a compound of formula [I]: [ka] The compound of formula [II] or formula [I] is bound to the immunogenic carrier via a linker. In one embodiment, the linker is bound to the compound of formula [I] via an amine group.

[0082] In one embodiment, the compound of formula [I] has the structure of a compound of formula [III]: [ka] wherein R is selected from H and C1-C6 alkyl.

[0083] Three such antibodies of the invention have been extensively characterized and are designated herein as "B11 scFv," "E6 scFv," and "G4 scFv" (and may also be referred to herein as "B11," "E6," and "G4"). The polypeptide sequences of each of the three CDRs of the variable domains of the VL and VH chains of each of B11 scFv, E6 scFv, and G4 scFv are described herein.

[0084] The VH CDR1, VH CDR2, and VH CDR3 of B11 scFv correspond to the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively. The VL CDR1, VL CDR2, and VL CDR3 of B11 scFv correspond to the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

[0085] The VH CDR1, VH CDR2, and VH CDR3 of E6 scFv correspond to the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively. The VL CDR1, VL CDR2, and VL CDR3 of E6 scFv correspond to the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0086] The VH CDR1, VH CDR2, and VH CDR3 of G4 scFv correspond to the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively. The VL CDR1, VL CDR2, and VL CDR3 of G4 scFv correspond to the amino acid sequences of SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively.

[0087] The antibody or antigen-binding fragment thereof can comprise a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2, 11, or 20.

[0088] The antibody or antigen-binding fragment thereof can comprise a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:6, 15, or 24.

[0089] The antibody or antigen-binding fragment thereof can comprise a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 6. These are the VH and VL amino acid sequences of B11 scFv.

[0090] The antibody or antigen-binding fragment thereof can comprise a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 15. These are the VH amino acid sequence and VL amino acid sequence of E6 scFv.

[0091] The antibody or antigen-binding fragment thereof can comprise a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 24. These are the VH amino acid sequence and VL amino acid sequence of G4 scFv.

[0092] Also disclosed are scFvs comprising or consisting of the amino acid sequence of SEQ ID NO:1 (B11 scFv) or SEQ ID NO:10 (E6 scFv) or SEQ ID NO:19 (G4 scFv).

[0093] A detailed analysis of the amino acid sequences of B11, E6, and G4 scFv according to Kabat numbering is shown in Table 1.

[0094] The polynucleotide sequences of each of the variable domains of the VL and VH chains of each of B11 scFv, E6 scFv and G4 scFv are also described herein.

[0095] The VH of the B11 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 29. The VL of the B11 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 30.

[0096] The VH of the E6 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 32. The VL of the B11 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 33.

[0097] The VH of the G4 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 35. The VL of the B11 scFv may be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO: 36.

[0098] The antibody or antigen-binding fragment thereof may comprise a VH encoded by a polynucleotide sequence comprising, or consisting of, SEQ ID NO:29 or SEQ ID NO:32 or SEQ ID NO:35.

[0099] The antibody, or antigen-binding fragment thereof, may comprise a VL encoded by a polynucleotide sequence comprising, or consisting of, SEQ ID NO:30, SEQ ID NO:33, or SEQ ID NO:36.

[0100] Also disclosed are scFVs that can be encoded by a polynucleotide sequence comprising or consisting of SEQ ID NO:28 (B11 scFv) or SEQ ID NO:31 (E6 scFv) or SEQ ID NO:34 (G4 scFv).

[0101] In certain embodiments, B11 and G4 are preferred. In certain embodiments, B11 is preferred.

[0102] As will be appreciated by those skilled in the art, there are various models for assigning / identifying CDR sequences in antibody VL / VH chains. The most common / widely accepted versions are the Chothia model and the Kabat model, but other models exist, such as the ABM model and the CONTACT model. The CDR sequences presented herein were determined using the Kabat model (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md.), as is conventional in the art.

[0103] Any one of a variety of immunogenic carrier proteins may be used in the conjugates of the present invention, e.g., proteins, peptides, oligonucleotides, or polymers. The immunogenic carrier protein is attached to a linker. Specific examples are albumins, such as bovine serum albumin (BSA), globulins, thyroglobulin, hemoglobin, hemocyanin, polylysine, polyglutamic acid, lysine-glutamic acid copolymers, and copolymers containing lysine or ornithine. Suitable classes of proteins include enterotoxins, outer membrane proteins, and excreted toxins of pathogenic bacteria, non-toxic or "toxoid" forms of such excreted toxins, bacterial toxins, and non-toxic proteins antigenically similar to other proteins. The use of viral proteins is also contemplated. In a preferred embodiment, the immunogenic carrier is a protein, preferably BSA or bovine thyroglobulin (BTG).

[0104] In one aspect, a method for preparing the above-mentioned conjugate is disclosed, comprising the steps of: (a) activating an immunogenic carrier; and (b) coupling the activated immunogenic carrier obtained in step (a) to a compound of formula (I) via a linker.

[0105] In the present invention, the compound of formula (I) is linked to an immunogenic carrier protein, which involves the use of the primary amine (-NH2) group of the compound of formula (I). There are numerous synthetic chemical groups that form chemical bonds with primary amines. These include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters. The majority of these conjugates couple with amines either by acylation or alkylation.

[0106] In one embodiment, the linker disuccinimidyl suberate (DSS) is used, which is a non-cleavable and membrane-permeable crosslinker that contains an amine-reactive N-hydroxysuccinimide (NHS) ester at each end of an 8-carbon spacer arm. The NHS ester reacts with primary amines at pH 7-9 to form stable amide bonds with release of an N-hydroxysuccinimide leaving group. DSS is first dissolved in an organic solvent such as DMF or DMSO and then added to the aqueous crosslinking reaction.

[0107] In another embodiment, the linker ethylene glycol bis(succinimidyl succinate) (EGS) is used. EGS is a water-insoluble homobifunctional N-hydroxysuccinimide ester (NHS ester). The spacer arm contains two cleavable ester sites that can be destroyed with hydroxylamine.

[0108] This results in two fragments with terminal amide bonds and release of ethylene glycol. Accessible α-amine groups present on the N-termini of proteins and peptides, including the ε-amine of lysine, react with NHS esters at pH 7-9 to form covalent amide bonds. The reaction results in the release of N-hydroxysuccinimide. NHS ester crosslinking reactions are most commonly performed in phosphate, carbonate / bicarbonate, HEPES, and borate buffers.

[0109] For preparation of conjugates, one of skill in the art can readily determine a suitable coupling method, e.g., as found in Hermanson, GT (1996) Bioconjugate Techniques 1st ed. Academic Press: San Diego, California, Vol. 1.

[0110] In one embodiment, the conjugate (immunogen) comprises a compound of formula [I]: [ka] wherein the compound of formula [I] is linked to BTG via an EGS linker that is attached to the amine group of the compound of formula [I]. This compound is referred to herein as "BTG-EGS-CEMA." In another embodiment, the conjugate (immunogen) comprises a compound of formula [I]: [ka] wherein the compound of formula [I] is conjugated to BTG via a DSS linker that is attached to the amine group of the compound of formula [I]. This conjugate is referred to herein as "BTG-DSS-CEMA."

[0111] The compounds of formula [I] ensure an intact CEMA structure after binding to a protein.

[0112] In an embodiment of the present invention, the antibody of the present invention is preferably an antigen-binding fragment. Suitably, the antigen-binding fragment is an scFv. According to the present disclosure, the scFv was identified by screening ELISA phages against the CEMA conjugate described herein, and among 96 colonies selected, 28 "hits" with signals were identified as positive. DNA was extracted and sequenced, and eight non-redundant sequences were identified, which were then used to generate eight scFvs, which were tested in ELISA and lateral flow immunoassays for CEMA binding.

[0113] 1) Three scFvs (herein designated G4 scFv, B11 scFv, and E6 scFv) were selected based on the highly favorable properties of reactivity with the CEMA carrier hapten. As can be seen from Figure 1, G4, B11, and E6 have better reactivity with BTG-EGS-CEMA and BTG-DSS-CEMA compared to CEMA-BTG and BTG alone in the ELISA assay.

[0114] 2) Binding inhibition in competitive ELISA using BTG-EGS-CEMA and free CEMA. As can be seen from Figure 2, G4, B11, and E6 have acceptable % inhibition at 10 μg / mL free CEMA with 1 ug / ml free CEMA distinguishing sensitivity.

[0115] 3) Binding inhibition in competitive ELISA using BTG-DSS-CEMA and free CEMA. As can be seen from Figure 3, G4, B11, and E6 have acceptable % inhibition at 10μg / mL free CEMA and 1ug / ml free CEMA with 0.1ug / ml free CEMA discriminating sensitivity.

[0116] 4) Reactivity testing of scFv via lateral flow immunoassay. As can be seen from Figure 4, G4, B11, and E6 are recognized by immunogens BTG-EGS-CEMA and BTG-DSS-CEMA. scFv G12 and G9 were not recognized by either immunogen, and scFv G8 had low recognition. Acceptable reactivity testing was obtained for scFv E6, F8, G6, B11, and G4.

[0117] 5) Cross-reaction with urine. As can be seen from Figure 5, good inhibition (showing positive signal) is achieved until 100ng / mL urine with B11, E6 and G4 on immunogen BTG-EGS-CEMA. The signal is slightly reduced in the presence of urine, but is not inhibited. As can be seen from Figure 6, good inhibition (showing positive signal) is achieved until 100ng / mL urine with B11, E6 and G4 on immunogen BTG-DSS-CEMA. The signal is slightly reduced in the presence of urine, but is not inhibited. As can be seen, B11, E6 and G4 allow detection of all positive urines with CEMA above 100ng / mL.

[0118] 6) As can be seen in FIG. 7, B11, E6 and G4 allow the detection of all positive urines with CEMA above 100 ng / mL.

[0119] 7) As can be seen from Figure 8, no cross-reactivity is seen with cotinine or compounds with structures close to CEMA, including 2-hydroxyethyl mercapturic acid (HEMA), monohydroxybutenyl mercapturic acid (MHBMA), 3-hydroxypropyl mercapturic acid (3-HPMA), or dihydroxybutyl mercapturic acid (DHBMA), using a tested concentration of 1 μg / ml. G4, B11 are highly specific for CEMA.

[0120] Based on this experimental data, it was concluded that G4, B11, and E6 are superior scFvs for detecting CEMA, especially in urine. Thus, in a preferred embodiment, the antibody is an antigen-binding fragment thereof. In a more preferred embodiment, the antigen-binding fragment is an scFv fragment. In a more preferred embodiment, the antigen-binding fragment is scFv G4, scFv B11, and scFv E6 as defined herein.

[0121] The F(ab')2 (110,000 dalton) fragment contains two antigen-binding domains linked at a hinge via disulfides. This fragment lacks most, but not all, of the Fc domain.

[0122] Fab' (55,000 dalton) fragments can be generated by reduction of the F(ab')2 fragment. Fab' fragments contain a free sulfhydryl group which can be alkylated or utilized in conjugation with enzymes, toxins, or other proteins of interest. Since Fab' is derived from F(ab')2, it may contain a small portion of Fc.

[0123] Fab (50,000 daltons) is a monovalent fragment that can be produced from IgG and / or IgM consisting of the VH, CH1 and VL, CL domains linked by intramolecular disulfide bonds.

[0124] Fv (25,000 daltons) is the smallest fragment produced from IgG and / or IgM that contains an intact antigen-binding site. Fv fragments have the same binding properties as Fab and similar three-dimensional binding properties. The VH and VL chains of the Fv fragment are held together by non-covalent interactions. Because these chains tend to dissociate upon dilution, methods have been developed to cross-link the chains via glutaraldehyde, intermolecular disulfides, or peptide linkers.

[0125] scFv is a single chain Fv and can be conveniently produced recombinantly. It has a molecular weight of about 28,000 Da. It consists of the VH and VL variable regions of immunoglobulins, connected with a short linker peptide of 10-25 amino acids. Each VH and VL domain contains three CDRs. To generate scFv, mRNA is first isolated from hybridomas (or spleen, lymphocytes, and bone marrow) followed by reverse transcription into cDNA, which serves as a template for antibody gene amplification, for example using PCR. Using this methodology, large libraries with a diverse range of antibody VH and VL genes can be generated. Biopanning, a procedure for selecting binding partners from a phage display library, is used to obtain scFvs with the best affinity and specificity. Once the antibody genes have been successfully cloned and sequenced, the scFv fragments can be easily expressed in a suitable expression system. Suitable purification tags are typically added to the C-terminus of antibody scFv fragments, such as ploy-histidine tags, FLAG-tags, HA-tags, and Myc-tags. Protease cleavage sizes can be designed to allow tag removal after purification. Alternatively, the tag can be retained and utilized in immunoassays. Further details regarding scFvs can be found in Ahmad et al. (2012) Clinical and developmental immunology (2012), Biosensors and Bioelectronics (2016) 85, 32-45, MAbs (2010) 2(1) 77-83, and US 4,946,778.

[0126] Also provided herein are isolated nucleic acids encoding antibodies and antigen-binding fragments thereof, vectors and host cells containing the nucleic acids, and recombinant techniques for the production of antibodies or antigen-binding fragments thereof. The antibodies described herein may be produced by recombinant expression. Nucleic acids encoding light and heavy chain variable regions are optionally linked to constant regions and inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector that ensure the expression of immunoglobulin polypeptides. Expression control sequences include, but are not limited to, promoters, signal sequences, enhancer elements, and transcription termination sequences. Once the expression vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the cross-reactive antibodies or antigen-binding fragments thereof. Generally, expression vectors contain a selectable marker that allows for the detection of those cells transformed with the desired DNA sequence. Antibodies or antigen-binding fragments thereof can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides, preferably signal sequences or other polypeptides with a specific cleavage site at the N-terminus of the polypeptide that is not delivered to the mature protein. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. When the heavy and light chains are cloned on separate expression vectors, the vectors are co-transfected to obtain expression and assembly of intact immunoglobulins. Once expressed, whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms can be purified. Substantially pure immunoglobulins of at least about 90-95% homogeneity are preferred, with 98-99% or more homogeneity being most preferred.

[0127] When using recombinant techniques, the antibody or antigen-binding fragment thereof can be produced intracellularly, in the lumen of the endoplasmic reticulum, or directly secreted into the medium. If the antibody or antigen-binding fragment thereof is produced intracellularly as a first step, particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., (1992) Bio / Technology 10:163-167, describes a procedure for isolating antibodies secreted into the lumen of the endoplasmic reticulum of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the antibody or antigen-binding fragment thereof is secreted into the medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants. Antibodies or antigen-binding fragments thereof prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody or antigen-binding fragment thereof to be recovered.

[0128] Immunoassays can be used to detect one or more of the metabolites described herein, including CEMA, using the antibodies or antigen-binding fragments thereof of the present invention. Immunoassay techniques are known in the art and include direct ELISA (enzyme-linked immunosorbent assay), indirect ELISA, sandwich ELISA, competitive ELISA, multiplex ELISA, radioimmunoassay (RIA) techniques, fluorescent immunoassays, chemiluminescent immunoassays, DRI immunoassays, quantitative immunoassays, lateral flow immunoassays, microfluidic immunoassays and agglutination immunoassays, as well as other similar techniques known in the art. The principles of these immunoassay methods are described, for example, in "The ELISA Guidebook" by John R. Crowther (1st ed., Humana Press 2000, ISBN 0896037282). Additional information on practical immunochromatography can be found in the handbook "Lateral flow immunochromatography assays" by PJ Davies et al., published March 15, 2008 (Wiley Online Library).

[0129] Direct ELISA uses a labeled primary antibody, or antigen-binding fragment thereof, to bind to and thereby quantitate a target antigen in a sample immobilized on a solid support, such as a microwell plate.

[0130] Indirect ELISA uses an unlabeled primary antibody or antigen-binding fragment thereof that binds to the target antibody and a labeled secondary antibody or antigen-binding fragment thereof that allows the antigen-binding primary antibody or antigen-binding fragment thereof to be recognized and quantified.

[0131] In sandwich ELISA, a target antigen is captured from a sample using an immobilized "capture" antibody that binds to one antigenic site within the antigen, and after removal of unbound metabolites, the so-captured antigen is detected using a "detection" antibody that binds to another antigenic site within said antigen, which may be directly labeled or indirectly detectable as described above.

[0132] Competitive ELISA is a preferred immunoassay, which uses a labeled "competitor" that can be either the primary antibody or the target antigen. In an embodiment, an immobilized unlabeled primary antibody is incubated with the sample, the reaction is allowed to reach equilibrium, and then the labeled target antigen is added. The latter binds to the primary antibody wherever its binding site is not already occupied by an unlabeled target antigen from the sample. Thus, the amount of detected labeled bound antigen is inversely correlated with the amount of unlabeled antigen in the sample.

[0133] Multiplex ELISAs allow for the simultaneous detection of two or more metabolites in a single compartment, usually at multiple sequence addresses (for further guidance see, e.g., Nielsen & Geierstanger 2004. J Immunol Methods 290:107-20, and Ling et al. 2007. Expert Rev Mol Diagn 7:87-98).

[0134] As will be appreciated, labels in ELISA techniques are often enzyme conjugated and endpoints are usually colorimetric, chemiluminescent or fluorescent, magnetic, piezoelectric, pyroelectric, and others.

[0135] Immunoassay devices are generally suitable for one-time use and home use, as they are simple and quick to use, and results can be visualized by the naked eye. In some situations, for example, when interpretation by the naked eye is impossible or causes uncertainty, more complex devices may be required to determine the presence and / or quantity of metabolites. Such devices are described in WO2016 / 075405.

[0136] Radioimmunoassays (RIAs) can be used to detect one or more of the metabolites described herein, including CEMA. This is a competition-based technique that involves the use of a known quantity of radioactively labeled (e.g., 125 I or 131I-labeled) target antigen is mixed with an antibody or fragment thereof, followed by the addition of unlabeled or "cold" antigen from the sample and measuring the amount of displaced target antigen (see "An Introduction to Radioimmunoassay and Related Techniques" edited by Chard T, Elsevier Science 1995, ISBN0444821198).

[0137] Agglutination immunoassays can be used to detect one or more of the metabolites described herein, including CEMA. These assays utilize the binding and agglutination (clumping) of antibodies or fragments thereof to antigen-DNA conjugates, allowing linkage of the DNA strands and subsequent quantification by methods including quantitative polymerase chain reaction (qPCR).

[0138] Advantageously, immunochromatography can be used to detect one or more of the metabolites described herein, including CEMA. This is also known as lateral flow immunochromatography assay. Immunochromatography can be integrated into a simple device for detecting the presence (or absence) of metabolites in a sample, such as urine, without the need for specialized expensive equipment. Immunochromatography can also be integrated into a portable lateral flow immunoassay device, as discussed below.

[0139] The general principle of immunochromatography is based on a liquid sample containing or suspected to contain one or more metabolites to be detected that migrates by capillary action without the aid of an external force through various zones of a lateral flow test strip. A lateral flow test strip generally comprises (i) a sample pad, (ii) a conjugate pad, (iii) a detection zone, and (iv) an optional absorbent pad. The sample pad and the optional absorbent pad are located at opposite ends of the lateral flow test strip. Typically, the conjugate pad is adjacent to the sample pad, the detection zone is adjacent to the conjugate pad, and the optional absorbent pad is adjacent to the detection zone.

[0140] The first element of the lateral flow test strip is a porous element for the sample, referred to herein as the sample pad. It acts as a sponge and holds excess sample fluid. It is usually made of cellulose or glass fibers, or a combination of both. Its function is to transport the sample to the other components of the lateral flow test strip. The sample pad must be smooth, continuous, and homogeneous to transport the sample. The sample pad can be impregnated with solutions such as buffer salts and surfactants, if necessary. Upon immersion, the fluid migrates to the second element of the lateral flow test strip.

[0141] The second element of the lateral flow test strip is a porous element for the conjugate, referred to herein as the conjugate pad. This is the case when labeled antibodies or antigen-binding fragments thereof are present, including those that can bind individually to CEMA (or, in the specific assay format, CEMA analogs) and, optionally, other metabolites. Glass fiber, cellulose, and polyester are typical examples of materials used to make the conjugate pad. The labeled antibodies or antigen-binding fragments thereof may be present in the conjugate pad in a dry format, for example, in a matrix, such as a salt-sugar matrix. In certain embodiments, when it is desired to detect metabolites in addition to CEMA, two or three of four or more different antibodies or their antigen-binding fragments may be used. The amounts may be adjusted as needed to fine-tune the sensitivity of the assay for each metabolite. Typically, the amount of labeled antibodies or their antigen-binding fragments used will be different for different metabolites. The labeled antibodies or their antigen-binding fragments may be labeled with the same or different labels as needed. The sample fluid solubilizes the labeled antibody or antigen-binding fragment thereof, and in one combined transport action, the sample and the labeled antibody or antigen-binding fragment thereof mix as they flow through the conjugate pad to form a labeled antibody-metabolite conjugate of the antigen-binding fragment. Lateral flow test strips have one or more detection zones, such as test lines, where another molecule is immobilized. The detection zones are usually nitrocellulose membranes. The exact configuration of the detection zones depends on the format of the assay. Determination of a positive or negative result can be done using the naked eye, or with a reader, if a numerical or automated result is required. For example, the reader can be a camera, such as a charge-coupled device camera, or an optical sensor, such as a confocal optical sensor.

[0142] Competitive formats are typically used for smaller metabolites such as CEMA, which have fewer binding sites, and are therefore generally preferred formats according to the present invention. The sample first encounters a labeled or tagged antibody against the target metabolite. In embodiments where a tagged antibody is used, detection is permitted by the tagged antibody, which is labeled. The test line comprises the target metabolite immobilized on the surface. If the target metabolite is not present in the sample, the unbound antibody will bind to these immobilized metabolite molecules, meaning that a visual marker is indicated. Conversely, if the target metabolite is present in the sample, it will bind to the antibody, preventing binding to the immobilized metabolites in the test line, and therefore no visual marker is indicated. In one embodiment of the device configured for use in competitive format, (a) the conjugate pad comprises or consists of a labeled antibody against the target metabolite disposed thereon, and (b) the detection zone comprises or consists of a target metabolite immobilized on the surface, which may comprise CEMA or a CEMA conjugate as described herein. In another embodiment of the device configured for use in a tag-based competitive format, (a) the conjugate pad comprises or consists of a labeled anti-tag antibody disposed thereon, (b) the intermediate pad contains an antibody or antigen-binding fragment thereof tagged to a target metabolite disposed thereon, said tagged antibody comprising a tagged CEMA antibody or antigen-binding fragment thereof as described herein, and (b) the detection zone comprises or consists of a target metabolite immobilized on a surface, which may comprise CEMA or a CEMA conjugate as described herein.

[0143] Preferably, the intermediate pad contains only CEMA tagged antibody or antigen binding fragment thereof therein. The separate location of the intermediate pad allows fine tuning of the amount of CEMA tagged antibody or antigen binding fragment thereof separate from the conjugate pad when more than one metabolite is detected.

[0144] After passing through the conjugate pad and detection zone, the fluid can enter a final porous element, referred to herein as the absorbent pad. This serves as a waste container and is an optional feature. Various portions of the lateral flow test strip are secured or attached onto a backing card, which serves as a support and facilitates handling of the strip.

[0145] Multiplex formats can also be used when detecting multiple metabolites with a device. Multiplex detection formats can be constructed in various ways, for example, by increasing the length of the lateral flow test strip or the length of the detection zone. Multiplex formats can be used in conjunction with competitive formats. In certain embodiments, multiplex competitive formats are preferred.

[0146] A signal is formed when the metabolite level in the sample is higher than a certain predefined threshold level, or reference value, or baseline value, to obtain a qualitative or semi-quantitative result. Depending on the assay format, the color or signal intensity may be compared to a reference color or signal chart. Alternatively, the amount or intensity of the color or signal may be measured by an electronic device, for example, with an absorbance sensor or light emission meter, resulting in a numerical value of the signal intensity or color absorption formed. This embodiment may be suitable for monitoring the aforementioned metabolite levels in a subject over a period of time. If necessary, it is possible to measure the intensity of the detection zone to determine the quantity of metabolites in the sample. Handheld diagnostic devices known as lateral flow readers are used by several companies to provide fully quantitative assay results. One such handheld lateral flow device platform is made by Detekt Biomedical LLC.

[0147] Lateral flow test strips can be configured to include a positive control that demonstrates that the test is effective for subjects who do not have any metabolites in their urine (e.g., using a protein specific to urine, such as creatine, albumin, or Tamm-Horsfall Protein (THP) as a marker). In one embodiment, the use of a lateral flow immunoassay is preferred. In another embodiment, the use of a competitive format lateral flow immunoassay is preferred.

[0148] In another embodiment, the present disclosure provides a portable lateral flow immunoassay device, such as a urine test strip, for detecting one or more of the metabolites described herein, the device using the principles of immunochromatography described above.

[0149] Portable lateral flow immunoassay devices typically comprise a lateral flow test strip contained in a housing, such as a liquid-tight or liquid-impermeable housing, to allow immersion of the device in a sample, wetting only those necessary elements of the lateral flow test strip. The devices typically have an elongated shape, the dimensions of which can vary depending on the actual use of the device, with exemplary dimensions being 6-8 cm in length and 3-6 mm in width.

[0150] Examples of portable lateral flow immunoassay devices are described in WO2007 / 023372, EP1657550, US2015 / 168397.

[0151] FIG. 10 shows a preferred portable lateral flow immunoassay device or urine test strip 100 configured in a competitive assay format. The device or urine test strip 100 is configured to detect CEMA and cotinine in this embodiment. In other embodiments, the device or urine test strip 100 may be configured to detect metabolites other than CEMA and cotinine. In other embodiments, the device or urine test strip 100 may be configured to detect only CEMA. The device 100 is made of a PVC support 109 to provide rigidity and has a nitrocellulose membrane 110 attached thereon. Starting at the proximal end, there is a sample pad 102 for deposition of the sample to be tested, and moving towards the distal end of the device, there is shown a conjugate pad 103 adjacent to the sample pad, an intermediate pad 104 adjacent to the conjugate pad, a cotinine test line 105, a CEMA test line 106, and a control test line 107. Those skilled in the art will understand that the conjugate pad and intermediate pad can be positioned as shown or replaced as needed. Those skilled in the art will also understand that the test lines can be arranged in any order, if desired. The lateral flow device or urine test strip 100 terminates at the distal end with an absorbent pad 108 to serve as a waste receptacle. The conjugate pad 103 contains a porous material that includes (i) a dried monoclonal tagged (e.g., anti-FLAG tag) antibody bound to a label (colloidal gold) and (ii) a polyclonal anti-cotinine antibody bound to the same label (colloidal gold). Different labels can be used if desired. The middle pad 104 is made of a porous material that contains a dried anti-CEMA scFv with a tag (e.g., FLAG tag). The FLAG tag is a polypeptide tag that can be added to proteins with the amino acid sequence motif DYKDDDDK (Hopp et al. (1988) Bio / Technology. 6(10):1204-10). The CEMA test line 106 includes a CEMA conjugate as described herein. The presence of anti-CEMA scFv on the CEMA test line 106 is detected using an anti-tag antibody bound to colloidal gold that was originally present on the conjugate pad.The cotinine test line 105 contains a cotinine-protein conjugate. The presence of cotinine is detected using anti-cotinine antibodies bound to the same label (colloidal gold) originally present in the conjugate pad. Although not shown, the device 100 can be housed in a liquid-tight or impermeable housing, allowing for immersion of the device 100 in a liquid sample. The housing can include or consist of plastic. The sample can be a liquid sample, such as urine. The device 100 has an elongated shape, the dimensions of which can vary depending on the actual use of the device, with exemplary dimensions being 6-8 cm in length and 3-6 mm in width.

[0152] The results of an exemplary competitive immunoassay test format are shown in FIG. 10, where a positive test for cotinine and CEMA indicates that the test subject is a smoker. In one embodiment of the present invention, the detection limit of the test is 200 ng / mL for the cotinine test line and 160 ng / mL for the CEMA test line. If the amount of cotinine in the test sample is greater than 200 ng / mL, a positive signal is generated, and if the amount of cotinine in the test sample is less than 200 ng / mL, a negative signal is generated. If the amount of CEMA in the test sample is greater than 160 ng / mL, a positive signal is generated, and if the amount of CEMA in the test sample is less than 160 ng / mL, a negative signal is generated. In the competitive format, a positive result for cotinine or CEMA means that the test line is not present, and a negative result for cotinine or CEMA means that the test line is present. A negative test for cotinine and CEMA indicates that the test subject is a non-smoker. A positive test for cotinine and a negative test for CEMA indicates that the test subject is a switcher from a smoker to a consumer of RRPs. The absence of a control line indicates an invalid test. A positive test indicates that the threshold of detection has been crossed. Because the test configuration shown in FIG. 10 is a competitive assay, the result is positive if there is no CEMA or cotinine test line, such that the absence of a signal at the test line is indicative of the presence of metabolites, while the appearance of a signal indicates the absence of metabolites. In certain embodiments, competitive formats are preferred for use in the present disclosure.

[0153] The present disclosure further provides kits for the detection of metabolites comprising means for detecting the level of one or more metabolites in a sample from a subject, in preferred embodiments, such kit or kits are ideally designed for use by a physician at home or in general practice.

[0154] Further disclosed are kits, particularly kits for determining the smoking status of a subject as taught herein, comprising (i) means for measuring metabolites as taught herein, particularly in a sample from the subject, and (ii) optionally, a reference value for one or more metabolites, or means for establishing said reference value, said reference value representing detection of the metabolite.

[0155] A home test kit may provide the subject with a readout that the subject can communicate to a physician, after which appropriate action can be taken. Non-limiting examples include systems with specific binding molecules for the required metabolites attached to a solid phase, such as urine test strips, for example portable lateral flow immunoassay devices. One non-limiting example is the use of lateral flow test strips and labeled antibodies or antigen-binding fragments thereof, a combination that does not require any membrane washing. Lateral flow test strips are well known in the field of pregnancy test kits, for example, where a first anti-hCG antibody is present on a support and is carried by the urine flow to a second immobilized anti-hCG antibody that complexes with hCG and allows visualization. Other non-limiting examples of such home testing devices, systems, or kits can be found, for example, in the following U.S. Patents: 6,107,045, 6,974,706, 5,108,889, 6,027,944, 6,482,156, 6,511,814, 5,824,268, 5,726,010, 6,001,658, or U.S. Patent Application Nos. 2008 / 0090305 or 2003 / 0109067.

[0156] Also disclosed is a kit for determining the smoking status of a subject, comprising: (i) a first device adapted to detect the presence of CEMA in a biological sample; and (ii) a second device adapted to detect the presence of cotinine in the biological sample, and optionally, a set of instructions for determining the smoking status of the subject.

[0157] The present invention further provides a nucleic acid construct comprising a polynucleotide as described herein. Typically, the construct is an expression vector that allows expression in a suitable host of a polypeptide encoded by the polynucleotide. The construct may comprise one or more of the following: a promoter active in the host, an enhancer, an origin of replication, and a marker, preferably a selectable marker. The host may be a eukaryotic or prokaryotic host. The construct may comprise a polynucleotide encoding a polypeptide encoding an scFv. The construct may comprise a polynucleotide encoding a polypeptide comprising three light chains or three heavy chains. Alternatively, the polynucleotide may encode a polypeptide comprising three heavy chains and three light chains linked by a suitably flexible linker of suitable length. Another possibility is that a single construct may comprise polynucleotides encoding two separate polypeptides, one comprising a light chain and one comprising a heavy chain. The separate polypeptides may be expressed independently or may form part of a single common operon. The construct may comprise one or more regulatory features, such as an enhancer, an origin of replication, and one or more markers (selectable or otherwise). The construct may be provided in liquid or solid form, preferably as a lyophilized powder, which is typically rehydrated with a sterile aqueous liquid prior to use.

[0158] Vectors include expression vectors, transformation vectors, and shuttle vectors.

[0159] An expression vector refers to a construct capable of in vivo or in vitro expression.

[0160] A transformation vector is a construct that can be transferred from one entity to another, which may be a species or a different species. If the construct can be transferred from one species to another, such as from an E. coli plasmid to a bacterium such as Bacillus, the transformation vector may be called a shuttle vector. It may also be a construct that can be transferred from an E. coli plasmid to an Agrobacterium to a plant.

[0161] The vector may be transformed into a suitable host cell as described below to provide for expression of a polypeptide encompassed by the invention. Thus, in a further aspect, the invention provides a process for preparing a polypeptide for use in the invention, comprising culturing a host cell transformed or transfected with an expression vector as described above under conditions that provide for expression by the vector of a coding sequence encoding the polypeptide, and recovering the expressed polypeptide. The vector may be, for example, a plasmid, virus, or phage vector provided with an origin of replication, optionally a promoter for expression of the aforementioned polynucleotide, and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes as are well known in the art. Many known heavy and light chain expression vectors are commercially available. The skilled artisan may select a vector that expresses the same constant region subtype as the original antibody. The sequences of the heavy and light chain variable regions are then readily placed in the vector accordingly. There is a wide range of known vectors commercially available for scFv expression.

[0162] The invention further provides a host cell, such as an in vitro host cell, comprising a polynucleotide or construct described herein. The host cell may be, for example, a bacterium, a yeast or other fungal cell, an insect cell, a plant cell, or a mammalian cell. In certain embodiments, the host cell is a bacterium.

[0163] E. coli is one prokaryotic host that may be used and is preferred in certain embodiments. Other microbial hosts include bacilli, such as Bacillus subtilis, and other Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts, expression vectors can be made, which typically contain expression control sequences compatible with the host cell (e.g., origin of replication). In addition, there are any number of different well-known promoters, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system from phage lambda. The promoter typically controls expression, optionally with an operator sequence, to initiate and complete transcription and translation, and has a ribosome binding site sequence, etc.

[0164] Other microbes such as yeast may also be used for expression. Saccharomyces is a preferred yeast host, optionally with a suitable vector having expression control sequences (e.g., promoters), origin of replication, termination sequences, etc. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization.

[0165] In addition to microorganisms, mammalian tissue cell cultures may be used to express and produce the antibodies or fragments thereof described herein (see Winnacker, From Genes to Clones, VCH Publishers, NY, NY (1987)). Several suitable host cell lines capable of secreting heterologous proteins (e.g., intact immunoglobulins) have been developed in the art and include CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, or transformed B cells or hybridomas. CHO cells are preferred in certain embodiments.

[0166] Alternatively, the antibody coding sequence can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Patent No. 5,741,957). Suitable transgenes include light and / or heavy chain coding sequences operably linked to a promoter and enhancer from a mammary gland-specific gene such as casein or beta-lactoglobulin.

[0167] Alternatively, the antibodies or antigen-binding fragments thereof described herein can be produced in transgenic plants such as tobacco, corn, soybean, and alfalfa. Improved "plant antibody" vectors (see Hendy et al. (1999) J. Immunol. Methods 231:137-146) and purification strategies, coupled with an increasing number of transformable crop species, make these methods practical and efficient ways to produce recombinant immunoglobulins. Furthermore, antibodies produced in plants have been shown to be safe and effective.

[0168] Full length antibodies or antigen-binding fragments or antibody fusion proteins can be produced in bacteria. Production in E. coli is faster and more cost-effective. For expression of antigen-binding fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523.

[0169] The present invention is further described in the following examples, which are provided to describe the invention in greater detail. These examples describe preferred modes currently contemplated for carrying out the invention and are intended to illustrate, but not to limit, the invention. EXAMPLES

[0170] Example 1 - Synthesis of CEMA-linked conjugates Cyanoethyl mercapturic acid (CEMA) is conjugated with beta-thyroglobulin (BTG) as a carrier protein using disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) linkers. First, CEMA and BTG are diluted to 20 mg / ml in PBS (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 aqueous solution). EGS or DSS are separately dissolved at 15 mM in dry DMSO for 2 hours at room temperature, followed by centrifugation at 2000 rpm for 2 minutes and collecting the resulting supernatant. The supernatant is then added to the CEMA-BTG mixture in 10x molar excess and incubated at room temperature for four hours, followed by quenching with 1 M Tris buffer (pH 7.4) for 30 minutes to a final concentration of 20 mM. The mixture is then centrifuged at 2000 rpm for 2 minutes and the supernatant is collected and then dialyzed for 2 hours in Tris / borate / EDTA (TBE) buffer (10X buffer: 1 M Tris base, 1 M boric acid, 0.02 M EDTA in RNase-free water, diluted to 1X for use) to remove residual DMSO.

[0171] Example 2 - Immunization and anti-CEMA library construction Three-month-old New Zealand rabbits are subcutaneously injected with 1 mg of BTG-DSS-CEMA or BTG-EGS-CEMA immunogen (prepared according to Example 1), diluted in a 1:1 ratio in incomplete Freund's adjuvant. Rabbits are injected every two weeks for a total of up to five injections. Antibody titers are tested one week after the third to fifth injections by obtaining a 5 mL blood sample serum from an ear vein. It is then allowed to clot for one hour at room temperature before being centrifuged at 3200 rpm for 15 minutes, and the serum layer is pipetted into a new tube. The antibody titers of the collected serum are then tested using an indirect ELISA assay against BSA-DSS-CEMA, BSA-EGS-CEMA, and BSA as a negative control. To do this, 96-well plates are coated overnight at 4°C with 1 μg / ml of conjugated antigen (in PBS, pH 7.5). After washing three times with PBS, the plates are then blocked with 2.5% Milk in PBS (pH 7.4) and washed three more times with PBS. Then, 50 μL of primary serum (diluted 1:100 and serially diluted 1 / 10) is added per well and the plates are incubated for 1 hour at 37°C before being washed three times with PBS. Then, 50 μL of anti-rabbit IgG antibody conjugated to HRP (Jackson 111-036-046) is added at 1 / 5000 dilution and incubated for 1 hour at 37°C before being washed three times again with PBS. Finally, the wells are incubated for 10 minutes with 50 μL of TMB (KPL 52-00-01) before being stopped by adding 50 μL of acid stop solution (H2SO4 0.1M). The signal is then measured at 450 nm in a BioteK EL808 reader.

[0172] The titer of the response is calculated as the dilution giving 50% of the highest signal in the ELISA. The rabbit with the highest antibody titer (1 / 170000) is selected and given one further injection of 1 mg of BTG-DSS-CEMA or BTG-EGS-CEMA diluted in incomplete Freund's adjuvant at a 1:1 ratio, before being euthanized and the spleen removed.

[0173] Spleens are harvested, cut into small pieces in Trizol reagent, pulverized using a dispersing device (IKA T18D Ultra-Turrax), and then mRNA is extracted by standard Trizol / BCP protocol. To do this, pulverized rabbit spleens in 30 ml of TRI reagent are incubated at room temperature for 5 minutes and then centrifuged (4° C., 2500 g, 10 minutes). 3 mL of BCP is then added to the supernatant in a new tube and incubated at room temperature for 15 minutes. The tube is centrifuged (4° C., 17500 g, 15 minutes), 12 mL of isopropanol is added to the supernatant, vortexed (15 seconds), and incubated at room temperature for 10 minutes. Then, after centrifugation (4° C., 17500 g, 10 minutes), the pellet of total RNA is washed using 1.5 mL of ethanol 75%, centrifuged (4° C., 17500 g, 10 minutes), and then dried at room temperature. For quantification, the RNA pellet is dissolved in molecular biology grade water and quantified on a spectrometer (Nanodrop). Total RNA is then analyzed on a 0.8% (w / v) agarose gel, thereby confirming the 18S and 28S rRNA bands, and the remaining RNA is stored at a temperature of -20°C.

[0174] For immune library construction, 20 μg of total RNA is reverse transcribed using SuperScript III First Strand Synthesis (SuperMix (Invitrogen)) according to the manufacturer's instructions. In the first step, 20 μg of isolated mRNA is mixed with 1 μL of annealing buffer and 1 μL (50 μM) of oligo dT, incubated at 65° C. for 5 min, and placed on ice for 1 min. Then, still on ice, 10 μl of 2× First Strand Reaction Mix and SuperScript™ III (Thermo Fisher Scientific) / RNaseOUT™ Enzyme Mix (Thermo Fisher Scientific) are added according to the manufacturer's instructions. After mixing, the reaction is placed at 50° C. for 50 min. The reaction is terminated by incubation at 85° C. for 5 min. After termination, the reaction mixture is placed on ice or stored at −20° C. depending on future needs, since cDNA is more stable than RNA for storage. PCR is performed to amplify the cDNA encoding the variable domains VLκ, VLλ, and VH using rabbit specific primers VLκ, VLλ, and VH forward and reverse primers (Ridder, R., Schmitz, R., Legay, F. et al. Generation of Rabbit Monoclonal Antibody Fragments from a Combinatorial Phage Display Library and Their Production in the Yeast Pichia pastoris. Nat Biotechnol 13, 255-260 (1995)). For a 50 μL total volume PCR reaction, 2 μL of cDNA product, 10× PCR buffer (100 mM Tris-HCl, 15 mM MgCl2, and 50 mM KCl, pH 8.3), 0.2 μM primers, 0.2 mM dNTPs, 1 mM MgCl2, and 5 units of Taq DNA polymerase are introduced into a reaction vessel, and the final volume of the reaction mixture is adjusted with sterile triple distilled water.The PCR reaction consisted of 35 cycles, each cycle including heating to denatured DNA for 5 min at 94° C. (pre-denaturation), 30 s at 94° C. (denaturation), 30 s at 55° C. (annealing), and 60 s at 72° C. (polymerization). After the final cycle, the PCR reaction was terminated by heating for an additional 10 min at 72° C. After PCR, 5 μL of each PCR product was analyzed on an agarose gel (0.8%) and only VLκ, VLλ, and VH reactions showing amplification were pooled.

[0175] The cDNA fragments encoding VL (VLκ and VLλ) and VH, respectively, are electrophoresed on a 1.5% agarose gel, extracted / purified using a gel extraction kit (Illustra GFXR, GE) according to the manufacturer's instructions, and cloned into the pGemT vector (Promega) as backup material by preparing the ligation reaction as follows: [Table 1]

[0176] The three ligation reactions are incubated overnight at 4°C and stored at -20°C.

[0177] The heavy and light chain PCR products are successively cloned into the pTH1 phagemid vector (Biotem, France) as follows.

[0178] In the first step, pTH1 domain vector and VL domain vector are prepared for library cloning. Both vectors are digested for VL cloning as follows: [Table 2]

[0179] The digest is incubated for 2 hours at 37° C. and controlled by 1.5% TAE agarose gel electrophoresis. The enzyme is inactivated at 65° C. for 10 minutes, 0.5 μL of CIP (1 U / μL) is added, and incubated for 30 minutes at 37° C. Both the pTH1 and VL domains are purified using a PCR purification kit according to the manufacturer's instructions and eluted in 50 μL of elution buffer or water.

[0180] The VL (approximately 380 bp) is used for ligation in the vector pTH1 as follows. [Table 3]

[0181] Incubate overnight at 16 °C, precipitate the ligation product with 10 μL 3 M sodium acetate pH 5.2 and 250 μL ethanol, incubate for 2 min at room temperature and centrifuge for 5 min at 16000 g at 4 °C. The pellet is washed with ethanol and dissolved in 35 μL dH2O after centrifugation for 2 min at 16000 g at 4 °C. For DNA amplification, the ligation reaction is incubated with electrocompetent XL1-Blue MRF' cells before electroporation using a 1.7 kV pulse. The pulse time is 4-5 ms for optimal electroporation efficiency. Immediately add 1 mL 37 °C pre-warmed SOC medium, store in a 2 mL cap and shake for 1 h at 600 rpm and 37 °C. To determine the amount of transformants, aliquot 10 μL (=10 -2 Dilution) to 10 -6 Perform serial dilutions up to dilution 10 and plate on 2xYT-GAT agar plates. -6 Plate the dilution and incubate overnight at 37 °C. Plate the remaining 990 µL onto a 2xYT-GAT agar plate and incubate overnight at 37 °C. Then, pick the colonies on the plate using a drigalsky spatula in 40 mL of 2xYT medium and use 5 mL of the bacterial solution for midi plasmid preparation according to the manufacturer's instructions.

[0182] In the second cloning step, the pTH1-VL and pGemT-VH repertoires are digested as follows. [Table 4]

[0183] The digest is incubated for 2 hours at 37°C and controlled by 1.5% TAE agarose gel electrophoresis. The enzyme is inactivated at 65°C for 10 minutes, 0.5 μL of CIP (1 U / μL) is added and incubated at 37°C for 30 minutes. Both pTH1-VL domain and VL domain are purified using a PCR purification kit according to the manufacturer's instructions and eluted in 50 μL of elution buffer or water. The vectors pTH1-VL (approximately 4610 bp) and VH (approximately 380 bp) are ligated as follows: [Table 5]

[0184] Incubate overnight at 16 °C, precipitate the ligation product with 10 μL 3M sodium acetate pH 5.2 and 250 μL ethanol, incubate for 2 min at room temperature and centrifuge for 5 min at 16000 g at 4 °C. The pellet is washed with ethanol and dissolved in 35 μL dH2O after centrifugation for 2 min at 16000 g at 4 °C. For DNA amplification, the ligation reaction is incubated with electrocompetent XL1-Blue MRF' cells before electroporation using a 1.7 kV pulse. The pulse time is 4-5 ms for optimal electroporation efficiency. Immediately add 1 mL 37 °C pre-warmed SOC medium, store in a 2 mL cap and shake for 1 h at 600 rpm and 37 °C. To determine the amount of transformants, 10 μL of the transformation (=10-2 dilution) is used to make a 10 -6 Perform serial dilutions up to dilution 10 and plate on 2xYT-GAT agar plates. -6Plate the dilution and incubate overnight at 37 °C. Plate the remaining 990 µL onto a 2xYT-GAT agar plate and incubate overnight at 37 °C. Then, pick the colonies on the plate in 40 mL of 2xYT medium using a drigalsky spatula, use 5 mL of the bacterial solution for midi plasmid preparation according to the manufacturer's instructions, and use 800 µL of the bacterial solution for glycerol stocks. Make 5 to 25 glycerol stocks out of the library and store at -80 °C.

[0185] To package the library, inoculate 400 mL of 2xTY-GA in a 1 L Erlenmeyer flask with 1 mL of antibody gene library stock and incubate at 37 °C and 250 rpm until the OD is ~0.5.

[0186] Bacterial culture (about 1.25*10 10 cells), 2.5*10 11 Mix with 10 ...

[0187] Example 3 - Panning For the selection of phage-displayed antibody fragments, panning is performed. To do this, 96-well Nunc Maxisorp round-bottom microtiter plates (Thermo Fisher Scientific) are coated overnight at 4 °C with 4 mL of 10-100 μg / mL antigen solution (BTG-EGS-CEMA, BTG-DSS-CEMA in 0.1 M NaHCO3 buffer, pH 8.6). The coated wells are washed and blocked with 300 μL of PBS containing 4% skim milk (4% PBSM), washed twice with PBS / 0.1% Tween 20 (Sigma) and washed twice with PBS. Then, 7 × 10 7 100 μL of freshly prepared phage containing phage is added to each well and the plate is incubated at 37° C. for 2 hours with gentle rocking. Each well is washed five times with 200 μL of PBS containing 0.5% Tween 20. Bound phage in each well is eluted using 0.05% trypsin (Thermo Fisher Scientific). The eluted phage is then re-amplified and titered by transformation of E. coli (XL1 blue MRF') and incubation with M13K07 helper phage (as described above). New phage is used for further panning until significant enrichment of CEMA-specific phage is achieved. A total of seven biopanning rounds are performed against CEMA to enrich for bound phage.

[0188] Example 4 - Identification of the scFv with the strongest binding After panning, specific antigen binding is determined by ELISA to identify the phages / scFv with the strongest binding. To do this, ELISA-phage is performed by coating 96-well plates with BTG-EGS-CEMA or BTG-DSS-CEMA diluted in PBS (pH 7.5) overnight at 4°C. The plates are then washed three times with PBS, followed by blocking with 1% BSA in Tris-buffered (15 mM) saline (TBS, pH 7.4) and washing three more times with PBS. 50 μL of selected colonies are then added per well, and the plates are incubated for one hour at 37°C, followed by washing three times with PBS. 50 μL of anti-M13KO7 antibody conjugated to HRP is then added at a dilution, incubated for one hour at 37°C, followed by washing again three times with PBS. Finally, the wells are incubated with 50 μL of TMB substrate for 5 minutes, followed by stopping by adding 50 μL of acid stop solution. The signal is measured at 450 nm in a BioteK EL808 reader.

[0189] The 28 antibodies with the highest BTG-EGS-CEMA / BTG (signal / background) ratio are considered positive. DNA extraction is performed on these 28 clones and the eight non-redundant sequences identified by sequence alignment. These eight scFvs are then produced as soluble scFvs and tested by ELISA and LFIA.

[0190] Example 5 - Generation of selected scFvs and comparison of sensitivity using EGS or DSS in ELISA For soluble scFv production, the cloned DNA is used to transform E. coli HB2151, a production-specific E. coli strain. The scFv expressed as soluble scFv is purified using a HIS-Tag on an NI-NTA column (Qiagen) according to the manufacturer's instructions.

[0191] In competitive ELISA, unlabeled primary scFvs are incubated with the target antigen. To do this, 96-well ELISA plates are coated with 1 μg / ml BTG-EGS-CEMA or BTG-DSS-CEMA coating overnight at 4° C., washed three times the next day, and incubated with blocking buffer (PBS, pH 7.4 containing 2.5% milk) for 1 h at 37° C. to reduce non-specific binding. Unlabeled primary scFvs R4C-B11, R5C-G4, R5C-F8, R5C-G6, R5C-G9, and R5C-E6 are then added to the scFv-CEMA mixture wells of the pre-coated ELISA plate and incubated for 1 h at 37° C. The wells are then washed three times with PBS, followed by an additional hour of incubation at 37° C. with anti-Flag secondary antibody conjugated to HRP, followed by washing again with PBS and incubation with TMB substrate for 5 min. The reaction is then stopped by adding a stop solution and the signal is measured at 450 nm for light absorbance (OD value). A standard curve is generated using the same method, but instead of adding samples, a series of dilutions of recombinant CEMA of known concentrations are added to 6-8 wells. The OD is then used to calculate the amount of the molecule of interest in each well by comparing each sample well to the standard curve.

[0192] As shown in Figure 2, R4C-B11, R5C-G4, R5C-F8, R5C-G6, and R5C-E6 had high inhibition rates of 77-97% in the presence of 10 μg / mL free CEMA. However, at 1 μg / mL free CEMA, it is possible to identify the scFv with the best sensitivity. The most sensitive scFv to BTG-EGS-CEMA is as follows: R4C-B11>R5C-F8>R5C-G6>R5C-E6=R5C-G4.

[0193] Similarly, Figure 3 shows that R4C-B11, R5C-G4, R5C-F8, and R5C-E6 are effective in inhibiting the binding of BTG-DSS-CEMA in the presence of 10 μg / mL of free CEMA. However, at 0.1 μg / mL of free CEMA, the inhibition rates show that the scFvs with the best sensitivity to BTG-DSS-CEMA are as follows: R4C-B11>R5C-G6>R5C-F8>R5C-E6>R5C-G4.

[0194] Example 6 - Comparison of sensitivity of selected scFv prepared using EGS or DSS in lateral flow immunoassays In an exemplary lateral flow immunoassay, the lateral flow test strip includes (i) a sample pad, (ii) a conjugate pad, (iii) an intermediate pad, (iv) a detection zone, and (v) an optional absorbent pad (see FIG. 9). The conjugate pad (cellulose fiber) is prepared by saturating the pad with Tween 20, 0.5% BSA (wt / vol) in Hepes, pH 8, diluting a monoclonal anti-FLAG tag antibody conjugated to colloidal gold at an optical density 530 nm (OD) of 5, and diluting a polyclonal anti-cotinine antibody conjugated to colloidal gold at an OD of 0.5. The pad is dried overnight at 37° C. The intermediate pad is prepared by soaking the pad in Hepes, pH 8, with Tween 20, 0.5% BSA (wt / vol), and a 10 μg / ml dilution of anti-CEMA scFv. The detection zone contains two test lines microsprayed with 0.25 mg / ml free BSA-DSS-CEMA or BSA-EGS-CEMA or 0.25 mg / ml free cotinine-BSA on a nitrocellulose membrane, which is dried overnight at 37° C. Thus, when the sample pad of the lateral flow test strip is immersed in a sample liquid (e.g., urine) or when a sample is added to the sample well of the cassette using a pipette, the urine is drawn through the strip by capillary action through each pad, liberating the dried anti-cotinine antibody and anti-FLAG tag antibody to the middle pad, where the anti-CEMA scFv binds to CEMA in the sample and the anti-FLAG tag antibody binds to the anti-CEMA scFv. Similarly, the anti-cotinine antibody binds to cotinine in urine. These immunogen-antibody complexes are then drawn further down the lateral flow test strip onto the test line, and the antibodies bound to cotinine or CEMA in the urine are unable to bind to the cotinine or CEMA at the test line and therefore do not produce a line. However, if the urine sample is negative, the anti-CEMA and anti-cotinine antibodies are free to bind to the cotinine and CEMA on the test line and produce a colored line. The test results are read 5 minutes after addition of the urine sample, with a control line consisting of goat anti-mouse antibodies (see Figures 9 and 10).

[0195] When performing an LFIA, lateral flow test strips are prepared with each of the eight anti-CEMA scFvs described in Example 4 on an intermediate pad.

[0196] As shown in Figure 4, the scFvs are slightly better at binding to the BTG-EGS-CEMA immunogen than the BTG-DSS-CEMA immunogen. Similarly, Figure 4 shows that scFVs R5C-G12 and R5C-G9 do not recognize either immunogen. ScFV R5C-G8 also appears to recognize CEMA poorly.

[0197] Example 7 - Inhibition profile in urine Since scFVs R5C-G12, R5C-G9, and R5C-G8 show insufficient reactivity in Example 6, only R4C-B11, R5C-G4, R5C-F8, R5C-G6, and R5C-E6 are used to determine the urinary inhibition profile in the lateral flow immunoassay test in this example.

[0198] Lateral flow immunoassay testing was performed (as described in Example 6) using urine containing 10-500 ng / mL CEMA, with 1x PBS and CEMA-negative urine as negative controls on the BTG-EGS-CEMA coated on the test line. As shown in Figure 5, scFv R5C-G6 and R5C-F8 are slightly inhibited in the presence of negative urine and therefore may not be suitable for use in urine lateral flow testing due to the risk of false positives. The inhibition is believed to be due to the high ionic content of urine.

[0199] However, as shown in Figure 5, scFvs R4C-B11, R5C-E6, and R5C-G4 show negative results for both 1x PBS and negative urine samples, but show strong inhibition in the presence of 500 ng / mL and 100 ng / mL CEMA in the urine samples. R4C-B11 also shows strong inhibition in the 50 ng / mL CEMA urine sample. As shown in Figure 5, scFvs R4C-B11, R5C-E6, and E5C-G4 all show negative responses in the presence of very small amounts of CEMA in the urine samples (10-20 ng / mL).

[0200] Therefore, only scFvs R5C-G4, R4C-B11, and R5C-E6 are used to perform lateral flow immunoassays on the BTG-DSS-CEMA coating (see Figure 6). Each of the three selected scFvs shows good inhibition in 50-500 ng / mL CEMA urine samples.

[0201] Thus, scFv R5C-G4, R4C-B11, R5C-E6 are assayed at dilutions of CEMA ranging from 0.488 to 535 ng / mL (Figure 7). Each of scFv R5C-G4, R4C-B11, R5C-E6 allows the detection of positive urine samples containing >100 ng / mL. Samples below 35 ng / mL appear negative.

[0202] Example 8 - Specificity of selected scFvs Based on the results of Examples 5, 6, and 7, the specificity of scFvs R5C-G4, R4C-B11, and R5C-E6 is tested using a lateral flow immunoassay with other molecules on the test line that have a structure similar to that of CEMA, such as HEMA, MHBMA, 3-HPMA, DHBMA, etc., at a concentration of 1 μg / mL.

[0203] As shown in Figure 8, there is no cross-reactivity of scFvs R5C-G4, R4C-B11, and R5C-E6 with any of the similar molecules, and thus, scFvs R5C-G4, R4C-B11, and R5C-E6 are highly specific for CEMA.

[0204] Example 9 - Rapid test kit for the qualitative detection of CEMA and cotinine in human urine A rapid lateral flow immunochromatographic assay kit is designed for the qualitative detection of cotinine and CEMA in human urine. The test kit is aimed at distinguishing between cigarette smokers, RRP (reduced risk product) users, and non-smokers.

[0205] The kit is a competitive immunochromatographic competitive assay for the detection of cotinine and CEMA in human urine. If CEMA and / or cotinine are not present in the sample, a test line appears, but if CEMA or cotinine are present in the sample, the signal is inhibited and no test line appears. During the test, the urine sample reacts with colloidal gold nanoparticles conjugated to either a polyclonal antibody directed against cotinine or a monoclonal antibody or antigen-binding fragment thereof directed against CEMA. The mixture then migrates through the membrane by capillary action and fills the cotinine-BSA and CEMA-BSA conjugate molecules printed on test lines 1 and 2, respectively. If cotinine and / or CEMA are present in the sample, they inhibit the binding of the antibodies and prevent the colored test line from appearing. Conversely, if the sample does not contain cotinine and / or CEMA, the conjugated antibodies recognize the CEMA or cotinine conjugate molecules on the test line, resulting in the appearance of a test line. A colored line should always be above the control line marked "C" indicating that sufficient volume or urine was applied and the test transfer was performed properly.

[0206] Specimen Collection: Collect urine in the provided collection cup and identify the sample by writing the donor's name and urine collection date next to it. With the provided pipette, collect 100 μL of urine and press the top of the reservoir to dispense the total volume of liquid contained within the pipette tube into the device's specimen well. Read results for 5-10 minutes. Do not read after 10 minutes.

[0207] Negative result: Three lines appear, one in the control area (C) and one in each test line area cotinine (1) and CEMA (2). This should be interpreted as a non-smoker urine. A negative result can also be obtained if the cotinine and CEMA concentrations are below the detection limit (low number of cigarettes per day).

[0208] Positive result: One line appears in the control zone (C). This should be interpreted as a smoker's urine (both CEMA and cotinine in the urine). Two lines appear in the control zone (C) and the CEMA test line (2). This should be interpreted as a switcher's urine (only cotinine in the urine).

[0209] Invalid Result: If the control line (C) is not visible, the test result is invalid. Repeat the procedure with a new test.

[0210] Further aspects of the disclosure are described in the following numbered paragraphs.

[0211] 1. An antibody or antigen-binding fragment thereof capable of binding (i) N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA) and (ii) a conjugate comprising a compound of formula [II], [ka] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C1-C6 alkyl, preferably a compound of formula [I]: [ka] An antibody or antigen-binding fragment thereof, wherein the compound of formula [II] or formula [I] is attached to an immunogenic carrier via a linker, preferably the linker is attached to the compound of formula [I] via an amine group.

[0212] 2. The antibody or antigen-binding fragment thereof described in paragraph 1, wherein the immunogenic carrier is a protein, preferably bovine serum albumin or bovine thyroglobulin.

[0213] 3. The antibody or antigen-binding fragment thereof described in paragraph 1 or paragraph 2, wherein the linker is glycol bis(succinimidyl succinate) (EGS) or disuccinimidyl suberate (DSS).

[0214] 4. Suitably, the antibody or antigen-binding fragment thereof according to any of paragraphs 1 to 3, wherein the antibody is a monoclonal antibody, and preferably the antigen-binding fragment thereof is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, Fv, rIgG, or a diabody, more preferably an scFv.

[0215] 5. The antibody or antigen-binding fragment thereof of any of paragraphs 1 to 4, wherein the antibody does not bind to cotinine or 2-hydroxyethyl methacrylate (HEMA) or monohydroxybutenyl-mercapturic acid (MHBMA) or 3-hydroxypropyl mercapturic acid (3-HPMA) or dihydroxybutyl mercapturic acid (DHBMA).

[0216] 6. The antibody or antigen-binding fragment thereof described in any of paragraphs 1 to 5, wherein the inhibition rate by urine is 20% or less, 10% or less, 5% or less, or no inhibition.

[0217] 7. The antibody or antigen-binding fragment thereof described in any of paragraphs 1 to 6, wherein the antibody or antigen-binding fragment thereof has a detection limit of 160 ng / mL of CEMA in urine in an immunoassay.

[0218] 8. An antibody or an antigen-binding fragment thereof, comprising VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9, respectively; or VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; or VH CDR1, VH CDR2 and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:23, respectively, and VL CDR1, VL CDR2 and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27, respectively, or an antibody or an antigen-binding fragment thereof.

[0219] 9. The antibody or antigen-binding fragment thereof described in paragraph 8, wherein the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2, or 11, or 20.

[0220] 10. The antibody or antigen-binding fragment thereof of paragraph 8 or paragraph 9, wherein the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 6, or 15, or 24.

[0221] 11. The antibody or antigen-binding fragment thereof according to paragraph 8, wherein the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 6, or wherein said antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 15, or wherein said antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 24.

[0222] 12. The antibody or antigen-binding fragment thereof according to any of paragraphs 8 to 12, wherein the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, an Fv, an rIgG, or a diabody, more preferably an scFv.

[0223] 13. The antibody or antigen-binding fragment thereof according to paragraph 12, wherein the antigen-binding fragment is an scFv, and said scFv comprises the amino acid sequence of SEQ ID NO: 1 or 10 or SEQ ID NO: 19.

[0224] 14. A polynucleotide encoding the antibody or antigen-binding fragment thereof according to any one of paragraphs 1 to 13, or a polynucleotide complementary thereto.

[0225] 15. The polynucleotide according to paragraph 14, comprising one or more polynucleotide sequences selected from the group consisting of SEQ ID NO:28, SEQ ID NO:31, and SEQ ID NO:34, or a polynucleotide complementary thereto.

[0226] 16. A vector comprising a polynucleotide sequence according to paragraph 14 or paragraph 15.

[0227] 17. The vector according to paragraph 16, further comprising an expression control sequence operably linked to the nucleic acid encoding the variable heavy chain domain and / or the variable light chain domain.

[0228] 18. A host cell containing a vector according to paragraph 16 or paragraph 17.

[0229] 19. The host cell according to paragraph 18, wherein the host cell is a eukaryotic or prokaryotic cell.

[0230] 20. The host cell according to paragraph 19, wherein the eukaryotic cell is a Chinese Hamster Ovary (CHO) cell.

[0231] 21. The host cell according to paragraph 19, wherein the host cell is an E. coli cell.

[0232] 22. A method for producing an antibody or antigen-binding fragment thereof, comprising incubating a host cell according to any of paragraphs 18 to 20 such that the encoded variable heavy chain domain and / or variable light chain domain is expressed by the cell, and recovering the expressed antibody or antigen-binding fragment thereof.

[0233] 23. The method of paragraph 22, further comprising isolating and / or purifying the recovered antibody or antigen-binding fragment thereof.

[0234] 24. A method for producing an antibody according to any one of paragraphs 1 to 13, comprising immunizing a non-human animal with a conjugate comprising a compound of formula [II]: [ka] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C1-C6 alkyl, preferably a compound of formula [I]: [ka] The aforementioned compound of formula [II] or formula [I] is bound to the immunogenic carrier via a linker, preferably the linker is bound to the compound of formula [I] via an amine group.

[0235] 25. A device for determining the presence or absence of CEMA in a sample, comprising an antibody or antigen-binding fragment thereof according to any of paragraphs 1 to 13 immobilized on a solid phase of the device.

[0236] 26. The device according to paragraph 25, which is a portable lateral flow immunoassay device, preferably a urine test strip.

[0237] 27. The device described in paragraph 25 or paragraph 26, wherein the device comprises: (i) a sample pad for receiving a sample; (ii) a conjugate pad in fluid communication with the sample pad; (iii) at least one detection zone in fluid communication with a distal end of the conjugate pad; and (iv) an adsorbent pad in fluid communication with the distal end of the detection zone.

[0238] 28. The device described in paragraph 27, wherein the conjugate pad comprises an antibody or antigen-binding fragment thereof described in any of paragraphs 1 to 13, wherein the antibody or antigen-binding fragment thereof is labeled and, optionally, is a labeled antibody or antigen-binding fragment thereof capable of binding to cotinine.

[0239] 29. A device according to paragraph 28, wherein the labeled antibody or antigen-binding fragment thereof according to any of paragraphs 1 to 13 and the labeled antibody or antigen-binding fragment thereof capable of binding to cotinine are contained in separate pads which are in fluid communication with each other.

[0240] 30. A device described in paragraph 28, wherein the labeled antibody or antigen-binding fragment thereof is separately contained in an intermediate pad of the device located adjacent to the conjugate pad, and optionally, a labeled antibody or antigen-binding fragment thereof capable of binding to cotinine is separately contained in the conjugate pad.

[0241] 31. A device according to any of paragraphs 25 to 30, wherein the detection zone comprises CEMA and, optionally, cotinine immobilised thereon, preferably wherein the CEMA is in the form of a conjugate according to paragraph 1 or paragraph 2.

[0242] 32. A method for detecting CEMA in a sample, comprising the use of a device described in any of paragraphs 25 to 31, or a method for detecting CEMA and cotinine in a sample, comprising the use of a device described in any of paragraphs 25 to 31.

[0243] 33. The method of paragraph 32, comprising: (i) applying an aliquot of a liquid biological sample, preferably urine, to the sample pad, whereby the liquid biological sample is moved by capillary action along a flow path defined by the sample pad, the conjugate pad, the detection zone, and the sorbent pad; and (ii) determining the presence or absence of CEMA in the detection zone, and optionally determining the presence or absence of cotinine in the detection zone.

[0244] 34. Use of an antibody or antigen-binding fragment thereof according to any of paragraphs 1 to 13, or a device according to any of paragraphs 25 to 31, for detecting CEMA in a sample.

[0245] Any publications cited or described herein provide relevant information disclosed prior to the filing date of this application. Nothing herein should be construed as an admission that the inventors are not entitled to antecedent to such disclosure. All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the present invention will become apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the present invention has been described in connection with certain preferred embodiments, it will be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described methods for carrying out the invention which are obvious to those skilled in the art of immunology, cell or molecular biology, or related fields, are intended to be within the scope of the following claims.

[0246] TIFF2024532139000021.tif232170 TIFF2024532139000022.tif250169 TIFF2024532139000023.tif245170 TIFF2024532139000024.tif249170 TIFF2024532139000025.tif86170

Table 6

Claims

1. An antibody or antigen-binding fragment thereof, VH CDR1, VH CDR2, and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and VL CDR1, VL CDR2, and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively; or VH CDR1, VH CDR2, and VH CDR3 consisting of the amino acid sequences of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively, and VL CDR1, VL CDR2, and VL CDR3 consisting of the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or An antibody or antigen-binding fragment thereof, comprising VH CDR1, VH CDR2, and VH CDR3 consisting of the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and VL CDR1, VL CDR2, and VL CDR3 consisting of the amino acid sequences of SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively.

2. the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 2, 11, or 20; and / or The antibody of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 6, 15, or 24.

3. the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO:2 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:6; or the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 11 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 15; or The antibody of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence consisting of the amino acid sequence of SEQ ID NO: 20 and a VL amino acid sequence consisting of the amino acid sequence of SEQ ID NO:

24.

4. The antigen-binding fragment may be a Fab fragment, a Fab' fragment, or a F(ab') 2 fragments, scFv, Fv, rIgG, and diabodies, preferably scFv, more preferably The antibody or antigen-binding fragment thereof of claim 1, wherein the antigen-binding fragment is an scFv and the scFv comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 10, or SEQ ID NO:

19.

5. The antibody or antigen-binding fragment thereof described in claim 1, wherein the antibody does not bind to cotinine or 2-hydroxyethyl methacrylate (HEMA) or monohydroxybutenyl mercapturic acid (MHBMA) or 3-hydroxypropyl mercapturic acid (3-HPMA) or dihydroxybutyl mercapturic acid (DHBMA).

6. 10. A device for determining the presence or absence of N-acetyl-S-[2-carboxyethyl]-L-cysteine ​​(CEMA) in a sample, comprising the antibody or antigen-binding fragment thereof described in claim 1 immobilized on a solid phase of the device.

7. wherein the device is a portable lateral flow immunoassay device, preferably a urine test strip, and more preferably The device, (i) a sample pad for receiving a sample; (ii) a conjugate pad in fluid communication with the sample pad; (iii) at least one detection zone in fluid communication with the distal end of the conjugate pad; and 7. The device of claim 6, further comprising: (iv) an absorbent pad in fluid communication with the distal end of the detection zone.

8. the conjugate pad comprises the antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof is labeled, and optionally further comprises a labeled antibody or antigen-binding fragment thereof capable of binding to cotinine; or The device described in claim 7, wherein the antibody or antigen-binding fragment thereof described in claim 1 is labeled and separately contained in an intermediate pad of the device located adjacent to the conjugate pad, and optionally, a labeled antibody or antigen-binding fragment thereof capable of binding to cotinine is separately contained in the conjugate pad.

9. 7. The device of claim 6, wherein the detection zone comprises CEMA and, optionally, cotinine immobilized thereon.

10. The CEMA Compound of formula [II]: 【Chemical 1】 [II] wherein n is selected from 0 to 4 (i.e., 0, 1, 2, 3, or 4), and each R is independently selected from H or C 1 -C 6 alkyl, preferably a compound of formula [I]: 【Chemistry 2】 [I] and in the form of a conjugate comprising The compound of formula [II] or formula [I] is bound to an immunogenic carrier via a linker, and preferably, the linker is bound to the compound of formula [I] via an amine group.

10. The apparatus of claim 9.

11. The device described in claim 10, wherein the immunogenic carrier is a protein, preferably bovine serum albumin or bovine thyroglobulin; and / or the linker is glycol bis(succinimidyl succinate) (EGS) or disuccinimidyl suberate (DSS).

12. A method for detecting CEMA in a sample, comprising the use of a device according to claim 6, or a method for detecting CEMA and cotinine in a sample, comprising the use of a device according to claim 6.

13. 10. Use of the antibody or antigen-binding fragment thereof of claim 1 or the device of claim 6 for detecting CEMA in a sample.

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