# US 2014/0154257 A1 · Materials and methods for diagnosing and treating asthma and dietary Fru-AGEs related disorders including auto-immune and other diseases found to be associated with elevated RAGE. The specification describes methods to identify and make dietary derived advanced glycation end-products, known as Fru-AGEs, Fru-AGE-haptens, and Fru-AGE immune complexes, and to make monoclonal and polyclonal antibodies to this plurality of bio-molecules for use in immunoassays and for use as therapeutic agents

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- Number: US 2014/0154257 A1
- Document ID: US20140154257A1
- Status: Published application
- Kind: A1
- Application number: 13/908,750
- Filed: 2013-06-03
- Published: 2014-06-05
- Earliest claimed: 2012-06-07
- Inventors: Luanne Robalo DeChristopher
- Web page: https://patentgrep.com/patent/US20140154257A1

## CPC

- G01N 33/6893: {related to diseases not provided for elsewhere}
- G01N 33/6854: {Immunoglobulins}
- G01N 2440/38: addition of carbohydrates, e.g. glycosylation, glycation
- G01N 2800/122: Chronic or obstructive airway disorders, e.g. asthma COPD
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

Present invention is directed to materials and methods useful in diagnosing gut derived advanced glycation end-products (“Fru-AGEs”) associated with asthma, juvenile arthritis and other pro-inflammatory chronic diseases. Fru-AGEs arise from the interaction [fructosylation] between elevated gastro-intestinal excess free fructose and other food/bio-molecules including peptides, proteins, lipids, lipo/glycoproteins and others in the digestive tract. Such Fru-AGEs may further interact with moieties of the systemic circulation of those at risk [fructose malabsorbers], i.e. with acute phase proteins, heat shock proteins, immunoglobulins, esRAGE, and sRAGE. Specification describes methods to identify and/or make such excess-free-fructose derived immunogens, i.e. Fru-AGEs, Fru-AGE-haptens, Fru-AGE immune complexes, and to make monoclonal/polyclonal antibodies to such bio-molecules for use as therapeutic agents and/or for use in immuno-assays including fluorescence enzyme immunoassay, microarray specific immunoglobulin/antibody testing, fluids detection [blood and urine] of GI Fru-AGE/haptens/immune-complexes.

## Claims

### Claim 1

Peptide containing food fragments of food proteins, lipoproteins, and/or glycoproteins that contain post-translational modifications, for example N-epsilon-(carboxy-ethyl)lysine (CEL) or N-epsilon-(carboxy-methyl)lysine (CML), also known as Fru-AGEs (dietary) for use in immuno-assays.

### Claim 2 (depends on 1)

The plurality of fragments of claim 1 that bind the receptor of advanced glycation end products (RAGE), its isoforms, or other immune bio-molecules.

### Claim 3 (depends on 1)

The plurality of claim 1 wherein food fragments contain post-translational modifications CEL and/or CML that arise from food processing.

### Claim 4 (depends on 1)

The method of claim 1 where fragments are post translationally modified, i.e. fructosylated (fructated) to incorporate modifications (CML/CEL) to their native sequences as may occur in the digestive tract via the well-characterized non-enzymatic Maillard reaction.

### Claim 5 (depends on 1)

The plurality of Fru-AGEs from claim 1 are bound to proteins of the systemic circulation as haptens, including for example heat shock proteins, hemoglobin, albumin, or others for use in immuno-assays.

### Claim 6 (depends on 1)

The plurality of Fru-AGEs from claim 1 complexed with proteins of the immune system including for example immunoglobulins/IgG/IgE/IgA, endogenous soluble RAGE (esRAGE), soluble RAGE (sRAGE), or others—all herein referred to as Fru-AGE-immuno complexes and/or Fru-AGE-macro-molecules for use in immuno-assays.

### Claim 7 (depends on 1, 5, 6)

Monoclonal and polyclonal antibodies to the plurality of Fru-AGEs, Fru-AGE haptens, and Fru-AGE-immune complexes (macro-molecules) from claims 1, 5 and 6 for use in immuno-assays.

### Claim 8 (depends on 1, 5, 6)

Monoclonal and polyclonal antibodies to the plurality of Fru-AGEs, Fru-AGE haptens, and Fru-AGE-immune complexes (macro-molecules) from claims 1, 5 and 6 for use as therapeutic agents for symptoms management.

### Claim 9 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of claims 1-8 comprising detecting asthma, or neutrophilic asthma, or COPD, or food associated asthma in the subject.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of claims 1-8 comprising biomarker assessment and disease detection known to be associated with elevated RAGE and inflammation including arthritis, rheumatoid arthritis; systemic lupus Erthematosus (SLE); Alzheimer's; atherosclerosis; heart disease; peripheral vascular disease (PVD); diabetic retinopathy, neurophathy, and nephropathy; lung cancer and fibrosis; irritable bowel disease; ulcerative colitis; Chron's disease; psoriasis; cancer; and others.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8)

The method of claims 1-8 comprising use of monoclonal and polyclonal antibodies as therapeutic agents in the management of pro-inflamatory diseases including asthma; COPD; pulmonary disorders; arthritis; rheumatoid arthritis; SLE; Alzheimer's; atherosclerosis; heart disease; peripheral vascular disease (PVD); diabetic retinopathy, neurophathy, and nephropathy; lung cancer; fibrosis; irritable bowel disease; ulcerative colitis; Chron's disease; psoriasis; cancer; and others.

### Claim 12

Diagnostic kits wherein the plurality of Fru-AGEs, Fru-AGE macro-molecules, and plurality of monoclonal and polyclonal antibodies to the aforementioned are used for well characterized fluorescence enzyme linked immunoassay (FEIA), ELISA, multiple and portable ELISA, micro-array or other immunoassay methods.

### Claim 13 (depends on 1, 5, 6, 7)

Diagnostic kits for immunoassay testing wherein bio-molecules of claims 1, 5, 6 and 7 are arrayed on a solid support for example microarray or wells.

### Claim 14 (depends on 1, 5)

Diagnostic kits for molecular immunogens, “allergens” diagnosis comprising as reagent—Fru-AGEs, Fru-AGE-haptens, and/or Fru-AGE-immune complexes of claims 1, 5, and 6.

### Claim 15 (depends on 7, 8)

Diagnostic kits for molecular immunogens/“allergens” diagnosis comprising as reagent—antibodies of claims 7 and 8.

### Claim 16 (depends on 1, 5, 7, 12)

The method of claim 12 wherein the plurality of Fru-AGE (claim 1); Fru-AGE macro-molecules (claims 5 & 6); monoclonal and polyclonal antibodies (claim 7); anti-immunoglobulin/anti-IgE/anti-IgG/anti-IgA; (e) sRAGE, anti-(e)sRAGE—are labeled and/or enzyme linked.

### Claim 17 (depends on 1, 5, 6, 7, 8)

The method of claims 7 and 8 wherein Fru-AGEs of claims 1, 5 and 6 are used for animal inoculations for production of polyclonal and monoclonal antibodies by conventional, hybridoma or other methods.

### Claim 17 (depends on 1, 5, 6, 7, 8)

The method of claims 1, 5, 6, 7 and 8 that includes the use of blocking proteins or buffers designed to block the portions of wells or microarrays that are still reactive after well coating or array printing to prevent non-specific binding.

### Claim 18 (depends on 5)

The method of claim 5 is distinguished from methods relating to antibodies production wherein a carrier protein serves primarily as adjuvant to elicit an immune response during animal inoculation.

### Claim 18 (depends on 1, 5, 6)

The method of claims 1, 5, 6, and 7 wherein antigens and/or antibodies are organized and arrayed in groups, for example by food type; or by sequence homology across food types, for example profilins in fruits, or tropomyosin in meats; or organized and arrayed as may be more likely observed in young children, or in adults, or as may be more likely observed in specific disease states.

### Claim 19 (depends on 1, 5, 6, 15)

The method of claims 1, 5, and 6 where fragments are identified, selected and lengths chosen such that lysines and/or arginines are preceded by a hydrophobic residue for example phenylalanine, isoleucine, tryptophan, leucine, valine, methionine, tyrosine, cysteine, or alanine.
a) The method of claims 1, 5, and 6 wherein the fragment contains a minimum of four residues to the right of the post-translationally modified lysine, and/or arginine.
b) The method of claims 1, 5, and 6 wherein fragments are a minimum of 5 amino acids in length and may extend to any length, for example, 5, 6, 7, 8, 9, 10 . . . N residues, wherein N may be any number that is a subset of the complete protein, or LP, or GP.
c) The method of claims 1, 5 and 6 wherein amino acid position one of the fragment begins with the CML/CEL left flanking N-terminus hydrophobic residue is the first residue in the sequence.
d) The method of claims 1, 5 and 6 wherein the next possible Fru-AGE shifts the beginning of the sequence one amino acid residue to the left, thereby beginning the sequence at the penultimate to the hydrophobic residue with the effect of dropping one residue off at the right or carboxy-terminus end of the previous fragment to arrive at a new fragment length.
e) The method of claims 1, 5 and 6 wherein shifts to the left may continue for each dietary Fru-AGE by dropping one residue from the right until there is a minimum of four residues to the right (carboxy-side) of the CML/CEL residue.
f) A method of claims 1, 5 and 6 wherein fragments are arrayed on a solid support as a series wherein the amino acid sequence of the next fragment in the series shifts by one amino acid to the left as per claims 15 a-e.

### Claim 19 (depends on 1, 5, 6)

The method of claims 1, 5 and 6 which comprises contacting wells or micro-arrays coated with a plurality of Fru-AGE antigens and/or Fru-AGE macro-molecules with serum obtained from a test subject and determining the amount of serum immunoglobulin (antibodies) or esRAGE or sRAGE bound using labeled anti-antibodies and anti-RAGE antibodies for example anti-IgE, anti-IgG, anti-sRAGE, anti-esRAGE.
a) The method of claims 1, 5 and 6 wherein patient samples are introduced into each well plate.
b) The method of claims 1, 5 and 6 wherein patient's antibodies present within the samples bind to well coating antigens.
c) The method of claims 1, 5 and 6 wherein a washing step clears unbound antibodies from the well.
d) The method of claims 1, 5 and 6 wherein labeled/enzyme labeled antibodies against the bound antibodies are added and incubated and another washing step follows to clear unbound enzyme labeled antibodies.
e) The method of claims 1, 5 and 6 wherein an incubation step with developing agent follows.
f) The method of claims 1, 5 and 6 wherein after incubation the signal is measured. The higher the signal, the higher the levels of antibodies present in the sample and the detected signal is proportional to the amount of epitope/immunoglobulin complex at each position in the array or wells.
g) The method of claims 1, 5 and 6, wherein in fluorescence, a type of electromagnetic spectroscopy which analyzes fluorescence from a sample, analysis is conducted using a beam of light, usually UV light that excites the electrons in the molecules of certain compounds causing them to emit light and fluorescence is then measured via devices known as fluoremeters.
h) The method of claims 1, 5 and 6 wherein, in non-fluorescence assays, other types of signals are possible and are measured using other techniques, for example absorption spectroscopy.

### Claim 20 (depends on 1, 5, 6, 20)

The method of claims 1, 5, 6 where a model digestion system is used to identify amino acid sequences and lengths of peptide containing food fragments.
a) The method of claim 20 wherein the model digestion system is one that mimics the biochemical environment of the digestive tract of different individuals.
b) The method of claim 20 wherein the biochemical environments reflect that of different age groups including for example young children, adolescents, and adults.
c) The method of claim 20 wherein the biochemical environments reflect that of individuals with different microbiome profiles, or with different disease diagnoses/profiles.
d) The method of claim 20 wherein adjustments are made to pH and/or to concentrations of proteases, phosphatidylcholine and bile salts for the purpose of “digesting” food proteins, lipoproteins, and glycoproteins into “model” peptide containing fragments for fructosylation into Fru-AGEs.

### Claim 20 (depends on 1, 5, 6)

The method of claims 1, 5, 6, and 7 wherein signal levels from immunoassays can be used as bio-markers of disease.

### Claim 21 (depends on 1, 5, 6, 7)

The method of claims 1, 5, 6 and 7 wherein each well or unique chip site contains a unique Fru-AGE, Fru-AGE macro-molecule, polyclonal antibody, monoclonal antibody, (e) sRAGE, or suitable control that is individually coating a well within the plate array, or region of the microchip.

### Claim 22 (depends on 7)

Diagnostic kits for antibody immunoassay wherein monoclonal and/or polyclonal antibodies of claim 7 are arrayed on a solid support for example wells or microarrays for fluids detection of Fru-AGEs and/or Fru-AGEs macro-molecules.
a) The method of claim 7 wherein antibodies are organized and arrayed by reactivity to immunogens by food type; or by sequence homology across food types, for example profilins in fruits, or tropomyosin in meats; or as may be observed in different age groups, for example children, adolescents, or adults; or as may be observed in specific disease states.
b) The method of claim 7 which comprises contacting wells or micro-arrays coated with the plurality of monoclonal and/or polyclonal antibodies with serum obtained from a test subject and determining the amount of serum Fru-AGEs and Fru-AGEs-immune complexes bound using labeled antibodies as per well characterized antibody microarray.

### Claim 23

Another aspect of the present invention is the provision of antibody kits for use in well characterized sandwich or competitive ELISA or more recently characterized multiple and portable (M & P) ELISA methods. wherein antibodies are bound to a solid support whether well plates or beads, or by other solid support methods and sample introduced either directly or as in M & P by multi-catcher devices with 8 or 12 immunosorbent ogival pins.
a.a) The key step in any of these assays is immobilization and detection of antigen in sample fluids. Antigen may be detected directly by antibodies of the present invention that may be designed as labeled primary antibody or indirectly by labeled secondary antibody which is an antibody to the primary antibody.
a.b) In the sandwich type ELISA, antigens contained within sample fluids are captured between two primary antibodies, for example a monoclonal capture antibody and a polyclonal detection antibody, both being part of the present invention.
a.c) Fluorescent or other type tags as known by those of skill in the art provide detection and a measure of the amount of antigen in the sample.

### Claim 24 (depends on 1, 5, 6, 7)

A method of claims 1, 5, 6 and 7 wherein Fru-AGEs, Model Fru-AGEs, Fru-AGE-haptens and Fru-AGE-immune complexes are synthesized or conjugated with labels or tags using a method as known by those of skill in the art for use in competition assays.
a) In this ELISA variation, unlabeled antigen from samples and the labeled antigen compete for binding to the capture antibody (monoclonal or polyclonal antibody of the present invention) that coats well plates. A decrease in signal from the labeled Fru-AGEs, Model Fru-AGEs, Fru-AGE-haptens, or Fru-AGE-immune complex indicates the presence of such antigens in samples when compared to assay wells with labeled targets only.

### Claim 25 (depends on 1, 5, 6)

A method of claims 1, 5 and 6 wherein peptide containing fragments may be derived from natural sources.

### Claim 26 (depends on 1, 5, 6)

A method of claims 1, 5 and 6 wherein peptide containing fragments may be obtained from any one of a number of well characterized recombinant protein expression methods, or peptide synthesis methods as utilized and known by those skilled in the art.

### Claim 27 (depends on 1, 5, 6)

A method of claims 1, 5 and 6 wherein peptide containing fragments may be obtained by chemical synthesis, using many protocols including automated methods as known by those of skill in the art.

### Claim 28 (depends on 1, 5, 6, 7, 8)

A method of claims 1, 5, 6, 7 and 8 where in certain embodiments, labels are incorporated into the Fru-AGEs, Model Fru-AGEs, Fru-AGE-haptens, Fru-AGE-immune complexes, monoclonal and polyclonal antibodies by methods well known by those of skill in the art.

### Claim 29 (depends on 1, 5, 6, 7, 8)

The methods of claims 1, 5, 6, 7 and 8 for purification and determination of molecule authenticity. Methods may include chromatographic purification, purification by mass separation, affinity column, histidine tags/metal binding, immunoaffinity chromatography, HPLC, reverse-phase HPLC, polyacrylamide gel electrophoresis, or amino acid analysis by microwave hydrolysis, or other known methods. If by recombinant protein expression methods, said proteins may need to be subjected to acid and enzymatic digestion steps.

### Claim 30 (depends on 1, 5, 6)

The method of claims 1, 5, 6, and 7 wherein immobilization onto a solid support or substrate may utilize many rigid and semi-rigid materials and methods including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles, and capillaries. The substrate may have any one of a variety of surface forms including wells, trenches, pins, channels and pores.

### Claim 31 (depends on 1, 5, 6, 7)

The method of claims 1, 5, 6 and 7 wherein immobilization may also be by covalent means via the use of chemical bonding or UV or bound to a glass surface that has been modified to contain epoxide or aldehyde groups, or avidin. Other examples include all such molecules transported from solution to a given position on a substrate by electrical means.

### Claim 32 (depends on 1, 5, 6, 7)

The method of claims 1, 5, 6 wherein antigens, and of claim 7 wherein antibodies are immobilized onto a solid support binding indirectly through a linker group (of varying lengths) for example polyethylene glycol (PEG), or diamines, or diacids wherein the terminal ends of such linkers interact with reactive groups on the substrate surface and the other end of the linker is designed for binding to molecules of the present invention.

### Claim 33 (depends on 1, 5, 6, 7)

The method of claims 1, 5, 6 and 7 where it is possible that the epitopes may be in solution.

### Claim 34 (depends on 7, 8)

The method of claims 7 and 8 wherein monoclonal/ and or polyclonal antibodies preparation may be by the hybridoma method, or other known recombinant techniques including use of suitable adjuvants and animals inoculations of for example mice, sheep, rabbits, rats and guinea pigs.

### Claim 35 (depends on 7, 8)

The method of claims 7 and 8 wherein small Fru-AGEs that are to be used as antigens in animal inoculation are conjugated to carrier proteins either directly or through linkers to insure that they induce an immune response and production of antibodies. Well known carrier proteins may include key hole limpet hemocyanin (KLH), Blue Carrier Immunogenic protein, and others.

### Claim 36 (depends on 5, 7, 8)

The method of claims 5, 7 and 8 wherein anyone of a number of conjugation methods of hapten-carrier conjugation are used including use of chemical moieties for protection of functional groups which are removed in the final steps of processing.