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WO2025111485A1 - Dispositif de point d'intervention pour la détection du cancer buccal - Google Patents

Dispositif de point d'intervention pour la détection du cancer buccal Download PDF

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Publication number
WO2025111485A1
WO2025111485A1 PCT/US2024/056932 US2024056932W WO2025111485A1 WO 2025111485 A1 WO2025111485 A1 WO 2025111485A1 US 2024056932 W US2024056932 W US 2024056932W WO 2025111485 A1 WO2025111485 A1 WO 2025111485A1
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Prior art keywords
healthy
suspect
sample
bdi
subject
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WO2025111485A9 (fr
Inventor
Umut A. Gurkan
Santosh K. GHOSH
Aaron Weinberg
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Case Western Reserve University
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Case Western Reserve University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers

Definitions

  • HNC Head and neck cancer
  • ⁇ 65,000 new cases were estimated to have been diagnosed in 2020.
  • OSCC Oral squamous cell carcinoma
  • the present disclosure relates generally to diagnostic point-of-care (POC) devices, systems, and methods for detecting oral cancer and, in particular, to POC devices, systems, and methods based on a lateral flow assay for detecting the presence of oral cancer in a subject.
  • POC point-of-care
  • One aspect of the present disclosure can include a POC, lateral flow assay device for detecting oral cancer in a subject.
  • the device can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells.
  • Another aspect of the present disclosure can include a system for detection of oral cancer in a subject.
  • the system can comprise a POC, lateral flow assay device and a mobile device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI).
  • the device can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells.
  • Another aspect of the present disclosure can include a method for detecting oral cancer in a subject.
  • the method can comprising the steps of: (a) providing a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (BD
  • Another aspect of the present disclosure can include a method for detecting and treating oral cancer in a subject.
  • the method can comprise the steps of: (a) providing a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; (BD
  • kits for detection of oral cancer in a subject can comprise: a POC, lateral flow assay device, wherein the device includes a housing that comprises: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells; and instructions for using the device for detection of oral cancer in the subject; optionally, means for obtaining healthy and suspect samples from the subject; optionally, separate cartridges for receiving healthy and suspect samples, each of which contains a cell lysis solution; optionally, means for delivering lysed suspect and healthy samples to the device; optionally, a mobile electronic device configured to access a communications network and
  • Fig.1A is a schematic illustration showing a point-of-care, lateral flow assay device for detecting oral cancer in a subject, constructed in accordance with one aspect of the present disclosure
  • Fig.1B is a schematic illustration showing an exemplary implementation the point-of-care, lateral flow assay device in Fig.1A
  • Figs.2A-E show immunofluorescence microscopy and flow-cytometry of DEFB103/hBD-3 and DEFB4/hBD-2.
  • FIG.3A-F show cytological and flow-cytometric analysis of non-invasively collected cytobrushed cells.
  • the cancer cells in Fig.3A (outlined in red) are present as three- dimensional clusters with marked variation in nuclear size and shape (blue arrows), and single cells and small clusters with abnormal cytoplasmic shapes (green arrows), and nuclear enlargement and multi-nucleation in occasional cells (black arrows).
  • Representative flow-cytometric data showing hBD-3 and hBD-2 expressing cells among E-cadherin+ epithelial cell population in non-invasively collected cytobrushed cells from cheek and tongue of a healthy subject (Fig.3C).
  • p value was calculated by non-parametric Mann-Whitney test) (Fig.3D).
  • FIG.3B Representative FACS data of one of the OSCC patients from Fig.3B (Pink: Cells collected from lesional site; Green: Cells collected from contralateral site; Maroon: Cells isolated from excised tumor of the same patient)
  • BDI Beta Defensin Index
  • BDI values were determined by biopsy followed by pathology review.
  • BDI values as determined in Fig.4A, were calculated following ELISA (p value was calculated by non-parametric Mann-Whitney test) (Fig.4B).
  • Figs.5A-D show the BDI discovery and validation study.
  • Black horizontal line for each cohort is the median BDI values.
  • Figs.6A-D show one example of a microfluidic intact cell analysis (MICA) device.
  • MICA microfluidic intact cell analysis
  • FIG.6A Schematic illustration of the MICA device with embedded micropillar arrays forming narrow openings from 160 ⁇ m down to 20 ⁇ m along the flow direction for cell aggregate filtering and individual cell retention
  • Fig.6B Photograph of an assembled MICA device
  • Cytobrush samples were collected from the lesion and the contralateral normal sites of patients, and pumped through micropillar arrays within respective troughs of the MICA device.
  • Representative microscopic image showing typical distribution of the retained cells in the MICA microchannel (Fig.6C).
  • Figs.7A-C show MICA-BDI proof of concepts. Representative results of phase-contrast, hBD-2 and -3 fluorescence and DAPI staining of nuclei of the cells collected from a healthy participant’s right and left cheek. A violin plot showing no significant difference in hBD-3 to hBD-2 ratio in cells collected from left vs right side of the healthy participant (Fig.7A). Representative results of phase-contrast, hBD-2 and -3 fluorescence and DAPI staining of nuclei of the cells collected from a cancerous patient’s lesional and contralateral side.
  • Fig.7B A violin plot showing a significantly higher hBD-3 to hBD-2 ratio in cells collected from the lesional side compared to the contralateral side.
  • OSCC oral squamous cell carcinoma
  • Figs.8A-D are a series of graphs showing TCGA data analysis. Expression of DEFB103 (Fig.8A) and DEFB4 (Fig.8B) across all TCGA cancer types (tumor (red) and normal (blue)) (as TPM, Transcript per million) in HNSC compared to normal samples in TCGA database).
  • Fig.8C Differential expression of DEFB103 across different subtype of HNSC
  • Fig.8D Differential expression of DEFB103 transcript across 4 different grades of HNSC and compared to normal samples. [* p ⁇ 0.05]
  • Fig.9D a series of plots showing DEFB103 expression and correlation analysis. Expression of DEFB103 based on cancer stage (Fig.9A), patient’s gender (Fig.9B), age (Fig.9C) and race (Fig.9D). [*p ⁇ 0.05].
  • Correlation of DEFB103 transcript with other genes in HNSC (Figs.9E-F). Differential expression of LCE3D and LCE3E between HNSC (T) and normal (N).
  • Fig.9G Gene ontology (GO) analysis (Fig.9H), using rho value >0.7, to identify 38 positively correlated genes with DEFB103 (Fig.9F);
  • Fig.11C Pie charts showing the percentage of OSCC (left panel) and non-cancerous (right panel) lesions based on anatomical sites within the oral cavity where they appeared (Fig.11C).
  • the BDI based on anatomical locations (Cheek, tongue, and others).
  • Fig.11D BDI values with respect to cancer stages.
  • Fig.11E Figs.12A-E are a series of charts showing BDI benchmarking.
  • Fig.12A shows BDI benchmarking against published OSCC biomarkers and in use (and/or published) OSCC biomarker platforms: IL-8: Interleukin 8; IL-1B: Interleukin 1 Beta, DUSP: Dual-specificity phosphatase; S100P: Calcium-Binding Protein S100P, solCD44: Soluble CD44, CPLANE1: Ciliogenesis and planar polarity effector complex subunit 1; NUS1+RCN1: Nuclear Undecaprenyl Pyrophosphate Synthase 1 + Reticulocalbin 1; GAS6: Growth Arrest Specific 6, LBC: Liquid based cytology, CDOT: Cancer Detect for Oral & Throat cancer POCOCT: Point Of Care Oral Cytology Test.
  • IL-8 Interleukin 8
  • IL-1B Interleukin 1 Beta
  • DUSP Dual-specificity phosphatase
  • S100P Calcium-Binding Protein S100
  • Detection methods for each of the biomarkers/platforms are described in Table 7.
  • Figs.12B-E show a comparison of sensitivity and specificity values (with 95% confidence intervals, when available) of BDI with published OSCC biomarkers and in use (and/or published) OSCC biomarker platforms.
  • Data for IL-8, IL-1B, S100P, DUSP1, mullite, velscope and CDx are from multiple single studies, POCOCT and BDI are single multi-center studies, while the rest are from single center studies. Note: In Figs.12B and 12C, black circles represent salivary biomarkers and black squares represents serum biomarkers.
  • Figs.12D and 12E green circles represent commercially available/in use platforms;
  • Fig.13 shows detection of epidermal growth factor receptor (EGFR) by MICA. Representative phase-contrast and fluorescent images of the cells collected from either the lesion site or the contralateral normal site of patients. Scale bars represent a length of 100 ⁇ m;
  • Fig.14 is a flow chart of image analysis and BDI generation; and
  • Fig.15 is a schematic illustration showing another exemplary implementation of a single lane or channel of the point-of-care, lateral flow assay device in Figs.1A-B.
  • each step comprises what is listed (unless that step includes a limiting term such as “consisting of”), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
  • the term “about”, when expressed as from “about” one particular value and/or “about” another particular value, also specifically contemplated and disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise.
  • phrases such as “from about X to Y” can mean “from about X to about Y”.
  • phrases such as “from about X to Y” can mean “from about X to about Y”.
  • references to a structure or feature that is disposed “adjacent” another feature may have portions that overlap or underlie the adjacent feature.
  • Spatially relative terms such as “under”, “below”, “lower”, “over”, “upper” and the like, may be used herein for ease of description to describe one element or feature’s relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms can encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if the device in the figures is inverted, elements described as “under” or “beneath” other elements or features would then be oriented “over” the other elements or features.
  • the terms “first,” “second,” etc. should not limit the elements being described by these terms. These terms are only used to distinguish one element from another. Thus, a “first” element discussed below could also be termed a “second” element without departing from the teachings of the present disclosure.
  • the sequence of operations (or acts/steps) is not limited to the order presented in the claims or figures unless specifically indicated otherwise.
  • the terms “optionally” and “optional” can mean that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
  • diagnosis can encompass determining the nature of disease in a subject, as well as determining the severity and probable outcome of disease or episode of disease and/or prospect of recovery (prognosis). “Diagnosis” can also encompass diagnosis in the context of rational therapy, in which the diagnosis guides therapy, including initial selection of therapy, modification of therapy (e.g., adjustment of dose and/or dosage regimen), and the like.
  • epithelial cell can refer to a cell comprising epithelial tissue in the body of a subject, and which may be found, for example, on the outside surface of skin, the linings of all digestive organs, the lining of the heart, the inner and outer membranes of the respiratory system, vessel walls, and the internal body cavities.
  • the types of epithelial tissue, and thus epithelial cells include simple squamous, stratified squamous, simple cuboidal, stratified cuboidal, simple columnar, and stratified columnar.
  • Unique types of epithelial tissue include pseudostratified epithelium and transitional epithelium. Epithelial cells tightly connected to each other via various types of junctions.
  • epithelial cells have an apical surface that is exposed to a lumen or outside environment. They have a basal surface that is connected to basement membrane, which adheres them to the underlying structure.
  • Non-limiting functions of epithelial cells include absorbing nutrients, providing mechanical protection, secreting various substances, and providing a matrix to house sensory receptor cells.
  • the term “intact epithelial cell” can refer to an epithelial cell that is mechanically intact, not disrupted, nor collapsed or burst.
  • oral cancer means any of the following: oral carcinogenesis, oral squamous cell carcinoma (OSCC), oral cancer, pre-cancer, pre- malignant lesion, oral adenocarcinoma, squamous cell carcinoma of the head and neck (SCCHN), any cytological or genetic abnormality of an oral cell, and any disease or disorder of oral cells.
  • OSCC oral squamous cell carcinoma
  • SCCHN squamous cell carcinoma of the head and neck
  • epithelial cancer can refer to any cancer derived from epithelial cells. This group includes many of the most common types of cancer, include nearly all those developing in the skin, head and neck, breast, prostate, lung, pancreas, and colon.
  • a common type of epithelial cancer is squamous cell carcinoma.
  • the methods of the present disclosure are directed to detecting epithelial cancer at a site of origin, while in other embodiments of the methods are directed to detecting epithelial cancer that has migrated as a result of metastasis.
  • the methods of the present disclosure are directed to detecting epithelial cancer selected from the group consisting of head and neck cancer, oral cancer, esophageal cancer, and skin cancer.
  • Head and neck cancer is cancer that starts in the lip, oral cavity (mouth), nasal cavity (inside the nose), paranasal sinuses, pharynx, larynx or parotid glands. Most head and neck cancers are biologically similar.
  • head and neck cancers are squamous cell carcinomas, so they are called head and neck squamous cell carcinomas (HNSCC). These cancers commonly originate from the mucosal lining (epithelium) of these regions.
  • Oral cancer or mouth cancer is a type of head and neck cancer, and includes any cancerous tissue growth located in the oral cavity. Typically, oral cancer is oral epithelial cancer.
  • methods of the present disclosure can also be used to detect epithelial skin cancer, which is epithelial cancer that arises from the skin, including basal-cell carcinoma and squamous-cell carcinoma.
  • the terms “subject” and “patient” can be used interchangeably and refer to a vertebrate, such as a mammal (e.g., a human). Mammals can include, but are not limited to, humans, dogs, cats, horses, cows, and pigs.
  • BD can refer to a class of cationic antimicrobial peptides that play an important role in innate and adaptive immunity, as well as other non-immunological processes (Machado L R, Ottolini B, Front Immunol., 6:115 (2015)). Defensins are an ancient and diverse family of proteins, and are present in most multicellular organisms.
  • a beta defensin detectable by the devices and methods of the present disclosure can be associated with any one of a variety of species including, but not limited to, humans, birds, fish, pigs, dogs, cats, and other livestock.
  • POC Point-of-care
  • lateral flow assay devices As shown in Fig.1A, one aspect of the present disclosure can include a POC, lateral flow assay device 10 for detecting oral cancer in a subject.
  • a lateral flow assay may be employed in a POC device 10 to detect the presence or absence of beta definsin-2 (hBD-2) (e.g., human BD-2 or hBD-2) and beta definsin-3 (BD-3) (e.g., human BD-3 or hBD-3) within a test or biological sample.
  • hBD-2 e.g., human BD-2 or hBD-2
  • BD-3) e.g., human BD-3 or hBD-3
  • the readout may be done visually, i.e., presence or absence of one or more colored test lines (also referred to as test stripes), and the confirmation/validation of the test may be done by the presence and/or absence of one or more colored indicator lines/stripes.
  • the test may be qualitative (presence or absence) as well as quantitative, and the detection/quantification may be aided by reading equipment, or can be purely visual detection by the eye of the user of the lateral flow assay.
  • a reading device such as an optical reader may be used in some embodiments to measure the intensity of the binding ligands.
  • the actual configuration and structure of the optical reader may generally vary depending on the binding ligands, which are to be measured.
  • optical detection techniques that may be utilized include, but are not limited to, luminescence (e.g., fluorescence, phosphorescence, etc.), absorbance (e.g., fluorescent or non- fluorescent), diffraction, and so on.
  • hBD-2 and hBD-3 may be achieved in accordance with the present disclosure.
  • the amount of hBD-2 and hBD-3 may be quantitatively or semi-quantitatively determined by using the intensities of the signals produced by binding ligands bound to hBD-2 and hBD-3 present in a test or biological sample.
  • the device 10 can comprise a housing 12 that includes: a healthy channel 14 and a suspect channel 16, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator 18; a first inlet 20 in fluid communication with the suspect channel; and a second inlet 22 in fluid communication with the healthy channel; wherein the channels do not include binding ligands that specifically bind to and immobilize intact epithelial cells.
  • the housing 12 may be based on, or made of, a capillary bed (such as porous paper or sintered polymer) or, in some instances, on a series of capillary beds in fluid communication with each other.
  • the capillary beds have the capacity to transport fluid by action of capillary forces.
  • the housing 12 is configured to obtain and receive healthy and suspect samples, which means that the housing should be capable of absorbing the healthy and suspect samples, and any one or combination of materials capable of doing so may be suitable.
  • Such materials used for the housing may include, but are not limited to, natural, synthetic or naturally occurring materials that are synthetically modified, such as polysaccharides (e.g., cellulose materials such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose), polyether sulfone, polyethylene, nylon polyvinylidene fluoride (PVDF), polyester, polypropylene, cotton, or cloth.
  • polysaccharides e.g., cellulose materials such as paper and cellulose derivatives, such as cellulose acetate and nitrocellulose
  • PVDF nylon polyvinylidene fluoride
  • the housing 12 can have a planar, sheet-like configuration.
  • the housing 12 can have a thickness equal to or less than 4 mm (such as less than 4, 3, 2, 1 mm), and a width and a length both greater than the thickness.
  • the width and length of the housing 12 are both greater (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 50 times greater or up to 4, 5, 6, 7, 8, 9, 10, 50 times greater) than the thickness.
  • the housing 12 is square-shaped or rectangular, and in other embodiments the housing is circular. If the housing 12 is an irregular shape, i.e., different from a square or rectangle, then the width, length and thickness refers to the maximum values for such an irregular shape. For example, the width of a circle will be the diameter.
  • the housing 12 may optionally comprise a backing layer (not shown) adhered thereto, which is liquid-impermeable so that fluid flowing through device 10 does not leak through the housing.
  • suitable materials for the backing layer include, but are not limited to, glass; polymeric materials, such as polystyrene, polypropylene, polyester, polybutadiene, polyvinylchloride, polyamide, polycarbonate, epoxides, methacrylates, and polymelamine.
  • the housing 12 includes a healthy channel 14 and a suspect channel 16.
  • the healthy channel 14 includes a second inlet 22 in fluid communication therewith and is adapted to receive a biological sample comprising a healthy sample.
  • the suspect channel 16 includes a first inlet 20 in fluid communication therewith and is adapted to receive a biological sample comprising a suspect sample.
  • Each of the healthy channel 14 and the suspect channel 16 includes a first labeled binding ligand that specifically binds to BD-2 (e.g., hBD-2), a second labeled binding ligand that specifically binds to BD-3 (e.g., hBD-3), and a control indicator 18.
  • binding ligands are known to those skilled in the art, such as antibodies, antibody fragments, and aptamers.
  • the first and second labeled binding ligands are antibodies.
  • Antibodies include polyclonal and monoclonal antibodies, as well as antibody fragments that contain the relevant antigen binding domain of the antibodies.
  • the term “antibody” as used herein refers to immunoglobulin molecules or other molecules which comprise at least one antigen-binding domain.
  • antibody as used herein is intended to include whole antibodies, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, multi-specific antibodies, single chain antibodies, epitope- binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd, Fvs, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, and totally synthetic and recombinant antibodies.
  • the antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
  • Monoclonal antibodies may be produced in animals such as mice and rats by immunization.
  • B cells can be isolated from the immunized animal, for example from the spleen.
  • the isolated B cells can be fused, for example with a myeloma cell line, to produce hybridomas that can be maintained indefinitely in in vitro cultures.
  • Hybridoma cells can be isolated by dilution (single cell cloning) and grown into colonies. Individual colonies can be screened for the production of antibodies of uniform affinity and specificity.
  • Hybridoma cells may be grown in tissue culture and antibodies may be isolated from the culture medium.
  • Hybridoma cells may also be injected into an animal, such as a mouse, to form tumors in vivo (such as peritoneal tumors) that produce antibodies that can be harvested as intraperitoneal fluid (ascites).
  • the lytic complement activity of serum may be optionally inactivated, for example by heating.
  • Biological analytes e.g., BD-2 or BD-3) may be used to generate antibodies.
  • polypeptides used for immunization will vary based on a number of factors, including the animal which is immunized, the antigenicity of the polypeptide selected, and the site of injection.
  • the polypeptides used as an immunogen may be modified as appropriate or administered in an adjuvant in order to increase the peptide antigenicity.
  • polypeptides, peptides, haptens, and small compounds may be conjugated to a carrier protein to elicit an immune response or may be administered with and adjuvant, e.g., incomplete Freund’s adjuvant.
  • Protocols for generating antibodies including preparing immunogens, immunization of animals, and collection of antiserum may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, N.Y., 1988) pp.55-120, and A. M. Campbell, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1984).
  • antibody fragment as used herein is intended to include any appropriate antibody fragment that comprises an antigen-binding domain that displays antigen binding function. Antibodies can be fragmented using conventional techniques.
  • F(ab′)2 fragments can be generated by treating the antibody with pepsin.
  • the resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab 1 fragments.
  • Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab′ and F(ab′)2, scFv, Fv, dsFv, Fd, dAbs, T and Abs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art.
  • Antibody fragments may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CH1, CH2, and CH3 domains.
  • Antibodies are designed for specific binding, as a result of the affinity of complementary determining region of the antibody for the epitope of the biological analyte.
  • An antibody “specifically binds” when the antibody preferentially binds a target structure, or subunit thereof, but binds to a substantially lesser degree or does not bind to a biological molecule that is not a target structure.
  • the antibody specifically binds to the target analyte (e.g., BD-2 or BD- 3) with a specific affinity of between 10 ⁇ 8 M and 10 ⁇ 11 M.
  • an antibody or antibody fragment binds to BD-2 or BD-3 with a specific affinity of greater than 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, or 10 ⁇ 11 M, between 10 ⁇ 8 M-10 ⁇ 11 M, 10 ⁇ 9 M- 10 ⁇ 10 M, and 10 ⁇ 10 M-10 ⁇ 11 M.
  • specific activity is measured using a competitive binding assay as set forth in Ausubel FM, (1994), Current Protocols in Molecular Biology (Chichester: John Wiley and Sons).
  • antibodies present in the healthy and suspect channels 14 and 16 can be fluorescently-labeled antibodies.
  • the binding ligand is an aptamer. Aptamers can be used as an alternative to antibodies for immunoassays (see Chen A1, Yang S2, Biosens Bioelectron.71, 230-42 (2015).
  • An aptamer is a nucleic acid that binds with high specificity and affinity to a particular target molecule or cell structure, through interactions other than Watson-Crick base pairing.
  • Aptamer functioning is unrelated to the nucleotide sequence itself, but rather is based on the secondary/tertiary structure formed, and are therefore best considered as non-coding sequences.
  • Aptamers of the present disclosure may be single-stranded RNA, DNA, a modified nucleic acid, or a mixture thereof.
  • the aptamers can also be in a linear or circular form. Accordingly, in some embodiments, the aptamers are single-stranded DNA, while in other embodiments they are single-stranded RNA.
  • the length of the aptamer of the present disclosure is not particularly limited, and can usually be about 10 to about 200 nucleotides, and can be, for example, about 100 nucleotides or less, about 50 nucleotides or less, about 40 nucleotides or less, or about 35 nucleotides or less.
  • the total number of nucleotides present in the aptamer is smaller, chemical synthesis and mass- production will be easier and less costly.
  • the various structural motifs that are involved in the non-Watson-Crick type of interactions involved in aptamer binding can be formed in nucleic acid sequences of 30 nucleotides or less.
  • the aptamers are capable of specifically binding to biological analytes, such as BD-2 and BD-3. Specific binding refers to binding which discriminates between the selected target and other potential targets, and binds with substantial affinity to the selected target.
  • Substantial affinity represents an aptamer having a binding dissociation constant of at least about 10 ⁇ 8 M, but in other embodiments, the aptamers can have a binding dissociation constant of at least about 10 ⁇ 9 M, about 10 ⁇ 10 M, about 10 ⁇ 11 M, or at least about 10 ⁇ 12 M.
  • Aptamers can include structural analogs of the original aptamer. Examples of structural analogs include aptamers modified at the 2′-position hydroxyl group of pyrimidine or purine nucleotides with a hydrogen atom, halogen, or an —O- alkyl group. Wild-type RNA and DNA aptamers are not as stable as would be preferred because of their susceptibility to degradation by nucleases.
  • Each of the healthy and suspect channels 14 and 16 includes a control indicator 18, which can function as a control to verify that the lateral flow assay has been conducted properly and/or to provide a standard value (e.g., a standard fluorescent value) for comparison to detected BD-2 and BD-3 levels.
  • a control indicator 18 can function as a control to verify that the lateral flow assay has been conducted properly and/or to provide a standard value (e.g., a standard fluorescent value) for comparison to detected BD-2 and BD-3 levels.
  • each of the healthy and suspect channels 14 and 16 do not include binding ligands that specifically bind to and immobilize intact epithelial cells.
  • binding ligands are not present in the healthy and suspect channels 14 and 16 is advantageous because the device 10 and associated lateral flow detection assay of the present disclosure uses lysed cells, thereby obviating the conventional need for binding ligands to bind whole cells.
  • each of the healthy and suspect channels 14 and 16 is free of a filter.
  • the fact that each of the healthy and suspect channels 14 and 16 is free of a filter is advantageous because all cells subject to the device 10 and associated assay of the present disclosure undergo lysis, obviating the need for filtration.
  • Each of the healthy and suspect channels 14 and 16 further include a detector zone 24.
  • Each detector zone 24 includes a first line or stripe 26 where one or more binding ligands that specifically bind BD-3 has been immobilized, and a second line or stripe 28 where one or more binding ligands that specifically bind BD- 2 has been immobilized.
  • each of the first and second stripes 26 and 28 changes color when, and if, BD-3 and BD-2 (respectively) are present in the test or biological samples and flowed through the channels 14 and 16.
  • the control indicator 18 can be located upstream or downstream of the first and second stripes 26 and 28.
  • the stripes 26 and 28 in the detector zone 24 may be disposed in a direction that is substantially perpendicular to the lateral flow (L) of the healthy and suspect samples. In some embodiments, the stripes 26 and 28 may be positioned in a direction that is substantially parallel to the flow (L) of the healthy and suspect samples.
  • the stripes 26 and 28 in the detector zone 24 do not need to be lines or stripes, and can also be other shapes, such as dots or patterns.
  • Another aspect of the present disclosure can include a method for detecting oral cancer in a subject.
  • a first step of the method can comprise providing a POC, lateral flow assay device 10, such as the one illustrated in Fig.1A and described above.
  • the method can include the step of obtaining a healthy sample and a suspect sample from a subject (e.g., a human subject).
  • a subject e.g., a human subject.
  • the present method involves determining the levels of BD-2 (e.g., hBD-2) and BD-3 (e.g., hBD-3) in biological samples.
  • a “biological sample”, as used herein, can refer to any tissue or fluid obtained from a subject that includes at least one epithelial cell.
  • Biological samples can include, but are not necessarily limited to, bodily fluids such as saliva, urine, and blood-related samples (e.g., whole blood, serum, plasma, and other blood-derived samples), cerebral spinal fluid, bronchoalveolar lavage, and the like.
  • Biological samples can also include a skin sample or a mucosal sample (e.g., derived or obtained from an oral cavity, a cervix, or an anus). Because the present disclosure is directed to diagnosing oral cancer, in some embodiments, the suspect sample and the healthy sample both include epithelial cells.
  • Biological samples can be obtained by any known means including needle stick, needle biopsy, swab, and the like.
  • the biological sample is a mucosal sample (e.g., from an oral cavity, such as the inside of a cheek), which may be obtained for example by using a cytobrush.
  • the head of the cytobrush is sized to correspond to the size of the suspect tissue or lesion.
  • a biological sample may be fresh or stored (e.g., blood or blood fraction stored in a blood bank). Samples can be stored for varying amounts of time, such as being stored for an hour, a day, a week, a month, or more than a month.
  • the biological sample may be a skin sample or a mucosal sample expressly obtained for the assays of this disclosure or a sample obtained for another purpose, which can be sub-sampled for the assays of this disclosure.
  • the biological samples of the present disclosure can be distinguished based on the role they play in the method.
  • the biological sample can be a suspect sample or a healthy sample. When carrying out the method, both a suspect sample and a healthy sample are used.
  • the site may be suspect as a result of visible irregularities, pain, or as the result of other diagnostic tests, or it may simply be the site where testing is being carried out.
  • the suspect sample is obtained from an oral lesion, while in other embodiments the suspect sample is obtained from a skin lesion. If the method indicates that the subject has, or is at increased risk of having, oral cancer, the cancer would be present at the suspect site.
  • the method of the present disclosure also involves obtaining a healthy sample, which is a biological sample obtained from a tissue site where there is no reason to suspect that oral cancer exists, or preferably where the tissue is clearly healthy and non-cancerous based on other available information.
  • the suspect sample and the healthy sample are obtained from the same subject, which can provide an advantage in terms of serving as an internal standard for more reliable diagnosis.
  • the suspect sample and the healthy sample may be obtained from different subjects.
  • the suspect sample and the healthy sample can be obtained from different tissue sites. Accordingly, in one embodiment, for example, where oral cancer is being diagnosed, the suspect sample is obtained from an oral lesion, while the healthy sample is skin, whole blood, serum, plasma, or mucosal tissue from an oral or cervical region that appears healthy.
  • an agent is added to the biological sample that reduces electrostatic interaction between ⁇ -defensin and negatively-charged moieties in the sample without affecting binding of detection antibodies and/or fragments thereof to the ⁇ -defensins (see U.S. Patent Publication No. 2010/0022025).
  • Negatively-charged moieties in the biological sample can include anionic glycoproteins, such as mucins, a family of large, heavily glycosylated proteins, and calprotectin, a calcium-binding protein secreted predominantly by neutrophils.
  • the agent can include a positively-charged moiety and/or ions capable of reducing the electrostatic interaction between ⁇ -defensin and negatively-charged moieties in the biological sample.
  • the positively-charged moiety and/or ions can be provided in the sample by administering a salt to the sample that upon addition can readily dissociate and form cations or positively charge electrolytes that are capable of associating with the negatively-charged moieties.
  • the salt upon dissociation can form divalent cations capable of associating with the negatively- charged moiety. Examples of salts capable of forming divalent cations are MgCl2 and CaCl2.
  • MgCl2 and CaCl2 upon addition to a biological sample can dissociate and form Mg 2+ and Ca 2+ cations.
  • the agent can be added to the biological sample at an amount effective to reduce electrostatic interaction between ⁇ -defensin and negatively-charged moieties in the sample without affecting binding of detection antibodies and/or fragments thereof to the ⁇ -defensins.
  • the electrostatic interaction between ⁇ -defensin and negatively-charged moieties in the bodily sample can be reduced by adding CaCl2 to a biological sample at a concentration of about 50 mmol/L to about 250 mmol/L.
  • Positively-charged moieties can be added to the biological sample before antibodies are contacted with the sample and/or simultaneously with antibody contact.
  • the suspect sample is obtained from a lesional site and the healthy sample is obtained from a corresponding contralateral normal site.
  • the suspect sample is obtained from a lesional site comprising skin or mucosa and the healthy sample is obtained from a corresponding contralateral normal skin or mucosal site.
  • each of the samples is contacted with a cell lysis solution for a time and in an amount to ensure that any epithelial cells present in the samples are lysed. In other words, following contact with the cell lysis solution, the suspect and healthy samples do not include intact epithelial cells.
  • cell lysis solutions are known in the art and routinely contain, for example, mild detergents (e.g., SDS), protease inhibitors and chelating agents.
  • a cell lysis solution employed by the assay of the present disclosure is cOmplete Lysis-M (Roche, Cat. No.04719956001).
  • the suspect and healthy samples are delivered to the first and second inlets 20 and 22 of the device 10, respectively.
  • the amount of healthy and suspect samples delivered to the device 10 can be readily determined by one skilled in the art and can be done, for example, using a pipette, syringe, etc.
  • the samples can migrate through the healthy and suspect channels 14 and 16, respectively, to the detector zones 24 where HB-2 (e.g., hBD-2) and HB-3 (e.g., hBD-3) in the healthy sample can bind the immobilized binding ligands (e.g., antibodies), and HB-2 and HB-3, if present in the suspect sample, can bind the immobilized binding ligands. Binding of HB-2 and HB-3 to the respective binding ligands (e.g., fluorescent antibodies) results in the production of a fluorescence emission, which can be detected (as discussed below) where the antibodies used to detect BD-2 and BD-3 emit at different fluorescent wavelengths so that their fluorescent signals can be easily distinguished.
  • HB-2 e.g., hBD-2
  • HB-3 e.g., hBD-3
  • BDI beta defensin index
  • this can be done, for example, by: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI.
  • determining the BDI can be done using the following formula: [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane).
  • the comparison of beta defensin levels or between ratios can be conducted by any suitable method known to those skilled in the art.
  • the comparison can be carried out mathematically or qualitatively by an individual operating an analytic device or by another individual who has access to the data provided by the analytic device.
  • the steps of determining and comparing the levels of BD-2 and BD-3 can be carried out electronically (e.g., by an electronic data processor).
  • the method can also include the step of providing a report indicating that the subject is in need of oral cancer therapy if the BDI is greater than one, for example.
  • the present method may also be useful for determining if and when therapy for treating epithelial cancer should be administered to a subject.
  • the method includes providing an oral cancer therapy to the subject if the BDI is greater than one.
  • BDI BDI
  • methods for treating oral cancer are known to those skilled in the art. Examples of therapy for oral cancer include surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy.
  • One example of the method is illustrated in Fig.1B and described below with reference to Steps 1-8.
  • Step 1 wash the mouth of a subject using, e.g., water.
  • Step 2 place two cartridges (e.g., red and green) on a flat surface, each of which includes a cell lysis solution.
  • Step 3 peel open a first bag containing a first cytobrush and remove. Rub and rotate the brush on the suspected lesion site (e.g., 20 times with pressure). Pick up red cartridge and hold steady in on hand. Slowly rotate the brush into the liquid of the cartridge (e.g., about 15 times, with occasional flicking between rotations, while holding the top of the cartridge). The purpose is to dislodge collected cells from the brush head into the liquid in the cartridge. Remove and discard the first cytobrush.
  • Step 4 peel open a bag containing second cytobrush and remove. Rub and rotate the brush on the contralateral site (e.g., about 15 times, opposite the suspected lesional site). Pick up the green cartridge and hold steady in one hand.
  • Step 5 keep both the red and green cartridges at room temperature for about 5 minutes.
  • Step 6 after about 5 minutes, flick both the cartridges, one by one, with finger until it is evident that liquid has accumulated at the bottom of each cartridge.
  • Step 7 place about 3 drops of liquid from red cartridge onto the inlet marked “L” on the device 10. Place about 3 drops of liquid from green cartridge onto the inlet marked “C” on the device 10. Wait for about 10 minutes.
  • Step 8 if all six lines or stripes are present as shown in Case #1-5, determine fluorescence intensities (FI) of the hBD-2 and hBD-3 bands using a portable fluorescence reader. Calculate BDI value using the following formula: [HBD-3FI/HBD-2FI]/(L-lane)/HBD-3FI/HBD-2FI](C-lane).
  • FI fluorescence intensities
  • the system can comprise a POC, lateral flow assay device 10 (e.g., as shown in Fig.1A and described above), as well as a mobile device (not shown) configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine the beta defensin index (BDI).
  • a POC lateral flow assay device 10
  • a mobile device not shown
  • a processor configured to access a camera configured to acquire fluorescence intensity values and determine the beta defensin index (BDI).
  • BDI beta defensin index
  • the processor is operatively coupled to the camera and programmed to: acquire fluorescence intensity (FI) values for the suspect sample (L-lane) and the healthy sample (C-lane); generate a BDI value using the formula, [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane); and output the generated BDI value to a display device (e.g., a computer, handheld electronic device (e.g., cell phone), etc.).
  • a display device e.g., a computer, handheld electronic device (e.g., cell phone), etc.
  • the diagnostic kit can include any one or combination of the following components: a POC, lateral flow device 10 (such as the one shown in Fig.1A and described above); means (e.g., cytobrushes) for obtaining healthy and suspect samples from a subject; cartridges for receiving healthy and suspect samples and further containing a cell lysis solution; means (e.g., syringes, pipettes) for delivering lysed suspect and healthy samples to the device; a mobile electronic device (as described above, e.g., being configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine the beta defensin index (BDI); and instructions for using the device (e.g., according to the above- described method) for detection of oral cancer in a subject.
  • a POC lateral flow device 10
  • means e.g., cytobrushes
  • cartridges for receiving healthy and suspect samples and further containing a cell lysis solution
  • means e.
  • a POC, lateral flow assay device for detecting oral cancer in a subject can comprise a housing that includes: a healthy channel and a suspect channel, each channel including a first labeled binding ligand that specifically binds to BD-2, a second labeled binding ligand that specifically binds to BD-3, and a control indicator; a first inlet in fluid communication with the suspect channel; and a second inlet in fluid communication with the healthy channel; wherein neither the healthy channel nor the suspect channel include binding ligands that specifically bind to and immobilize intact epithelial cells.
  • Aspect 2 The device of Aspect 1, wherein each of the healthy and suspect channels is free of a filter.
  • Aspect 3 The device of any one of Aspects 1-2, wherein the first and second labeled binding ligands are antibodies.
  • Aspect 4 The device of Aspect 3, wherein the antibodies are fluorescently-labeled antibodies.
  • Aspect 5 A system for detection of oral cancer in a subject, comprising the device of Aspect 1 and a mobile device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI).
  • BDI beta defensin index
  • Aspect 6 The system of Aspect 5, wherein the processor is operatively coupled to the camera and programmed to: acquire fluorescence intensity (FI) values for the suspect sample (L-lane) and the healthy sample (C-lane); generate a BDI value using the formula, [BD-3FI/BD-2FI]/(L-lane)/BD-3FI/BD-2FI](C-lane); and output the generated BDI value to a display device.
  • FI fluorescence intensity
  • a method for detecting oral cancer in a subject comprising: (a) providing the POC, lateral flow assay device of Aspect 1; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; and (e) characterizing the subject as having oral cancer based on the BDI; wherein at least step (c) is performed at about room temperature.
  • BDI beta defensin index
  • Aspect 8 The method of Aspect 7, wherein step (d) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI.
  • Aspect 9 The method of any one of Aspects 7-8, wherein the suspect sample is obtained from a lesional site and the healthy sample is obtained from a corresponding contralateral normal site.
  • Aspect 10 The method of Aspect 9, wherein the suspect sample is obtained from a lesional site comprising skin or mucosa.
  • Aspect 11 The method of Aspect 9, wherein the suspect and healthy samples are not saliva.
  • Aspect 12 The method of any one of Aspects 7-11, wherein the suspect and healthy samples delivered to the first and second inlets at step (c) do not include intact epithelial cells.
  • Aspect 13 The method of any one of Aspects 7-12, wherein: at step (b), the healthy and suspect samples are obtained non-invasively by cytobrushing and comprise healthy and suspect cells, respectively; and following step (b), the healthy and suspect cells are lysed prior to step (c).
  • a method for detecting and treating oral cancer in a subject comprising the steps of: (a) providing the device of Aspect 1; (b) obtaining a healthy sample and a suspect sample from the subject; (c) delivering the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (d) determining a beta defensin index (BDI) based on detected levels of BD-2 and BD-3 by the device; (e) characterizing the subject as having oral cancer if the BDI is greater than 1; and (f) treating the oral squamous cell carcinoma with a therapy selected from the group consisting of surgery to remove the cancer, freezing cancer cells, localized heat to destroy cancer cells, chemotherapy, radiation therapy, and targeted drug therapy; wherein at least step (c) is performed at about room temperature.
  • BDI beta defensin index
  • a kit for detection of oral cancer in a subject comprising: a point-of-care, lateral flow device as recited in Aspect 1; and instructions for using the device for detection of oral cancer in the subject; optionally, means for obtaining healthy and suspect samples from the subject; optionally, separate cartridges for receiving healthy and suspect samples, each of which contains a cell lysis solution; optionally, means for delivering lysed suspect and healthy samples to the device; optionally, a mobile electronic device configured to access a communications network and having a processor configured to access a camera configured to acquire fluorescence intensity values and determine a beta defensin index (BDI).
  • BDI beta defensin index
  • Aspect 16 The kit of Aspect 15, wherein the instructions include the following steps: (a) obtain a healthy sample and a suspect sample from the subject; (b) deliver the suspect sample and the healthy sample to the first and second inlets of the device, respectively; (c) determine the BDI based on detected levels of BD-2 and BD-3 by the device; and (d) characterize the subject as having oral cancer based on the BDI; wherein at least step (b) is performed at about room temperature.
  • Aspect 17 The kit of any one of Aspects 15-16, wherein step (c) further comprises the steps of: (i) comparing the level of BD-3 to BD-2 determined in the suspect sample to obtain a suspect BD-3/BD-2 ratio; (ii) comparing the level of BD-3 to BD-2 determined in the healthy sample to obtain a healthy BD-3/BD-2 ratio; and (iii) dividing the suspect BD-3/BD-2 ratio by the healthy BD-3/BD-2 ratio to obtain the BDI.
  • Aspect 18 The kit of any one of Aspects 15-17, wherein the instructions further recite: at step (a), obtain the healthy and suspect samples non-invasively by cytobrushing so that the healthy and suspect samples comprise healthy and suspect cells, respectively; and following step (a), lyse the healthy and suspect cells prior to step (b).
  • step (a) obtain the healthy and suspect samples non-invasively by cytobrushing so that the healthy and suspect samples comprise healthy and suspect cells, respectively; and following step (a), lyse the healthy and suspect cells prior to step (b).
  • EXAMPLE 1 [00129] An experiment was performed in which the inventors: (i) analyzed the TCGA database to understand and annotate the expression of hBD-3 transcript (DEFB103) in the context of head and neck squamous cell carcinoma (HNSCC); (ii) validated the expression of hBD-3 and hBD-2 in retrospectively collected carcinoma- in-situ (CIS) and OSCC tissue using immunofluorescence microscopy; (iii) demonstrated differential hBD-3 and -2 expression in cell populations from excised OSCC tumors by fluorescent-activated cell sorting (FACS); (iv) showed by FACS that cytobrush collected cells from OSCC lesions have similar defensin expression profiles as cells excised by biopsy from OSCC tumor tissue; (v) designed an ELISA- based BDI assay platform to discriminate OSCC from benign lesions; (vi) validated the BDI platform through multi-center clinical studies of three non-overlapping cohorts of subjects; and (vii) shown the BDI in a
  • each section (5 ⁇ m) was de-paraffinized in xylene and hydrated with serially diluted ethanol, followed by antigen retrieval at 98°C in a high pH Target Retrieval Solution (Dako Co) and blocking with 10% donkey serum containing 0.25% Triton X-100, overnight at 4°C. After washing with PBS, each section was incubated with the respective (anti-hBD-3; NOVUS Biologicals, anti- hBD-2; Santa Cruz Biotechnology) primary antibody (1 h, room temperature), washed in PBS (3 ⁇ 10 min), and then stained with the compatible fluorescent dye- conjugated secondary antibody. For double immunofluorescence, consecutive staining by different primary and secondary antibodies was performed.
  • Isotype controls were conducted using isotype-matched IgGs, corresponding to each primary antibody. Sections were mounted on slides using VECTASHIELD Fluorescent Mounting Media (Vector Lab Inc., Burlingame, CA) containing DAPI to visualize nuclei. Immunofluorescent images were generated using a Leica DMI 6000B fluorescence microscope (Leica Microsystems, Bannockburn, IL) or an Olympus BX51 fluorescence microscope mounted with the Olympus DP71 camera (Olympus America Inc., Center Valley, PA). [00136] Immunofluorescence images were processed using the NIH ImageJ program (Collins, T.J. ImageJ for microscopy. Biotechniques 43, 25-30 (2007)).
  • hBD-2 and hBD-3 were acquired in 16-bit gray scale, respectively. Fluorescent densities on each of the antibody treated sections were measured with ImageJ as described previously (Collins, T.J. ImageJ for microscopy. Biotechniques 43, 25-30 (2007); Kawsar, H.I. et al., Oral Oncol.45, 696-702 (2021)).
  • the expression of hBD-2 and hBD-3 was represented as the ratio of relative fluorescence intensity of hBD-2 and hBD-3 over that of nuclei, respectively.
  • Cytobrush sample collection procedure [00137] All cytobrush samples were obtained using ROVERS ORCELLEX BRUSH (Rovers Medical Devices, Oss, Netherlands) for cytobrushing oral mucosa based on the Kajun et. al., J Oral Sci 60, 45-50 (2016) report showing good quality mucosal cell collection with this cytobrush. Each participant was first asked to rinse his/her mouth with water. Before performing a biopsy, the clinician applied the cytobrush onto the mucosal surface of the suspicious lesion and, with pressure, turned the brush head 10 turns to collect as much of the entire thickness of the lesion as possible. The brush was then inserted into a sample collection tube (containing 2 ml of PBS) and placed on ice.
  • xE whole exome sequencing of paired cytobrush samples was performed by Tempus Labs, Inc., (Chicago, IL, USA ) (Lau, D. et al., Nat Commun 13, 4053 (2022); Beaubier, N. et al., Oncotarget 9, 25826-25832, (2016); Beaubier, N. et al., Nat Biotechnol 37, 1351-1360 (2019); Leibowitz, B.D. et al., BMC Cancer 22, 587 (2022)).
  • Tempus xE is a whole exome next-generation sequencing proprietary assay which analyzes the entire coding region (exome) of the patient’s genome, combined with whole transcriptome RNA sequencing. This was done as a tumor:normal matched assay for each tumor and contralateral normal cytobrush specimen, sequenced to an average depth of 250x and 150x, respectively.
  • Whole transcriptome RNA-seq was 50 million reads encompassing 19,396 genes covering ⁇ 39 Mb of genomic space.
  • the cytobrush samples from OSSC lesions were matched to corresponding contralateral normal samples to ensure fidelity of somatic variant calling. From DNA sequencing, somatic and single nucleotide variants, insertions and deletions and copy number variant data were generated.
  • Cytobrush sample processing for flow cytometry of defensins [00142] For flow cytometry staining, single-cell suspensions were obtained from cytobrush samples by resuspending the samples in a PBS-based, enzyme-free cell dissociation buffer (Gibco) at room temperature for 5 minutes, followed by passing suspensions through 100 uM cell-strainers. Excised tumors were processed by collagenase digestion (0.5mg/ml) for 8 minutes at room temperature. The cells were then centrifuged in PBS/BSA before sequential flow-cytometry staining for E- cadherin, hBD-2 and hBD3 proteins.
  • PBS-based, enzyme-free cell dissociation buffer Gibco
  • Excised tumors were processed by collagenase digestion (0.5mg/ml) for 8 minutes at room temperature.
  • the cells were then centrifuged in PBS/BSA before sequential flow-cytometry staining for E- cadherin, hBD-2 and hBD3 proteins.
  • the cells were washed after surface E-cadherin staining, fixed, permeabilized and stained with antibodies for intracellular hBD2 and hBD3 proteins.
  • Live-Dead viability staining (Life Technologies/Thermofisher) was used to remove dead cells in the flow cytometry analysis. Data was acquired using a BD Fortessa cytometer and analyzed using FlowJo 9.8 or 10.5.3 software.
  • PE-conjugated anti-human ⁇ -E-cadherin polyclonal antibody was purchased from R&D systems. Alexa-flour 647 conjugated hBD2, and FITC conjugated hBD3 polyclonal antibodies were purchased from BIOSS (Woburn, MassachusettsU.S.A.).
  • Beta Defensin ELISA and determination of BDI was collected and used for the ELISA assay.
  • 96-well immunoplates (R&D Systems, City, MN) were coated with 50 ⁇ l anti-hBD-2 or hBD-3 antibodies diluted to 1 ⁇ g/ml, 4°C, overnight, followed by blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS).
  • BSA bovine serum albumin
  • PBS phosphate-buffered saline
  • the plate was then incubated at room temperature (RT) for 1 h.
  • the wells were washed 3 times with PBS containing 0.1% Tween 20 and incubated with 50 ⁇ l of the biotinylated secondary antibody (Peprotech, NJ) diluted to 0.1 ⁇ g/ml, at RT, 30 min.
  • Each well was washed 3 times, 50 ⁇ l/well of streptavidin-peroxidase (R&D Systems, 1:200 in wash buffer) was added to each well and incubated for 20 minutes at RT.
  • MICA Microfluidic Intact Cell Analysis
  • the wafer was then UV-patterned under a photomask, post-expo baked (95°C, 10 min), developed in a photoresist solvent (propylene glycol monomethyl ether acetate, Sigma Aldrich, St. Louis, MO), and hard-baked at 110°C overnight.
  • a photoresist solvent propylene glycol monomethyl ether acetate, Sigma Aldrich, St. Louis, MO
  • Surface passivation of the master wafer was performed under vacuum using trichloro (1H,1H,2H,2H-perfluorooctyl) saline (PFOCTS, Sigma Aldrich).
  • PFOCTS trichloro
  • a polydimethylsiloxane (PDMS, ThermoFisher Scientific) pre-polymer was mixed with the curing agent at a volume ratio of 10:1 and degassed to remove any air bubbles.
  • the mixture was then poured over the master wafer and cured at 80°C overnight. Thereafter, PDMS replicas were peeled off, cut into individual pieces, and inlet and outlet holes were punched. Excess saline was removed by sonicating in isopropanol. Finally, the PDMS replicas were covalently bonded on standard microscope glass slides using oxygen plasma to form the micro-channels. [00150] Following tubing assembly, the MICA microchannel was rinsed with 100% ethanol and PBS, and then blocked with 2% bovine serum albumin (BSA, ProSpec- Tany TechnoGene Ltd., East Brunswick, NJ) at 4°C overnight to prevent any non- specific cell adhesion to the microchannel walls.
  • BSA bovine serum albumin
  • Cytobrush samples collected from lesional and contralateral sites of each patient were placed on ice before testing.
  • the cytobrush sample was injected into the appropriate microchannel (lesion in one; contralateral in the other) under 40 mbar inlet pressure using a Flow-EZ pressure control unit (Fluigent, Lowell, MA) until a reasonable cell density was achieved in the microchannel.
  • Flow-EZ pressure control unit Flugent, Lowell, MA
  • the fixed cells were permeabilized with Triton X- 100 (Sigma Aldrich) (0.1% in PBS with 1% BSA) for 15 min at RT, and incubated in a mixture of 4’,6-diamidino-2-phenylindole (DAPI, 10 ⁇ g/mL final concentration) and hBD-3 and hBD-2 antibodies (Bioss Antibodies, Woburn, MA; Catalogue: bs-7378R- A488 and bs-4307R-A647) (1% in PBS with 1% BSA) in the dark at RT for 1 h.
  • Triton X- 100 Sigma Aldrich
  • DAPI 6-diamidino-2-phenylindole
  • hBD-3 and hBD-2 antibodies Bioss Antibodies, Woburn, MA; Catalogue: bs-7378R- A488 and bs-4307R-A647)
  • the microchannel was washed with PBS and imaged under 10 ⁇ objective with an inverted microscope (Olympus IX83) and a microscope camera (EXi Blue EXI-BLU-RF-M-14-C).
  • the retained cells were fluorescently labeled with an hBD-3 antibody (Alexa Fluor 488 conjugated) and an hBD-2 antibody (Alexa Fluor 647 conjugated).
  • Cell nuclei were also labeled with DAPI, and used to distinguish intact cells from debris.
  • Salivary levels of hBD-2 and hBD-3 were determined following the protocol as described by Ghosh, S.K. et al., Clin Chem 53, 757-765 (2007). Briefly, 96-well immunoplates (R&D Systems, MN) were coated with 50 ⁇ l anti-hBD-2 or hBD-3 antibodies (Peprotech, NJ) diluted to 1 ⁇ g/ml, 4°C, overnight, followed by blocking with 1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). The wells were washed 3 times with PBS containing 0.1% Tween 20.
  • BSA bovine serum albumin
  • the basal subtype of HNSCC is characterized by genes associated with; (a) epidermal development, (b) ErbB signaling (ErbB; a family of four receptor tyrosine kinases, of which epidermal growth factor receptor (EGFR) is the first discovered member), and (c) growth/transcription factor signaling (Walter, V. et al., PLoS One 8, e56823 (2013)).
  • DEFB103 expression was highest in grade 1; i.e., the “well-differentiated” HNSCC subtype, compared to the other HNSCC subtypes (Fig.8D), and its expression was also significantly higher in well-differentiated grade 1 HNSCC compared to normal/healthy sites (p ⁇ 0.00003) (Fig.8D).
  • DEFB103 expression was significantly higher in Stage 1 and 3 cancers compared to normal.
  • gender both male and female DEFB103 are significantly higher in HNSC compared to normal (Fig.9B).
  • transcript, protein and now flow cytometry from three independent studies confirm the overexpression of hBD-3 and reduced expression of hBD-2 in CIS and OSCC lesions.
  • hBD-3 vs. hBD-2 in oral CIS and in OSCC lesions we hypothesized that by collecting epithelial cells from a suspicious lesion and comparing its hBD-3/hBD-2 ratio to cells collected from the same subject’s contralateral healthy oral mucosa, a reliable biomarker for OSCC could be established.
  • Non-invasively collected (cytobrushed) mucosal cells can be used to collect cancer cells and to assay for the expression of human beta defensins
  • our board-certified pathologist conducted cytological analysis of cytobrush samples (Figs.3A-B). We identified cancerous cells in the lesional site (Fig.3A) but not in the contralateral site (Fig.3B) of the same patient.
  • Whole exome sequencing xE whole exome, TEMPUS, Chicago
  • Beta-Defensin-Index (BDI) and defined it as the ratio of hBD-3/hBD-2 in the lesional site over the ratio of hBD-3/hBD-2 in the contralateral site of the same patient ( Figure 3A).
  • BDI Beta-Defensin-Index
  • Demographic information e.g., age, gender, and smoking status
  • lesion location in the oral cavity and diagnosis based on pathology review is included for every subject in the proof-of-principle, discovery, and validation studies (Table 4 and Table 6).
  • Table 6 Demographic and clinical parameters (stages) of 92 subjects [Discovery and Validation cohorts]
  • smokin DI values correlated with smoking status (e.g., never or non-smokers; former and current smokers).
  • the BDI values of both current and former smokers were significantly higher than those of non-smokers (Fig.11A).
  • male participants had significantly higher BDI values compared to female participants (Fig.11B) consistent with the literature, showing that males smoke more than females and OSCC is more prevalent in males (Chinwong, D. et al., J Addict 2018; Lee, Y-C. et al., Medicine 100, e27674 (2021)).
  • MICA Microfluidic Intact Cell Assay
  • MICA includes micropillar arrays embedded into the microchannel forming narrow openings along the flow direction, coupled with two 400- ⁇ m wide side passageways. MICA is designed such that large cell aggregates are retained upstream within the coarse openings and individual cells are retained downstream within the narrower openings. The side passageways are designed to prevent clogging of the near-inlet portion of the micropillar arrays.
  • the MICA microchannel overall dimensions are 27 mm ⁇ 4 mm ⁇ 120 ⁇ m (length ⁇ width ⁇ height).
  • a photograph of MICA is shown in Fig.6B.
  • the microscopic phase-contrast image shown in Figs.6C-D represents a typical cell distribution in the MICA microchannel and confirms that cell aggregates were being filtered upstream while individual cells were retained downstream.
  • EGFR epidermal growth factor receptor
  • EGFR was selected as a proof-of-principle marker as it is overexpressed in up to 90% of all OSCC patients (Grandis, J.R. et al., Cancer Res 53, 3579-3584 (1993); Xu, M.J. et al., Cancer Metastasis Rev 36, 463- 473 (2017); Rubin Grandis, J. et al., J Natl Cancer Inst 90, 824-832 (1998)), and its activation results in hBD-3 expression (Feng. Z. et al., Infect Immun 82, 4458-4465 (2014); Bengal, J.S. et al., Pathog Dis 74, (2016); Shuyi, Y.
  • this Example illustrates one configuration of a single lane or channel comprising a device 10 of the present disclosure.
  • a device 10 of the present application can be constructed having two or more lanes or channels, each of which being constructed as shown in Fig.15 and described herein.
  • FIA fluorescence immunoassay
  • the device is designed to quantitatively determine hBD-2 and hBD-3 levels in lesion and healthy site cytobrush lysate samples.
  • Development and Analytical Validation [00193] Material selection for device development [00194] A validated and tested nitrocellulose membrane (Sartorius, Germany) is designed on which two test lines and one control line is printed. Pore size and membrane properties determine the sample flow rate and sensitivity, which is considered in selection.
  • a conjugated pad comprises a glass fiber or polyester pad (Ahlstrom, Pennsylvania) that holds and releases a fluorescent label (e.g., Europium) Thermo Scientific), which is conjugated to anti-human hBD-2&3 detection antibodies (polyclonal, Thermo Scientific).
  • Sample pad is a diagnostic-grade, hydrophilic spunbonded polyester membrane (Ahlstrom, Grade 6614) that is highly pure when received. Sample pad is inert and does not interact with cationic peptides.
  • Absorbent pad is cellulose paper (Ahlstrom 222) placed at a first end of the nitrocellulose membrane to keep the flow consistent by absorbing excess reagents.
  • Europium fluorescent beads are used for signal detection, which produce robust detectable signals that are read using portable readers (Hemex Health, Portland, OR). Europium beads are conjugated to detection antibodies as well as rabbit IgG (Thermo Scientific) for control band generation.
  • Cytobrush (Rovers Orcellex Brush, Netherlands) cell collection device provides reliable, reproducible, easy-to-use, fast, comfortable, non-invasive cell collection of high cellularity (e.g., about 55,000 cells).
  • Reagent preparation [00196] Capture antibodies are printed (KinBio, Rehovot, Israel) on the nitrocellulose membrane to form hBD-2 and hBD-3 test lines and a control line. Polyclonal antibodies naturally bind to different epitopes on the target, which allows the use the same antibody for both detection and capture. If monoclonal antibodies are used, detection antibodies are different than the capture antibodies to achieve capture and detection on distinct epitopes of the target molecules.
  • Europium beads are conjugated with selected detection antibodies, and their stability is determined at different temperatures (e.g., 25°C, 37°C, and 45°C). Binding efficiency is confirmed by serial dilution of purified antigens in composite samples by measuring the line intensity to determine limit of detection, limit of quantitation, and dynamic range.
  • Device assembly [00198] Capture antibodies are printed on the nitrocellulose membrane using a dispenser, followed by a drying step. The nitrocellulose membrane is assembled with the conjugate pad (e.g., impregnated with the fluorescent conjugate bound to the detection antibodies), sample pad, and absorbent pad onto a backing card (Lohmann, Florida) for stability (shown as bottom layer in Fig.15).
  • Assay optimization Test concentrations of the capture and detection antibodies, buffer conditions, and sample volume to ensure >90% sensitivity and >90% specificity are carefully tested. Flow rate is optimized by adjusting the sample pad and absorbent pad properties. Nitrocellulose paper is selected by considering both flow rate and protein binding capacity, which affect the limit of detection (LOD). [00201] Limit of Detection (LOD) and Limit of Quantification (LOQ) [00202] The lowest concentration of the hBD-2 and hBD-3 analytes that can be detected, but not necessarily quantitated, is determined as an exact value. LOD is determined by repeatedly measuring blank samples and samples with very low concentrations.
  • LOQ in the low pg/ml range, is determined as the lowest concentration at which the analyte can not only be detected but also measured with acceptable accuracy and precision.
  • Analytical testing To evaluate analytical sensitivity and specificity as part of the assay development process, the device is tested with known positive and negative samples to assess its performance. Cross-reactivity analysis using similar analytes is performed to ensure specificity. Since the device is a fluorescent assay, a fluorescence reader is used, which is calibrated with known standards to quantify the fluorescent intensity correlating to analytical concentration. [00205] Other considerations [00206] Simple visual and/or audible results can be added to make the device more accessible and to let any frontline healthcare worker operate the device.
  • the Hemex Reader for example, has connectivity to upload test results to medical records to report to a clinician directly. An internal control line confirms that the test and the reader work as intended. Shelf-stable reagents are used to maximize the shelf-life, considering the temperature, storage, and shipping conditions of resource limited settings. Cytobrush sample collection efficiency and sample desorption into the testing buffer is monitored. Regulatory-compliant protective substances in the cytobrush buffer are used to maximize shelf-life in room temperature storage. [00207] Validation to Ensure Accuracy and Precision [00208] Accuracy and precision of device is evaluated to ensure that the test is both reliable in terms of true results (accuracy) and reproducible in terms of consistent results (precision).
  • Construction and testing of a device and assay that demonstrates a sensitivity and specificity greater than 90% to ensure accuracy, and CV ⁇ 25% to ensure precision is intended. Potential failure modes of the tests which may lead to inconclusive or repeat tests are analyzed. Standardization of room temperature lysis of cytobrush samples is achieved. Testing and analytical validation of the device using cytobrush cell lysates from normal subjects is performed. Batch testing (e.g., 1,200 devices) is performed in multiple (e.g., three) batches (e.g., 400 devices per batch).
  • Analytical performance is assessed by following the BEST (Biomarkers, EndpointS, and Other Tools Resource) definitions and the FDA “Bioanalytical Method Validation” guidelines, including, standard curve creation, sensitivity, specificity, precision, limit of detection, and limit of quantification.
  • Statistical design and data analysis is based on published guidelines. A sample size of about 200 tests for an adequately powered (significance at 0.05, power at 90%) study design is used. Typical measurement for normal and lesion sites, and the clinically meaningful difference expected in a cancerous lesion is used. These procedures ensure that the device provides accurate results that are close to the true value (accuracy) and that repeated tests yield similar results (precision).
  • Pre-commercial manufacturing, packaging, and stability Manufacturing, packaging, and accelerated stability testing is performed to determine shelf life. Moving from small-scale production to larger batch processes to ensure consistency is envisioned. For this purpose, three different pilot batches are produced for mixed testing. Rigorous quality control measures by following ISO 13485 standards to ensure each batch meets the required standards are developed and implemented. As part of packaging, each device is accompanied with a reader and a mobile app for fluorescence detection, and clear instructions for use. [00211] Preparation of samples with known concentrations [00212] Lysate samples are prepared with known concentrations of hBD-2 and hBD-3 to cover the expected range of concentrations in real-world samples.
  • cytobrush samples from healthy (non-cancerous) are collected in 2 ml lysis buffer, hBD-3 is 6.8 ⁇ 6 pg/ml, and hBD-2 is 16 ⁇ 12.6 pg/ml, which are concentrated 20-fold during centrifugation steps.
  • Concentration samples for hBD-2 and hBD-3 are prepared as follows: 5 pg/ml; 50 pg/ml; 100 pg/ml; 200 pg/ml; 500 pg/ml; 1000 pg/ml; and 4000 pg/ml.
  • calibration coefficients are the coefficient of a polynomial that normalizes the intensity of each test line with what was observed on ELISA.
  • the calibration coefficients are encoded on a machine readable QR code that is on each test device or cartridge. This allows the Reader to automatically compensate for lot-to-lot variability.

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Abstract

Un procédé de détection d'un cancer buccal chez un sujet peut comprendre : (a) la fourniture d'un dispositif de dosage à écoulement latéral POC comprenant : un canal sain et un canal suspect, chaque canal comprenant des premier et second ligands de liaison marqués qui se lient spécifiquement à BD-2 et BD-3, respectivement, et un indicateur de commande ; des première et seconde entrées en communication fluidique avec les canaux suspects et sains, respectivement ; les canaux ne comprenant pas de ligands de liaison qui se lient spécifiquement à et immobilisent des cellules épithéliales intactes ; (b) l'obtention d'un échantillon sain et d'un échantillon suspect provenant du sujet ; (c) la distribution des échantillons suspect et sain aux première et seconde entrées du dispositif, respectivement ; (d) la détermination d'un indice de bêta-défensine (BDI) sur la base des niveaux détectés de BD-2 et BD-3 par le dispositif ; et (e) la caractérisation du sujet comme présentant un cancer buccal sur la base du BDI ; au moins l'étape (c) étant effectuée à environ la température ambiante.
PCT/US2024/056932 2023-11-21 2024-11-21 Dispositif de point d'intervention pour la détection du cancer buccal Pending WO2025111485A1 (fr)

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