ES2366971T3 - TESTS OF CELLS T. - Google Patents

Abstract

A method for measuring a collaborating T cell response to a test substance comprising the following steps: (a) isolating antigen presenting cells (APCs) and T cells from a sample obtained from an organism; (b) deplete regulatory T cells from isolated cells; (c) incubating said APCs and depleted regulatory T cell cells obtained in item (b) with the test substance; and (d) test the T cell responses to the test substance.

Description

The present invention relates to new T cell test procedures, in particular where T cell responses to test antigens are increased by the elimination of regulatory T cells. The present invention also relates to new assays where incubation time with antigens or other samples is varied to optimize the detection of T-cell responses. In particular, the invention relates to T-cell assays with proteinaceous samples where optimal detection of T cell epitopes are achieved by utilizing multiple time point measurements of T cell proliferation or cytokine release. In addition, the invention relates to T cell assays where the incubation time with antigen with antigen presenting cells (APCs) or T cells or both APCs and T cells is varied to optimize the detection of T cell epitopes. The invention particularly refers to T-cell assays with immunomodulatory or toxic samples that directly affect APCs, T-cells or both APCs and T-cells. The invention has particular application for the measurement of human T-cell responses to pharmaceuticals, allergens, irritants or others. substances contacted by man.

T-cell assays provide an effective procedure to measure T-cell responses to antigens and other samples, especially in humans. Such assays are considered as "ex vivo" assays where blood samples are taken from donors and processed in such a way that primary cultures of blood cells are used directly in said assays. For proteinaceous and peptide samples, ex vivo human T cell assays have been used to detect human T cell epitopes for various purposes including the evaluation of the potential immunogenicity of such samples in man (Jones et al., L., J. Interferon Cytokine Res., Volume 24 (2004) p560-572), which defines epitopes of T cells within a protein sequence for subsequent inclusion in vaccines, and what defines epitopes of T cells within a protein sequence for elimination later to avoid immunogenicity (Jones et al., J., Interferon Cytokine Res., volume 24 (2 (2004) page 560572, and Jones et al, Ja Thromb. Haemost., volume 3 (2005) page 1-10) Current T-cell assay procedures broadly include incubating proteinaceous or peptide samples with a mixture of APCs and T cells prior to the measurement of T-cell responses, or incubating proteinaceous or peptide samples with APCs and then s add T cells prior to the measurement of T cell responses. In both types of assay, multiple blood samples are used individually for the parallel assay of each individual proteinaceous or peptide sample, and the T cell responses are then routinely measured in A single point of time. T cell responses are typically measured by the incorporation of a radioactive tag pulse such as tritiated thymidine (3HTdR) into proliferating T cells ("Proliferation of T cells" or by the release of cytokines such as IL-2 from activated T cells ("cytokine release").

Current T-cell assay procedures for detecting T-cell epitopes are limited by one or both of poor sensitivity and / or interference due to toxic or immunomodulatory samples that inhibit, stimulate or otherwise modify APCs, T cells or both. APCs and T-cells As such, current T-cell assay procedures may not detect some or all of T-cell epitopes in certain proteinaceous and peptide samples and may not be applicable to the measurement of T-cell responses to toxic samples. or immunomodulators including proteinaceous and peptide samples, non-proteinaceous samples including organic molecules, and formulations of non-proteinaceous and proteinaceous samples where the same formulation can be immunomodulatory or toxic.

In relation to sensitivity, a primary cause of poor sensitivity in ex vivo T-cell assays may be related to factors in the assay mixture that reduce T-cell responses or test antigens including cell types or factors within the test culture. or by the test antigen or the same test samples. Other causes of poor sensitivity in ex vivo T cell assays may be related to the kinetics of T cell responses to T cell epitopes within proteinaceous or peptide samples whereby individual T cell epitopes can induce T cell responses. at different times For T cell proliferation where a single time point is used, T cell proliferation with the addition of certain samples may, on the other hand, be initially rapid but then reduced at the time when a radioactive tag pulse is added in such a way that no significant proliferation response is detected. On the other hand, the proliferation of T cells with the addition of certain other samples may be initially slow at the time when a radioactive tag pulse is added such that no significant proliferation response is detected even if the subsequent proliferation becomes significant. . For the release of cytokine where a single time point is used, the production of cytokines with the addition of certain samples, on the one hand, may be initially rapid but these cytokines can subsequently be consumed by the cells within the test mixture of such that no cytokine is detected at the single test time point. On the other hand, cytokine release may be initially slow so that no proliferation response is detected at the single test time point even if the subsequent release of cytokines becomes significant. The kinetics of cytokine proliferation or release can be influenced by a range of factors such as allotypic variation in T-cell responses between different blood samples, the efficiency and kinetics of APC uptake and processing, proteolysis efficiency of proteinaceous samples or peptides within APCs, strength and frequency of T cell epitopes within a proteinaceous or peptide sample, binding affinity of T cell epitopes to specific MHC class II allotypes, recognition efficiency of MHC class II peptide complexes by T cell receptors, frequency and concentration of co-stimulatory cell surface molecules, costimulatory cytokine concentrations, stimulation of other cells in the test mixture such as CD8 + T cells or suppressor T cells, the presence of memory T cells , and the capacity of some samples such as p peptides small ones to be loaded directly into MHC class II molecules expressed on the surface of APCs.

In relation to the interference of the T-cell assay by the toxic or immunomodulatory samples, said samples may be directly bound or captured by APCs, T cells or both APC and T cells. Said samples may decrease or increase the normal immunological function of APCs and / or T cells such that T cell epitopes or T cell responses to samples are not detected. Other causes of T cell assay interference by immunomodulatory samples is through toxicity to APCs, T cells or both APCs and T cells. Other causes of T cell assay interference by immunomodulatory samples include the reduction or increase of subsets of APCs or T cells such as the increase of CD8 + T cells or suppressor T cells.

In order to usefully exploit T-cell assays for a range of applications especially in relation to human pharmaceuticals, there is a need for more sensitive T-cell assay procedures for optimal detection of T-cell epitopes and also a need. of T-cell assays that can be used with toxic or immunomodulatory samples.

WO 2004/050706 discloses CCR5 and FoxP3 molecules that may be molecules for depletion or recognition in accordance with the present invention. Similarly, Ep-A-1241249 discloses other molecules such as CTLA-4 and CD4. WO97 / 39721 discloses Th2 specific cytokine genes, where the activity of transcription factors thereof can be modulated in accordance with the present invention to regulate its expression. WO99 / 19484 discloses certain molecules associated with the T-cell receptor (TRAMS).

The present invention is based in part on the discovery that the removal of regulatory T cells from the T cell test mixtures results in substantial increases in collaborating T cell responses to test antigens. Thus, the present invention provides new T cell assay procedures for the optimal detection of T cell epitopes where the suppressor T cell is removed from the cultures resulting in an increase in T cell responses to test antigens. In addition, the present invention provides new T-cell assay procedures for the optimal detection of immunogenicity in proteins that modulate T cells and / or APCs, or proteins that have a toxic effect on T cells and / or APC. The present invention can also be applied to the detection of non-proteinaceous compounds that can stimulate T cells directly through the T-cell receptor, or by covalently binding to proteins, or by directly binding to peptides linked by MHC class II molecules, or by binding directly to MHC class II molecules. In particular, the invention provides methods where regulatory T cells that would normally reduce effector T cell responses are removed from cultures prior to measurement of the responses to test antigens. In addition, the invention provides methods with multiple measurement time points where the time points after incubation with antigens or other samples are optimized for the detection of T-cell responses.

In the first aspect, the present invention provides a method for measuring a helper T cell response to a test substance comprising the following steps:

(to)

isolate cells presenting antigens (APCs) and T cells from a sample obtained from an organism;

(b)

deplete regulatory T cells from isolated cells;

(C)

incubating said APCs and depleted regulatory T cell cells obtained in item (b) with the test substance; Y

(d)

test the T cell responses to the test substance.

APCs and T cells are usually obtained from a blood sample. However, different sources of T cells and / or APCs may be used in the invention including those obtained from tonsils, Peyer patches, tumors and cell lines. In a preferable embodiment, the process is carried out by the

5

fifteen

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use of human peripheral blood mononuclear cells (PBMCs).

As used herein the term "depletion" means the removal of some of the regulatory T cells. This can be done by physically removing the cells or by inhibiting or modulating the action of the T cells. Thus the activity of the recognized T cells is reduced. Preferably 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% of the activity of the recognized T cells is eliminated by the depletion process.

Those skilled in the art will understand that, as part of the present invention, a range of procedures for depletion or recognition of regulatory T cells could be used as alternatives to depletion of regulatory T cells under CD25hi. It will also be understood that the present invention will also include methods for modulating the effects of regulatory T cells in T cell assays. For depletion.

or recognition, molecules expressed on the surface of regulatory T cells can be used in conjunction with or as alternatives to CD25 for the depletion of these cells. Such molecules may include but are not limited to GITR, CTLA-4, CD103, chemokine receptor 4 CC, CD62L and CD45RA and may also include surface associated cytokines or surface forms of cytokines such as IL-10 and TGFP. Depletion can be achieved by various methods including binding to specific antibodies to absorb regulatory T cells in a solid phase, or to cause destruction or inhibition of said regulatory T cells, or otherwise separate regulatory T cells from other T cells to T-cell assays For modulation, molecules secreted by regulatory T cells can be prevented from such secretion or they can be blocked / inhibited / destroyed after secretion. Said molecules can include cytokines such as IL10, IL-4, IL-5 and TGFP and said molecules can be blocked by the use of organic or inorganic molecules that bind to said molecules, for example antibodies or soluble receptors, or by inhibitory nucleic acids such as siRNA antisense oligonucleotides, or other nucleic acids administered to regulatory or induced T cells within said cells. Modulation of regulatory T cell activity can also be achieved by locating receptors or other surface molecules in regulatory T cells including but not limited to GITR, CTLA-4. CD103, chemokine receptor 4 CC, CD62L and CD45RA in such a way as to break the suppressive function of these cells. Such inhibition of function can be achieved, for example, by specific antibodies in an agonist function or that can block the target-ligand interactions such that regulatory T cells are not eliminated but become non-functional. Modulation of the activity of regulatory T cells can also be achieved by blocking the white receptors of the molecules secreted by the regulatory T cells or by blocking mechanisms activated or reduced by said segregated molecules. Also for modulation, regulatory T cells can be directly inhibited, for example by blocking transcription factors such as foxp3 or blocking other functions or mechanisms related to regulatory T cells. Such inhibition or blockage can be achieved by organic or inorganic molecules, or by inhibitory nucleic acids such as siRNA, antisense oligonucleotides, or other nucleic acids administered to regulatory or induced T cells within said cells. In all cases where nucleic acid, inorganic or organic molecules are used to inhibit the action of or otherwise modulate regulatory T cells, where said same molecules interfere with T-cell assays, said molecules will preferably be removed from such assays. or modified to a form that will not interfere with such tests. For example, the specific antibodies or proteins used to remove molecules secreted by regulatory T cells will be selectively removed prior to T-cell assays or will be used in a specific manner that will not interfere with T-cell assays. For example, for Human T cell assays, a human form of an antibody or protein will be used to avoid T cell responses to the antibody or the same protein.

In the T-cell assays of the present invention with test antigens that do not modulate T cells and / or APCs (typically proteins or peptides but also non-proteinaceous compounds) the key steps are as follows;

or more time points for T cell proliferation and / or cytokine release

(one)

PBMCs are isolated from human blood samples

(2)

Optionally, CD8 + T cells are removed

(3)

T cells are depleted from CD25hi +

(4)

The cultures are incubated with the test antigens in one or more concentrations and are tested in one

The key steps in the T-cell assays of the present invention where the test antigens do modulate the T / or APCs are the following;

(one)

PBMCs are isolated from human blood samples 4

(2)

APCs are isolated, typically by plastic adhesion, APCs are induced to differentiate them by using cytokines and the test antigen is added to APCs

(3)

Autologous PBMCs, processed by prior removal of CD25hi + T cells and optionally CD8 + T cells, are mixed with APCs

(4) The cultures are incubated with the test antigens in one or more concentrations and tested in one

or more time points for T cell proliferation and / or cytokine release.

When the test substances are proteinaceous and peptide samples or non-proteinaceous samples that are not immunomodulatory or toxic to APCs or T cells, blood can be used as a source of CD4 + T cells and APCs (in the form of monocyte dendritic cells). Typically, a donor cohort is selected that best represents the number and frequency of HLA-DR allotypes expressed in the world population or in the population under study. The allotypes expressed in the cohort are typically> 80% of those expressed in the population with all major HLA-DR alleles (individual allotypes with a frequency> 5% expressed in the world population) being well represented. Alternatively, allotypes expressed in the cohort are chosen to overrepresent or understand HLA allotypes that are thought to be associated with a particular disease under study. In a preferable embodiment of the present invention, the PBMCs are prepared from blood samples by fractionation in density gradients and are then depleted from the CD8 + T cells and CD25hi T cells such that the remaining PBMC mainly comprises the CD4 + T cells (~ 70%) and APCs (10-20% monocytes and 1-3% dendritic cells). Said CDM + CD25hi depleted PBMCs are established in the cell culture or more proteinaceous or peptide samples or non-proteinaceous samples are added and the cultures are incubated.

The measurement of T-cell responses can then be conducted at a fixed time point, or at multiple time points. These time points can be predetermined by measuring the kinetics of T cell responses to similar samples or an optimization substance.

The optimal conditions for an assay can be determined by using an optimization substance. An "optimization substance" as used herein is a compound that is known to induce T-cell responses, such as total toxic / immunomodulatory peptides / proteins, which have a size and structure similar to the samples that they must be tested or with properties similar to the test substance. For proteinaceous or peptide samples or non-proteinaceous samples, one or more peptides (typically 9-40 amino acids in length) or total proteins or non-proteinaceous compounds with a size and structures similar to the samples to be tested as a sample may be used. optimization substance Optimization substances are tested and the results are used to define the kinetics of typical T-cell responses. For example, T-cell responses are measured at various time points, much more commonly between days 4 and 9 after the addition of the sample by using one or more of a range of different alternative tests. Once the kinetics of the T-cell responses to the optimization substance is established, a set of test time points can be defined for the subsequent test of the samples. In this way, T-cell responses to test samples can be tested at one or more appropriate time points. Alternatively, or in addition, two or more concentrations can be used to establish the kinetics of the T-cell responses to the optimization substance, and the samples can then be tested at these concentrations.

The T cell response can be measured by the use of a number of different assays such as T cell proliferation by incorporating a 3HTdR pulse (or other radioactive, fluorescent or chemiluminescent compounds captured by proliferating T cells), cytokine release such such as IL-2 or IFNy, mRNA transcription changes, increased transcription of the mRNA activation marker, Ca2 + flux, and changes in phenotypic markers especially markers for activated T cells. Typically, for proteinaceous or peptide samples, T-cell responses will be measured by incorporating a 3HTdR pulse on days 5, 6, 7, and 8 after sample addition or by measuring cytokine release. (especially IL-2) at 8 days after sample addition (or by incorporation of 3HTdR and cytokine release measurements). As an alternative, especially for peptides with highly overlapping sequences (for example 15mers of a protein sequence with 12 amino acid overlays), the incorporation of a 3HTdR pulse and / or the measurement of cytokine release at a single point of time, typically on day 7 after the addition of the test peptide. Adjacent overlapping peptides possibly contain T cell epitopes that together enhance sensitivity for the detection of T cell epitopes.

When the proteinaceous or peptide samples are immunomodulatory or toxic to the APCs or T cells, the sample obtained from the organism is processed and the APCs are separated from the other cells. This is typically accomplished by adhesion to the plastic, and the proteinaceous or peptide sample is then incubated with these APCs. APCs can be incubated with cytokines such as interleukin 4, macrophage colony stimulating factor and granulocytes, tumor necrosis factor alpha and interleukin 1 alpha to induce a mature APC phenotype. Samples in standard T-cell assays with pre-fractionated APCs will usually require a sample incubation time: APC of up to 48 hours. Preferably, semi-mature APCs are generated by incubation in growth medium containing interleukin 4 and colony stimulating factor of macrophages and granulocytes for up to 4 days. Samples including toxic or immunomodulatory samples are then added to the half-ripe APCs and incubated for a short period. Depending on the toxicity or immunomodulatory function of the sample, incubation times with half-ripe APCs may vary from 3 to 10 hours. After incubation of the sample: APC, the exogenous sample is removed by repeated washing of the half-ripe APCs. The APCs used from the mature sample are then generated by incubation with a pro-inflammatory stimulus such as tumor necrosis factor or interleukin 1 or CD40 ligand or lipopolysaccharide. Autologous T cells, typically CD4 + CD8-CD25hi depleted T cells prepared from PBMCs as above, are added to the pulsed APCs of the mature sample. Depleted CD4 + CD8-CD25hi T cells are incubated with pulsed APCs of mature sample for an additional incubation time point range. An optimization substance as described above can be used to establish the kinetics of the responses with different APC incubation time points and / or different T cell incubation time points. The results obtained with the optimization substance they can be used to define a set of time points for incubation of APCs and / or incubation of T cells for subsequent sample testing. In this way, the T-cell responses to the test samples are detected at one or more of the test time points. Alternatively, or in addition, two or more concentrations can be used to establish the kinetics of the T-cell responses to the optimization substance and the samples can then be tested at these concentrations.

When the sample to be tested is non-proteinaceous, any of the above procedures (i.e., procedures for proteinaceous or peptide samples with or without immunomodulatory or toxic properties) may be used depending on whether the non-proteinaceous sample is immunomodulatory or toxic to APCs , T cells or both.

For non-proteinaceous or proteinaceous samples that are immunomodulatory with respect to APCs, T cells or both, an additional optional step is to test directly for the reduction or increase of phenotypic markers of, for example, the activation of T cells or differentiation of APCs. Typical markers of T cell activation include changes in the expression of CD69, CD25, CTLA4, GITR and the measurement of intracellular Ca2 + flux. Common phenotypic markers used to assess the differentiation of APCs include MHC class II, CD80 and CD86, which are highly expressed in mature APCs. These additional steps may provide information about the kinetics of T-cell responses to test samples that assist in the definition of test time points for optional testing of T-cell responses to test samples.

The new ex vivo T-cell assay procedures of the present invention have a range of applications especially in relation to pharmaceuticals for human use. For proteins for expected use as pharmaceuticals, the T-cell assays of the present invention can be used to identify T-cell epitopes within the protein sequence by testing overlapping peptides of the protein sequence. The location and strength of said T cell epitopes can then be used for the evaluation of the potential immunogenicity of the protein in man. Alternatively, T cell epitopes within the protein can be subsequently removed by mutation of the protein sequence prior to use in man. T cell epitopes within certain proteins can also be identified by methods of the present invention and can then be incorporated into vaccines by including the sequence of T cell epitopes (or variant thereof) within a protein vaccine. for the addition to other components as part of a vaccine.

The new T-cell assays of the present invention can be used for the evaluation of the potential immunogenicity of a range of types of molecules including peptides, proteins and non-proteins, including organic molecules, lipids, carbohydrates or molecules composed of two or more different moieties. including conjugates, mixtures and formulations. The T-cell assays of the present invention have wide application both in research, development, manufacturing and in clinical trials of pharmaceutical products. In research, for example, T cell responses to different analogues of active molecules can be used to assess the potential immunogenicity of these analogs in man. Such T-cell responses can thus be used as criteria for the selection of major pharmaceutical products for further development. In development, for example, T cell responses to different formulations of the same molecule can be determined to assess the potential immunogenicity of these formulations in man. Such T-cell responses can thus be used as criteria for the selection of the optimal formulation for clinical trials. In manufacturing, for example, the T cell responses to the manufacturing batches of the same molecule can be determined to assess the potential immunogenicity of these batches and also to evaluate any changes in the molecule between batches. Such T-cell responses can be used as a quality test for manufacturing. In the clinical trial, for example, T-cell responses can be determined by using the patient's blood, for example, to evaluate the immunogenicity to the pharmaceutical product that goes through the trials. The T-cell assays of the present invention could also be used in clinical trials to determine any MHC restriction of the T-cell responses to the pharmaceutical product.

As an alternative to the use in the detection of T-cell epitopes, the T-cell assay procedures of the present invention can be used to evaluate potential adverse reactions to pharmaceuticals, preferably for human use. These adverse reactions including hypersensitivity, allergy, irritant character, immunosuppression, hyperimmune stimulation and injection site reactions. The T-cell assay procedures of the present invention can also be used to evaluate potential adverse reactions to treatments with non-pharmaceutical products such as transplantation, to environmental agents such as grass pollen allergens, to food, to cosmetics, and a range of industrially produced reagents such as detergents and enzymes.

Those skilled in the art will understand that a range of variations can be used in the T-cell test procedures of the present invention, since these variations will fall within the scope of the invention, for example by using multiple test time points in cell response analysis

T. For example, it will be understood that within the scope there is a range of different procedures known in the art for the analysis of T-cell responses including procedures such as peptide binding to MHC that determines the individual steps towards a cell response. T. T-cell assays can be carried out with enriched APCs for Langerhan cells, different subsets of macrophages or different subsets of APCs, and / or through the use of or enrichment of different subsets of T cells. Cytokines will also be understood as cytokines. could be added to (or removed from) the test mixtures of the T-cell assays of the present invention, for example, to enhance sensitivity or to reduce or increase specific APCs or T-cells. Different formats of cell assays can be used T in the invention, for example memory assay formats where T cells are primed by the presentation of APC of a protein or peptide and then again tested for a related or equal protein or peptide.

The following examples are provided to illustrate the invention and should not be considered as restrictive of the scope of the invention.

Example 1: Effect of CD25 + T cell depletion on T cell responses

Peripheral blood mononuclear cells were isolated from leukocyte layers of healthy community giver (from blood drawn within 24 hours) obtained from the National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK) and in accordance with the approval granted by the Local Research Ethics Committee of the Addenbrooke Hospital. PBMCs were isolated from leukocyte layers by Ficoll density centrifugation (GE Healthcare, Chalfont St Giles, RU) and CD8 + T cells were depleted using CD8 + RossetteSepTM (StemCell Technologies, Vancouver, Canada). The donors were characterized by identifying HLA-DR haplotypes using a tissue typing kit based on SSP-PCR Allset ™ (Dynal, Wirral, UK) as well as determining the T cell responses to a control hemocyanin antigen extracted of mollusk called Californian lapa (KLH) (Pierce, Cramlington, UK), Tetanus toxoid (Aventis Pasteur, Lyon, France) and epitope of control influenza HA peptide (C32, pages 307-319).

The depletion of the CD25hi T cells was carried out through the use of Miltenyi Biotech anti-CD25 micro-accounts (Guildford, UK) using the standard magnet protocol and provider. 10 vials of each donor were thawed and the cells were resuspended in 30mls of 2% inactive human serum / PBS (Autogen Bioclear, Calne, Wiltshire, UK). 5x107 cells were transferred to 3 x 15ml tubes with the remaining cells maintained as total PBMCs. A mixture of anti-CD25 micro-account dilution was performed using 300μl of beads + 4200μl of separation buffer (0.5% human serum / 2mM EDTA / PBS). The 15ml tubes were centrifuged and resuspended in 500 µl of micro-account dilution mixture. The tubes were then kept at 4 ° C for 5, 10 or 20 minutes before separation in the column. The columns were placed upwards by placing the column in the magnet supported on a platform, adding 2mls of separation buffer to the column and allowing it to drip. After incubation with beads, 10ml of separation buffer was added and the tubes were centrifuged at 1500rpm for 7 minutes. The cells were then resuspended in 500μl of separation buffer and added to the column followed by 2 x 1ml washes with separation buffer. Flow through the column was collected in 15ml tubes and contained the depleted fraction of CD25hi T cells. These cells were centrifuged at 1500rpm for 7 minutes and resuspended in 3ml of AIMV medium (Invitrogen, Paisley, UK) before of the count.

Cells were stained for CD4 and CD25 and cell numbers were detected by FACS. 5-10x105 cells from each cell population were placed in a well of a 96-well U-based plate (Greiner Bio-One, Frickenhausen, Germany). The plate was centrifuged at 1200rpm for 4 minutes. The supernatant was expelled and the cells were resuspended in 50 μl of antibody dilution. The antibody dilution consisted of a 1/50 dilution of FITC-labeled anti-CD4 antibody (R&D Systems, Minneapolis, USA) + 1/25 dilution of PE-labeled anti-CD25 antibody (R&D Systems, Minneapolis, USA) in buffer FACS (1% human serum / 0.01% sodium azide / PBS). Control wells were also stained, stained with isotype controls or stained with once labeled antibody.

The plates were incubated on ice for 30 minutes in the dark. The plates were then centrifuged at 1200 rpm for 4 minutes. The supernatant was expelled and the cells were resuspended in 200μl FACS buffer. This was repeated twice and the cells were then transferred to the FACS tubes. The cells were run through a Calibur FACS (Becton Dickinson, Oxford, UK), and the data was collected and analyzed based on size, granularity and fluorescent labels.

Proliferation assays were carried out as follows. Total CD8 + T cell depleted PBMCs and CD8 + CD25hi depleted PBMCs were added at 2 x 105 per well in 100μl of AIMV. When using 96-well flat-base plates, triplicate cultures were established for each test condition. For each peptide 100μl were added to the cell cultures to give a final concentration of 5μM. The cells were incubated with peptides and protein antigens for 7 days before pressing each well with 1mCl / ml of 3HTdR (GE Healthcare, Chalfont St Giles, UK), for 18 hours.

For the proliferation test, a threshold of a stimulation index equal to or greater than 2 (SI≥2) was used whereby peptides that induce proliferative responses above this threshold were considered positive (dotted line). All data were analyzed to determine the coefficient of variance (CV), standard deviation (SD) and significance (p <0.05) using an unpaired T test of a student address. All responses shown with SI≥2 were significantly different (p <0.05) from the mean untreated controls.

The results are shown in Figure 1 representing proliferative T cell responses in PBMCs from three human donors (475, 440 and 462) to a series of weak T cell epitopes or boundaries (peptides 1 (PGQTATITCSGHALG), 2 (GDKFVSWYQQGSGQS) , 6 (IKPEAPGCDASPEELNRYYASLRHYLNLVTRQRY), 9 (QSISNWLNWYQQKPG), 13 (KGLEWLVVIWSDGSS), 17 (AASGFTFSSFGMSWV), 20 (DTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQ -GTQV), 24 (HQSLVIKLMPNITLL) and a pair of strong T cell epitopes (peptides 25 (PKYRNMQPLNSLKIAT) and 26 ( TVFYNIPPMPL) and the KLH antigen The results show an increase in T cell responses for all peptides after depletion of CD25hi T cells Maximum responses were determined for all peptides after 10 or 20 depletion minutes of CD25hi T cells These results demonstrated strong increases in T cell responses after depletion of CD25hi T cells which, in the peptide examples os such as peptides 1 and 2, allowed the detection of T-cell epitopes in peptides previously classified as limits or negative for T-cell responses.

Example 2 - Peptide T cell temporal sequence assays

The depleted peptides of T-cell and wild-type (WT) epitopes (HLRHCLSCSKCRKEM and HARHCLSCSKCRKEM, respectively) obtained from the sequence of human TNFR1s were synthesized (Pepscan Systems, Leystad, The Netherlands) and tested using the procedure of example 1 in that the depleted PBMCs of the CD8 + CD25hi T cells were used to compare the peptides obtained from TNFR-1s in terms of the ability to stimulate the T cell responses of twenty healthy donors. Bulk cultures were established by adding 1ml of 2-4x106 / ml of PBMC depleted CD8 + CD25hi T cells in AIM V culture medium to each well of a 24-well plate (Greiner Bio-One, Frickenhausen, Germany) . Each peptide was tested separately against each donor by adding 1ml of 10μM peptide to each bulk culture (final concentration of 5μM for 2ml per bulk culture). For comparison, additional bulk cultures were established for untreated and positive controls (KLH). Replicated samples (from T blasts) were removed from bulk cultures on days 6-9 and proliferation in round-well 96-well plates was evaluated. Data were used to assess the magnitude and kinetics of T cell responses to each peptide on days 6, 7, 8 and 9 after stimulation. In addition, the same twenty healthy donors used in the temporal sequence proliferation assay were tested for IL-2 production after 8 days of culture with TNFR1 peptides using an Elispot IL-2 assay. Elispot plates were pre-moistened with 70% ethanol then coated with IL-2 capture antibody (R&D systems, Minneapolis, USA) overnight at 4 ° C. The plates were washed twice with PBS (Invitrogen, Paisley, RU) then blocked in 1% BSA / PBS for 2 hours at room temperature. The plates were washed in PBS prior to the addition of depleted PBMC from CD8 + CD25hi T cells to 4X105 cells per well and the test samples in a final concentration of 5μM. After 7 days at 37 ° C / 5% CO2 the plates developed. After first water washing after PBS, the IL-2 detection antibody (R&D systems, Minneapolis, USA) in 1% PBS / BSA was added for 2 hours at 37 ° C. After further washing with PBS, streptavadine-AP (R&D systems, Minneapolis, USA) was added for 1.5 hours, the plates were washed again, then BCIP / NBT chromogen (R&D systems, Minneapolis, USA) was added for 30 minutes . The plates were washed with water, dried, then stain counts were analyzed using Immunospot Elispot analyzer, software version 3 (Cleveland, Ohio, USA).

For both T-cell and Elispot IL-2 proliferation assays, responses that exceeded an SI threshold of 2 (dotted line) and are significantly (p <0.05) different than the background (*) were considered positive. The results shown in Figure 2 indicate that the WT peptide gave responses in the same three human donors (3, 8 and 11) in both proliferation assays and Elispot IL-2m indicating that this peptide contains a cell epitope T. The temporal sequence of proliferation indicated that for these three donors, peak proliferation responses were detected 7 days after the addition of and, in each case, the TNFR1 WT peptide would have been classified negative as a T-cell epitope if Proliferation responses would have been measured at time points 8 or 9 days after peptide addition. These results also show a strong correlation between T cell responses measured by proliferation and Elispot IL-2.

Example 3 - Total protein T-cell temporal sequence assays

Human TNFR1s proteins from wild-type mutant and T-cell epitope (WT) were prepared as human Fc fusion proteins as described in WO / 2004/113387 with the epitope depleted protein possessing I10Q, T20R mutations , H23P, L56A, L108T, L110H and L149D. Proliferation assays and Elispot IL-2 were carried out as in Example 2 except that 1ml of TNFR1s proteins were added to a final concentration of 10μg / ml. The data shown in Figure 3 indicates that, for the proliferation assay, significant T-cell responses were detected in donors 13 and 17 for WT but not the depleted protein of the mutant T-cell epitope. Peak responses were observed on days 8 and 9 and neither donor 13 or 17 showed any significant response on day 6. For the Elispot IL-2 trial, both donors 13 and 17 were again positive for T-cell responses to WT but not the mutated epitope depleted protein. In addition, the donor gives a significant response to the WT protein in this assay. As with Example 2, the results also demonstrate the usefulness of the temporal sequence assay in the detection of T-cell responses, in this case to total proteins. As with Example 2, the results show a good correlation between the T-cell responses measured by proliferation and Elispot IL-2.

Example 4 - Temporary sequence assays of immunomodulatory protein T cells

This example illustrates the invention when used to measure T-cell responses to an immunomodulatory protein, human beta interferon which is known to increase inhibitory molecules in dendritic cells such as HLA-G (Mitsdoerffer M et al J Neuroimmunol. 2005 159: 155-64) and B7-1H (Schreiner B et al J Neuroimmunol. 2004 155: 172-82). To test whether the epitopes of linear T cells present in the IFN beta sequence could stimulate T cells in vitro, a modified method was developed to load the antigen into dendritic cells obtained from monocytes in which the biological effects of IFN beta in dendritic cells (DC) and CD4 + T cells were minimized.

PBMC monocytes were isolated by adhesion to tissue culture plastic (> 90% CD14 +) and grown in 24-well plates in AIM V medium with 5% hot inactive human AB serum (Autogen Bioclear, Calne, Wiltshire, UK ) (growth medium) at a density of approximately 1x106 per well (24-well plate). Monocytes were incubated in growth medium containing human IL-4 (Peprotech, Rocky Hill, NJ, USA) and GM-CSF (Peprotech, Rocky Hill, NJ, USA) for 3 days. On day 3, 44 μg / ml of Betaferon (Schering AG, Berlin, Germany) were added in 0.5 ml of assay buffer plus 3% hot inactive human AB serum and 25mM (final concentration) HEPES pH 8. The wells of control containing 50μg / ml of KLH or no antigen (untreated cells) were incubated in 1ml of PBS + 0.01% Tween 20 plus 3% hot inactive human AB serum (standard buffer). The DCs were incubated with antigen for 6 hours after which the DCs were washed 6 times to remove exogenous beta IFN. The cells were then resuspended in growth medium containing TNF alpha (Peprotech, Rocky Hill, NJ, USA), GM-CSF and IL-4 overnight.

On day 4, autologous CD8 + CD25 + T cells depleted were isolated by negative selection of PBMC (Dynal, Wirral, UK human CD4 + negative isolation kit) and then added to DCs at 1x105 per well in both plates. Proliferation and Elispot. Elispot plates were incubated for 6 days before development (as in example 2) and proliferation plates were incubated for 7 days before proliferation was measured by incorporation of 3HTdR (or 6 hour pulse at 1 μCl / well).

As in example 2, for the proliferation and Elipsot tests, an empirical threshold of stimulation index ≥2 was selected where the responses above this threshold were considered positive. In addition, statistical analysis was also carried out to determine if the responses were significantly deferential (p <0.05) of the untreated control (*). Additional analysis to determine the degree of intraassay variation included the coefficient of variance (CV).

The results, as shown in Figure 4, indicate significant T cell responses in 4 of the 29 donors for the proliferation assay and the same 4 of the 29 donors for the Elispot IL-2 assay. These data show that T cell responses could be demonstrated reproducibly even with an immunomodulatory protein.

Example 5 - Small-molecule T-cell temporal sequence assays

Carbamazepine (Novartis Pharmaceuticals, RU) and an N-acetyl iminoestilbeno (an analogue of carbamazepine synthesized in accordance with Ying et al Journal of Allergy and Clinical Immunology 2006; 118: 233-241) were compared in terms of the ability to stimulate T cell responses in a panel of healthy givers. Both compounds were tested at 25μg / ml in separate bulk cultures for each donor in accordance with the procedure of Example 2. In summary, bulk cultures were established by using 2-4x106 of depleted PBMC of CD8 + CD25hi T cells in each well of a 24-well plate. Replicated samples (from T blasts) were removed from bulk cultures on days 5-8 and proliferation in 96-well plates was evaluated. Data were used to assess the magnitude and kinetics of T cell responses to each compound.

As for example 2, an SI≥2 was used as a threshold for positive responses and the data was also analyzed to determine the coefficient of variance (CV), standard deviation (SD) and significance (p <0.05) using the use of parametric or non-parametric statistical analysis. Any given compound was considered to be immunogenic only if the response is statistically significant (p <0.05) with an SI≥2.

The results show that the N-acetyl iminoestilbene of the carbamazepine metabolite stimulates fewer donors than carbamazepine (known to be a potent inducer of delayed allergic responses in patients) when tested at a concentration range by using the test procedure of temporal sequence T cells. It is clear that the use of a single-time T-cell assay in a large number of T-cell responses would not have been detected. Instead, most T-cell responses against carbamazepine are induced on day 5 with only one additional response detected on days 6 and 7. The evaluation of T-cell responses against N-acetyl and carbamazepine on days 6, 7 or 8 I would not have discriminated against any level of immunogenicity between these two compounds.

Claims (30)

1. A method for measuring a collaborative T cell response to a test substance comprising the following steps:

(to)

isolate cells presenting antigens (APCs) and T cells from a sample obtained from an organism;

(b)

deplete regulatory T cells from isolated cells;

(C)

incubating said APCs and depleted regulatory T cell cells obtained in item (b) with the test substance; Y

(d)

test the T cell responses to the test substance.

2.

The method of claim 1 wherein said method further comprises: (ai) separating antigen presenting cells (APCs) from other cells; and step (c) comprises incubating the test substance with the separate APCs prior to the subsequent addition of

depleted cells of regulatory T cells.

3.

The method of claim 2 wherein the APCs are treated with cytokines prior to the addition of the test substance.

Four.

The method of claim 1 to 3 wherein the APCs and T cells are obtained from peripheral blood mononuclear cells (PBMCs).

5.

The method of any one of claims 1 to 4 wherein the APCs and T cells are human.

6.

The method of any one of claims 1 to 5 wherein the regulatory T cells are depleted from the CD25hi + T cells.

7.

The method of any one of claims 1 to 6 wherein the T cells are depleted from CD8 + T cells.

8.

The method of any one of claims 1 to 7 wherein the T cell responses are assayed by measuring any or more of the T cell proliferation, cytokine releases, T cell transcription changes, and / or other markers associated with T cell activation

9.

The method of claim 8 wherein the proliferation of T cells is measured by tritiated detimidine uptake.

10.

The method of claim 8 wherein the cytokine release is measured by the release of IL2 and / or IFNy.

eleven.

The method of any one of claims 1 to 10 wherein the T cell responses are assayed at more than one time point during incubation.

12.

The method of any one of claims 2 to 11 wherein the APCs are incubated with the test substance for more than a length of time prior to the addition of said depleted T-cell cells.

13.

The process of claim 1-12 wherein the test substance is tested in more than one concentration.

14.

The method of any one of claims 1 to 13 wherein an optimization substance is tested to determine the optimal time / s and / or concentration / is to test the test substance.

fifteen.

The method of any one of claims 1 to 13 wherein the test substance is a protein.

16.

The method of any one of claims 1 to 13 wherein the test substance is a peptide

17.

The method of any one of claims 1 to 13 wherein the test substance is a non-protein.

18.

The method of claim 17 wherein the test substance is an organic molecule, a lipid, a carbohydrate or a molecule composed of two or more moieties including conjugates, mixtures and formulations.

19.

The method of any one of claims 1 to 16 wherein the test substance is immunomodulatory or toxic to T cells and / or APCs.

twenty.

The method of any one of claims 4 to 13 wherein donor PBMCs expressing HLA allotypes representing> 80% of the expression in the world population of the study population are used.

twenty-one.

The method of any one of claims 4 to 13 wherein donor PBMCs are used to represent the HLA allotypes connected to a disease under study.

22

The method of any one of claims 1 to 15 wherein the overlapping peptides of a protein sequence are tested to identify T cell epitopes in the protein sequence.

2. 3.

The method of any one of claims 15 to 18 wherein a series of molecules are individually tested to assess relative immunogenicity.

24.

The method of claim 23 wherein the relative T-cell responses are used as the basis for selecting the main pharmaceutical products for further development.

25.

The method of any one of claims 15 to 18 wherein a test substance is analyzed to assess the potential immunogenicity.

26.

The method of any one of claims 15 to 18 in which different formulations of a test substance are analyzed to assess relative immunogenicity.

27.

The method of any one of claims 15 to 18 wherein different manufacturing batches of a test substance are analyzed to assess the potential immunogenicity.

28.

The method of any one of claims 15 to 18 wherein a test substance is analyzed by using patient blood as a source of T cells to assess immunogenicity to the test substance.

29.

The use of a method of any one of claims 1 to 15 to identify T cell epitopes in a protein sequence.

30

Use of a method of any one of claims 1 to 18 to evaluate the immunogenicity of a test substance.