GB2499180A

GB2499180A - Method of identifying sensitizers or allergens using measurements of T-cell proliferation and/or IFN Gamma

Info

Publication number: GB2499180A
Application number: GB201115537A
Authority: GB
Inventors: Anne Mary Dickinson
Original assignee: Alcyomics Ltd
Current assignee: Alcyomics Ltd
Priority date: 2011-09-08
Filing date: 2011-09-08
Publication date: 2013-08-14
Also published as: GB201115537D0

Abstract

An in vitro method of identifying chemical compounds that are sensitizers or non-sensitizers and/or allergens or non-allergens is disclosed, the method comprising separating monocyte-derived dendritic cells from a blood sample comprising T-cells, incubating the dendritic cells with a test compound, incubating the compound-treated dendritic cells with the aforementioned separated blood sample, and measuring T-cell proliferation and/or IFN-g in the incubated mixture, wherein the level of T-cell proliferation and/or IFN-g expression correlates with a defined grade of histological change observed in a skin explant treated with said test compound, and determining whether the test compound is a sensitizer or allergen based on a comparison of the measured T-cell proliferation or IFN-g measurements to control measurements. Related kits for identifying chemical compounds that are sensitizers or allergens are disclosed. The method of the invention is of particular utility in the identification and prediction of skin sensitizers that may cause allergic contact dermatitis.

Description

1

Assay

The present invention relates to a method of predicting immunogenicity and hypersensitivity or allergic reactions to potential therapeutic compounds, cosmetics and 5 chemical sensitizers. The method provides an in vitro assay employing blood derived cells and is of particular utility in the identification and prediction of skin sensitizers and in particular agents that may cause allergic contact dermatitis. The assay of the present invention provides inter alia methods of screening library compounds for sensitizing activity and kits therefor.

10

BACKGROUND

The delayed-type hypersensitivity reaction of Allergic Contact Dermatitis (ACD) can be acquired when a sensitized individual later becomes challenged with the same small 15 molecule. ACD manifests itself during the phase of elicitation; following penetration of the epidermis and acquisition/processing by an antigen presenting cell (APC)- a specialized cell within the skin, which presents the allergen or antigen to other cells known as T cells recruited by chemokines to the skin, causing their activation and the production of high levels of lymphokines. These molecules give rise to a secondary response with skin 20 inflammation and keratinocyte (skin cell) apoptosis. Distinct from its near relative, Irritant Contact Dermatitis (ICD), which is caused by irritants (e.g. soap, detergents, perfumes etc) and which can affect anyone who succumbs to sufficient exposure, ACD is influenced by environmental and genetic factors and may take many years to manifest, long after initial contact. With approximately 20% of the general adult population believed to be allergic to 25 one or more chemical sensitizers, and with a growing list of novel cosmetic and pharmaceutical products becoming available, ACD threatens to be an increasing future occupational and consumer health problem. Developing suitable and sensitive methods for the assessment of a chemical's potential to cause ACD will be a crucial step in combating this disease. As regards drug allergies, these are rarely detected in non-clinical 30 studies and are usually only observed in Phase 3 clinical trials or during commercialization when larger populations are exposed to the drug. Although the number of drugs that elicit allergic reactions is relatively low, the potential impact is very high due to the late stage of development in which it is detected. Therefore, non-clinical methods to predict for the potential to produce allergic reactions are needed to help in compound selection.

35

Identifying chemicals that have the potential to induce hypersensitivity skin reactions is a mandatory component of new product discovery by pharmaceutical and cosmetic

2

industries. Historically, predictive testing has exclusively relied on in vivo animal testing. In the traditional guinea pig test, the product is painted on the body and the guinea pig is then injected with an additional chemical to help accentuate the effect of the test chemical in developing dermatitis. Alternatively in the mouse ear swelling test, the mouse's ears are 5 painted with the test substance and its immunological response is determined by examination of lymph node tissue. However, with an imposing EU ban on animal testing nearing its full implementation, there is a pressing need for the development of alternative predicative in vitro and in silico techniques. Although it is known from the prior art to gage up and/or down regulation of gene products such as cytokines these assays are laborious 10 and results are inconsistent. No validated in vitro model currently exists to predict immunogenicity and hypersensitivity or allergic reactions to potential therapeutic compounds, cosmetics and chemical sensitizers.

There is therefore a need for an in vitro assay to discriminate between sensitizers and non-15 sensitizers and/or allergens and non-allergens for predicting the sensitizing nature of novel pharmaceutical, cosmetic and chemical products. There is a need for a simple, robust, accurate assay for testing novel compounds for hypersensitivity and allergic reactions.

BRIEF SUMMARY OF THE DISCLOSURE

20

According to a first aspect of the invention there is provided an in vitro method of identifying chemical compounds that are sensitizers or non-sensitizers and /or allergens or non-allergens, the method comprising:

(i) separating a population of monocyte-derived dendritic cells from a donor 25 blood sample comprising a population of T cells;

(ii) incubating the monocyte-derived dendritic cells with a test compound;

(iii) incubating the compound treated monocyte-derived dendritic cells of (ii) with the separated donor blood sample of (i);

(iv) determining the level of T cell proliferation and / or IFN-y expression in the 30 sample of (iii); and

(v) comparing the level of T cell proliferation in the sample with the level of T cell proliferation in at least one control sample treated with a control sensitizer compound and at least one control sample treated with a control non-sensitizer compound and /or at least one control sample treated with a control allergen compound and at least one control

35 sample treated with a control non-allergen compound; or

(vi) comparing the level of IFN-y expression in the sample with the level of IFN-Y expression in at least one control sample treated with a control sensitizer compound and

3

at least one control sample treated with a control non-sensitizer compound and /or at least one control sample treated with a control allergen compound and at least one control sample treated with a control non-allergen compound,

wherein comparison of T cell proliferation and/or IFN-y expression in the sample 5 with T cell proliferation and/or IFN-y expression in the control samples identifies the test compound as a sensitizer or non-sensitizer and/or an allergen or non-allergen.

By comparing the level of T cell proliferation and/or the level of IFN-y expression induced by a test compound to levels induced by a sensitizing and non-sensitizing control 10 compound (or allergen or non-allergen control compound), the method allows prediction of the sensitizing capability of the test compound. The use of sensitizing and non-sensitizing control compounds (or allergen or non-allergen control compound), advantageously provides thresholds values for sensitizing and non-sensitizing and / or allergic and non-allergic levels of T cell proliferation and IFN-y expression.

15

Preferably, the level of T cell proliferation determined in (iv) correlates with a defined grade of histological change observed in a skin explant treated with said compound. More preferably, the level of T cell proliferation in the skin explant treated with said compound correlates with an LLNA class observed in a LLNA mouse model treated with said 20 compound.

Preferably, the level of IFN-y expression determined in (iv) correlates with a defined grade of histological change observed in a skin explant treated with said compound. More preferably, level of IFN-y expression in the skin explant treated with said compound 25 correlates with a LLNA class observed in a LLNA mouse model treated with said compound.

Preferably, each of said control compounds is administered in a concentration such that at least 70% of treated cells remain viable 24 hours after exposure to the compound. More 30 preferably, at least 75% of treated cells or at least 80% of treated cells remain viable 24 hours after exposure to the compound. Still more preferably, 85, 90 or 95% of treated cells remain viable 24 hours after exposure to the compound.

Preferably, the percentage of viable cells is determined using a cell viability assay. 35 Preferably the cells of the cell viability assay are peripheral blood mononuclear cells, more preferably blood mononuclear-derived monocytes, and more preferably monocyte-derived dendritic cells.

4

Preferably, the control sensitizer compound is selected from a group consisting of dinitrochlorobenzene, nickel sulphate, eugenol, cinnamaldehyde, and 2-mercaptobenzothiazole. More preferably, dinitrochlorobenzene is administered at a 5 concentration of 0.1 |jM, nickel sulphate is administered at a concentration of 0.1 |jM, eugenol is administered at a concentration of 15 |jM, cinnamaldehyde is administered at a concentration of 0.1 |jM and 2-mercaptobenzothiazole is administered at a concentration of 0.1 |jM.

10 Preferably, the control non-sensitizer compound is selected from a group consisting of sodium dodecyl sulphate, triton-X-100, zinc sulphate, dimethyl sulfoxide, and glutamic acid. More preferably, sodium dodecyl sulphate is administered at a concentration of 10 ng/ml, triton-X-100 is administered at a concentration of 0.0001%, zinc sulphate is administered at a concentration of 0.1 |jM, dimethyl sulfoxide is administered at a 15 concentration of 0.0001%, and glutamic acid is administered at a concentration of 0.1 |jM.

Preferably, the control allergen compound is selected from a group consisting of penicillin, amoxicillin, lamotrigine, neomycin, and benzocaine. More preferably, penicillin is administered at a concentration of 100 jjg/ml, amoxicillin is administered at a concentration 20 of 100 jjg/ml, lamotrigine is administered at a concentration of 100 jjg/ml, neomycin is administered at a concentration of 10 jjg/ml, and benzocaine is administered at a concentration of 10 jjg/ml.

Preferably, the control non-allergen compound is selected from a group consisting of 25 simvastatin, methotrexate and metformin. More preferably, simvastatin is administered at a concentration of 10 |jg/ml, methotrexate is administered at a concentration of 10 jjg/ml, and metformin is administered at a concentration of 10 jjg/ml.

Preferably, the level of T cell proliferation is determined by [3H] thymidine incorporation. 30 Alternative methods of T cell proliferation can be by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2'-deoxyuridine (BrdU) incorporation.

Preferably, IFN-y expression is determined by flow cytometry. Alternative methods of 35 measuring IFN-y production can be by ELISA, ELISPOT and real-time RT-PCR.

Preferably, the donor blood sample is a human blood sample.

5

Preferably, the monocyte-derived dendritic cells are obtained from peripheral blood mononuclear cells (PBMC). More preferably, the monocyte-derived dendritic cells are generated from CD14+ monocytes from said PBMCs.

5 For example and without limitation, the T cell and monocyte-derived dendritic cells may be separated from PBMC by magnetic activated cell sorting or similar techniques.

Dendritic cells are generated from CD14+ monocytes from PBMCs using standard methods and the remaining monocyte depleted PBMC population is used as the "T cell" 10 preparation of step (iii). Dendritic cells (DCs) are antigen-presenting cells with the ability to induce primary T-cell responses and are routinely produced by culturing monocytes in the presence of IL-4 and GM-CSF for 5-7 days (Standard DC). Alternatively they may be produced by a modified protocol described hereinafter for differentiation of human monocytes into mature DCs within 48 hours (Fast DC). The modified protocol comprises 15 an incubation after 24 hours with maturation cytokines such as TNF-a (10ng/ml), IL-13 (10ng/ml), IL-6 (10ng/ml), 1uM PGE2, Resiquimod (2.5jjg/ml), CD40L (1|jg/ml) and LPS (0.1|jg/ml). It will be appreciated that either method of producing DCs is applicable to the present invention but that generation of Mature Fast DCs may be preferred to reduce the overall period for performing the assay of the present invention.

20

Preferably, the dendritic cells are either standard or fast matured dendritic cells.

Preferably, the first incubating step of step (ii) is for between 2 to 24 hours. Typical incubation conditions are carried out at 37°C in a humidified 5% C02air incubator, a typical 25 culture medium is Roswell Park Memorial Institute 1640 (RPMI 1640, Gibco UK) containing 100 lU/ml penicillin, 100|jg/ml streptomycin (Gibco UK) and 2 mM L-glutamine (Gibco UK) supplemented with 10% v/v heat inactivated foetal calf serum (FCS, Sera Lab), or Ex Vivo (Gibco UK) serum free medium. The culture conditions are non-limiting in so far as other variations in conditions that allow for growth and maintenance of the cells are 30 equally applicable.

Preferably, the incubation of step (ii) comprises incubation with 10% heat inactivated fetal calf serum.

35 Preferably, the second incubating of step (iii) is for between 3-7 days. Preferably, the second incubating of step (iii), comprising incubating DCs with a population of T cells isolated in step (i) is for between 3-7 days using the same culture conditions as for step (ii)

6

except that 10% heat inactivated autologous serum is used and replaces foetal calf serum. In the instance where a control comprises DCs with a population of T cells isolated in step (i) having been exposed to a non-sensitizer, this further or second set of cells is incubated in identical conditions to the test mixture.

5

Preferably, the incubation of step (iii) comprises incubation with 10% heat inactivated autologous serum.

Preferably, the control sample comprises a further or second set of monocyte-derived 10 dendritic cells that have been incubated in step (ii) with the control compound(s). More preferably, said further or second set of monocyte-derived dendritic cells are separated from the donor blood sample of step (i). Alternatively the control may be monocyte-derived dendritic cells incubated with no additional chemical compounds at all.

15 Preferably, said at least one control sample treated with a control sensitizer compound of the method comprises:

(i) a first control sample treated with DNCB; and

(ii) a second control sample treated with NiS04.

20 Preferably, said at least one control sample treated with a control non-sensitizer compound of the method comprises:

(i) a first control sample treated with Trton-X; and

(ii) a second control sample treated with ZnS04.

25 More preferably, said at least one control sample treated with a control sensitizer compound of the method comprises:

(i) a first control sample treated with DNCB; and

(ii) a second control sample treated with NiS04, and said at least one control sample treated with a control non-sensitizer compound of the 30 method comprises:

(i) a first control sample treated with Trton-X; and

(ii) a second control sample treated with ZnS04.

Preferably, the first and second control samples may be derived from the same donor 35 blood sample. More preferably each control sample of step (v) or (vi) is derived from the same donor blood sample. Most preferably, each control sample of step (v) or (vi) is derived from the same donor blood sample as used in step (i).

7

Preferably, the level of T cell proliferation and / or IFN-y expression in a control sample treated with a control sensitizer compound, a control non-sensitizer compound, a control allergen compound or a control non-allergen compound correlates with a defined grade of 5 histological change observed in a skin explant treated with said compound.

Preferably, the level of T cell proliferation and / or IFN-y expression in a control sample treated with a control sensitizer compound, a control non-sensitizer compound, a control allergen compound or a control non-allergen compound correlates with a LLNA class 10 observed in a LLNA mouse model treated with said compound.

In a second aspect, the invention provides a kit for identifying chemical compounds that are sensitizers or non-sensitizers and /or allergens or non-allergens, the kit comprising:

(i) at least one control sensitizer compound and at least one control non-sensitizer 15 compound and/or at least one control allergen compound and at least one control non-

allergen compound; and

(ii) instructions for use of the control compounds in the method according to the invention.

Preferably, the kit further comprises a means for correlating T cell proliferation with a 20 defined grade of histological change. More preferably, the kit further comprises a means for correlating IFN-y expression with a defined grade of histological change.

Preferably, the defined grade of histological change correlates with a LLNA class.

25 Preferably, each of said control compounds is provided in a concentration such that at least 70% of cells exposed to the compound remain viable 24 hours after exposure thereto. More preferably, wherein at least 75% of cells exposed to the compound or at least 80% of cells exposed to the compound remain viable 24 hours after exposure thereto. Still more preferably, 85, 90 or 95% of treated cells remain viable 24 hours after 30 exposure to the compound.

Preferably, the control sensitizer compound is selected from a group consisting of dinitrochlorobenzene, nickel sulphate, eugenol, cinnamaldehyde, and 2-mercaptobenzothiazole. More preferably, dinitrochlorobenzene is provided at a 35 concentration of 0.1 |jM, nickel sulphate is provided at a concentration of 0.1 |jM, eugenol is provided at a concentration of 15 |jM, cinnamaldehyde is provided at a concentration of 0.1 |jM, and 2-mercaptobenzothiazole is provided at a concentration of 0.1 |jM.

8

Preferably, the control non-sensitizer compound is selected from a group consisting of sodium dodecyl sulphate, triton-X-100, zinc sulphate, dimethyl sulfoxide, and glutamic acid. More preferably, sodium dodecyl sulphate is provided at a concentration of 10 ng/ml, 5 triton-X-100 is provided at a concentration of 0.0001%, zinc sulphate is provided at a concentration of 0.1 |jM, dimethyl sulfoxide is provided at a concentration of 0.0001%, and glutamic acid is provided at a concentration of 0.1 |jM.

Preferably, the control allergen compound is selected from a group consisting of penicillin, 10 amoxicillin, lamotrigine, neomycin, and benzocaine. More preferably, penicillin is provided at a concentration of 100 jjg/ml, amoxicillin is provided at a concentration of 100 jjg/ml, lamotrigine is provided at a concentration of 100 jjg/ml, neomycin is provided at a concentration of 10 |jg/ml, and benzocaine is provided at a concentration of 10 jjg/ml.

15 Preferably, the control non-allergen compound is selected from a group consisting of simvastatin, methotrexate and metformin. More preferably, simvastatin is provided at a concentration of 10 |jg/ml, methotrexate is provided at a concentration of 10 jjg/ml, and metformin is provided at a concentration of 10 |jg/ml.

20 Preferably, the kit further comprises means for separating monocytes from a blood sample and instructions for use thereof. More preferably, said means for separating monocytes comprises a CD14+ cell separation kit.

Preferably, the kit further comprises a means for fast dendritic cell maturation and 25 instructions for use thererof.

Preferably, the at least one control sensitizer compound and at least one control non-sensitizer compound and/or at least one control allergen compound and at least one control non-allergen compound of the kit are located in defined positions on a solid 30 support. More preferably, said solid support is a 6, 12, 24, 48 or 96 well plate.

Preferably, the kit comprises at least two control sensitizer compounds, wherein said compounds are DNCB and NiS04.

35 Preferably, the kit comprises at least two control non-sensitizer compounds, wherein said compounds are Trton-X and ZnS04.

9

Still more preferably, the kit comprises at least two control sensitizer compounds, wherein said compounds are DNCB and NiS04 and at least two control non-sensitizer compounds, wherein said compounds are Trton-X and ZnS04.

5 Reference herein to a "sensitizer" includes any chemical compound or chemical agent or antibody that causes a substantial proportion of exposed people or animals to develop an allergic reaction in normal tissue after single or repeated exposure to the said compound, antibody or chemical agent.

10 Reference herein to an "allergen" and "allergenic" includes any foreign substance such as an environmental substance or chemical that is capable of inducing allergy or a specific hypersensitive reaction in the body. Common allergens include plant pollens, spores of mold, animal dander, house dust, foods, feathers, dyes, soaps, detergents, cosmetics, plastics, and drugs. Allergens can enter the body by, for example, being inhaled, 15 swallowed, touched, or injected.

Reference herein to a "chemical compound" is intended to include a chemical, therapeutic, pharmaceutical or cosmetic agent, substance, preparation or composition.

20 Reference herein to "autologous" means that the blood derived products and skin explants are derived or collected from the same individual.

BRIEF DESCRIPTION OF THE DRAWINGS

25 Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows histopathological changes for different grades of skin graft versus host reaction (GVHR).

30

Figure 2 shows a T cell proliferation response to sensitizers and non-sensitizers;

Figure 3 shows a T cell proliferation response to allergens and non-allergens;

35 Figure 4 shows IFN-y expression levels in response to allergens and non-allergens;

Figure 5 shows IFN-y expression levels in response to sensitizers and non-sensitizers;

10

Figure 6 is a scatterplot showing correlation between histological grade and IFN-y expression response;

5 Figure 7 is a scatterplot showing correlation between histological grade and T cell proliferation.

Figure 8 is a scatterplot showing correlation between histological grade and IFN-y expression response;

10

Figure 9 is a scatterplot showing correlation between histological grade and T cell proliferation.

DETAILED DESCRIPTION

15 The present invention conveniently provides a non-artificial, human in vitro assay and methods which allows for the study of primary and secondary immune responses in the presence of potential sensitizing compounds thereby advantageously reducing the need for extensive animal testing.

20 This approach is unique and gives insight into the use of a blood based assay for predicting response to chemical sensitizers and to investigate their potential allergic / inflammatory signals. The present invention provides a blood based assay that improves on the current techniques and provides a novel means of testing novel drugs for hypersensitivity and allergic reactions.

25

The assay of the present invention provides the advantage over a three dimensional skin equivalent model as it uses blood with autologous immune cells enabling immune responses to be studied and cellular and molecular targets identified thus aiding in drug discovery, improving drug design and optimisation for drug dosage prior to a clinical trial.

30

Features ascribed to the first aspect of the invention are applicable mutatis mutandis to all other aspects of the invention.

Identification of control compounds

35 A variety of sensitizers, non-sensitizers, allergens and non-allergens were tested to provide control compounds for use in the methods and kits of the present invention, details of which are provided in table 1.

11

5 Table 1

Sensitizers

Abbr.

Concentration

Dinitrochlorobenzene

DNCB

0.1uM

Nickel Sulphate

Nl S04

0.1uM

Eugenol

Eug

2.5ng/ml (15uM)

Cinnamaldehyde

Cinn

0.1uM

2-mercaptobenzothiazole

2Mercap

0.1uM

Non -sensitizers

Sodium Dodecyl Sulfate

SDS

10ng/ml

Triton-X-100

Trton-X

0.0001%

Zinc Sulphate

Zn S04

0.1uM

Dimethyl sulfoxide

DMSO

0.0001%

Glutamic Acid

LGA

0.1uM

AHergerss

Penicillin

Pen

100ug/ml

Amoxicillin

Amox

100ug/ml

Lamotrigine

Lam

100ug/ml

Neomycin

Neo

10ug/ml

Benzocaine

Benz

10ug/ml

Non-Ailergens

Simvastatin

SimV

10ug/ml

Methotrexate

MTX

10ug/ml

Metformin

Met

10ug/ml

A viability assay was performed using concentrations of both allergens and non-allergens and sensitizers and non-sensitizers to determine the optimal concentration for tests. Concentrations subsequently selected for use in the modified proliferation assay are noted 10 in table 1.

12

Viability assay

A dye exclusion method can be used to investigate cell viability. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan 5 blue, eosin, or propidium, whereas dead cells do not. Cells are treated with different concentrations of the test substance for a period of 24 hours. Cells are harvested and an aliquot of the cell suspension is mixed with trypan blue (1:1) and then visually examined to determine whether cells take up or exclude dye. The trypan blue chromopore is negatively charged and cannot react with a cell unless the cell membrane is damaged, therefore a 10 viable cell has a clear cytoplasm whereas a non-viable cell has a blue cytoplasm. A total of 100 cells are counted. The unstained (viable) and stained (non-viable) cells are counted separately using a haemocytometer and viability recorded. By culturing cells in the presence of the test reagents any adverse effect of the reagents on cell viability can be observed. Cell viability of 70% or more is regarded as adequate for the methods of the 15 present invention. Preferably, the cell viability is 75% or more or 80% or more.

Preparation of Peripheral Blood Mononuclear Cells (PBMC)

Peripheral Blood Mononuclear cells (PBMC) from blood obtained from healthy volunteers was prepared by density-gradient centrifugation using Lymphoprep™ solution (Axis-20 Shields) and diluted 1:6 in Phosphate Buffered Saline (PBS) (Lonza BioWhittaker, Belgium). Mononuclear cells were collected from the density mediunrplasma interface and washed three times in cold PBS and counted using an Improved Neubauer cell counting chamber (Weber Scientific International Ltd., UK). Cell viability was assessed by trypan blue (Gibco).

25

Separation ofCD14+ Monocytes using the MACS® technology

The MACS® (Magnetic-activated cell sorting) technology (Miltenyi Biotec) uses columns filled with magnetic particles to separate magnetically labelled cells. For the separation process these columns are placed in a strong magnetic field (QuadroMACS® separator). 30 Required amount of PBMC were transferred to a fresh 50ml falcon tube, topped up with PBS and strained through a 100)jm nylon filter to remove any clumps. 100 x 106 mononuclear cells were washed and re-suspended in 800|jl cold MACS buffer (PBS containing 0.5% FCS and 1mM ethylenediaminetetraacetic acid (EDTA)). The cells were incubated at 2-8°C for 20 minutes with 100|jl CD14 antibody coupled with magnetic 35 microbeads. The cell suspension was added to the column allowing the negative cells to pass through for collection (as the "T cell" fraction and the positive cells (CD14+) were then collected and assessed for purity by flow cytometry analysis.

13

Generation of monocyte-derived dendritic cells (moDC)

CD14 positive monocytes purified by MACS® separation were cultured in a 24 well plate at a density of 0.5 x 106/ml in culture medium with 50ng/ml GM-CSF and 50ng/ml IL-4. 5 After 3 days 400|jl of the medium were carefully removed and 500|jl fresh medium containing 50ng/ml GM-CSF and 50ng/ml IL-4 (Immunotools) were added and left for a further 3 days. After 6 days immature antigen presenting cells dendritic cells (DC) were either collected or allowed to mature by adding lipopolysaccharides (LPS) (0.1 |jg/ml, Sigma), IL-13 (10ng/ml, Immunotools) and TNFa (10ng/ml, Immunotools) for a further 24 10 hours.

Generation of Mature Fast DC

CD14 positive selection cells were put into culture (0.5x106 cells per well in 24 well plate) with RP-10 medium supplemented with IL-4 (50ng/ml) and GM-CSF (50ng/ml). After 24 15 hours maturation cytokines TNF-a (10ng/ml), IL-13 (10ng/ml), IL-6 (10ng/ml), 1uM PGE2, Resiquimod (2.5jjg/ml), CD40L (1 jjg/ml) and LPS (0.1 jjg/ml) were added to each well for a further 24 hours.

Flow Cytometric Analysis

20 7-Aminoactinomycin D (7-AAD) was used to assess viability and CD3 for the presence of T cells. Samples were incubated for 20 minutes at 4°C. Subsequently, the cells were washed again as described above and then re-suspended in 300|jl FACS buffer. In instances where 7-AAD was used, this was added prior to Flow Analysis. Cells were analyzed using the FACSCalibur™ flow cytometer (BD Biosciences) coupled with BD CellQuest™ 25 software. All final data interpretation was performed using FlowJo™ software (version 7.6). The CBA Flex BD kit was used to carry out the preparation for the measurement of IFN-y. Preparation procedures for standards and samples using mixed capture beads were followed according to the manufacturer's instructions provided with the kit. 50|jl of mixed capture beads were added to each test tube, along with 50|jl of serial standard solution to 30 the standard tubes or 50|jl of the sample to the sample vials. The tubes were mixed and incubated in the absence of light for 1 hour. After the incubation 50|jl of mixed detection reagent was added to each tube and they were incubated for a further 2 hours in the absence of light. 1 ml of wash buffer was added to each tube and they were spun at 400g for 5 minutes, the supernatant was discarded and 300|jl of wash buffer was added. Cells 35 were then passed through a flow cytometer (Facs Canto ll)(BD Biosciences) running BD CellQuest™ software. Data interpretation was carried out using FlowJo™ software (v7.6).

14

T Cell Proliferation Assays

Immature cells from both allogeneic and autologous sources in triplicate at a ratio of 1:10 (200|jl total volume) in 96-well round-bottomed plates for 5 days at 37°C in a humidified 5% C02 in air incubator. After 5 days, 40|jl of supernatant was removed from the top of 5 each triplicate well and stored at -20°C for further cytokine analysis. [3H]-Thymidine (used at a concentration of 3.7MBq/ml) was then added to each well using appropriate radiation protection methods and allowed to incubate for 16-18 hours at 37°C in a humidified 5% C02 in air incubator. Cells were harvested and subsequently counted using the 1450 MicroBeta TriLux Microplate Scintillation and Luminescence Counter (PerkinElmer®). Data 10 was interpreted using Graphpad Prism® software.

Treatment of moDC with sensitizers and non-sensitizers / allergens and non-sensitizers

T cell proliferation response to sensitizers and non-sensitizers 15 Sensitizing chemicals, NiS04, 2Mercap, Cinn, DNCB and Eugenol, were chosen. The concentrations of chemicals used was 0.1 |jM DNCB, 15|jM Eugenol, 0.1 |jM NiS04, 0.1 |jM Cinn, and 0.1 |jM 2mercap. Chemicals were dissolved following supplier instructions on solubility. DNCB was dissolved in DMSO (LabScan, Dublin, Ireland), Eugenol was dissolved in 70% ethyl alcohol (Fisher Scientific, UK). NiS04, Cinn, 2Mercap were 20 prepared in dH20.

Non-sensitizing chemicals SDS, LGA, Trton-X, ZnS04, DMSO were chosen. The concentration of chemicals used was 10ng/ml SDS, 0.1 |jM LGA, 0.0001% Trton-X, 0.1 |jM ZnS04, 0.0001% DMSO. SDS, DMSO and Triton X-100 were dissolved in RPMI (Gibco 25 UK). ZnS04, LGA was prepared in dH20.

FastDC's where treated with sensitizers (black) and non-sensitizers (grey) for 24 hours before co-culturing with autologous cells for five days. Cells were pulsed with [3H] thymidine on day five and harvested after 18 hours.

30

As illustrated in Figure 2, untreated FastDC's (negative control) (n=15) and non-sensitizers SDS (n=13) Triton-X (n=6), ZnS04 (n=5), LGA (n=5) and DMSO (n=5) induced low autologous T cell proliferation. Sensitizers Eugenol (n=10), NiS04 (n=6), DNCB (n=6), 2 Mercap (n=4) and Cinnamaldehyde (n=4) induced significantly higher levels of proliferation 35 of autologous T cells in comparison to the negative control (p=0.0005, p=0.002, p=0.01, p=0.006 and p=0.01, respectively). Comparison of the non-sensitizers to sensitizers showed SDS induced significantly lower proliferation of autologous T cells to sensitizers

15

Eugenol, NiS04, DNCB, 2 Mercap and Cinn (p=1.88E-05, p=2.19E-05, p=2.84E-06, p=0.02, and p=0.0005). Triton-X induced significantly lower proliferation of autologous T cells compared to sensitizers Eugenol, NiS04, DNCB, 2 Mercap, Cinn (p=0.0003, p=0.0001, p=9.44E-05, p=0.03 and p=0.001, respectively). Glutamic acid (LGA) induced 5 significantly lower proliferation of autologous T cells compared to sensitizers Eugenol, NiS04 and DNCB and Cinn (p=0.01, p=0.007, p=0.002 and p=0.02). Zinc sulphate (ZnSo4) induced significantly lower proliferation of autologous T cells compared to sensitizers Eugenol, NiS04 and DNCB (p=0.01, p=0.01 and p=0.005). The non-sensitizer DMSO induced significantly lower proliferation of autologous T cells to sensitizers DNCB (p= 10 0.05). Untreated FastDC's cultured with allogenic T cells (positive control) (n=13) showed significantly higher proliferation of T cells compared to all conditions.

T cell proliferation response to allergens and non-allergens

Allergens Lam, Amox, Pen, Neo and Benz were chosen. The concentrations of chemicals 15 used was 100 jjg/ml Lam, 100 jjg/ml Amox, 100 jjg/ml Pen, 10 jjg/ml Neo and 100 jjg/ml Benz. Lam was prepared in DMSO. Amox was prepared in 1M NH40H. Pen was prepared in dH20. Neo was prepared in RPMI (Gibco UK). Benz was prepared in ethanol.

Non-allergens SimV, Met, and MTX were chosen. The concentration of chemicals used 20 was 10|Jg/mI for each of SimV, Met, and MTX. MTX was prepared in 0.1 M Sodium hydroxide. Met was dissolved in and SimV was prepared in RPMI (Gibco UK).

FastDC's drug treated with allergens (black) and non-allergens (grey) for 24 hours induce proliferation of autologous T cells. Drug treated FastDC's where co-cultured with autologous cells for five days. Cells were pulsed with [3H] thymidine on day five and 25 harvested after 18 hours.

As illustrated in Figure 3, untreated FastDC's (negative control) (n=13) and non-allergens Metformin (n=6), MTX (n=5) and Simvastatin (n=4) induced low autologous T cell proliferation. Allergens Penicillin (Pen) (n=10), Amoxicillin (Amox) (n=10) and Lamotrigine 30 (Lam) (n=10), Neomycin (Neo) (n=5) and Benzocaine (Benz) (n=4) induced significantly higher levels of proliferation of autologous T cells in comparison to the negative control (p=3.47E-11, p=9.38E-08, p=1.08E-13, p=7.02E-13 and p=1.93E-15, respectively). Comparison of the non-allergen to allergens showed Lamotrigine induced significantly higher proliferation of autologous T cells compared to Metformin (p=2.22E-05), MTX 35 (p=1.07E-11) and Simvastatin (p=0.01). Amoxicillin induced significantly higher proliferation of autologous T cells compared to Metformin (p=0.04) and MTX (p=0.0001). Penicillin induced significantly higher proliferation of autologous T cells compared to

16

Metformin (p=0.0007) and MTX (p=3.99E-08). Neomycin induced significantly higher proliferation of autologous T cells compared to Metformin (p=2.02E-05), MTX (p=5.60E-11) and Simvastatin (p=0.0004). Benzocaine induced significantly higher proliferation of autologous T cells compared to Metformin (p=8.19E-07), MTX (p=1.92E-12) and 5 Simvastatin (p=2.37E-06). Untreated FastDC's cultured with allogenic T cells (positive control) (n=12) showed significantly higher proliferation of T cells compared to all non-allergens and all allergens with exception to Benzocaine.

IFN-y expression response to allergens and non-allergens 10 Allergens Lam, Amox, Pen, Neo and Benz were chosen. The concentrations of chemicals used was 100 jjg/ml Lam, 100 jjg/ml Amox, 100 jjg/ml Pen, 10 jjg/ml Neo and 10 jjg/ml Benz.

Non-allergens SimV, Met, and MTX were chosen. The concentration of chemicals used 15 was 10|Jg/mI for each of SimV, Met, and MTX.

Drug treated FastDC's with allergens (black) and non-allergens (grey) were co-cultured with autologous cells for five days. Supernatants were collected and Interferon-y levels were measured by FACs analysis (flow cytometry).

20

As illustrated in Figure 4, untreated FastDC's (negative control) (n=13) and non-allergens Metformin (n=6), MTX (n=5) and Simvastatin (n=4) produced low levels of IFN-y. FastDC's treated with allergens Lamotrigine (n=9), Amoxicillin (n=6), Penicillin (n=8), Neomycin (n=5) and Benzocaine (n=4) produced significantly higher levels of IFN-y compared to the 25 negative control (p=0.04, p=3.91E-06, p=0.003, p=9.42E-06 and p=2.67l-06). Comparison between the allergens and non-allergens showed Amoxicillin produced significantly higher IFN-y levels when compared to Metformin, MTX and Simvastatin (p=0.004, p=0.003, p=0.004). Penicillin produced significantly higher IFN-y levels when compared to Metformin (p=0.05). , Neomycin produced significantly higher IFN-y levels when compared 30 to Metformin, MTX and Simvastatin (p=0.03, p=0.006 and p=0.008) and Benzocaine produced significantly higher IFN-y levels when compared to MTX and Simvastatin (p=0.002 and p=0.009). Untreated FastDC's cultured with allogenic T cells (positive control) (n=9) showed significantly higher IFN-y levels compared to untreated FastDC + autologous cells (p=0.006).

35

IFN-y expression to sensitizers and non-sensitizers

17

Sensitizing chemicals, NiS04, 2Mercap, Cinn, DNCB and Eugenol, were chosen. The concentrations of chemicals used was 0.1 |jM DNCB, 15|jM Eugenol, 0.1 |jM NiS04, 0.1 |jM Cinn, and 0.1 |jM 2mercap. Chemicals were dissolved following supplier instructions on solubility. DNCB was dissolved in DMSO (LabScan, Dublin, Ireland), Eugenol was 5 dissolved in 70% ethyl alcohol (Fisher Scientific, UK). NiS04, Cinn, 2Mercap were prepared in dH20.

Non-sensitizing chemicals SDS, LGA, Trton-X, ZnS04, and DMSO were chosen. The concentration of chemicals used was 10ng/ml SDS, 0.1 |jM LGA, 0.0001% Trton-X, 0.1 |jM 10 ZnS04, 0.0001% DMSO. SDS and Triton X-100 were dissolved in RPMI (Gibco UK). SDS and Triton X-100 and DMSO were dissolved in RPMI (Gibco UK). ZnS04, LGA was prepared in dH20.

Drug treated FastDC's with sensitizers (black) and non-sensitizers (grey) were co-cultured 15 with autologous cells for five days. Supernatants were collected and Interferon-y levels were measured by FACs analysis (flow cytometry).

As illustrated in Figure 5, T cells activated in response to sensitizers produced significantly higher IFN-y levels compared to non-sensitizers. Eugenol produced significantly higher 20 IFN-y levels compared SDS, Triton-X and ZnS04(p=0.02, p=0.05 and p=0.04) while sensitizer NiS04 produced significantly higher IFN-y levels compared to SDS, Triton-X and ZnS04 and LGA (p=0.02, p=0.004, p=0.0001 and p=0.003). Sensitizer DNCB produced significantly higher IFN-y levels compared to all the non-sensitizers (SDS p=0.0006, Triton-X p=8.90E-05, LGA p=0.0003, ZnS04 p=3.52E-05, and DMSO p=0.02). Cinnemaldehyde 25 produced significantly higher IFN-y levels compared to Triton-X and ZnS04 (p=0.02 and p=0.001). No IFN-y production was detected for2-Mercap.

18

Correlation of T cell proliferation and IFN-y expression with histological changes in skin explants

Skin Explant Assay

The results obtained using the methods and kits of the present invention relating to T cell 5 proliferation and IFN-y expression correlate to results obtained using a skin explants assay. The skin explant assay consists of co-incubating the treated and untreated DC cells with T cells from the same donor for 7 days. After this time the T cells are added in 96 well plates to sections from a 4mm skin biopsy from the same donor. The skin is co-incubated for three days and then routinely stained for histopathology. Skin incubated with medium 10 alone or autologous cells alone are used as controls. The skin is then routinely sectioned and stained for histopathological damage (grades 0-IV) using a criteria which is very similar to that used and observed in the clinical setting with distinct pathological damage observed from grades 1 -IV (Figure 1).

15 Histopathological grading criteria are described in Table 2 below.

Table 2

Grade 0

Normal Skin

Grade 1

Mild vacuolization of the epidermal cells with occasional dyskeratotic bodies.

Grade II

Diffuse vacuolization of basal cells with scattered dyskeratotic bodies.

Grade III

Sub-epidermal cleft formation.

Grade IV

Complete epidermal separation.

Correlation between levels of IFN-y production and levels of histological damage 20 As illustrated in Figure 6, a positive correlation was observed between increased levels of IFN-y and the level of positive grades in the Skin explants assay. Correlation coefficient was determined as 0.57 with a significance of p=0.00 (n=54). These data were generated using control samples treated with the following sensitizers - Eugenol, DBCB, 2 Mercap, NiS04, Cinn; non-sensitizers- SDS, Triton-X, DMSO, LGA, ZnS04; and allergens -25 Pencillin, Amoxicillin, Lamotrigine and Neomycin.

Correlation between levels of T cell proliferation and levels of histological damage As illustrated in Figure 7, a significant positive correlation was observed between increased levels of T cell proliferation and positive grades in the Skin explant assay. 30 Correlation coefficient was determined as 0.53 with a significance of p=0.00 (n=57). These data were generated using control samples treated with the following sensitizers Eugenol,

19

DBCB, 2 Mercap, NiS04 and Cinn; non-sensitisers- SDS, Triton-X, DMSO, LGA, ZnS04; and allergens- Pencillin, Amoxicillin, Lamotrigine and Neomycin.

Correlation between levels of IFN-y production and levels of histological damage 5 As illustrated in Figure 8, a positive correlation was observed between increased levels of IFN-y and the level of positive grades in the Skin explant assay. Correlation coefficient was determined as 0.918 with a significance of p=0.000 (N=16). These data were generated using control samples treated with DNCB, control samples treated with NiS04, control samples treated with Triton-X and control samples treated with ZnS04.

10

Correlation between levels of T cell proliferation and levels of histological damage As illustrated in Figure 9, a significant positive correlation was observed between increased levels of T cell proliferation and positive grades in the Skin explant assay. Correlation coefficient was determined as 0.769 with a significance of p=0.000 (N=70). 15 These data were generated using control samples treated with DNCB, control samples treated with NiS04, control samples treated with Trton-X and control samples treated with ZnS04.

Correlation between levels of IFN-y expression and LLNA mouse model data 20 The mouse local lymph node assay (LLNA) is now in widespread use for the evaluation of skin sensitization potential. The test measures cellular proliferation as a function of in vivo radioisotope incorporation into the DNA of dividing lymphocytes. It provides classification of test compounds as either non-sensitizer, weak, moderate, strong or extreme sensitizers.

25 Table 3 demonstrates the correlation between IFN-y expression, histological explants grading and LLNA classification for sensitizers and non-sensitizers.

Table 3

Chemical Sensitizers

L i..

Skin Explant

Result

INF-y Mean

DNCB

Extreme

Grade III

x4

367.23

Grade II

x2

Cinnamaldehyde

Moderate

Grade III

x1

135.23

Grade 1

X2

MBT

Moderate

Grade III

x1

0

Grade II

x1

Pencillin g

Weak

Grade III

x3

391.45

20

Grade II

x1

Engenol

Weak

Grade III

x3

289.16

Grade II

x1

Nickel Sulphate

Negative

Grade III

x1

234.06

Grade II

x3

Grade 1

x1

Nors Senzitisers

Zinc Sulphate

Negative

Grade 1

x2

13.92

Gradel/ll x1

SDS (Sodium Dodecl Sulphate)

Grade 0/1

x3

60.03

Grade l/ll x1

Grade II

x2

LGA (glutamic Acid)

Grade 1

x2

63.15

DMSO

Grade 1

X2

164.40

Grade II

x1

Triton-x

Grade 1

x4

41.85

The above data clearly demonstrates that the novel and inventive methods and kits of the invention provide data confirming the sensitizing and/or allergenic properties of a test compound which is comparable and correlates with data generated using skin explants 5 and animal models. The method of the invention has proven it is a viable in vitro alternative to the traditional in vivo model and effectively assesses the potential a chemical has to induce hypersensitivity responses, the potential a drug has to induce allergenicity responses and the potential to predict immune responses. Moreover the fact that the methods and kits do not require skin explants but rather can be performed on blood 10 samples provides a clear technical advantage.

The assay of the present invention provides, to our knowledge, the first demonstration that a sensitivity and/or allergic reactions in blood samples can be used to discriminate between sensitizers and non-sensitizers and/or allergens and non-allergens.

15

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the

21

plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

5 Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or 10 process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel 15 combination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

20

22

Claims

1. An in vitro method of identifying chemical compounds that are sensitizers or non-sensitizers and /or allergens or non-allergens, the method comprising:

5 (i) separating a population of monocyte-derived dendritic cells from a donor blood sample comprising a population of T cells;

(ii) incubating the monocyte-derived dendritic cells with a test compound;

10 (iv) determining the level of T cell proliferation and / or IFN-y expression in the sample of (iii), wherein the level of T cell proliferation and / or IFN-y expression correlates with a defined grade of histological change observed in a skin explant treated with said test compound; and

(v) comparing the level of T cell proliferation in the sample with the level of T 15 cell proliferation in at least one control sample treated with a control sensitizer compound and at least one control sample treated with a control non-sensitizer compound and /or at least one control sample treated with a control allergen compound and at least one control sample treated with a control non-allergen compound; or

(vi) comparing the level of IFN-y expression in the sample with the level of IFN-20 y expression in at least one control sample treated with a control sensitizer compound and at least one control sample treated with a control non-sensitizer compound and /or at least one control sample treated with a control allergen compound and at least one control sample treated with a control non-allergen compound,

wherein comparison of T cell proliferation and/or IFN-y expression in the sample 25 with T cell proliferation and/or IFN-y expression in the control samples identifies the test compound as a sensitizer or non-sensitizer and/or an allergen or non-allergen.

2. The method according to claim 1, wherein the level of T cell proliferation in the skin explant treated with said compound correlates with an LLNA class observed in a LLNA

30 mouse model treated with said compound.

3. The method according to claim 1, wherein the level of IFN-y expression in the skin explant treated with said compound correlates with a LLNA class observed in a LLNA mouse model treated with said compound.

35

23

4. The method according to any one of claims 1 to 3, wherein each of said control compounds is administered in a concentration such that at least 70% of treated cells remain viable 24 hours after exposure to the compound.

5 5. The method according to claim 4, wherein at least 75% of treated cells or at least 80% of treated cells remain viable 24 hours after exposure to the compound.

6. The method according to any preceding claim, wherein the control sensitizer compound is selected from a group consisting of dinitrochlorobenzene, nickel sulphate,

10 eugenol, cinnamaldehyde, and 2-mercaptobenzothiazole.

7. The method according to claim 6, wherein dinitrochlorobenzene is administered at a concentration of 0.1 |jM.

15 8. The method according to any one of claims 6 or 7, wherein nickel sulphate is administered at a concentration of 0.1 |jM.

9. The method according to any one of claims 6 to 8, wherein eugenol is administered at a concentration of 15 |jM.

20

10. The method according to any one of claims 6 to 9, wherein cinnamaldehyde is administered at a concentration of 0.1 |jM.

11. The method according to any one of claims 6 to 10, wherein 2-25 mercaptobenzothiazole is administered at a concentration of 0.1 |jM.

12. The method according to any preceding claim, wherein the control non-sensitizer compound is selected from a group consisting of sodium dodecyl sulphate, triton-X-100, zinc sulphate, dimethyl sulfoxide, and glutamic acid.

30

13. The method according to claim 12, wherein sodium dodecyl sulphate is administered at a concentration of 10 ng/ml.

14. The method according to any one of claims 12 or 13, wherein triton-X-100 is 35 administered at a concentration of 0.0001%.

24

15. The method according to any one of claims 12 to 14, wherein zinc sulphate is administered at a concentration of 0.1 |jM.

16. The method according to any one of claims 12 to 15, wherein dimethyl sulfoxide is 5 administered at a concentration of 0.0001%.

17. The method according to any one of claims 12 to 16, wherein glutamic acid is administered at a concentration of 0.1 |jM.

10 18. The method according to any preceding claim, wherein the control allergen compound is selected from a group consisting of penicillin, amoxicillin, lamotrigine, neomycin, and benzocaine.

19. The method according to claim 18, wherein penicillin is administered at a 15 concentration of 100 jjg/ml.

20. The method according to any one of claims 18 or 19, wherein amoxicillin is administered at a concentration of 100 |jg/ml.

20 21. The method according to any one of claims 18 to 20, wherein lamotrigine is administered at a concentration of 100 jjg/ml.

22. The method according to any one of claims 18 to 21, wherein neomycin is administered at a concentration of 10 jjg/ml.

25

23. The method according to any one of claims 18 to 22, wherein benzocaine is administered at a concentration of 10 jjg/ml

24. The method according to any preceding claim, wherein the control non-allergen 30 compound is selected from a group consisting of simvastatin, methotrexate and metformin.

25. The method according to claim 24, wherein simvastatin is administered at a concentration of 10 |Jg/mI.

35 26. The method according to any one of claims 24 or 25, wherein methotrexate is administered at a concentration of 10 jjg/ml.

25

27. The method according to any one of claims 24 to 26, wherein metformin is administered at a concentration of 10 jjg/ml.

28. The method according to any preceding claim, wherein the level of T cell 5 proliferation is determined by [3H] thymidine incorporation.

29. The method according to any preceding claim, wherein IFN-y expression is determined by flow cytometry.

10 30. The method according to any preceding claim wherein the donor blood sample is a human blood sample.

31. The method according to any preceding claim, wherein the monocyte-derived dendritic cells are obtained from peripheral blood mononuclear cells (PBMC).

15

32. The method according to claim 31, wherein the monocyte-derived dendritic cells are generated from CD14+ monocytes from said PBMCs.

33. The method according to any preceding claim wherein the dendritic cells are 20 either standard or fast matured dendritic cells.

34. The method according to any preceding claim wherein the first incubating step of step (ii) is for between 2 to 24 hours.

25 35. The method according to any preceding claim, wherein the incubation of step (ii) comprises incubation with 10% heat inactivated fetal calf serum.

36. The method according to any preceding claim, wherein the second incubating of step (iii) is for between 3-7 days.

30

37. The method according to any preceding claim, wherein the incubation of step (iii) comprises incubation with 10% heat inactivated autologous serum.

35

38. The method according to any preceding claim, wherein the control sample comprises a further or second set of monocyte-derived dendritic cells that have been incubated in step (ii) with the control compound(s).

26

39. The method according to claim 38, wherein said further or second set of monocyte-derived dendritic cells are separated from the donor blood sample of step (i).

40. A kit for identifying chemical compounds that are sensitizers or non-sensitizers 5 and /or allergens or non-allergens, the kit comprising:

(i) at least one control sensitizer compound and at least one control non-sensitizer compound and/or at least one control allergen compound and at least one control non-allergen compound; and

(ii) instructions for use of the control compounds in the method according to any one of 10 claims 1 to 39.

41. The kit according to claim 40, wherein the kit further comprises a means for correlating T cell proliferation with a defined grade of histological change.

15 42. The kit according to claim 40, wherein the kit further comprises a means for correlating IFN-y expression with a defined grade of histological change.

43. The kit according to any one of claims 40 to 42, wherein the defined grade of histological change correlates with a LLNA class.

20

44. The kit according to any one of claims 40 to 43, wherein each of said control compounds is provided in a concentration such that at least 70% of cells exposed to the compound remain viable 24 hours after exposure thereto.

25 45. The kit according to claim 44, wherein at least 75% of cells exposed to the compound or at least 80% of cells exposed to the compound remain viable 24 hours after exposure thereto.

46. The kit according to any one of claims 40 to 45, wherein the control sensitizer 30 compound is selected from a group consisting of dinitrochlorobenzene, nickel sulphate,

eugenol, cinnamaldehyde, and 2-mercaptobenzothiazole.

47. The kit according to claim 46, wherein dinitrochlorobenzene is provided at a concentration of 0.1 |jM.

35

48. The kit according to any one of claims 46 or 47, wherein nickel sulphate is provided at a concentration of 0.1 |jM.

27

49. The kit according to any one of claims 46 to 48, wherein eugenol is provided at a concentration of 15 |jM.

5 50. The kit according to any one of claims 46 to 49, wherein cinnamaldehyde is provided at a concentration of 0.1 |jM.

51. The kit according to any one of claims 46 to 50, wherein 2-mercaptobenzothiazole is provided at a concentration of 0.1 |jM.

10

52. The kit according to any one of claims 40 to 51, wherein the control non-sensitizer compound is selected from a group consisting of sodium dodecyl sulphate, triton-X-100, zinc sulphate, dimethyl sulfoxide, and glutamic acid.

15 53. The kit according to claim 52, wherein sodium dodecyl sulphate is provided at a concentration of 10 ng/ml.

54. The kit according to any one of claims 52 or 53, wherein triton-X-100 is provided at a concentration of 0.0001%.

20

55. The kit according to any one of claims 52 to 54, wherein zinc sulphate is provided at a concentration of 0.1 |jM.

56. The kit according to any one of claims 52 to 55, wherein dimethyl sulfoxide is 25 provided at a concentration of 0.0001%.

57. The kit according to any one of claims 52 to 56, wherein glutamic acid is provided at a concentration of 0.1 |jM.

30 58. The kit according to any one of claims 40 to 57, wherein the control allergen compound is selected from a group consisting of penicillin, amoxicillin, lamotrigine, neomycin, and benzocaine.

59. The kit according to claim 58, wherein penicillin is provided at a concentration of 35 100 (jg/ml.

28

60. The kit according to any one of claims 58 or 59, wherein amoxicillin is provided at a concentration of 100 jjg/ml.

61. The kit according to any one of claims 58 to 60, wherein lamotrigine is provided at 5 a concentration of 100 jjg/ml.

62. The kit according to any one of claims 58 to 61, wherein neomycin is provided at a concentration of 10 jjg/ml.

10 63. The kit according to any one of claims 58 to 62, wherein benzocaine is provided at a concentration of 10 jjg/ml.

64. The kit according to any one of claims 40 to 63, wherein the control non-allergen compound is selected from a group consisting of simvastatin, methotrexate and metformin.

15

65. The kit according to claim 64, wherein simvastatin is provided at a concentration of 10 jjg/ml.

66. The kit according to any one of claims 64 or 65, wherein methotrexate is provided 20 at a concentration of 10 jjg/ml.

67. The kit according to any one of claims 64 to 66, wherein metformin is provided at a concentration of 10 jjg/ml.

25 68. The kit according to any one of claims 40 to 67, further comprising means for separating monocytes from a blood sample and instructions for use thereof.

69. The kit according to claim 68, wherein said means for separating monocytes comprises a CD14+ cell separation kit.

30

70. The kit according to any of claims 40 to 69, further comprising a means for fast dendritic cell maturation and instructions for use thererof.

71. The kit according to any one of claims 40 to 70, wherein the at least one control 35 sensitizer compound and at least one control non-sensitizer compound and/or at least one control allergen compound and at least one control non-allergen compound are located in defined positions on a solid support.

29

72. The kit according to claim 71, wherein said solid support is a 6, 12, 24, 48 or 96 well plate.

5 73. The kit according to any one of claims 40 to72 wherein said kit comprises at least two control sensitizer compounds, wherein said compounds are DNCB and NiS04.

74. The kit according to any one of claims 40 to73 wherein said kit comprises at least two control non-sensitizer compounds, wherein said compounds are Trton-X and ZnS04.

10

75. The method according to any one of claims 1 to 39 wherein said at least one control sample treated with a control sensitizer compound comprises:

(i) a first control sample treated with DNCB; and

(ii) a second control sample treated with NiS04.

15

76. The method according to any one of claims 1 to 39 and 75, wherein said at least one control sample treated with a control non-sensitizer compound comprises:

(i) a first control sample treated with Trton-X; and

(ii) a second control sample treated with ZnS04.

20

77. The method according to any one of claims 1 to 39, 75 and 76, wherein the level of T cell proliferation and / or IFN-y expression in a control sample treated with a control sensitizer compound, a control non-sensitizer compound, a control allergen compound or a control non-allergen compound correlates with a defined grade of histological change

25 observed in a skin explant treated with said compound.

78. The method according to any one of claims 1 to 39, 75 and 76, wherein the level of T cell proliferation and / or IFN-y expression in a control sample treated with a control sensitizer compound, a control non-sensitizer compound, a control allergen compound or a

30 control non-allergen compound correlates with a LLNA class observed in a LLNA mouse model treated with said compound.

79. An in vitro method of identifying chemical compounds that are sensitizers or non-sensitizers and /or allergens or non-allergens substantially as described herein with

35 reference to the accompanying drawings and examples.

30

80. A kit for identifying chemical compounds that are sensitizers or non-sensitizers and /or allergens or non-allergens substantially as described herein with reference to the accompanying drawings and examples.

10

•.'????.• INTELLECTUAL

*.*. .V PROPERTY OFFICE

31

Application No: GB1115537.1 Examiner: Dr Philip Mountjoy

Claims searched: 1-80 Date of search: 6 January 2012

Patents Act 1977: Search Report under Section 17

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