Diagnostic method for determining the presence and amount of human interleukin-3 in a sample using novel IL-3 antibodies

Abstract

For determining the presence and amount of human interleukin 3 (IL-3) in a sample, the present invention provides a diagnostic method, wherein an anti-IL-3-antibody, fragment or construct thereof is added to said sample under conditions which allow for binding said antibody, fragment or construct thereof to IL-3 and detecting the amount of antibody bound IL-3 in said sample, wherein the anti-IL-3-antibody is clone 13. Further subject matter of the present invention are the novel antibody clone 13, a nucleic acid encoding said antibody and a hybridoma cell line which produces antibody clone 13. A diagnostic assay kit contains all necessary reagents and materials for performing such assay, preferably an ELISA assay and especially preferably contains antibody clones 13 and 11.

Claims

1. A nucleic acid encoding anti-IL3 antibody clone 13 produced by the hybridoma deposited as Deutsche Sammlung von Mikroorganism (DSM) ACC3164.

2. A vector comprising the nucleic acid of claim 1.

3. A host cell comprising the vector of claim 2.

Description

(1) The following examples and figures further illustrate and describe the present invention but are not intended to limit the scope thereof.

(2) FIG. 1 shows the amino acid sequence homology of IL-3 of various species;

(3) FIG. 2 shows in a Western blot the ability of monoclonal antibodies to bind to IL-3.

(4) FIG. 3 shows the relative IL-3 affinity of antibodies determined using varying concentrations of antibodies at a constant amount of IL-3 which was bound to the solid phase in an ELISA assay.

(5) FIG. 4 shows the results of tests performed to detect a possible cross-reactivity of anti-IL-3 antibodies with other human cytokines. In the tests, binding of the antibodies to IL-3, GM-CSF and IL-5 was compared.

(6) FIGS. 5 and 6 show the results of tests performed to detect possible cross-reactivity of the various anti-IL-3 antibodies with IL-3 from other species. In the test depicted in FIG. 8, also a commercially available anti-IL-3 antibody was included.

(7) FIG. 7 to 10 show the results of ELISA assays which were performed using differing combinations of coating and detection antibodies selected from the antibody clones 8, 11, 13, 44 and 47 as well as using a commercially available test kit. The results indicate that best performance can be achieved by using a combination of clones 13 and 11 and that these tests work tremendously well to detect and quantitate IL-3, even when body fluids (plasma, serum) are used as test samples.

(8) FIG. 11 to 14 show the results of ELISA assays performed to investigate the stability of tests performed inter alia with plasma and serum and using as the coating (solid-phase bound) antibody clone 13 and as detection antibody HRP-labelled clone 11. Plasma and serum samples containing IL-3 were stored for the given periods of time at various temperatures. The test results confirmed very good stability and performance for the test formats.

(9) FIGS. 15 and 16 show clinical data regarding the plasma IL-3 levels of patients with or without active RA, as well as data showing an analysis of plasma IL-3 levels of randomly picked patients presenting at the University Hospital Regensburg. The results indicate that in general only a small percentage of randomly picked patients show IL-3 levels above 20 pg/ml, while more than 50% of patients with active RA have IL-3 levels above 20 pg/ml. Among confirmed active RA patients, there are two subgroups only one of which shows high plasma levels of IL-3.

(10) FIGS. 17 and 18 show clinical data regarding the plasma IL-3, IL-6 and TNF- levels of patients with or without active RA (FIG. 17), as well as data showing an analysis of cytokine levels in patients with diagnosed RA (FIG. 18). The results indicate that IL-3 but not IL-6 or TNF- can separate between RA and non-RA types of arthritis (FIG. 17). Furthermore, what can be concluded from the data presented in FIG. 18 is that IL-3 and IL-6 but not TNF- correlate with disease activity in patients with RA.

(11) FIG. 19 shows clinical data regarding the plasma IL-3, IL-6 and TNF- levels of RA-patients treated with DMARD and/or biologicals. The data indicate that 63% of RA patients not responding to DMARDs/biologicals express high IL-3 levels. Patients with high IL-3 levels are more frequent among those patients that did not respond to current therapies. These patients would qualify for treatment with anti-IL-3-antibodies.

EXAMPLE 1

Generation of Monoclonal Anti-IL-3 Antibodies

(12) Anti-IL-3 antibodies were produced by immunizing Balb/c mice using at least 6 i.p. injections of human eukaryotic glycosylated IL-3 in alumn at four week intervals. Two days before cell fusion, IL-3 in PBS was injected intraperitoneally. Antibody-producing splenocytes obtained from the immunized mice (HGPRT positive, able to grow on HAT medium) were fused with the myeloma cell line X63Ag8.6.5.3 in the presence of polyethylene glycol (PEG) and a selection of hybridomas performed in an HAT-selection medium. Hybridomas were cultivated in RPMI-1640 medium supplemented by 10% FCS (neat inactivated, HIA), P/S and glutamine (1:100). Obtained cells are able to grow in suspension and are splitted every three days in a ratio of 1:4.

(13) For storage purposes hybridoma cells are transferred from a cell culture bottle into 50 ml or 15 ml cell culture flasks (BD Falcon). After centrifugation at 1400 rpm for 5 minutes at room temperature, the supernatant is completely removed. Cells are resuspended in a freezing medium (90% FCS (HIA)+10% DMSO) and 1.5 ml aliquots are filled into vials. The cells are prefrozen in a freezing container in a freezer at 80 C. and after 1-2 days transferred to a liquid nitrogen storage tank.

(14) Cloning and recloning of the obtained hybridoma cell lines are performed using limited dilution to provide long-term stable sources for monoclonal antibodies.

(15) Obtained antibodies are shown in table 1.

(16) For determining the isotypes of the antibodies, ELISA assays were performed using hIL-3 coated plates to which the antibodies were added. Bound antibodies were detected using isotype specific secondary antibodies. For further analyses, only antibodies of isotype IgG were used.

(17) TABLE-US-00001 TABLE 1 Overview of mAbs against human IL-3 Original clone First cloning Second cloning Isotype Clone 2 2.28 2.28.11 IgM, kappa Clone 3 3.47 3.47.20 IgG1, kappa Clone 5 5.3 5.3.2 IgM, kappa Clone 6 6.38 6.38.14 IgG1, kappa Clone 7 7.42 7.42.45 IgM, kappa Clone 8 8.36 8.36.38 IgG1, kappa Clone 10 10.12 10.12.4 IgG1, kappa Clone 11 11.14 11.14.6 IgG1, kappa Clone 13 13.47 13.4.4 IgG1, kappa Clone 36 36.26 36.26.10 IgG1, kappa Clone 38 38.18 38.18.5 IgG1, lambda Clone 41 41.28 41.28.4 IgG1, kappa Clone 42 42.47 42.47.36 IgG1, kappa Clone 43 43.14 43.14.28 IgG1, kappa Clone 44 44.16 44.16.16 IgG1, kappa Clone 45 45.14 45.14.27 IgG1, kappa Clone 46 46.21 46.21.1 IgG1, kappa Clone 47 47.28 47.28.15 IgG1, kappa

EXAMPLE 2

Determination of the Amount of IgG1 in the Hybridoma Supernatants

(18) Several of the obtained antibodies of the type IgG1 were isolated from hybridoma clones and their concentration determined. The determination of the concentration was performed according to following method: 96-wellplates are coated overnight at room temperature with anti-mouse IgG (1:100 in PBS) in a concentration of 100 l/well. Blocking is performed by adding 100 l per well of 2% BSA in PBS and incubation at room temperature for two hours. After the blocking reaction, the plates are washed twice. Two samples and blanks, respectively, of supernatants of clones 3.47.20, 6.38.14, 8.36.38, 10.12.4, 11.14.6 are incubated undiluted, as well as with dilutions of 1:3, 1:9, 1:27, 1:81, 1:243, 1:729 and 1:2187 (100 l per well, dilution in 2% BSA in PBS) at room temperature. Mouse IgG1 in a starting concentration of 1 mg/ml is used as standard, whereas a concentration of 20 ng/ml is applied in dilutions of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128.

(19) The plate is washed three times and then incubated with biotinylated anti-mouse IgG1 (diluted by 1:250 in 2% BSA in PBS) for one hour at room temperature with 100 l per well. After washing the plate a further three times, streptavidin-HRP (1:1000 in 2% BSA in PBS) is added for one hour at room temperature and in the dark. The concentration of the antibodies is determined after adding ABTS and incubating for further 30 minutes and measuring the signal at 405 and 490 nanometers on a spectrophotrometer. Based on this determination, a desired amount of the antibodies tested is applied for the further tests.

EXAMPLE 3

Detection of IL-3 by Monoclonal Antibodies in a Western-Blot Assay

(20) For preparing the gel and performing the western-blot analysis, standard methods are used. A 12% PAA resolving gel is poured, overlayed with about 1-2 ml of water and polymerisation conducted for 30 to 45 min until a recognizable line is formed. The water is removed, a stacking gel poured onto the resolving gel and a Teflon comb is inserted. Polymerisation is performed for 30 min, then the comb is carefully removed.

(21) Samples of IL-3 are prepared by mixing of recombinant human IL-3 1:1 with Laemmli buffer and heating the samples at 60 C. for 5 min. An amount of 1 g per lane of IL-3 as well as a usual standard for determining molecule sizes is loaded onto the gel. The gel is then mounted in a SDS-PAGE gel electrophoresis apparatus which already contains a running buffer. The inserted gel is then cautiously overlayed with additional running buffer and electrophoresis performed at 20 to 25 mA with voltage adjusted to infinite for approximately 1.5 hours. When the run is completed, the gel is retrieved from the apparatus and the stacking gel is removed.

(22) Six layers of Whatman paper that has been presoaked in transfer buffer, and a PVDF membrane are cut to fit the size of the gel. The transfer stack is adjusted in the usual way and transfer effected by semi-dry blotting for 40 min at 20-25 mA and voltage adjusted to infinite. The membrane is then incubated overnight at 4 C. on a shaking apparatus with a blocking solution (5% powdered skim milk in PBS) and the membrane washed three times for 5 min each with PBS at room temperature.

(23) Antibody clones are incubated at a concentration of 5 g/ml in blocking solution for 2 hours at room temperature under agitation on the shaking apparatus. After three washing steps, HRP labelled anti-mouse immune-globulin (1:1000 in blocking solution) is added and incubation is conducted for 1 hour at room temperature while shaking. After three further washing steps, a detection solution (1:1 mixture of solutions A and B of the Western-blotting Luminal Reagent obtained from NALGENE) is added and incubated for 1 min at room temperature. Films are then adjusted on the membranes with different times of expositions and developed in the dark room.

(24) FIG. 2 shows the results of binding of antibody clones 2, 3, 5, 6, 7, 8, 10, 11 and 13. Binding to IL-3 at the given concentration was detected for clones 8, 11 and, to a lesser extent, for clone 13.

EXAMPLE 4

Analysis of the IL-3 Affinity and Specificity of Monoclonal Antibodies

(25) a) Affinity of the Antibodies for IL3

(26) The affinity of the obtained antibodies for IL-3 was measured in an ELISA assay. ELISA plates were coated overnight with 1 g/ml of anti-human IL-3 antibody (RD, goat IgG anti-human IL-3 AF-203-NA). For each concentration, duplicates were used (212 wells). For this purpose, the first concentration (2 g/ml) is diluted in PBS, further dilutions are made in PBS containing 2 g/ml control goat IgG to keep the total concentration of IgG constant. Blocking with 2% BSA is performed for 2 hours at room temperature, followed by 5 washing steps using PBS.

(27) The wells are then incubated with hIL-3 (0.25 g/ml in PBS) for 2 hours at room temperature, for the control group no hIL-3 is added. After five further washing steps with PBS, the wells are incubated overnight at 4 C. with serial (1:3) dilutions of antibodies clone 8 and 11 obtained in example 1, the antibodies being used in PBS buffer containing 2% BSA and with a starting concentration of 20 g/ml.

(28) After five further washing steps, bound antibody is detected using goat-anti-mouse-HRP antibody (1:500 in PBS with 2% BSA) and incubation for 1 hour at room temperature. After five further washing steps, ABTS (ROCHE, 1 mg/ml) is added as substrate and the optical density measured in a spectrometer at 405 nm.

(29) FIG. 3 shows the results of tests including antibodies clone 3, 8, 10, 11 and 13. The tests were performed in the manner as described with different concentrations/dilutions of antibodies as shown in the figure I.

(30) b) Cross-reactivity with Other Cytokines

(31) To determine the usefulness of the obtained monoclonal antibodies for diagnostic assays, it is important to be able to exclude cross-reactivities with closely related cytokines which are also present in blood, plasma, serum or other body fluids of patients. To this end, wells of ELISA plates were coated by adding 100 l/well of human IL-3 (1 g/ml), GM-CSF (1 g/ml) or IL-5 (1 g/ml) in PBS. As negative control PBS was used (100 l/well). For each tested antibody, different dilutions were tested mandatorily on a common plate with hIL-3, hGM-CSF, hIL-5 and PBS.

(32) The cytokine coated plates were washed three times and blocking performed for 2 hours at room temperature using 2% BSA in PBS. After three further washing steps, antibodies clone 3.47.20, 8.36.38, 10.12.4, 11.14.6, 13.4.4 and just medium (RPMI1640 containing 10% FCS) as control were added at a concentration of 40 g/ml and 1:5 and 1:25 dilutions thereof in a volume of 100 l/well and incubated for 1 hour at room temperature. On each plate a negative control is used.

(33) After three washing steps, a secondary HRP-labelled rabbit anti-mouse IgG (DAKO-Cytomation P260 (1:2000 in 2% BSA in PBS, 100 l/well) was added and the plates incubated at room temperature for 1 hour in the dark. After another three washing steps, ABTS (ROCHE, 1 mg/ml) was added and spectrometry performed at 405 and 490 nm after 30 min.

(34) The results are shown in FIG. 4 indicating some weak cross-reactivity for clones 8 and 10, but no significant cross-reactivity for clones 11 and 13.

(35) c) Cross-reactivity with IL-3 from Other Species

(36) As a further property of the monoclonal antibodies, their cross reactivity with IL-3 from other species was determined. For a respective assay, the wells of ELISA plates were coated with human, murine, rat and rhesus IL-3 (1 g/ml) in PBS as well as with PBS as background with 100 l/well and incubated overnight in a refrigerator. For each antibody, different dilutions were tested mandatorily on a common plate with hIL-3, murine IL-3, rat IL-3, rhesus IL-3 and PBS negative control.

(37) The IL-3 coated plates were washed three times and blocking performed for 2 hours at room temperature with 2% BSA in PBS. After three washing steps, antibody clones 3.47.20, 8.36.38, 10.12.4, 11.14.6, 13.4.4 in certain concentrations as indicated in FIGS. 7 and 8, and 1:5, 1:25 and 1:125 dilutions thereof were added at volumes of 100 l/well. R&D monoclonal anti-IL-3 antibody clone 4806 (R&D Systems, Inc., catalogue No. MAB203) was used (100 l/well) in concentrations of 40 g/ml, 20 g/ml, 10 g/ml, 5 g/ml and 2.5 g/ml and, as negative control, medium (100 l/well) without antibody (RPMI 1640 containing 10% FCS) was used. On each plate a negative control was used.

(38) After three washing steps, a secondary HRP-labelled rabbit anti-mouse IgG (DAKO-Cytomation P260 (1:2000 in 2% BSA in PBS, 100 l/well) was added and the plates incubated at room temperature for 1 hour in the dark. After another three washing steps, ABTS (ROCHE, 1 mg/ml) was added and spectrometry performed at 405 and 490 nm after 30 min.

(39) Results are shown in FIGS. 5 and 6, as mentioned above, indicating that albeit a faint cross reactivity of clone 10, none of the antibodies of example 1 showed detectable cross-reactivity. The R&D antibody on the other hand, showed some cross-reaction with rhesus IL-3.

EXAMPLE 5

Development of a Highly Sensitive and Specific ELISA Assay

(40) Anti-IL-3 antibody clones 8, 11, 13 and further antibody clones 44 (44.16.16, DSM ACC3166) and 47 (47.28.15, DSM ACC3167) were analysed for their use in the development of a highly sensitive and specific ELISA assay for the determination of IL-3, especially for diagnostic purposes in blood, plasma or serum, as well as other body fluids.

(41) To this end, ELISA plates were incubated with 5 g/ml of anti-IL-3 antibody overnight at room temperature to coat the plates. After three washing steps, blocking is performed using 1% BSA in PBS at 100 l/well for 1 hour at room temperature. After further three washing steps, samples are incubated with 60 l/well of IL-3 in various concentrations in PBS, plasma and serum. After another three washing steps, detection of solid-phase bound IL-3 is performed by adding 60 l/well of a different and HRP labelled anti-IL-3 antibody at a concentration of 400 ng/ml and incubation for 2 hours at room temperature, followed by three washing steps and addition of TMB buffer (10 ml TMB buffer, 1 tablet of TMB, 3 l H.sub.2O.sub.2) (0.1 mg/ml, SIGMA-ALDRICH). The reaction is stopped by adding 100 l/well of H.sub.2SO.sub.4 (12.5% in H.sub.2O). The results are obtained by spectrometry at 450 nm and shown in FIGS. 7 to 10.

(42) Labelling of the anti-IL3 antibody clones was performed using the Lightning-Link HRP Conjugation Kit (Innova Biosciences) using the following protocol: For each of the purified antibody clones 8, 11, 13, 44 and 47 100 l solutions with a concentration of 1 g/l (in PBS) were produced. To each antibody solution, 10 l of LL-modifying reagent were added and the obtained solution mixed carefully. For each antibody solution a Lightning Link mix bottle (100 g reagent) was opened and the antibody solution including the LL-modifying agent added directly onto the reagent powder. Mixing was performed very cautiously by up- and down-pipetting of the solution. The lid was readjusted on the bottle of the Lightning Link mix and the bottles incubated for 3 hours at room temperature whereupon 10 l LL-quencher reagent were added and incubated for a further 30 min at room temperature. After this treatment the antibodies were stored at 20 C. for further use.

(43) As a comparative assay, analogue tests were performed using the Quantikine Human IL-3 ELISA test kit provided by R&D Systems, Inc., Catalogue No. Dy 203.

(44) The results of this experiment are shown in FIG. 7 to 10 and indicate that antibody clone 8 is not suitable as either coating or detection antibody, whereas antibody clones 11 and 13 are both suitable as coating and detection antibodies and the best results are achieved using clone 13 as coating and clone 11-HRP as detection antibody. It is furthermore observed that the commercially available IL-3 ELISA test kit obtainable from R&D Systems shows a remarkably lower sensitivity as an ELISA test kit according to the present invention with antibody clones 13 and 11 (FIG. 7). It was also observed that the IL-3 ELISA test kit of R&D Systems showed high background signals and therefore was not sufficiently reliable and sensitive when plasma or serum samples are used. An inventive test kit with clones 13/11, on the other hand, retained the same sensitivity as when using PBS or PBS/BSA samples also for plasma and serum (comparison shown in FIG. 10). Tests performed with different plasma samples (Plasma EDTA, Plasma Citrat) and serum could also be shown to be stable for at least 24 hours at room temperature (FIG. 11) and there was also no detectable signal loss after freezing and thawing of the samples (FIGS. 12-14).

EXAMPLE 6

Analysis of IL-3 Level in Plasma of Patients

(45) An ELISA assay with clone 13 as coating and clone 11-HRP as detection antibody was performed for patients with inflammatory joint diseases. Test conditions and reagents were as described in Example 7. The results are shown in FIG. 15.

(46) It was observed that in patients with non-active RA (DAS28<2.6, N=9) the mean plasma level of IL-3 was significantly lower (12 pg/ml) than for patients with active RA (DAS282.6, N=45; IL-3=73 pg/ml). For patients suffering from a different form of arthritis (non-rheumatoid arthritis, n=10), also significantly lower mean IL-3 plasma levels (IL-3=1 pg/ml) were observed than for patients with active RA.

(47) Remarkably, there were two groups of patients with active RA (DAS2.6). About half of the patients (N=21) showed very low IL-3 plasma levels (mean value IL-3=1.3 pg/ml, SEM (standard error of the mean)=0.27 pg/ml), whereas the second group (N=24) showed very high IL-3 levels (mean value IL-3=136 pg/ml, SEM=35 pg/ml). The ability to perform the test according to the present invention and to gain reliable and specific information about the IL-3 levels in the patients allows for a stratification of active-RA patients for a therapeutic IL-3 blocking treatment into subgroups with high and with low IL-3 levels. Patients with high IL-3 levels can be considered as a target group that will greatly benefit from such treatment.

(48) For a further experiment, random plasma samples from patients treated at the University Hospital Regensburg were analyzed. The diagnosis of these patients was not known as the samples were analyzed in an anonymous way. The data obtained are shown in FIG. 16 and indicate that only a very low percentage of patients (4.7%) treated at the University Hospital express high levels of IL-3 while most of the patients express no IL-3 or very low levels thereof.

(49) In further experiments plasma IL-3, IL-6 and TNF- levels have been analysed in patients suffering from arthritis/arthralgia (no rheumatoid arthritis; n=87) or from rheumatoid arthritis (n=108) (FIG. 17). The obtained data clearly demonstrate that IL-3 but not IL-6 or TNF- can separate between RA and non-RA types of arthritis.

(50) Within the group of RA patients it was found that IL-3 and IL-6 but not TNF- levels were strongly increased in patients with active RA (DAS28 >2.6; n=93) compared to patients with non-active RA (DAS282.6; n=15). Still, the IL-6 levels were clearly decreased in comparison to the IL-3 levels (FIG. 18).

(51) As shown in FIG. 19 >60% of the patients not responding to DMARDs/biologicals express high IL-3 levels. Among those patients that did not respond to current therapies patients with high IL-3 levels were more frequent. These patients would qualify for treatment with anti-IL-3 antibodies since the data indicate that patients with high IL-3 levels obviously do not respond to other kinds of therapies like, e.g., DMARDs or biologicals.