# US 2017/0285010 A1 · Analytical Methods and Arrays for Use in the Same

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- Number: US 2017/0285010 A1
- Document ID: US20170285010A1
- Status: Published application
- Kind: A1
- Application number: 15/518,580
- Filed: 2015-11-27
- Published: 2017-10-05
- Earliest claimed: 2014-11-28
- Assignees: Senzagen AB
- Applicants: Senzagen AB
- Inventors: Malin Lindstedt; Carl A.K. Borrebaeck; Henrik Johansson; Ann-Sofie Albrekt; Andrew Forreryd
- Web page: https://patentgrep.com/patent/US20170285010A1

## CPC

- G01N 33/5047: {Cells of the immune system}
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q 1/6883: for diseases caused by alterations of genetic material
- G01N 33/5023: {on expression patterns}
- C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
- C12Q 2600/158: Expression markers
- C12Q 2600/178: miRNA, siRNA or ncRNA
- G01N 2333/70578: NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30 CD40 or CD95
- G01N 2333/924: acting on glycosyl compounds (3.2)
- G01N 2800/12: Pulmonary diseases
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

The present invention relates to a method for identifying agents capable of inducing respiratory sensitization in a mammal and arrays and diagnostic kits for use in such methods. In particular, the methods include measurement of the expression of the biomarkers listed in Table A(i), Table A(ii) and/or Table A(iii) in cells exposed to a test agent.

## Claims

### Claim 1

A method for identifying agents capable of inducing respiratory sensitization in a mammal comprising or consisting of the steps of:
a) exposing a population of dendritic cells or a population of dendritic-like cells to a test agent; and
b) measuring in the cells the expression of one or more biomarker(s) selected from the group defined in Table A(i) and/or Table A(ii);
wherein the expression in the cells of the one or more biomarkers measured in step (b) is indicative of the respiratory sensitizing effect of the test agent.

### Claim 2 (depends on 1)

The method according to claim 1 further comprising:
c) exposing a separate population of the dendritic cells or dendritic-like cells to one or more negative control agent that is not a respiratory sensitizer in a mammal; and
d) measuring in the cells the expression of the one or more biomarker(s) measured in step (b)
wherein the test agent is identified as a respiratory sensitizer in the event that the presence and/or amount in the test sample of the one or more biomarker measured in step (d) differs from the presence and/or amount in the control sample of the one or more biomarker measured in step (b).

### Claim 3 (depends on 1, 2)

The method according to claim 1 or 2 further comprising:
e) exposing a separate population of the dendritic cells or dendritic-like cells to one or more positive control agent that is a respiratory sensitizer in a mammal; and
f) measuring in the cells the expression of the one or more biomarker(s) measured in step (b)
wherein the test agent is identified as a respiratory sensitizer in the event that the presence and/or amount in the test sample of the one or more biomarker measured in step (f) corresponds to the presence and/or amount in the positive control sample of the one or more biomarker measured in step (b).

### Claim 4

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of one or more biomarkers defined in Table A(ii) for example, at least 2 or 3 of the biomarkers defined in Table 1A.

### Claim 5

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of TNFRSF19.

### Claim 6

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of SNORA74A.

### Claim 7

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of SPAM1.

### Claim 8

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of TNFRSF19, SNORA74A and SPAM1.

### Claim 9

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring in step (b) the expression of one or more biomarkers defined in Table A(ii), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341 or 342 of the biomarkers defined in Table A(ii).

### Claim 10

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table A(ii).

### Claim 11

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of one or more of the biomarkers defined in Table A(iii), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 of the biomarkers defined in Table A(iii).

### Claim 12

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table A(iii).

### Claim 13

The method according to any one of the preceding claims wherein step (b) comprises or consists of measuring the expression of all of the biomarkers defined in Table A.

### Claim 14

The method according to any one of the preceding claims wherein step (b) comprises measuring the expression of a nucleic acid molecule encoding the one or more biomarker(s).

### Claim 15 (depends on 14)

The method according to claim 14 wherein the nucleic acid molecule is a cDNA molecule or an mRNA molecule.

### Claim 16 (depends on 14)

The method according to claim 14 wherein the nucleic acid molecule is an mRNA molecule.

### Claim 17 (depends on 14)

The method according to claim 14 wherein the nucleic acid molecule is a cDNA molecule.

### Claim 18 (depends on 14, 15, 16, 17)

The method according to any one of claims 14 to 17 wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using a method selected from the group consisting of Southern hybridisation, Northern hybridisation, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real-time PCR (qRT-PCR), nanoarray, microarray, macroarray, autoradiography and in situ hybridisation.

### Claim 19 (depends on 14, 15, 16, 17, 18)

The method according to any one of claims 14 to 18 wherein measuring the expression of the one or more biomarker(s) in step (b) is determined using a DNA microarray.

### Claim 20

The method according to any one of the preceding claims wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using one or more binding moieties, each capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table A.

### Claim 21 (depends on 20)

The method according to claim 20 wherein the one or more binding moieties each comprise or consist of a nucleic acid molecule.

### Claim 22 (depends on 21)

The method according to claim 21 wherein the one or more binding moieties each comprise or consist of DNA, RNA, PNA, LNA, GNA, TNA or PMO.

### Claim 23 (depends on 20, 22)

The method according to claim 20 or 22 wherein the one or more binding moieties each comprise or consist of DNA.

### Claim 24 (depends on 21, 22, 23, 24)

The method according to any one of claims 21 to 24 wherein the one or more binding moieties are 5 to 100 nucleotides in length.

### Claim 25 (depends on 21, 22, 23, 24, 25)

The method according to any one of claims 21 to 25 wherein the one or more nucleic acid molecules are 15 to 35 nucleotides in length.

### Claim 26 (depends on 21, 22, 23, 24, 25, 26)

The method according to any one of claims 21 to 26 wherein the binding moiety comprises a detectable moiety.

### Claim 27 (depends on 26)

The method according to claim 26 wherein the detectable moiety is selected from the group consisting of: a fluorescent moiety; a luminescent moiety; a chemiluminescent moiety; a radioactive moiety (for example, a radioactive atom); or an enzymatic moiety.

### Claim 28 (depends on 26)

The method according to claim 26 wherein the detectable moiety comprises or consists of a radioactive atom.

### Claim 29 (depends on 28)

The method according to claim 28 wherein the radioactive atom is selected from the group consisting of technetium-99m, iodine-123, iodine-125, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, phosphorus-32, sulphur-35, deuterium, tritium, rhenium-186, rhenium-188 and yttrium-90.

### Claim 30 (depends on 27)

The method according to claim 27 wherein the detectable moiety of the binding moiety is a fluorescent moiety.

### Claim 31 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)

The method according to any one of claims 1 to 22 wherein step (b) comprises or consists of measuring the expression of the protein of the one or more biomarker defined in step (b).

### Claim 32 (depends on 31)

The method according to claim 31 wherein measuring the expression of the one or more biomarker(s) in step (b) is performed using one or more binding moieties each capable of binding selectively to one of the biomarkers identified in Table A.

### Claim 33 (depends on 32)

The method according to claim 32 wherein the one or more binding moieties comprise or consist of an antibody or an antigen-binding fragment thereof.

### Claim 34 (depends on 33)

The method according to claim 33 wherein the antibody or fragment thereof is a monoclonal antibody or fragment thereof.

### Claim 35 (depends on 33, 34)

The method according to claim 33 or 34 wherein the antibody or antigen-binding fragment is selected from the group consisting of intact antibodies, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab′ fragments and F(ab)2 fragments), single variable domains (e.g. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).

### Claim 36 (depends on 35)

The method according to claim 35 wherein the antibody or antigen-binding fragment is a single chain Fv (scFv).

### Claim 37 (depends on 32)

The method according to claim 32 wherein the one or more binding moieties comprise or consist of an antibody-like binding agent, for example an affibody or aptamer.

### Claim 38 (depends on 32, 33, 34, 35, 36, 37)

The method according to any one of claims 32 to 37 wherein the one or more binding moieties comprise a detectable moiety.

### Claim 39 (depends on 38)

The method according to claim 38 wherein the detectable moiety is selected from the group consisting of a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, a radioactive moiety and an enzymatic moiety.

### Claim 40

The method according to any one of the preceding claims wherein step (b) is performed using an array.

### Claim 41 (depends on 40)

The method according to claim 40 wherein the array is a bead-based array.

### Claim 42 (depends on 41)

The method according to claim 41 wherein the array is a surface-based array.

### Claim 43 (depends on 40, 41, 42)

The method according to any one of claims 40 to 42 wherein the array is selected from the group consisting of: macroarray; microarray; nanoarray.

### Claim 44 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39)

An array for use in a method according any one of the preceding claims, the array comprising one or more first binding agents as defined in any one of claims 20 to 30 and 32 to 39.

### Claim 45 (depends on 44)

An array according to claim 44 comprising binding agents which are collectively capable of binding to all of the biomarkers defined in Table 1.

### Claim 46 (depends on 44, 45)

An array according to claim 44 or 45 wherein the first binding agents are immobilised.

### Claim 47

The method according to any one of the preceding claims for identifying agents capable of inducing a respiratory hypersensitivity response.

### Claim 48

The method according to any one of the preceding claims wherein the hypersensitivity response is a humoral hypersensitivity response.

### Claim 49 (depends on 47, 48)

The method according to claim 47 or 48 wherein the hypersensitivity response is a type I hypersensitivity response.

### Claim 50

The method according to any one of the preceding claims for identifying agents capable of inducing respiratory allergy.

### Claim 51

The method according to any one of the preceding claims wherein the population of dendritic cells or population of dendritic-like cells is a population of dendritic-like cells.

### Claim 52 (depends on 51)

The method according to claim 51 wherein the dendritic-like cells are myeloid dendritic-like cells.

### Claim 53 (depends on 52)

The method according to claim 52 wherein the myeloid dendritic-like cells are derived from myeloid dendritic cells.

### Claim 54 (depends on 53)

The method according to claim 53 wherein the cells derived from myeloid dendritic cells are myeloid leukaemia-derived cells.

### Claim 55 (depends on 54)

The method according to claim 54 wherein the myeloid leukaemia-derived cells are selected from the group consisting of KG-1, THP-1, U-937, HL-60, Monomac-6, AML-193 and MUTZ-3.

### Claim 56

The method according to any one of the preceding claims wherein the dendritic-like cells are MUTZ-3 cells.

### Claim 57 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56)

The method according to any one of the claims 2 to 56 wherein the one or more negative control agent provided in step (c) is selected from the group consisting of 1-Butanol; 2-Aminophenol; 2-Hydroxyethyl acrylate; 2-nitro-1,4-Phenylenediamine; 4-Aminobenzoic acid; Chlorobenzene; Dimethyl formamide; Ethyl vanillin; Formaldehyde; Geraniol; Hexylcinnamic aldehyde; Isopropanol; Kathon CG*; Methyl salicylate; Penicillin G; Propylene glycol; Potassium Dichromate; Potassium permanganate; Tween 80; and Zinc sulphate.

### Claim 58 (depends on 57)

The method according to claim 57 wherein at least 2 control non-sensitizing agents are provided, for example, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 control non-sensitizing agents.

### Claim 59 (depends on 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)

The method according to any one of claims 3 to 58 wherein the one or more positive control agent provided in step (e) comprises or consists of one or more agent selected from the group consisting of ammonium hexachloroplatinate, ammonium persulfate, glutaraldehyde, hexamethylen diisocyanate, maleic anhydride, methylene diphenol diisocyanate, phtalic anhydride, toluendiisocyanate and trimellitic anhydride.

### Claim 60 (depends on 59)

The method according to claim 59 wherein at least 2 control sensitizing agents are provided, for example, at least 3, 4, 5, 6, 7, 8, 9 or at least 10 control sensitizing agents.

### Claim 61

The method according to any one of the preceding claims wherein the method is indicative of the sensitizing potency of the sample to be tested.

### Claim 62 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39)

An array for use in a method according any one of the preceding claims, the array comprising one or more binding moieties as defined in any one of claims 20 to 30 and 32 to 39.

### Claim 63 (depends on 62)

An array according to claim 62 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table A(i).

### Claim 64 (depends on 62, 63)

An array according to claim 62 or 63 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table A(ii).

### Claim 65 (depends on 62, 63, 64)

An array according to claim 62, 63 or 64 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table A(iii).

### Claim 66 (depends on 62, 63, 64, 65)

An array according to any one of claims 62 to 65 wherein the binding moieties are capable of binding to all of the biomarkers defined in Table A.

### Claim 67 (depends on 62, 63, 64, 65)

An array according to any on of claims 62 to 65 wherein the binding moieties are immobilised.

### Claim 68

Use of two or more biomarkers selected from the group defined in Table A in combination for identifying respiratory hypersensitivity response sensitising agents.

### Claim 69 (depends on 68)

The use according to claim 68 wherein all of the biomarkers defined in Table A are used collectively for identifying hypersensitivity response sensitising agents.

### Claim 70 (depends on 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 34, 35, 36, 37, 38, 39)

Use of one or more binding moiety as defined in any one of claims 20 to 30 or 32 to 39 for identifying respiratory hypersensitivity response sensitising agents.

### Claim 71 (depends on 70)

The use according to claim 70 wherein all of the biomarkers defined in Table A are used collectively for identifying hypersensitivity response sensitising agents

### Claim 72 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67)

An analytical kit for use in a method according any one of claims 1 to 61 comprising:
A) an array according to any one of claims 62 to 67 and/or one or more binding moiety as defined in any one of claims 20 to 30 or 32 to 39; and
B) instructions for performing the method as defined in any one of claims 1 to 60 (optional).

### Claim 73 (depends on 72)

An analytical kit according to claim 72 further comprising one or more control samples.

### Claim 74 (depends on 73)

An analytical kit according to claim 73 comprising one or more non-sensitizing agent(s).

### Claim 75 (depends on 72, 73, 74)

An analytical kit according to claim 72, 73 or 74 comprising one or more sensitizing agent(s).

### Claim 76

A method of treating or preventing a respiratory type I hypersensitivity reaction (such as respiratory asthma) in a patient comprising the steps of:
(a) providing one or more test agent that the patient is or has been exposed to;
(b) determining whether the one or more test agent provided in step (a) is a respiratory sensitizer using a method provided in the first aspect of the present invention; and
(c) where one or more test agent is identified as a respiratory sensitizer, reducing or preventing exposure of the patient to the one or more test agent identified as a respiratory sensitizer and/or providing appropriate treatment for the symptoms of sensitization.

### Claim 77 (depends on 76)

The method according to claim 76 wherein the treatment of the symptoms of sensitization is selected from the group consisting of short-acting beta2-adrenoceptor agonists (SABA), such as salbutamol; anticholinergic medications, such as ipratropium bromide; other adrenergic agonists, such as inhaled epinephrine; Corticosteroids such as beclomethasone; long-acting beta-adrenoceptor agonists (LABA) such as salmeterol and formoterol;
leukotriene antagonists such as montelukast and zafirlukast; and/or mast cell stabilizers (such as cromolyn sodium).

### Claim 78

A computer program for operating the method defined in the first aspect of the invention.

### Claim 79 (depends on 78)

The computer program according to claim 78 wherein the computer program is recorded on a computer-readable carrier.

### Claim 80

A method or use substantially as described herein.

### Claim 81

An array, kit or computer program substantially as described herein.