METHODS AND KIT FOR DETECTION OF ANALYTES
20230056380 · 2023-02-23
Inventors
Cpc classification
A61B5/411
HUMAN NECESSITIES
Y02A90/10
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention provides for a kit and methods that detect certain analytes of interest potentially present in blood and bodily fluids of a living mammal. The methods and kit encompass a bioassay performed in vivo. Contact of the bioassay reagent with the analyte, if present, renders a response that can be clinically assessed visually or by reading instrumentation or by biosensor. In one embodiment, the invention may be used to detect the presence, absence, or amount of suspected analyte present in a patient test subject. The invention is particularly suited for point-of-care (POC) use, self-testing, large-scale implementation and for use with patients where limited sample volumes are available or accessible.
Claims
1. A method for determining a presence, absence, or amount of an analyte in a living mammal comprising method steps of: providing at least one affinity reagent construct comprising at least one Fc.epsilon.R1 receptor binding domain and at least one additional moiety capable of binding said analyte wherein the resulting construct binds both a mast cell and said analyte in any order; contacting at least one endogenous mast cell in situ and present within a target tissue of said mammal with said reagent construct to elicit a multivalent binding response when said analyte is present; and performing an assessment of said multivalent binding response to determine said presence or amount of said analyte.
2. The method of claim 1 wherein said contacting further comprises delivering said affinity reagent construct into a skin of said mammal.
3. The method of claim 1 wherein said contacting further comprises delivering said affinity reagent construct into tissue of said mammal wherein said affinity reagent is exposed to from about 125 to 20,000 mast cells per cubic millimeter.
4. The method of claim 3 wherein said delivering further comprising performing delivery with at least one device selected from the group consisting essentially of a needle prick, intradermal injection, solid needle, hollow needle, patch method or needleless system.
5. The method of claim 2 wherein said contacting step further comprises delivering said affinity reagent construct into the dermal layer.
6. The method of claim 1 wherein performing said assessment further comprises evaluating said target tissue for at least one physiological change in said target tissue.
7. The method of claim 6 further comprising evaluating a morphological change in said target tissue to render assessment of said multivalent binding response.
8. The method of claim 6 further comprising using a device to measure a morphological change.
9. The method of claim 6 further comprises using a sensor for evaluation of said target tissue to render assessment of said multivalent binding response.
10. The method of claim 7 further comprising performing a visual inspection for a wheal and flare reaction.
11. The method of claim 8 wherein said device measures a wheal and flare reaction.
12. The method of claim 1 wherein said constructing step further comprises using at least one technique selected from the group consisting essentially of hybridoma technology, covalent conjugation, non-covalent binding and genetic engineering methods.
13. The method of claim 1 wherein said constructing step further comprises fashioning said additional moiety using at least one member from the group consisting essentially of antibodies, recombinant antibodies, engineered antibodies, antibody fragments, synthetic antibodies, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor binding molecules, drugs, toxicants, toxins, pathogens, pathogen components, biomarkers, cell surface markers, ligands, RNA, or DNA.
14. The method of claim 1 wherein performing an assessment further comprises measuring a substance released during mast cell degranulation.
15. The method of claim 1 wherein said analyte is Mycobacterium tuberculosis and said additional moiety comprises at least one major histocompatibility complex protein complexed with at least one Mycobacterium tuberculosis antigen.
16. The method of claim 15 wherein said major histocompatibility complex protein is selected from the group consisting of MHC Class I and MCH Class II proteins.
17. The method of claim 15 wherein said Mycobacterium tuberculosis antigen is either Mtb secretory antigenic target ESAT-6 or 10-kDa culture filtrate protein CFP-10.
18. The method of claim 1 wherein said analyte is selected from SARS-CoV-2 antibodies and said additional moiety comprises an antigen derived from SARS-CoV-2 virus.
19. The method of claim 18 wherein said antigen is derived from a protein selected from the group consisting of SARS-CoV-2 receptor binding domain, spike protein, spike S 1 protein, spike S2 protein and SARS-CoV-2 nucleocapsid protein.
20. A method of testing for a suspected pathology in a living mammal comprising method steps of: selecting an analyte indicative of said suspected pathology; providing at least one affinity reagent construct comprising at least one Fc.epsilon.R1 receptor binding domain and at least one additional moiety capable of binding said analyte wherein the resulting construct binds both a mast cell and said analyte in any order; contacting at least one endogenous mast cell in situ and present within a target tissue of said mammal with said reagent construct to elicit a multivalent binding response when said analyte is present; and performing an assessment of said multivalent binding response to determine said presence or amount of said analyte.
21. A method of testing a living mammal for exposure to at least one substance selected from the group consisting essentially of chemicals, drugs, biowarfare agents, toxicants, toxins and pathogens, the method comprising method steps of: selecting an analyte indicative of exposure to said substance; providing at least one affinity reagent construct comprising at least one Fc.epsilon.R1 receptor binding domain and at least one additional moiety capable of binding said analyte wherein the resulting construct binds both a mast cell and said analyte in any order; contacting at least one endogenous mast cell in situ and present within a target tissue of said mammal with said reagent construct to elicit a multivalent binding response when said analyte is present; and performing an assessment of said multivalent binding response to determine said presence or amount of said analyte.
22. An affinity reagent for an in vivo bioassay, said reagent comprising at least one Fc.epsilon.R1 receptor binding domain and at least one additional moiety capable of bindingan analyte of interest, said reagent capable of binding both a mast cell and said analyte in any order.
23. The affinity reagent of claim 22 wherein the additional moiety is selected from the group consisting essentially of antibodies, recombinant antibodies, engineered antibodies, antibody fragments, synthetic antibodies, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor binding molecules, drugs, toxicants, toxins, pathogens, pathogen components, biomarkers, cell surface markers, ligands, RNA and DNA.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0058]
[0059]
DETAILED DESCRIPTION OF THE INVENTION
[0060] While the methods and affinity reagents of the present invention may vary from one another in some aspects, the general approach and procedural steps set forth herein are all designed to determine the presence, absence, or amount of an analyte of interest in a mammalian subject. At the outset, the artisan will appreciate that in order to practice the methods and bioassay kit of the present invention, an affinity reagent (AR) must be constructed based upon the analyte of interest as further explained herein. Such analytes will broadly include a broad spectrum of substances and are intended to include chemicals, peptides, proteins, lipids, carbohydrates, glycoproteins, nucleic acid sequences, or a combination thereof, the foregoing being illustrated by but not limited to substances such as antibodies, hormones, receptors, receptor binding molecules, antigens, drugs, toxicants, toxins, pathogens, biomarkers, biochemicals, cell surface markers, RNA, DNA and other molecules of interest.
[0061] It will be appreciated that one must select the appropriate analyte of interest as an indicator of diseases or disease states as well as the detection and sentinel monitoring of certain environmental and conflict based exposures that may result due to bio-warfare and terrorist events. Similarly, selecting the appropriate analyte of interest is also important to determining the absence, presence or amount of a substance such as hormones, growth factors, metabolites, etc. as an indication of a subject’s general health, fitness or emotional and/or physical state of being.
[0062] Generally, diseases and disease state as well as exposures to undesirable substances are typically hallmarked by the presence of some associated analyte, the presence or absence of which may be used an indicator of the same. For example, such an analyte may be a pathogen, a component of or by-product produced by a pathogen. Such an analyte may also be a biomarker representing a pathogen’s presence in the subject to be screened or monitored. Alternatively, such an analyte may be endogenously released by cells of the subject as a direct or indirect response to the presence of a pathogen or an acute or chronic disease state such as TB. Such analytes can also be present due to certain genetically inherited pathologies or latent conditions that develop over time. There are also analytes that can be used to indicate the physical health, emotional health or fitness status of a subject. Exposure of a subject to certain chemicals and other substances may also result in the presence or absence of certain analytes in the subject, whether it is those the substances themselves or some analyte endogenously produced and released in the subject as a response to such conditions or exposures. Accordingly, the artisan will appreciate that when practicing the present invention, the selection of the appropriate analyte or analytes will be dependent upon the purpose of the testing sought to be performed. Those skilled in the art will also appreciate that there are numerous resources readily available and well known in the arts of physiology, medicine, epidemiology and related health sciences that may serve as guides when attempting to select an appropriate analyte.
[0063] As a footnote to the foregoing, the artisan will appreciate that in cases where the present invention will be used for sentinel monitoring, it is intended that while the AR delivered into the subject will react with the mast cells in the target tissue at the site of affinity reagent administration, crosslinking across the mast cell membrane will not occur unless the analyte is present. Once binding of the AR to the Fc.epsilon.Rl receptors on the mast cells has occurred, mast cells are “sensitized” to the analyte and are incorporated by the present invention to signal the presence of the analyte in the subject, typically pursuant to some event such as an exposure of the subject to a pathogen, toxin or biowarfare agent or a change in the subject’s physiology such as a biomarker that is normally not present or actively circulating in the blood, interstitial fluid, or the lymphatic system or is merely circulating at negligible levels. Upon the arrival of the analyte of interest in the bodily fluids of the subject at the AR delivery site, analyte binding to the AR occurs. This induces the multivalent binding response.
[0064] Once an appropriate analyte is selected, an affinity reagent (AR) for binding such analyte should be constructed. As will be appreciated from the discussion below, the analyte specificity of the test is determined by the additional moiety. Nonetheless, before engaging in construction of the reagent, it would be beneficial to have the artisan become more familiar with the pertinent terminology and the underlying mechanisms upon which the present invention is based. Accordingly, the following section has been provided for the convenience of the reader although the information is readily ascertainable from a myriad of resources currently available and well known in the arts of immunology, molecular biology and biochemistry.
[0065] In immediate (type 1) hypersensitivity, B-cells are stimulated (by CD4+TH2 cells) to produce IgE antibodies specific to antigens such as classic allergens and certain parasites. The difference between a normal infectious immune response and a type 1 hypersensitivity response is that in type 1 hypersensitivity, the antibody is IgE instead of the IgA, IgG, or IgM produced by B-cells. IgE antibodies bind to what is known as the Fc.epsilon.Rl receptor on the surface of tissue mast cells. This receptor is sometimes referred to as a “high affinity” mast cell receptor. Once these mast cells become bound to IgE antibodies, the mast cells are said to be “sensitized”. Subsequent exposure of the sensitized mast cells to the same antigen induces cross-linking of the IgE bound to those sensitized mast cells, resulting in anaphylactic degranulation. This degranulation is characterized by the immediate and explosive release of pharmacologically active pre-formed mediators from storage granules present in the mast cells as well as the concurrent synthesis of inflammatory lipid mediators from arachidonic acid. Some of these mediators include histamine, leukotriene (LTC4 and LTD4), and prostaglandin, which act on proteins (e.g., G-protein coupled receptors) located on surrounding tissues. The principal effects of these products are vasodilation and smooth-muscle contraction. Typically, a wheal (edema)-and-flare (erythema) reaction appears at the site of the surrounding tissues that is often referred to as hives. As mentioned above in the summary of the invention, the term “mast cell” is intended to mean any cells that possess Fc.epsilon.Rl receptor on their plasma membranes one exemplification being basophils and eosinophils to the extent they possess such Fc.epsilon.Rl receptors.
[0066] More particularly, the plasma membrane of mast cells is endowed with receptors that bind to the Fc portion of the IgE molecule at what is known as the “Fc.epsilon.Rl receptor binding domain”. These receptors bind circulating IgE with very high affinity and retain it at the mast cell surface for extended periods of time. Activation of the mast cell results by way of cross linking between bound IgE molecules, thereby inducing the fusion of the granules with the cell surface membrane. This leads to the exocytosis of the granule contents and the onset of an immunological cascade of events typically associated with allergies. (Aalberse, R.C., J. Allergy Clin. Immunol. 106:228 (2000); Al-Muhsen et al., CMAJ 168(10):1279 (2003); Scholl et al., The J. Immunol 175:6645 (2005); Handlogten et al., J. Immunol. 192:2035 (2014); Handlogten et al., Chemistry & Biology 21(20):1445 (2014); Matsuo et al., Allergology International 64:332 (2015)).
[0067] In humans and other mammals, mast cells are found in highest concentration immediately beneath the epithelial surfaces of the skin and the mucosa. Their location at the host-environment interface suggests a central role for these cells in immune surveillance that has been supported by numerous studies examining the responses of mast cells to various pathogens. One of the earliest observations about mast cells, made by their discoverer, Paul Ehrlich, is that they frequently adopt a perivascular localization within tissues. Mast cells are also in close proximity to lymphatic vessels in connective tissue (Ovary, Z., Jpn. J. Allergol. 43 1375 (1994); Turner et al., Nature 402 B24 (1999); Marcelino da Silva et al., Journal of Histochemistry and Cytochemistry 62 698 (2014)).
Constructing the Affinity Reagent
[0068] The affinity reagent construct for the methods and bioassay kit of the present invention is comprised of at least one Fc.epsilon.Rl receptor binding domain and at least one additional moiety selected and fashioned for specifically binding to an analyte of interest. As mentioned above, the additional moiety or “analyte binding moiety” determines the analyte binding specificity of the present invention while the Fc.epsilon.Rl receptor binding domain binds the AR to the mast cell Fc.epsilon.Rl receptor. As mentioned above in the summary of the invention, this dualistic binding can also be referred to as “multivalent binding” and is responsible for what is termed herein as the “multivalent binding response” that occurs in the target tissue. The multivalent binding response occurs in the target tissue starting with the crosslinking at the Fc.epsilon.Rl receptors present on the mast cell(s) and leads to mast cell activation and other physiological changes or responses in the test subject that can be detected or measured.
[0069] Generally speaking, any molecule having analyte binding specificity to the analyte of interest can be fashioned to be incorporated into the reagent construct as an additional moiety during the construction of the affinity reagent of the present invention. For purposes of the present disclosure, the term “fashioning” or “fashioned” is intended to mean the preparation of the analyte binding moiety for incorporation into the reagent construct of the present invention and includes but is not limited to the removal of any extraneous materials unnecessary for incorporating the binding moiety into the reagent construct or for binding to the analyte of interest in order to leave intact the desired analyte binding specificity of the moiety. For example, a protein antigen may only require that portion of the molecule (i.e., a short amino acid sequence) necessary for the AR to bind the antibody of interest. Limiting unnecessary material can generally help to reduce cross reactivity of the AR. Fashioning or fashioned may also include the addition of materials not typically present on the analyte binding moiety but necessary for construction of the AR. For example, the addition of a linker to a drug may be required for the chemical conjugation of the drug to the F1.epsilon.R1 binding domain.
[0070] Some examples of suitable types of additional moieties used for the construction of affinity reagent of the present invention include antibodies, recombinant antibodies, engineered antibodies, antibody fragments, synthetic antibodies, engineered non-antibody binding proteins, antigens, chimeric molecules, fusion proteins, aptamers, hormones, receptors, receptor binding molecules, drugs, toxicants, toxins, pathogens, pathogen components, biomarkers, cell surface markers, ligands, RNA and DNA as well as biochemicals and chemicals that bind specifically substances. It will be appreciated by those skilled in the art that the moiety selected will be compatible in type and specificity to bind the analyte of interest.
[0071] Turning now to the drawings,
[0072] Alternatively,
[0073]
[0074] In
[0075] Importantly, the drawings are exemplary of only a limited number of embodiments for constructing the affinity reagent of the present invention. The analyte binding moiety of the present invention may be comprised of a peptide, protein, nucleic acid, lipid, carbohydrate or any combination thereof, for example. Moreover, the AR may be either homogeneous wherein the AR molecules that are delivered to the target tissue are composed of identical AR molecules or a heterogeneous combination of AR molecules that recognize different binding sites on the analyte of interest. Furthermore, the analyte binding moiety of the AR may be mono-specific requiring the analyte to possess two or more identical epitopes or binding sites for cross-linking. Alternatively, the binding moiety may be bi-specific, poly-specific, or a mixed population of ARs allowing crosslinking through binding of similar and dissimilar epitopes or binding sites on the analyte of interest. Importantly, those skilled in the art will readily appreciate that the AR repertoire for each analyte can be constructed in a myriad of ways to optimize performance of the assay and to recognize analyte variants or isotypes.
[0076] There are a multitude of available in vitro techniques and recombinant methods that the artisan can employ to construct an Fc.epsilon.Rl receptor binding domain with an analyte binding moiety to form the AR as contemplated pursuant to the present invention, examples of which include hybridoma techniques, covalent conjugation, non-covalent binding, genetic engineering methods, recombinant techniques, or a combination thereof. Genetic engineering methods, for example, employ recombinant DNA methods to form proteins not normally produced by cells and provide an avenue for those proteins to be expressed in large quantities in a variety of expression vectors. For purposes of the present invention, the terms “in vitro construction” and “constructing in vitro” are intended to have the ordinary and customary meanings attributed to them by those skilled in the art. Similarly, the terms “recombinant construction” and “constructing recombinantly” are also intended to have the ordinary and customary meanings attributed to them by those skilled in the art.
[0077] Additionally, the artisan will take note that the Fc.epsilon.Rl receptor binding domain of human IgE comprising C2, C3, and C4 regions is sufficient for binding to alpha chain of the high affinity receptor (Fc-epsilon-R1) mast cell receptor. Studies with human IgE Fc domains have demonstrated that the smallest fragment that shows Fc-epsilon-R1 binding activity spans amino acids 329-547 and lacks the entire C2 domain. Moreover, studies have also shown that Fc fragment 315-547 is an S-S-linked dimer and that Fc fragment 329-547 that forms a dimer without S-S bonding, both bind the Fc-epsilon-R1 receptor with high affinity. The presence of N-linked sugars did not appear to be necessary for high affinity binding (Basu et al., The Journal of Biological Chemistry 268:13118 (1993)). The Fc.epsilon.Rl receptor binding domain can be purified directly from proteolytic digest of IgE molecules or can be produced by recombinant methods. The Fc.epsilon.Rl receptor binding domain from IgE has been recombinantly produced and purified from a variety of expression vectors (Kamiya et al., Exp. Med. 180:297 (1996); Liu et al., Proc. Natl. Acad. Sci. USA 81:5369 (1984)). As such, construction of the Fc.epsilon.Rl receptor domain of the AR will typically include the C2, C3, and C4 regions. In an alternative embodiment, such construction will include at least the C3 and C4 regions, and at a minimum, amino acids 329-547.
[0078] Those familiar with the art will appreciate that Fc.epsilon.Rl receptor binding domains derived or modified from any species, recombinantly synthesized or otherwise constructed will generally be sufficient to bind to mammalian F1.epsilon.R1 mast cell receptors. Accordingly, the methods and kit of present invention may be readily modified to test any mammal that possesses mast cells, examples of which include humans, dogs, cats, horses, farm animals, and livestock provided that the design and adaption of the methods and kit for each species integrate proper consideration regarding sensitivity, cross reactivity, etc. as between the different species for which a test is being developed. To that end, there are various humanization protocols available to address those situations where a moiety derived from another species may be modified for use in humans.
[0079] As mentioned above, one embodiment of the present invention comprises antibodies or antibody fragments that may be fashioned for use as the additional moiety. Those skilled in the art will readily appreciate that antibodies available in the art as contemplated for use with the present invention may be fashioned using standardized tools and techniques readily available for designing and developing research products, diagnostics and therapies. Generally, antibody molecules may be engineered to include monospecific, bispecific or tri-specific antigen binding domains as well as being further manipulated to render non-humanized, humanized or chimeric molecules. Whether utilized in whole or in part, such antibodies exemplify suitable analyte binding moieties that may be fashioned for the AR molecule. Over the past three decades antibodies have been dissected into smaller antigen binding fragments, initially by proteolysis and later by genetic engineering to produce mono or multivalent fragments that include, but not limited to, Fab, F(ab).sub.2, Monospecific Fab.sub.2, Bispecific Fab.sub.2, Trispecific Fab.sub.3 fragments. Antibody fragments (e.g., single chain variable fragments (scFv), and V.sub.HH domains) and artificial affinity binders (e.g., Affibodies, Monobodies, DARPins, etc.) have been created and are developed by screening large gene libraries of potential binders with various panning technologies. Such antibody fragments also make suitable analyte binding moieties that may be fashioned for the AR molecule.
[0080] Additionally, various technologies have afforded the development of numerous protein scaffolds with unique affinity interaction domains that bind target epitopes (Groff et al, Biotechnology Advances 33:1787 (2015); Marx, V. Nature Methods 10(9):829 (2013). Antibody-antigen binding diversity can also be acquired by unorthodox mechanisms that represents a third layer of diversification of immune repertoires beyond the variability introduced by recombination and mutagenesis (as described by Kanyavuz et al., Nature Reviews, Immunology 19:355 DOI:org/10.1038/s41577-019-0126-7 (2019)). Furthermore, additional specificities may be acquired by using phage display technology which has been used extensively to generate large libraries of antibody fragments by exploiting the capability of bacteriophage to express and display biologically functional protein molecule on its surface. Combinatorial libraries of antibodies have been generated in bacteriophage lambda expression systems which may be screened as bacteriophage plaques or as colonies of lysogens (Marks et. al., Biotechnology 10 779 (1992). Accordingly, any of the foregoing or derivatives thereof would constitute suitable analyte binding moieties for constructing the affinity reagent of the present invention.
[0081] Aptamers are similar to antibodies in that they can bind to proteins and modulate their function, and they are often referred to as chemical antibodies due to their synthetic production (Groff et al, Biotechnology Advances 33:1787 (2015); Marx, V. Nature Methods 10:829 (2013). Aptamers also make suitable analyte binding moieties for the AR molecule. Aptamers are short, single-stranded DNA or RNA oligonucleotides that can bind to their targets with high specificity and affinity through van der Waals forces, hydrogen bonding, salt bridges, and hydrophobic and other electrostatic interactions. Aptamers have the ability to fold into complex and stable three-dimensional shapes, which allows them to fold within or around their targets. DNA aptamers have greater chemical stability, while RNA aptamers produce more structural shapes due to their greater flexibility. Aptamers have been identified against targets that include small organic and inorganic molecules, such as dyes, nucleotides, amino acids, and drugs; biopolymers, such as peptides, proteins, and polysaccharides; ions; phospholipids; nucleic acids; viruses; bacteria; cell fragments; and whole cells. Accordingly, aptamers are further examples of analyte binding moieties that can be incorporated into the AR of the present invention.
[0082] There are a number of molecules that can participate in molecular binding and include proteins, nucleic acids, carbohydrates, lipids, and small organic molecules such as drugs. Types of complexes that form as a result of molecular binding include: protein-protein, protein-DNA, protein-hormone, and protein-drug. These include proteins that form stable complexes with other molecules. Cellular receptors are structures composed of protein that receive and transduce signals which are typically integrated into biological systems. These signals are typically chemical messengers, which bind to a receptor and cause some form of cellular/tissue response, e.g. a change in the electrical activity of a cell. A molecule that binds to a receptor is typically referred to as a ligand. Ligands can be a proteins or peptides, or other small molecules such as hormones, pharmaceutical drugs, toxins, or parts of the outside of a virus or microbe. Receptors and receptor ligands are both additional examples of analyte binding moieties that can be incorporated into AR molecules by those skilled in the art.
[0083] Circulating nucleic acids (CNA) have been reported in a number of clinical disorders like cancer, stroke, trauma, myocardial infarction, autoimmune disorders, and pregnancy-associated complications. The term CNAs refers to segments of genomic, mitochondrial or viral DNA, RNA and microRNA (miRNA) found in the bloodstream. Scientists have now discovered disease-specific genetic aberrations, such as mutations, microsatellite alterations, epigenetic modulations (including aberrant methylation), as well as viral DNA/RNA from nucleic acids in plasma and serum. CNA has received special attention because of its potential application as a non-invasive, rapid and sensitive tool for molecular diagnosis and monitoring of acute pathologies and the prenatal diagnosis of fetal genetic diseases (Suraj et al., Biomedical Reports 6:8 (2017). Adapting complimentary sequences of CNAs associated with diseases and disease states will provide yet another analyte binding moiety for construction of the AR of the present invention.
[0084] The analyte binding moieties described above can be fashioned and incorporated into the AR of the present invention by a number of methods well known in the art including covalent conjugation and non-covalent binding methods. Additionally, molecular techniques can be employed to construct and produce AR molecules as fusion proteins wherein the nucleic acid sequence of both the Fc.epsilon.Rl receptor binding domain and the analyte binding moiety are recombinantly joined and inserted into an expression vector. Fusion proteins or chimeric proteins (literally, made of parts from different sources) are proteins created through the joining of two or more genes that originally coded for separate proteins. Translation of this fusion gene results in a single or multiple polypeptides with functional properties derived from each of the original proteins. Recombinant fusion or chimeric proteins are created artificially by recombinant DNA technology for use in biological research or therapeutics. The terms chimeric or chimera usually designate hybrid proteins made of polypeptides having different functions or physicochemical patterns. Accordingly, both fusion proteins and other chimeric molecules also provide suitable choices to be incorporated as the additional binding moiety of the present invention.
[0085] Additionally, monoclonal and polyclonal IgG, IgM, IgA, and IgD antibodies can of course be sequenced and produced recombinantly, providing an easy pathway for isotype switching from Fc gamma, Fc mu, Fc alpha, and Fc delta to Fc epsilon, respectively. AR molecules can also be generated by in vitro methods using hybridoma technologies, essentially producing IgE molecules possessing the Fc.epsilon.Rl binding domain and F(ab).sub.2 region specific to the analyte of interest. These techniques are all well known in the art. Pathogens sometimes recognize host cells through receptor binding proteins such as CD4, CD48, ACE2 or TLR2. These receptors and other pathogen components may be used as the analyte binding moiety on affinity reagents in order to assess disease states. For example, the number of CD4 cells are routinely measured in patients with HIV as an indicator of advanced HIV disease.
[0086] In light of the foregoing, it will be readily appreciated by the artisan that one may readily modify and substitute one AR for another AR to create a different kit embodiment. Accordingly, it will be further appreciated that standardized manufacturing SOPs embracing the present invention can be quickly modified to allow the rapid production of a new AR molecules and novel bioassay kits thereby providing an extensive platform for diagnostic testing. New AR-bioassays can be carried through similar consistent product development cycles to yield rapid and large-scale deployment in foreseen and unforeseen healthcare emergencies. Patient implementation programs can be standardized as well, thereby reducing the uncertainty typically associated with the introduction of new methods.
Contacting Mast Cells With AR
[0087] As mentioned above, the methods and kit of the present invention further include the step of sensitizing mast cells by contacting the affinity reagent in vivo with a plurality of endogenous mammalian mast cells via delivery of the AR to mast cells that are present within a target tissue of a living mammalian subject to elicit and enable a multivalent binding response when the analyte of interest is or becomes present. It will be appreciated by the artisan that the aforementioned delivery also contemplates selecting within a test subject an anatomical area and type of tissue having a concentration of endogenous mast cells ranging from about 125 to 20,000 mast cells per cubic millimeter for the species of mammal upon which the delivery is to be performed. For example, in most mammals, the skin is an acceptable target tissue and more specifically, the upper dermal layer of the skin. In humans, delivery sites include the skin of the upper arm or forearm and lower body extremities at approximately the same depth and manner as a typical allergy prick test or tuberculin skin test, the particulars of which are well known and established in the clinical arts. Notwithstanding the foregoing, any tissue wherein the affinity reagent is exposed to from about 125 to 20,000 mast cells per cubic millimeter would generally be suitable provided that the method for detecting the multivalent binding response can be effectively utilized and is reasonably practical for clinical purposes.
[0088] Physical delivery of the affinity reagent may be performed by any suitable means including but not limited to a needle prick, intradermal injection, solid needle, hollow needle, micro-needle, patch method or a needleless system. In one simple embodiment, the AR is delivered into the skin by a needle. The needle may be either solid or hollow. The AR is injected into the skin by hollow needle or the needle can be solid and coated with an AR coating, wherein the AR is released from the needle when the needle makes contact with bodily fluids.
[0089] For example,
[0090] Where applicable, the tests along with positive and negative controls can be applied together or separately and, in any order, and at any time. For example, when using commercially available or custom designed allergy skin test applicators, a single applicator having a single prong that includes one or more spike members to penetrate the skin can be used to apply the test as well as a positive control and a negative control sequentially as needed. Similarly, a dual prong applicator, with each prong having one or more spike members, can be used to apply the test and a control in parallel. In addition, a multiprong applicator with each prong having one or more spike members can be used to apply a variety of tests and controls in parallel. Each prong can also have a touch activator to reduce pain which is well known to practitioners who employ allergy testing devices in their practice.
[0091] Multiplex use of the invention provides a means of detecting an active disease signature, thereby preventing unnecessary testing and treatment. For example, instead of detecting antibodies from patients that have been previously exposed to HBV and have cleared the virus, this invention can be used to detect viral particles, DNA, RNA, and other constituents of the virus simultaneously without taking multiple samples in order to demonstrate the presence of active disease. (Liang, T.J., Hepatology 49(5 Suppl):S13-S21 (2009); Gerlich, W.H., Virology Journal 10(239) http://www.virologyj.com/content/10/⅟239 (2013); Venkatakrishnan et al., Annu. Rev. Virol. 3:451 (2016); Jiang et al., Journal of Virology 90(7):3330 (2016); Hu et al., Viruses 9 56 (2017)).
[0092] As shown below, in Table 1 in information from the Center for Disease Control, Atlanta, Ga, detecting both viral particles (HBsAg) and antibodies (HbsAb and HBcAb) at the same time using an AR applied in multiplex can provide a definitive diagnosis and treatment plan for hepatitis B patients which is not currently commercially available (Diepolder et al., Gastroenterology 116 650 (1999); Kimura et al., Journal of Clinical Micro. 41(5):1901 (2003); Tong, S., Int. J. Med. Sci. 2(1):2 (2005); y et al., Journal of Clinical Microbiology 44:2321 (2006); Gallagher et al., Virology 502:176 (2017)).
TABLE-US-00001 Hepatitis B Test Panel Interpretation and Action Needed HBsAg HBsAb HBcAb Not Immune - Get Vaccinated Neg Neg Neg Immune - Protected Neg Pos Pos Immune - Protected Neg Pos Neg Infected - Need More Testing Pos Neg Pos Could be Infected - Need More Testing Neg Neg Pos
[0093] As discussed, the AR can be delivered into an area of the skin in situ (or other applicable mast cell rich areas), singularly or in multiplex, by needle prick or intradermal injection or patch method, including needleless systems. Recent advances in intradermal delivery needle design have reduced the pain associated with injections. Smaller gauge and sharper needles reduce tissue damage and therefore decrease the amount of inflammatory mediators released (for reference see Zehrung et al., “Intradermal Delivery of Vaccines: A review of the literature and the potential for development for use in low- and middle-income countries” Program for Appropriate Technology in Health (PATH) Aug. 27, 2009; Ita, K., Pharmaceutics 7:90 (2015); Larraneta et al., Materials Science and Engineering R 104:1 (2016); Martin et al., Safety 3:25 D01:10.3390/safety3040025 (2017); Shrestha et al., Scientific Reports 8:13749 DOI:10.1038/s41598-018-32026-9 (2018).
[0094] Microneedles are typically less than 0.2 mm in width and less than 2 mm in length. They are usually fabricated from silicon, plastic or metal and may be hollow for delivery or sampling of substances through a lumen or the needles may be solid and coated with substances. By selecting an appropriate needle length, the depth of penetration of the microneedle can be controlled to avoid the peripheral nerve net of the skin and reduce or eliminate the sensation of pain. The extremely small diameter of the microneedle and its sharpness also contribute to reduced sensation during the injection.
[0095] The advantages of needle-free injection devices have been recognized for some time. Some of the advantages of needle-free devices and methods include the absence of a needle which can intimidate a patient and also present a hazard to healthcare workers. The injection jet generated by a needle-free device is generally smaller in diameter than a hypodermic needle and thus, in certain instances, a needle-free injection is less painful than an injection provided by a hypodermic needle device. Because of these and other advantages of needle-free injection many variations of pneumatic, electronic or spring activated needle-free injection devices have been designed to provide a single injection, or alternatively a series of injections to one or more patients. Most known needle-free injection devices operate by driving the injectable fluid through a nozzle with a powered piston to create a fine, high-pressure jet of fluid that penetrates the skin.
[0096] Additionally, there are pre-fillable delivery devices available for administering intradermal injections which include a pre-fillable container adapted to store a substance. The substance is expelled through a needle cannula having a forward tip adapted to administer the intradermal injection. A limiter surrounds the needle cannula having a generally flat skin engaging surface extending in a plane generally perpendicular to the needle cannula. An insert is centrally located within the skin engaging surface and is pierceable by the forward tip. A sleeve with a first end and a second end surrounds the pre-fillable container. The limiter is inserted through the first end and the second end is affixed to a depressible plunger for expelling the substance from the container. The limiter is movable between a first position and a second position thereby exposing the forward tip and is selectively movable between a third position and a fourth position thereby concealing the forward tip. These and other devices are available for pain-free and safe application of substances intradermally and the described methods and others are well known to those knowledgeable in the art.
[0097] It will be appreciated by the artisan that the AR can be applied alone or together with any of several well known pharmaceutical carriers suitable as a vehicle for adding constituents that may be used to influence AR binding kinetics, promote chemotaxis, or elicit substances from near-by cells that can be evaluated and measured. The vehicle can be added simultaneously with the AR or separately.
[0098] It will be further appreciated that there are a number of techniques currently available in the clinical laboratory and medical manufacturing arts for rendering a suitable vehicle for carrying the AR into the target tissue or for depositing it onto the delivery end of the device until the device is utilized to deliver the AR into the target tissue. In one embodiment, AR may be suspended in a liquid medium of suitable viscosity that is used, for example, to deposit the AR onto the delivery end of the device such as the end of a solid needle whereupon it is subsequently dried to render an AR layer deposited on the device until used on a subject. Alternatively, the AR may, for example, be delivered to the target tissue in a liquid medium that is delivered by hollow needle or needless system and injected into the target tissue. The selection of such medium should integrate factors such as storage conditions and shelf life. Various suitable substances are commercially available for use as coating mediums or vehicles by which to carry or deposit the AR provided that they are properly selected for the AR to be delivered, the particulars of which are further addressed below.
[0099] The vehicle or medium for applying, transporting, storing, using, etc. the AR may be dependent on the AR construct itself. The vehicle may be liquid or dry and may include buffers, salts, protease or nuclease inhibitors, bacteriostats, chemotactic agents, detergents, stabilizing reagents, etc., or may be composed of proprietary reagents provided by a 3.sup.rd party supplier. The list of additives is broad and well known to those practiced in the art. The AR is delivered in vivo and therefore resides in bodily fluids once delivered. It optimally performs within the physiologic pH range of 6 -9.
[0100] As mentioned above, the AR can be applied singularly without additional constituents or in a vehicle suitable for adding constituents that may be added to influence AR binding kinetics, promote chemotaxis, or may elicit substances from nearby cells that can be measured as contemplated by the methods of present invention. Moreover, the vehicle for delivery may be optimized to perturb the equilibrium between endogenous, preexisting IgE that is bound to mast cells and the newly available AR molecules in order to provide a method by which to increase the concentration of AR molecules on the mast cell. Equilibrium shifting towards AR binding may be accomplished by a variety of methods that are well known in the art. For example, the addition of excess AR, changes in pH, changes in salt concentration, and the addition of detergents may be employed for equilibrium shifting.
[0101] The delivery step of the methods of the present invention may also include a vehicle composed of a chemotactic or other constitutes that draw cells of interest to the target tissue. While the multivalent binding response can, in theory, be localized to many body regions, the most common site will consist of AR-sensitized mast cells located in interstitial fluid of the target tissue. This provides an opportunity to detect cells expressing specific cell surface markers such as distinct immune cell populations. Detection or measurement of receptor binding ligands such as LAG3 cleavage, CD8/Treg ratios, cytokines, chemokines can be used to select and manage patients on immunotherapy. Chemotactic factors can be introduced concurrently or synchronously with the AR to attract cells of interest to the target tissue.
[0102] The delivery vehicle may also allow the methods of the present invention to elicit and detect analytes secreted or released from surrounding cells. Delivery of the AR may optionally include a vehicle composed of constituents that cause release of analytes from neighboring cells, such as T-lymphocytes, known to be in close physical proximity to endogenous mast cells. The AR specific for the analyte released from the T-cells, would detect their presence. For example, Interferon-γ release assays (IGRA) are in vitro tests used in the diagnosis of some infectious diseases, especially tuberculosis. Interferon-γ (IFN-γ) release assays rely on the fact that T-lymphocytes will release IFN-γ when exposed to specific Mycobacterium tuberculosis (MTB) antigens. T-cells from most persons that have been infected with MTB will release INF-gamma when a patient’s sample is mixed with certain MTB antigens. Accordingly, MTB antigens could be delivered to the target tissue in addition to an AR specific for INF-gamma. MTB antigens included in the AR delivery medium would stimulate release of INF-gamma from adjacent pre-exposed T-cells. The INF-gamma could then be detected using a method wherein the AR binds to INF-gamma triggering mast cell degranulation. The presence of INF-gamma correlates to MTB infection. Additionally, the methods and kit of the present invention reduces the complexity of a test that is similar to the IGRA test but eliminates the need to remove a patient’s sample of blood and the necessity for performing the test on a benchtop under tissue culture conditions.
[0103] Further yet, there are numerous molecules released from mast cells during and after degranulation, any of which can be used to measure a response; in solo, in combination, or through enhanced secondary reactions and cascades. Accordingly, in another further embodiment of the invention, a probe placed in a target tissue may be retained there as a physicochemical detector. Biologically sensitive detectors may be created using biological engineering techniques known in the art. A transducer or the detector element deployed in the target tissue could be easily designed to transform a signal or change occurring as a function which occurs as a result of the multivalent binding response into a second signal for easy measurement and quantification. Alternatively, the delivery device may be used to deliver the AR to the target tissue while remaining positioned thereafter to operate as the detector as discussed above.
[0104] Delivery of active molecules into or via the skin (transdermal delivery) can offer significant advantages over the more conventional routes of delivery. The use of chemical penetration enhancers such as dimethylsulfoxide that interact with skin lipids to facilitate the transport of the AR across membranes into the dermal layer of the skin.
[0105] Dyes including but not limited to methylene blue, fluorescein and fluorescent peptides that can be detected visually or by device in the dermal layer of the skin may be used to enhance detection/assessment of the multivalent binding response of the present invention. For example, dyes such as methylene blue can be included in the application vehicle to provide an indicator that the affinity reagent was applied correctly to the dermal layer of the skin. There are many adaptations that can be envisioned where adding dyes or fluorescent compounds and other reagents to the application vehicle or AR that are visible by eye or detectable device could be utilized. Attaching a dye to the AR such a fluorescein would allow the monitoring of a positive reaction by way of an external fluorescent detector. Monitoring dyes can be used to determine parameters such as density of mast cells at the application site, the number of AR molecules occupying mast Fc.epsilon.Rl receptors and the rate of the reaction. Measurement of these parameters and others may be used singularly or combination to quantitatively or semi-quantitively measure analytes using the current invention.
Assessing the Multivalent Binding Response
[0106] The methods of the present invention further include performing an assessment of the multivalent binding response to determine the presence, absence, or amount of the analyte of interest. It will be appreciated that assessment of such a binding response presupposes providing adequate time for a binding response to occur. Binding response times are dependent upon the purpose or intended reason for performing the method as well as the concentration of the analyte in the target tissue at the time the AR is delivered.
[0107] The multivalent binding response of the present invention is a function of the multivalent binding discussed above and encompasses crosslinking of Fc.epsilon.Rl receptors across the mast cell membrane along with degranulation of the mast cell and any additional and secondary physiological changes to the target tissue that directly or indirectly result from such degranulation. Assessment of the multivalent binding response, if and when it that occurs, may be accomplished by various qualitative and quantitative approaches including a basic clinical evaluation of the target tissue at or near the delivery site by visual inspection to identify at least one morphological change in the target tissue that is associated with the binding response (e.g. a wheal and flare dermal reaction). Alternatively, a device capable of measuring at least one changes in the target tissue that is associated with the binding response may be employed to evaluate the tissue.
[0108] Since there are numerous molecules released from mast cells during degranulation, any of these molecules may be theoretically used to measure a response whether in solo, in combination, or through enhanced secondary reactions and cascades. As such, performing an assessment of the binding response may be accomplished by measuring a biochemical change to the target tissue such as a mast cell degranulation substance released by mast cell degranulation or by measuring an endogenous substance released in response to a mast cell degranulation substance. Alternatively, a wheal and flare response is a physiological tissue change that is visible and easily measured. Devices may also be employed to measure parameters associated with a wheal and flare reaction such as width, density or intensity, tensile changes in skin, or the heat generated from a positive reaction. Moreover, enhancing technologies such as secondary reagents and conjugates thereof provides an additional secondary mode of signal enhancement and measurement.
[0109] A sensor or biosensor may also be employed to assess the binding response by way of transducers or similar apparatus. As mentioned earlier in the disclosure, a probe may be placed in proximity of the AR after delivery to a target tissue and retained there as a physicochemical detector. Biologically sensitive elements may be created using techniques generally known in the arts of biomedical engineering and related fields. A transducer or the detector element deployed in the target tissue could be easily designed to transform a signal or change occurring as a result of mast cell degranulation. Three major techniques are based on their transduction mechanism, namely electrochemical, optical, and acoustic-based sensors. Electrochemical sensors are further divided into impedimetric, amperometric, potentiometric, and conductance-based sensors. Optical sensors include fluorescence- and chemiluminescence-based biosensors, surface-enhanced Raman spectroscopy (SERS), and surface plasmon resonance (SPR)-based sensors. Acoustic wave-based sensors have two subcategories, quartz crystal microbalance (QCM)-based and surface acoustic wave (SAW)-based sensors. The sensor reader device with the associated electronics or signal processors would lead to a display of the results in a user-friendly way applicable to connectivity networks, smartphones and similar displays and communication devices as well as user interface mechanisms. Sensor technology is a rapidly developing field and there are many variations known to those experienced in the art that could be employed. The applicator can both introduce the AR to mast cells and can act as the detector element providing a single step assay and result (Upasham et al, Advanced Health Care Technologies 4:1 https://doi.org/10.2147/AHCT.S138543 (2018); Turner et al., Oxford, UK: Oxford University Press. p770 (1987); Banica et al., UK: John Wiley & Sons p576 ISBN 9781118354230 (2012); Dincer et al., Advanced Materials 31(30) doi:10.1002/adma.201806739 (2019).
[0110] In situations where the method is being used to test a population or group for suspected exposure to a substance such as a pathogen, toxin, etc., the expected binding response time will generally be either immediate or will occur within a few seconds provided that the test subject has been exposed to the substance and that the analyte selected to hallmark that exposure is actively circulating within the test subject. Generally, if the analyte is actively circulating, it can be expected to be found in the interstitial fluid at the AR delivery site within the target tissue. In contrast to the foregoing, there are situations where the method is being used as a sentinel test to perform an ongoing monitoring function to detect a future exposure as further described in the section below. As expected, a multivalent binding response will not occur until exposure to a substance has taken place and the analyte selected to hallmark such an exposure has had time to circulate to the target tissue delivery site. In short, performing an assessment of the multivalent binding response is secondary to providing a sufficient opportunity for a detectable multivalent binding response to occur in the test subject. Where the binding response is immediate, assessment may, in turn, be done immediately.
[0111] It will be further appreciated that the multivalent binding response time may also vary depending upon the analyte of interest to be detected and/or the character of the analyte binding moiety utilized for the reagent construct. Binding response times may be adjusted based upon the construction and concentration of the AR, the vehicle used for contacting the mast cells with AR, and the detection method. Notwithstanding the foregoing, adequate detection times may be generally categorized as follows: a rapid test method would range from immediately (T.sup.1= 0) up to about two hours (T.sup.2 = 2 hrs); a sensitive test method would range from two hours (T.sup.1 = 2 hrs) to about 48 hrs (T.sup.2 = 48 hrs); and the AR-sentinel test would have an expiration date of ranging from about 12 weeks to about 6 months from the time of delivery of the AR to the target tissue.
[0112] In the example sections below, binding responses and the resulting tissue change ranged from 30 sec to 8 hours depending on the concentration of HBsAg in the circulatory system. As an example of a sensitive method, mice challenged with 20 ug or more of HBsAg were positive after 20 min. However, mice challenged with 10 ug could only be detected after 8 hours. Unlike all blood assays, a bioassay performed by the methods of the present invention affords continuous exposure of the assay to the analyte as it circulates throughout the body. This continuous exposure enables very low detection levels and imparts significant sensitivity to the assay. Accordingly, it will be appreciated that bioassays incorporating the methods of the present invention are generally not limited by the amount of analyte in a sample as are, for instance, in vitro assays.
[0113] In a further embodiment, the methods and kit of the present invention may be designed or adapted to monitor immunotherapy reagents, immune function, disease states and effects of treatment and therapy. While the AR may be delivered to a target tissue residing in various regions of the test recipient’s body, optimal delivery sites will include those that are populated with endogenous mast cells that are located within the interstitial fluid of the target tissue. This provides an opportunity to detect cells expressing specific cell surface markers such as distinct immune cell populations. Detection or measurement of receptor binding ligands such as LAG3 cleavage, CD8/Treg ratios, cytokines, chemokines can be used to select and manage patients on immunotherapy. Chemotactic factors can be introduced concurrently or synchronously with the AR to attract cells of interest. As mentioned above, the delivery medium can include a chemotactic or other constitutes that draw cells of interest into the proximity of the AR once it has been delivered.
[0114] Further to the aforementioned, another embodiment of the invention provides a tool for exploring interstitial fluid by affording a methodology for collecting critical information present within interstitial and lymph fluid as well as providing for the development of specialized assays or treatments. It is almost impossible to collect significant amounts of interstitial fluid. The time-consuming nature of currently available procedures for removing and testing interstitial and lymph fluid introduces risks to patients and the need for medical expertise and specialized equipment limits the use of these procedures to basic research. Interstitial fluid and lymph contain unique biomarkers useful for detecting and diagnosing diseases and disease states, monitoring treatment and recovery, and for tracking patient well-being. The present invention can be adapted to ideally accomplish those objectives by fashioning the analyte binding moiety from an appropriate biomarker.
[0115] In a further embodiment, the bioassay of the present invention can be used for point-of-care and self-testing where the AR-bioassay is applied to a localized area of the skin (or other applicable areas) as a homogeneous assay, singularly or in multiplex, by needle prick or any intradermal injection method, including needleless systems. A blood sample is not required, and results can be read in a few minutes visually, by device or sensor.
[0116] In yet another embodiment, the present invention may be used for immediate measurement of troponin, providing a real-time measurement that would allow first-responders to provide patients suffering a potential myocardial infarction with immediate and life-saving care on-site or in transit. Such a troponin test can be designed for visual interpretation or for interpretation by a biosensor or other detection modality. A further aspect of this embodiment may include a transmitter possessing a delivery needle and/or sensor, separate or one in the same, that is applied to a patient. Test results corresponding to the presence or amount of troponin present in a subject are transmitted in real-time via the transmitter to a communication device such as a cell phone thereby providing a first responder or a remote medical staff with immediate results and the ability to monitor results should they change with time. Many tests considered STAT where immediate results could impact a patient’s well-being could be adapted to the present invention in such a manner. The use of sensors and other means for detecting the multivalent binding response is discussed in more detail below.
[0117] An additional embodiment of the invention pretains to a test for M. tuberculosis (Mtb), wherein the test directly detects Mtb activated T cells. An affinity reagent is constructed that contains at least one Fc.epsilon.Rl receptor binding domain and at least one MHC Class I molecule. The MHC molecule portion of the affinity reagent is paired with an Mtb antigen, forming a complex. The MHC molecule can be complexed with any suitable M. tuberculosis antigen. Preferably, the antigens are choosen from the secretory ESAT-6 protein, 10-kDa culture filtrate protein (CFP-10), or any of the proteins present in PPD preparations (Hodapp, T. et al., Eur Respir J 2012; 40: 152-160. doi: 10.1183/09031936.00175611; Urdahl, K.B. et al., Nature Review, VOL 4, No 3, MAY 2011). The affinity reagent is applied to a subject by any of the methods described herein. The affinity reagent binds to T cell receptors on T cells that have been activated by the presence of Mtb resulting in mast cell degranulation and a wheal and flare reaction that corresponds to the presence of Mtb.
[0118] Another embodimenet of the invention is a test to detect superantigens. Superantigens are implicated in T cell driven cytokine release syndrome (CRS) accompanied by multiple organ dysfunction (MOD). Superantigens bind directly to TCR and MHC-II receptors outside the conventional antigen-binding site and stimulate large number of T cells. Superantigens are implicated in Multisystem Inflammatory Syndrome in Children (MIS-C), COVID-19 cytokine storm syndrome, sepsis, toxic shock syndrome, chimeric antigen receptor T-cell (CART) therapy, administration of certain T cell agonistic antibodies and immune check point inhibitors.
[0119] An affinity reagent is constructed that contains at least one Fc.epsilon.Rl receptor binding domain and at least one MHC Class II molecule. A second affinity reagent is constructed that has at least one Fc.epsilon.Rl receptor binding domain and at least one T Cell Receptor (TCR) molecule. The paired affinity reagents are applied to the skin. The presence of superantigens will result in the cross-linking the MHC and TCR moieties of the affinity reagents leading to mast cell degranulation followed by a measurable wheal and flare reaction.
Bioassay Kit
[0120] Minimally, the bioassay kit of the present invention includes the AR and a device for contacting mast cells in vivo with the AR. Use of the kit will be obvious to those skilled in the art upon reading the detailed description for the methods of the present invention. The kit may optionally include a sensor or other device for determining the presence, absence, or amount of an analyte of interest. The device utilized for contacting mast cells with the AR component of the kit can be any suitable device as described herein. The AR component of the kit may include any affinity reagents constructed in accordance with the methods set forth herein. The AR component will be pre-coated onto or pre-loaded into the delivery device as also described above. It is contemplated herein that in some cases, the AR may in be included in the kit in a lyophilized form for reconstitution with a suitable sterile carrier medium followed by subsequent application to the delivery end of the device just prior to initiating delivery of the AR to the target tissue of the subject. Optionally, the kit may include a device for detecting the multivalent binding response as described above.
Sentinel Monitoring
[0121] The present invention also provides a method for the sentinel monitoring of an individual, wherein the affinity reagent is applied to the subject before the subject has been exposed to the analyte of interest or while the analyte is at undetectable concentrations. In this embodiment, even though the AR is delivered into the subject and reacts with mast cells in situ thereby sensitizing them to the analyte, crosslinking will not occur in the absence of the analyte. The mast cells that have become sensitized by the AR will stay sensitized for an extended period of time. Resident mast cells are long-lived cells that can survive for up to 12 weeks in the skin of Wistar rats or longer (Kiernan J.A, J Anat 128:225-238 (1979)). If the sensitized mast cells are subsequently exposed to the analyte of interest over the following days, weeks or months, the crosslinking that induces degranulation of the sensitized mast cells and other additional physiological changes to the target tissue resulting therefrom will then occur constituting the multivalent binding response which can be assessed to determine the presence or amount of the analyte of interest. For example, an AR molecule specific for the Ebola virus is delivered to a healthcare worker in the skin on the forearm before he or she enters the field to treat patients. If the healthcare worker is exposed at any time to Ebola, the Ebola virus or particles from the virus will bind to the sensitized mast cells eliciting a multivalent binding response which, in turn, may be assessed by evaluating the tissue at the delivery site where a wheal and flare reaction appears to alert the worker.
[0122] Modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific examples described below are offered only to illustrate some embodiments of the present invention, such invention intended to be limited only by way of the appended claims, taken in conjunction with the full scope of equivalents to which such claims are entitled.
EXAMPLES
Example 1
CD-1 Mice
[0123] Female CD-1 mice, retired breeders (-30 grams) were purchased from Charles River, 251 Ballardvale St., Wilmington, MA 01887. The mouse has played a historically important role in the dissection of the mechanisms of anaphylaxis in humans. Antigen-induced cross-linking of mast cell-associated IgE that leads to mast cell degranulation with release of mediators is highly conserved in mammals. Those knowledgeable in the art, would understand that the anaphylactic-based examples as encompassed in this invention and carried out in mice would translate to humans and other mammals (Ovary Z., Arerugi 43(12):1375 (1994)).
Preparation of the HBsAg Affinity Reagent (HBsAg-AR)
[0124] The AR was constructed by recombinantly switching the Fc gamma region of an anti-HBsAg IgG antibody to that of an Fc epsilon region. Specifically, the HBsAg—AR was prepared from Anit-HBsAg 5C3 mouse IgG2a Fc silent antibody to contain the F1.epsilon.R1 receptor domain by switching the IgG isotype to IgE (mouse) and was carried out by Absolute Antibody Ltd, Wilton Centre, Redcar, Cleveland TS10 4RF, UK by proprietary methods. Anti-HBsAg 5C3 IgE was supplied in PBS with 0.2% Procline 300 and recognizes an antigenic determinant on HBsAg (Wands et al., Proc. Natl. Acad. Sci. USA 79:1277 (1982); Wands et al., Proc. Natl. Acad. Sci. USA 81:2237 (1984); Ben-Porath et al., J. Clin. Invest. 76:1338 (1985)).
Hepatitis B Surface Antigen (HBsAg)
[0125] Recombinant full length HBsAg antigen (Subtype adw) produced in Saccharomyces cerevisiae (containing plasmid pCGA7) was purchased from Fitzgerald Industries International, 30 Sudbury Rd., Suite 1A North, Action, MA 01720. HBsAg was supplied in 0.05M phosphate, 0.2M NaCl, pH 7.2. Expression of HBsAg in yeast results in spherical and particles identical in size and shape to those particles (20-22 nm) found in HBV-infected patient’s sera (Miyanohara et al., Proc. Natl. Acad. Sci. USA Vol 80, pp. 1-5, January 1983). HBsAg itself has a molecular mass of ~24 kDa and the glycosylated form has a molecular mass of 27 kDa while the 22 nm particle has a molecular mass of 2.18 MDa (Ono et al., Nucleic Acids Research 11(6):1747 (1983); Miyanohara et al., Proc. Natl. Acad. Sci. USA 80:1 (1983).; Gilbert et al.," Proc. Natl. Acad. Sci. USA 102(41):14783 (2005)).
Avertin Anesthetic
[0126] A stock solution was prepared from 99% 2,2,2-tribromoethanol (Acros Organics) and tert-amyl alcohol, reagent grade (Fisher Scientific). Ten ml tert-amyl alcohol was added to 10 g Avertin (2,2,2-tribromoethanol). The bottle in which the Avertin arrives is convenient for mixing. Drop in a stir bar and stir on a magnetic stirrer until Avertin is completely dissolved. This step took approximately 2 hours. The stock was kept in a dark bottle and tightly capped. It was stored at room temp. It should be noted that the Avertin stock is photosensitive and hydroscopic. Stock solution should be stable for 6-12 months. Working solution (1.2% Avertin) was prepared by adding 0.240 ml of stock to 19.76 ml of sterile water dropwise while water was being stirred by stir bar. Stirring was performed until the stock solution was completely dissolved. The stock solution was refrigerated and protected from light for use within 2-weeks. Avertin was administered IP using 0.800 ml per 30-gram mouse.
Evans Blue
[0127] Due to its water solubility and slow excretion, as well as its tight binding to serum albumin, Evans Blue has been widely used in biomedicine to determine vascular permeability. When the release of histamine and other vasodilators from mast cells result in a disruption of the barrier and increased vascular permeability, Evans Blue-bounded albumin extravasates from the circulation into neighboring tissues. Leakage of dye across the blood vessel signifies a disintegration of the barrier, and the accumulation of Evans Blue dye can be quantified. A stock solution was prepared by adding 1 gram Evans Blue to 30 mls of water.
Example 2
Method for Detecting Analyte
[0128] Contact of the AR with analyte resulted in a visible and measurable localized inflammatory response that is directly proportional to the amount of analyte present in the subject. To demonstrate this, mice were injected intradermally with the HBsAg—AR to mast cells in vivo and then challenged intravenously with various amounts of HBsAg. Whereas, wheal and flare measurements alone can be performed in humans, mouse skin is very thin and measuring the leakage of the Evans blue around a positive wheal and flare reaction provided an easy method to visualize a positive reaction.
[0129] Mice under Avertin anesthesia were injected intradermally with 100 ng (10 ul) of HBsAg—AR in phosphate buffered saline (PBS) in the right ear pinna; mice received 10 ul of PBS intradermally in the left ear pinna as a control. The next day, mice were challenged intravenously with 100 ul of HBsAg in 1% Evans blue. Mice were challenged with 100 ug, 50 ug, 20 ug and 10 ug of HBsAg. HBsAg challenges were administered by retro-orbital injections. Reactions were measure at 20 minutes. Mice were categorized base on the surface area and density of Evans blue dye in the ear (Table 2).
TABLE-US-00002 Challenge 100ug 50ug 20ug 10ug Reaction +++++ ++++ ++ Neg
Example 3
Enhanced Sensitivity
[0130] AR delivered to the target tissue was continually exposed to the analyte as the analyte circulated throughout the body until a measurable response occurred, thereby enhancing assay sensitivity. To demonstrate this, the mouse was challenged with 10 ug of HBsAg as described in Example 2 and was observed for the next 8 hours. The mouse registered as negative following 20 min to 4 hr exposure to the analyte but was clearly positive after 8 hours (Table 3).
[0130] TABLE-US-00003 Time 20 min 1 hour 2 hours 4 hours 8 hours Reaction Neg Neg Neg Neg ++
Example 4
Ease of Use and Simplicity
[0131] The method of the present invention was performed in one step without the necessity to process samples by separation or washing steps, providing an easy to use homogeneous type format. To demonstrate this, mice with circulating HBsAg were tested per the method of the present invention wherein the AR was delivered both intradermally and by needle prick. Mice under Avertin anesthesia were challenged with 100 ug HBsAg diluted in 100 ul of 1% Evans Blue as described above. HBsAg challenges were administered by retro-orbital injections. Injected HBsAg was allowed to pre-circulate for 10 min prior to the intradermally application of the HBsAg—AR in the right ear pinna as described above. HBsAg—AR was also pipetted onto the surface of the left ear and a needle was used to pierce the surface layer of the skin. Reaction times were observed and recorded in Table 4. Intradermal application of the AR resulted in a dense blue patch covering 30% to 40% of the ear. Needle prink resulted in small discrete and dense blue patches.
[0131] TABLE-US-00004 Time 30 sec 2 min 3 min 5 min Intradermal +/- +++ ++++ ++++ Needle Prick +/- +++ ++++ ++++
Example 5
Sentinel Test
[0132] To demonstrate this, Mice under Avertin anesthesia were injected intradermally with 100 ng (10 ul) of HBsAg—AR in PBS in the right ear pinna; mice received 10 ul of PBS intradermally in the left ear pinna as a control as described above. Seven days later, Mice under Avertin anesthesia were challenged with 100 ug HBsAg diluted in 100 ul of 1% Evans Blue as described above. After 16 days, anesthetized mice were challenged with 30 ug HBsAg diluted in 50 ul of 1% Evans Blue as described above. HBsAg challenges were administered by retro-orbital injections. Reactions were measured at 20 minutes. The results demonstrated that the method could be used to detect exposure to analytes 7 and 10-days after the AR mast cells, in situ, are exposed to AR (Table 5).
[0132] TABLE-US-00005 Time 7-Days 16-Days Reaction ++++ ++++
Example 6
Efficacy in Humans and Sentinel Test
[0133] A recombinantly produced affinity reagent that detected Ara h 2 was made by Absolute Antibody using proprietary methods. In this case, the affinity reagent was essentially an IgE antibody with specificity to an epitope on Ara h 2 allergen. The reagent possessed the required Fc.epsilon.Rl binding domain and the additional moiety that detected Ara h 2. The affinity reagent (1 mg/ml) was supplied in PBS, 0.2% Procline 300.
[0134] An intradermal injection was performed on the forearm of the applicant with 100 ng (10 ul) of Ara h 2-affinity reagent in PBS (protocol #1). Additionally, the affinity reagent was applied using a Unitest PC skin test applicator (Lincoln Diagnostics, Inc., Decatur, IL 62526 ) using 2 different procedures. In the first procedure, ~ 20 ul of affinity reagent was pipetted onto the skin in PBS, and the Unitest PC applicator administered through the affinity reagent into the dermal layer of the skin (protocol #2). In the second procedure, the affinity reagent was suspended in 50% glycerol in PBS allowing the affinity reagent to be absorbed onto the tip of the Unitest PC by capillary action. The Unitest PC applicator was then applied to the dermal layer of the skin in one step (protocol #3). A 50 ug/ml concentration of affinity reagent was used with the Unitest device.
[0135] Peanuts were consumed at 30 min intervals for 6 hours. The assay using protocol #1, #2, and #3 was applied to the forearm and the reaction measured after 20 min. Controls were included for each protocol consisting of the application vehicle without the affinity reagent. A distinct wheal and flare was clearly visible using all three protocols where the affinity reagent was applied, while negative control showed no sign reaction (Table 6).
[0136] Ara 2 h was eliminated from the applicant’s blood by waiting 24 hr after the last peanuts were consumed. The assay was then applied using protocol #2 and the reaction measured after 20 min. To demonstrate feasibility of a sentinel test, the assay was applied to the applicant prior to consuming peanuts. The assay was negative and remained negative for 24 hours. Because of the lag period between eating peanuts and the appearance of Ara 2 h in the blood, test results were measured 20 minutes after the first sign of a reaction (Table 7).
TABLE-US-00006 Protocol #1 (mm) Protocol #2 (mm) Protocol #3 (mm) Control 0.0 0.0 0.0 +Ara h 2 12.00 8.5 7.0
TABLE-US-00007 Protocol #2 (mm) -Ara h 2 0.0 +Ara h 2 (after 24 hrs) 7.0
Example 7
Human Sentinel Test
[0137] The affinity reagent (anti-Ara h 2, IgE) was prepared as described above and applied in ~5 to 10 ul of saline to the forearm of the applicant by intradermal injection creating a bubblelike area. The bubblelike area disappeared in a few hours and all traces of the injection site were gone by the next day. Peanuts were consumed by the applicant 1 week later. Within 30 min of consumption a tingle at the site was present, followed by a slight sting. With a few minutes a distinct wheal and flare reaction appeared and remained for approximately 2 hours. The wheal measured 20 mm within 20 min of the first signs of a reaction (Table 8).
[0137] TABLE-US-00008 Reaction -Ara h 2 0.0 mm +Ara h 2 (after 1 wk) 20.0 mm
Example 8
Application of Affinity Reagent Dried onto Applicator
[0138] The affinity reagent (anti-Ara h2, IgE) was dried onto the tip of a Unitest PC applicator. A 50 ul aliquot of a 1:10 dilution of stock affinity reagent into PBS was placed in a 1.5 ml microfuge tube. The Unitest PC applicator was placed in the microfuge tube with the tip submerged in the affinity reagent for 1 or 2 minutes. The applicator was removed and allowed to dry for 4 hours. The applicator was applied to the subject’s arm for 30 seconds. There was no blood visible, but the imprint of the applicator was present showing that the applicator had been properly applied to the forearm as indicated by the manufacturers user’s guide. All marks of the applied test had disappeared within 30 min. The subject consumed peanuts 24 hours later. Within 20 min a slight tingling occurred followed by a low-grade stinging sensation at which time a wheal and flare reaction was observed. The wheal and flare was at maximum intensity at 40 min post-consumption. A distinct wheal and flare reaction of 8 mm was present and persisted for over 2 hours (Table 9). After 2 hours the wheal and flare diminished and only a remnant of the reaction remained over the next 6 hours.
[0138] TABLE-US-00009 Reaction -Ara h 2 0.0 mm +Ara h 2 (applied dry) 8.0 mm
[0139] While the foregoing disclosure taken together with the references incorporated herein relates to and describes certain embodiments that are provided to enable and exemplify the concept of the invention as well as certain exemplary instruction as to reduction to practice, those skilled in the art will appreciate that changes and modifications to the foregoing may be made without departing from the spirit of the invention and without undue experimentation. The invention described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The terms and expressions are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Accordingly, it is intended to claim all such changes and modifications as fall within the true scope of the invention.