METHOD FOR THE PRODUCTION OF THYMOCYTE SUPERNATANT

Abstract

Herein is reported a method for producing a thymocyte supernatant comprising the steps of co-cultivating thymocytes and mononuclear cells at a cell ratio of at least 0.5:1.2 in the presence of phorbol-12-myristate-13-acetate and Phytohemagglutinin M for up to 60 hours, and separating the co-cultivation medium from the cells and thereby producing the thymocyte supernatant.

Claims

1. A method for producing a thymocyte supernatant comprising the following steps: co-cultivating thymocytes and mononuclear cells at a cell ratio of 0.5:1.2 or more in the presence of phorbol-12-myristate-13-acetate and phytohemagglutinin M for up to 60 hours, and separating the co-cultivation medium from the cells and thereby producing the thymocyte supernatant.

2. The method according to claim 1, wherein the thymocyte to mononuclear cell ratio is of from 0.5:1.25 to 0.5:4.

3. The method according to claim 2, wherein the ratio is about 0.5:2.

4. The method according to claim 1, wherein the thymocyte cell density is about 510.sup.5 cells/ml in the co-cultivating.

5. The method according to claim 1, wherein prior to the co-cultivating the thymocytes are incubated for up to 60 hours at 37 C. in cultivation medium.

6. The method according to claim 1, wherein the mononuclear cells are isolated from PBMCs by adherence to a solid surface at a cell density of 210.sup.6 cells/ml and the attached mononuclear cells are incubated for about 40 hours in cultivation medium prior to the co-cultivating with the thymocytes.

7. The method according to claim 1, wherein prior to the co-cultivating of the thymocytes and mononuclear cells the cultivation medium of the thymocytes is replaced by fresh medium containing 10 ng/ml phorbol-12-myristate-13-acetate (PMA) and 5 g/ml phytohemagglutinin M (PHA-M).

8. The method according to claim 1, wherein the co-cultivating is started by removing the cultivation medium from the mononuclear cells and adding the thymocyte suspension.

9. The method according to claim 1, wherein the medium is RPMI medium supplemented with 10% (v/v) FCS, 1% (w/v) of a 200 mM glutamine solution that comprises penicillin and streptomycin, 2% (v/v) of a 100 mM sodium pyruvate solution, and 1% (v/v) of a 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES) buffer, further comprising 0.05 M -mercaptoethanol.

10. A method for co-cultivating one or more B-cells comprising the step of co-cultivating the one or more B-cells with EL4-B5 cells in the presence of TSN produced with a method according to claim 1.

11. A thymocyte supernatant produced with a method according to claim 1.

12. Use of a thymocyte supernatant produced with a method according to claim 1 in the co-cultivation of B-cells and feeder cells.

Description

DETAILED DESCRIPTION

[0077] In the following the invention is exemplified using rabbit B-cells. This is an example and shall not be construed as a limitation. The invention can be practised with B-cells of any origin.

A. The Production of TSN Prior to the Current Invention

[0078] Rabbit specific cytokines were generated by the preparation of a rabbit thymocyte supernatant (TSN). Therefore rabbit T-cells and rabbit macrophages were used (see e.g. Weber, M., et al., J. Immunol. Meth. (2003); Steenbakkers, P. G., et al., Mol. Biol. Rep. (1994)). The rabbit T-cell precursors can be isolated from the thymus of 4-5 week-old rabbits (see e.g. WO 2011/147903; Seeber, S., et al., PLoS ONE (2014) e86184).

[0079] Briefly, the thymocytes are seeded at a cell density of 510.sup.5 cells/ml in cell culture flasks in a cultivation medium and incubated for about 48 h, at 37 C. PBMCs isolated from blood of adult rabbits are used to enrich monocytes/macrophages by adherence at 37 C. in cultivation medium at a cell density of 1-310.sup.6 cells/ml. Attached monocytes/macrophages are cultivated for about 48 h in a cultivation medium. T-cells and macrophages obtained from different rabbits are kept in separate flasks. Prior to the mixing of T-cells and macrophages, T-cells are centrifuged and resuspended in cultivation medium containing 10 ng/ml phorbol-12-myristate-13-acetate (PMA) and 5 g/ml phytohemagglutinin M (PHA-M) at a cell density of 510.sup.5 cells/ml. The medium is removed from the macrophage cultures and replaced by the T-cell suspension to have a final macrophage concentration of 110.sup.6 cells/ml. After co-cultivation for 48 hours, the T-cell/macrophage conditioned medium is removed and termed (TSN).

[0080] In the following Table the characteristics of TSN lots produced with the method as outlined above is shown (see also FIG. 1, left part; obtained with method according to example 12 using a TSN produced with a method according to example 10).

TABLE-US-00002 TABLE ratio IgG positive wells to ratio average productivity wells TSN lot reference to reference reference 1.00 1.00 1 0.80 0.75 2 0.83 0.89 3 0.84 0.65 4 0.78 0.42 5 1.04 0.49 6 0.88 0.56 7 0.96 0.64 8 0.94 0.69 9 1.11 0.79 10 0.83 0.66 11 1.11 0.39 12 1.04 0.78 13 0.95 0.48 average 0.93 0.63

[0081] Thus, TSN lots produced with the method as known from the art show a high lot to lot variability. As a consequence low performing TSN lots have to be identified and discarded or added in higher amounts in a B-cell co-cultivation, both leading, amongst other things, to increased costs.

B. The Production of TSN with Novel and Inventive Method as Reported Herein

[0082] Herein is reported an improved method for the production of thymocyte supernatant (TSN). The TSN produced with the method as reported herein can be used as additive in the co-cultivation of single deposited B-cells, which can be of any source, with feeder cells in a suitable co-cultivation medium.

[0083] The invention is based at least in part on the finding that the increase of the macrophage to T-cell ratio during the preparation of the TSN leads to a TSN preparation that, when used as additive in the co-cultivation of B-cells and feeder cells, results in higher overall IgG-concentrations. This, in turn, results in the beneficial effect that more assays can be performed per cultivation supernatant. In addition, this improvement reduces the discharge rate of the TSN lots.

[0084] One aspect as reported herein is a method for producing a thymocyte supernatant comprising the following steps: [0085] co-cultivating thymocytes and mononuclear cells at a cell ratio of 0.5:1.2 or more in the presence of phorbol-12-myristate-13-acetate (PMA) and phytohemagglutinin M (PHA-M) for up to 60 hours, and [0086] separating the co-cultivation medium from the cells and thereby producing the thymocyte supernatant.

[0087] In one embodiment the thymocyte to mononuclear cell ratio is of from 0.5:1.2 to 0.5:4. In one embodiment the ratio is of from 0.5:1.5 to 0.5:3. In one preferred embodiment the ratio is about 0.5:2.

[0088] In one embodiment the thymocyte cell density is about 510.sup.5 cells/ml (0.510.sup.6 cells/ml).

[0089] In one embodiment the thymocytes are obtained from the thymus of an (young) experimental animal. In one embodiment the thymocytes are T-cells.

[0090] In one embodiment prior to the co-cultivating the thymocytes are incubated for up to 60 hours at 37 C. in cultivation medium. In one embodiment this incubating is for about 30-46 hours. In one preferred embodiment for about 40 hours.

[0091] In one embodiment the mononuclear cells are isolated from the blood of an (adult) experimental animal. In one embodiment the mononuclear cells are isolated from the PBMCs of the (adult) experimental animal. In one embodiment the mononuclear cells are isolated by adherence to a solid surface from the PBMCs of the (adult) experimental animal. In one embodiment the isolation is at a cell density of 1-310.sup.6 cells/ml, in one preferred embodiment of about 210.sup.6 cells/ml. In one preferred embodiment the attached mononuclear cells are macrophages. In one embodiment the attached mononuclear cells are cultivation for up to 60 hours in cultivation medium prior to the co-cultivating with the thymocytes, in one preferred embodiment for about 40-48 hours.

[0092] In one embodiment prior to the co-cultivating of the thymocytes and mononuclear cells the cultivation medium of the thymocytes is replaced by fresh medium containing 10 ng/ml phorbol-12-myristate-13-acetate (PMA) and 5 g/ml phytohemagglutinin M (PHA-M).

[0093] In one embodiment the co-cultivating is started by removing the cultivation medium from the mononuclear cells and adding the thymocyte suspension.

[0094] In one embodiment the cultivation medium of the mononuclear cells is EL4-B5 medium. In one embodiment the medium is RPMI 1640 medium supplemented with FCS, glutamine/penicillin/streptomycin, Na-pyruvate, HEPES buffer and beta-mercapto ethanol.

[0095] It has been found that depending on the amount of macrophages present in the co-cultivation of thymocytes (T-cells) with mononuclear cells (macrophages), i.e. depending on the ratio of said cells, the produced TSN shows different properties. The respective results are shown in the following Table (total wells=4*84).

TABLE-US-00003 TABLE ratio average frequency average [*10.sup.6 cells/ml] average IgG of IgG positive productivity of all thymocytes: positive wells wells IgG positive wells mononuclear cells [n] [% of total wells] [g/ml] 0.5:1 33.3 2.3 39.6 2.7 0.35 0.05 0.5:1.25 38.8 4.8 46.1 5.7 0.81 0.10 0.5:1.5 34.5 5.5 41.1 6.6 1.22 0.12 0.5:2 37.3 1.8 44.4 2.1 1.32 0.23

[0096] In the following Table the characteristics of TSN lots produced with the method according to the current invention as reported herein is shown (see also FIG. 1, right part; obtained with single deposited B-cells according to Example 12 using a TSN produced with a method according to Example 9).

TABLE-US-00004 TABLE ratio IgG positive wells ratio average productivity wells TSN lot to reference to reference reference 1.00 1.00 14 0.83 0.52 15 0.96 0.96 16 0.99 1.55 17 1.04 1.15 18 0.98 1.21 19 1.33 1.53 20 0.81 1.37 21 1.05 1.54 22 1.01 1.09 23 1.14 1.26 24 1.23 1.02 25 1.03 1.38 26 1.03 0.96 27 1.04 1.31 28 1.17 1.52 29 1.10 1.46 30 1.00 1.95 31 0.97 2.60 32 0.94 1.27 33 0.94 1.15 34 0.85 1.05 35 0.96 1.32 average 1.02 1.33

[0097] Thus, TSN lots produced with the inventive method as reported herein show a reduced lot to lot variability. Additionally the secreted IgG concentration in the cultivation supernatant is increased.

C. B-Cell Cloning Process Using TSN Produced with the Inventive Method as Reported Herein

Immunization

[0098] For the generation of therapeutic antibodies either a non-human animal is immunized with the therapeutic target (either alone or in combination with an immunogenic stimulus) to elicit an immune response or synthetic approaches, such as phage display libraries are used. If a transgenic animal (i.e. having a human immune system) or a human phage display library is used human antibodies are obtained. Otherwise non-human animal antibodies are obtained that will be humanized thereafter. A rare possibility to obtain potential therapeutic antibodies is from the blood of a human being that has recovered from a disease.

[0099] Often non-human animals, such as mice, rabbits, hamster and rats, are used as animal model for evaluating antibody based therapies. Therefore, it is normally required to provide cross-reactive antibodies binding to the non-human animal antigen as well as to the human antigen.

[0100] In the methods as reported herein B-cells obtained from any source e.g. in one embodiment one or more human, mouse, hamster or rabbit B-cells, can be used.

[0101] In case of a rabbit B-cell the feeder cells can be either murine EL4-B5 cells or a mammalian cells, such as CHO cells or BHK cells or HEK cells, expressing rabbit CD40L. In one embodiment the rabbit is selected from the group consisting of New Zealand White (NZW) rabbits, Zimmermann-rabbits (ZIKA), Alicia-mutant strain rabbits, basilea mutant strain rabbits, transgenic rabbits with a human immunoglobulin locus, rbIgM knock-out rabbits, and cross-breeding thereof.

[0102] In case of a human B-cell the feeder cells can be either murine EL4-B5 cells or mammalian cells, such as CHO cells or BHK cells or HEK cells, expressing human CD40L.

[0103] In case of a murine B-cell the feeder cells can be either murine EL4-B5 cells or mammalian cells, such as CHO cells or BHK cells or HEK cells, expressing mouse CD40L. In one embodiment the mouse is an NMRI-mouse or a balb/c-mouse.

[0104] In case of a hamster B-cell the feeder cells can be either murine EL4-B5 cells or mammalian cells, such as CHO cells or BHK cells or HEK cells, expressing hamster CD40L. In one embodiment the hamster is selected from the group consisting of Armenian hamster (Cricetulus migratorius), Chinese hamster (Cricetulus griseus), and Syrian hamster (Mesocricetulus auratus). In one embodiment the hamster is the Armenia hamster.

[0105] In one embodiment the non-human animals, e.g. mice, hamster and rabbits, chosen for immunization are not older than 12 weeks.

Source and Isolation of B-Cells

[0106] Blood provides a high diversity of antibody producing B-cells. The therefrom obtained B-cell clones secrete antibodies that have almost no identical or overlapping amino acid sequences within the CDRs, thus, show a high diversity.

[0107] In one embodiment B-cells, e.g. from the blood, are obtained of from 4 days after immunization until at most 9 days after immunization or the most recent boost of the non-human animal. This time span allows for a high flexibility in the method as reported herein. In this time span it is likely that the B-cells providing for the most affine antibodies migrate from spleen to blood (see e.g. Paus, D., et al., JEM 203 (2006) 1081-1091; Smith, K. G. S., et al., The EMBO J. 16 (1997) 2996-3006; Wrammert, J., et al., Nature 453 (2008) 667-672).

[0108] B-cells from the blood, e.g. of a non-human animal or from human blood, may be obtained with any method known in the art. For example, density gradient centrifugation (DGC) or red blood cell lysis (lysis) can be used. Density gradient centrifugation compared to hypotonic lysis provides for a higher overall yield, i.e. number of B-cell clones. Additionally from the cells obtained by density gradient centrifugation a larger number of cells divide and grow in the co-cultivation step. Also the concentration of secreted antibody is higher compared to cells obtained with a different method. Therefore, in one embodiment the providing of a population of B-cells is by density gradient centrifugation.

Selection Steps Prior to Co-Cultivation

[0109] B-cells producing antibodies that specifically bind an antigen can be enriched, e.g. from peripheral blood mononuclear cells (PBMCs). Thus, in one embodiment of all methods as reported herein the B-cell population is enriched from peripheral blood mononuclear cells (PBMCs).

[0110] In one embodiment of all methods as reported herein the PBMCs are depleted of macrophages. This is advantageous for B-cells of rabbit origin for the co-cultivation step. Macrophages can be depleted from PBMCs by adhesion to the surface of the cell culture plate.

[0111] In one embodiment of the methods as reported herein the B-cells are from a protein-immunized animal and are depleted of macrophages prior to labeling.

[0112] Cells not producing an antibody binding the antigen or, likewise, cells producing an antibody binding to the antigen can be reduced or enriched, respectively, by using a panning approach. In panning the respective antigen is presented attached to a surface and cells binding thereto can be selectively enriched in/from a cell population (in this case the cells bound to the surface attached antigen are processed further).

[0113] The method as reported herein comprises in one embodiment prior to the single cell depositing a selecting step in which B-cells producing specific and/or non-cross-reactive antibodies are selected based on cell surface markers and fluorescence activated cell sorting/gating. In one embodiment mature B-cells are sorted/enriched/selected. For selection of B-cells from different non-human animal species different cell surface markers can be used.

[0114] With the labeling of non-target cell populations and non-specifically binding lymphocytes it is possible to selectively deplete these cells. In such a depletion step only a partial depletion can be achieved. Albeit the depletion is not quantitative it provides for an advantage in the succeeding fluorescence labeling of the remaining cells as the number of interfering cells is reduced. By performing a single cell depositing of mature B-cells (memory B-cells, affinity matured plasmablasts and plasma cells) using fluorescence activated cell sorting a higher number of Ig wells/number of single deposited cells can be obtained in the co-cultivation step.

[0115] Different cell populations can be labeled specifically by using different surface markers such as e.g. CD3.sup.+-cells (T-cells), CD19.sup.+-cells (B-cells), IgM.sup.+-cells (mature naive B-cells), IgG.sup.+-cells (mature B-cells), CD38.sup.+-cells (e.g. plasmablasts), and IgG.sup.+CD38.sup.+-cells (pre-plasma cells).

[0116] Immuno-fluorescence labeling for selection of mature IgW-B-cells, such as memory B-cells, plasmablasts, and plasma cells, is available. For a selection or enrichment of B-cells the cells are either single labeled, or double labeled, or triple labeled. Also advantageous is a labeling that results in a fraction of about 0.1% to 2.5% of labeled cells with respect to the total cell population.

[0117] In one embodiment B-cells are deposited as single cells selected by the labeling of surface molecules present on 0.1% to 2.5% of the B-cells in the population, in another embodiment on 0.3% to 1.5% of the B-cells of the population, in a further embodiment on 0.5% to 1% of the B-cells of the population.

[0118] In one embodiment the method is with the proviso that if the cells are of rabbit origin the labeling is not of IgG.sup.+-B-cells and/or CD138.sup.+-B-cells.

[0119] In one embodiment of all methods as reported herein IgG.sup.+CD19.sup.+-B-cells are deposited as single cells from the B-cells obtained from a non-immunized non-human animal or from a human.

TABLE-US-00005 TABLE Immuno-fluorescence labeling for the determination of mature mouse- (A-J), hamster- (K) and rabbit (L-N)-B-cells. B-cell origin sorting of B-cells with fraction of all viable cells (%) mouse IgG.sup.+CD19.sup.+ .sup.0.5 0.2 n =14 mouse IgG.sup.+CD38.sup.+ 0.8 0.5 n = 9 mouse IgG.sup.+CD138.sup.+ 0.06 0.07 n = 6 mouse IgG.sup.CD138.sup.+ 0.6 0.5 n = 6 mouse IgG.sup.+CD27.sup.+ 0.1 0.1 n = 8 mouse CD27.sup.+CD138.sup.+ 1.5 0.5 n = 2 mouse CD27.sup.+IgG.sup.+CD3.sup. 0.10 0.04 n = 3 mouse CD3.sup.CD27.sup.+ 1.33 n = 1 mouse IgG.sup.+CD268.sup.+ 0.8 n = 1 mouse CD38.sup.+CD3.sup. 12 7 n = 2 hamster IgG.sup.+IgM.sup. 0.6 0.1 n = 15 rabbit IgG.sup.+ 0.6 0.2, n = 5 rabbit IgG.sup.+IgM.sup. 0.4 0.2, n = 2 rabbit IgG.sup.+CD138.sup.+ 0.3 0.1,n = 5

[0120] In one embodiment the methods comprise the step of depleting the B-cell population of macrophages and enriching of B-cells of the B-cell population secreting antibody specifically binding a target antigen.

[0121] In one embodiment of all aspects the population of B-cells is a non-human animal B-cell population. In one embodiment the B-cell population is a mouse B-cell population, or a hamster B-cell population, or a rabbit B-cell population. In one preferred embodiment the B-cell population is a rabbit B-cell population.

[0122] In one embodiment the B-cell population is a human B-cell population.

[0123] In one embodiment of all aspects the B-cells are mature B-cells.

[0124] In one embodiment of all aspects the single cells are deposited (individually) into the wells of a multi-well plate.

[0125] In one embodiment of all aspects the deposited cells are labeled with one or three fluorescence dyes and the incubation is with two to four fluorescently labeled antibodies.

[0126] In one embodiment of all aspects the labeling of the B-cells of the population of B-cells results in labeling of 0.1% to 2.5% of the cells of the (total) B-cell population.

[0127] In one embodiment of all aspects the labeling is of B-cell surface IgG.

[0128] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG+IgM-B-cells).

[0129] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-light chain antibody (the labeling is of cell surface IgG and cell surface antibody light chain) and the selection is of cells positive for cell surface IgG and positive for cell surface antibody light chain (results in single cell deposition of IgG+LC+-B-cells).

[0130] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG+IgM-B-cells), whereby the population of B-cells has been incubated with (target) antigen, which is immobilized on a solid surface, and (only) B-cells bound to the immobilized antigen have been recovered and subjected to the incubation with the fluorescently labeled antibodies.

[0131] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-CD19 antibody (the labeling is of cell surface IgG and cell surface CD19) and the selection is of cells positive for cell surface IgG and also positive for cell surface CD19 (results in single cell deposition of IgG+CD19+-B-cells).

[0132] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-CD38 antibody (the labeling is of cell surface IgG and cell surface CD38) and the selection is of cells positive for cell surface IgG and also positive for cell surface CD38 (results in single cell deposition of IgG+CD38+-B-cells).

[0133] In one embodiment of all previous embodiment the incubation is in addition with a fluorescently labeled anti-light chain antibody (the labeling is of cell surface antibody light chain in addition to the other two labels) and the selection is of cells positive for cell surface antibody light chain (results in single cell deposition of LC+-B-cells).

[0134] In one embodiment of all aspects the B-cell population is a rabbit B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody (the labeling is of cell surface IgG) and the selection is of cells positive for cell surface IgG (results in single cell deposition of IgG+-B-cells).

[0135] In one preferred embodiment of all aspects the B-cell population is a rabbit B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG+IgM-B-cells).

[0136] In one embodiment of all previous embodiment the incubation of the rabbit B-cells is in addition with a fluorescently labeled anti-light chain antibody (the labeling is of cell surface antibody light chain in addition to the other two labels) and the selection is of cells positive for cell surface antibody light chain (results in single cell deposition of LC+-B-cells).

[0137] In one embodiment of all aspects the B-cell population is a hamster B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG+IgM-B-cells).

[0138] In one embodiment of all aspects the B-cell population is a murine B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody (the labeling is of cell surface IgG) and the selection is of cells positive for cell surface IgG (results in single cell deposition of IgG+-B-cells).

[0139] In one embodiment of all aspects the B-cell population is a murine B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-CD19 antibody (the labeling is of cell surface IgG and cell surface CD19) and the selection is of cells positive for cell surface IgG and also positive for cell surface CD19 (results in single cell deposition of IgG+CD19+-B-cells).

[0140] In one embodiment the animal is an experimental animal. In one embodiment the experimental animal is selected from mouse, hamster, and rabbit. In one embodiment the experimental animal is a rabbit.

Single Cell Depositing

[0141] The method as reported herein comprises the step of depositing the B-cells of a B-cell population as single cells. In one embodiment of all methods as reported herein the depositing as single cells is by fluorescence activated cell sorting (FACS). The surface marker used for the labeling required for the FACS single cell depositing can be with the specific marker combination as outlined herein.

[0142] The depositing is by introducing the labeled B-cell preparation into a flow cytometer and depositing those cells as single cells that have been labeled with one to three fluorescent labels. As it is possible to incubate the cells with more fluorescent dyes as those which are used for selecting the cells in the cell sorter the cells can be selected for the presence of specific surface markers and (optionally) simultaneously for the absence of other surface markers.

[0143] The labeling and single cell deposition is done in order to reduce the complexity of the B-cell population by depleting those B-cells that are not likely to produce an antibody having the intended characteristics. The labeled antibodies bind to a specific polypeptide displayed on the surface of B-cells and, thus, provide for a positive selection label. Likewise it is also possible to select cells that are only labeled with a reduced number of fluorescent dyes compared to the number of labeled antibodies with which the B-cell had been incubated, such as e.g. cells having one fluorescent label out of two (i.e. incubation with two fluorescently label antibodies has been performed but only one thereof binds to the B-cells). Based on the binding/non-binding of the fluorescently labeled antibodies to the individual B-cells of the B-cell population it is possible to identify and separate target B-cells using a microfluidic sorting apparatus. Concomitantly with the selection also the amount of the label can be determined.

[0144] An additional centrifugation step after the single cell depositing and prior to the co-cultivation can increase the number of antibody secreting cells and the amount of the secreted IgG.

[0145] In one embodiment of all methods as reported herein the method comprises the step of centrifuging the single deposited cells prior to the co-cultivation with feeder cells. In one preferred embodiment the centrifuging is for 5 min. at 300g.

Co-Cultivation

[0146] Single deposited B-cells can be co-cultivated with feeder cells in the presence of TSN produced with a method as reported herein as additive. In one embodiment the B-cells are co-cultivated with murine EL-4 B5 cells as feeder cells.

[0147] The co-cultivation step with feeder cells can be preceded and also succeeded by a number of additional steps.

[0148] By using the TSN produced with a method as reported herein after about seven (7) days, i.e. after 5, 6, 7, or 8 days, especially after 7 or 8 days, of co-cultivation high antibody concentrations in the cultivation supernatant can be obtained. With the thereby provided amount of antibody a high number of different analyses can be performed in order to characterize the antibody, e.g. regarding binding specificity, in more detail. With the improved characterization of the antibody at this early stage in the screening/selection process it is possible to reduce the number of required nucleic acid isolations and sequencing reactions that have to be performed. Additionally the B-cell clone provides an amount of mRNA encoding monoclonal light and heavy chain variable region allowing the use of degenerated PCR primer and obviates the requirement of highly specific primer. Also the required number of PCR cycles is reduced. Thus, in one embodiment the reverse transcriptase PCR is with degenerated PCR primer for the light and heavy chain variable domain.

[0149] In one embodiment of all B-cell co-cultivation methods as reported herein the thymocyte cultivation supernatant is obtained with a method as reported herein.

Characterization of Co-Cultivated Cells

[0150] For the (qualitative and quantitative) determination of secreted IgG after the co-cultivation generally all methods known to a person of skill in the art such as an ELISA can be used. In one embodiment of all methods as reported herein an ELISA is used.

[0151] Depending on the characterization results a B-cell clone can be obtained, i.e. selected. The term clone denotes a population of dividing and antibody secreting B-cells arising from/originating from a single B-cell. Thus, a B-cell clone produces a monoclonal antibody.

Further Methods Using the Method as Reported Herein:

[0152] One aspect as reported herein is a method for co-cultivating one or more B-cells (for the production of immunoglobulin) comprising the step of [0153] co-cultivating the one or more B-cells with feeder cells in the presence TSN produced with a method as reported herein (and thereby producing immunoglobulin).

[0154] In one embodiment the method further comprises the step of [0155] recovering the immunoglobulin from the cells or the cultivation medium and thereby producing the immunoglobulin.

[0156] In one embodiment the immunoglobulin is an antibody.

[0157] In one embodiment the co-cultivating is further in the presence of 1.5-7.25 ng/ml phorbol myristate acetate.

[0158] In one embodiment the co-cultivating is further in the presence of Staphylococcus aureus strain Cowan's cells (SAC).

[0159] In one embodiment the feeder cells are (murine) EL4-B5 cells.

[0160] In one embodiment the method is for the co-cultivation of one B-cell. In one preferred embodiment the one B-cell is a single deposited B-cell.

[0161] In one embodiment the co-cultivating is for 5 to 14 days.

[0162] The result of the co-cultivating is a B-cell clone, i.e. a population of B-cells that are the progeny of a single B-cell.

[0163] In one embodiment the method for co-cultivating one or more B-cells comprises prior to the co-cultivating step the following step: [0164] depositing those B-cells of a population of B-cells that have been labeled with one to three fluorescence dyes/fluorophores as single cells.

[0165] In one embodiment the method for co-cultivating one or more B-cells comprises prior to the co-cultivating step the following step: [0166] depositing those B-cells of a population of B-cells as single cells that have been contacted with two to four antibodies each specifically binding to a different B-cell surface antigen, whereby each antibody is conjugated to a different fluorescent dye, but labeled only with one to three fluorescence dyes.

[0167] The labeling is in one embodiment by contacting the B-cell population (sequentially or simultaneously) with two to four fluorescently labeled antibodies.

[0168] In one embodiment the method for co-cultivating one or more B-cells comprises the step of incubating the population of B-cells in the co-cultivation medium prior to the single cell depositing/deposition. In one embodiment the incubating is at about 37 C. In one embodiment the incubating is for 0.5 to two hours. In one embodiment the incubating is for about one hour. In one preferred embodiment the incubating is at about 37 C. for about one hour.

[0169] In one embodiment the method for co-cultivating one or more B-cells comprises after the depositing step and before the co-cultivating step the step of centrifuging the single cell deposited B-cells. In one embodiment the centrifuging is for about 1 min. to about 30 min. In one embodiment the centrifuging is for about 5 min. In one embodiment the centrifuging is at about 100g to about 1,000g. In one embodiment the centrifuging is at about 300g. In one preferred embodiment the centrifuging is for about 5 min. at about 300g.

[0170] In one embodiment the method for co-cultivating one or more B-cells comprises the following steps as first steps [0171] a) labeling the B-cells of a population of B-cells with one to three fluorescent dyes (optionally by incubating the B-cell population with two to four fluorescently labeled antibodies specifically binding to two to four different pre-determined B-cell surface markers), [0172] b) optionally incubating the cells in co-cultivation medium, [0173] c) depositing those B-cells of the population of B-cells that have been labeled with one to three fluorescent dyes (and optionally not labeled with the other fluorescent dye(s)) as single cells, [0174] d) optionally centrifuging the single deposited B-cells, [0175] e) (individually) co-cultivating each single deposited B-cell with feeder cells in a co-cultivation medium, which has been supplemented with a feeder mix, [0176] f) selecting a B-cell clone proliferating and secreting an antibody in step e).

[0177] One aspect as reported herein is a method for producing an antibody comprising the following steps: [0178] a) labeling the B-cells of a population of B-cells with one to three fluorescent dyes (optionally by incubating the B-cell population with two to four fluorescently labeled antibodies specifically binding to two to four different pre-determined B-cell surface markers), [0179] b) optionally incubating the cells in co-cultivation medium, [0180] c) depositing those B-cells of the population of B-cells that have been labeled with one to three fluorescent dyes (and optionally not labeled with the other fluorescent dye(s)) as single cells, [0181] d) optionally centrifuging the single deposited B-cells, [0182] e) (individually) co-cultivating each single deposited B-cell with feeder cells in a co-cultivation medium, which has been supplemented with a feeder mix, [0183] f) selecting a B-cell clone of step e) secreting an antibody, [0184] g) i) obtaining one or more nucleic acids encoding the secreted antibody's variable domains from the B-cell clone selected in step f), [0185] ii) if the B-cell clone is not a human B-cell clone humanizing the variable domains and providing the respective encoding nucleic acids, and [0186] iii) introducing the one or more nucleic acids in one or more expression vectors, [0187] h) cultivating a cell, which has been transfected with the one or more expression vectors of step g), and recovering the antibody from the cell or the cultivation supernatant and thereby producing the antibody.

[0188] In one embodiment the method for producing an antibody comprises the following steps [0189] a) labeling the B-cells of a population of B-cells with one to three fluorescent dyes (optionally by incubating the B-cell population with two to four fluorescently labeled antibodies specifically binding to two to four different pre-determined B-cell surface markers), [0190] b) optionally incubating the cells in co-cultivation medium, [0191] c) depositing those B-cells of a population of B-cells that have been labeled with one to three fluorescent dyes (and optionally not labeled with the other fluorescent dye(s)) as single cells, [0192] d) optionally centrifuging the single deposited B-cells, [0193] e) (individually) co-cultivating each single deposited B-cell with feeder cells in a co-cultivation medium, which has been supplemented with a feeder mix, [0194] f) determining the binding specificity of the antibodies secreted in the cultivation medium of the individual B-cells, [0195] g) obtaining one or more nucleic acids encoding the secreted antibody's variable domains from the B-cell clone by a reverse transcriptase PCR and nucleotide sequencing, (and thereby obtaining a monoclonal antibody variable light and heavy chain domain encoding nucleic acid,) [0196] h) if the B-cell is a non-human B-cell humanizing the variable light and heavy chain domain and providing a nucleic acid encoding the humanized variable domains, [0197] i) introducing the monoclonal antibody variable light and heavy chain variable domain encoding nucleic acid in one or more expression vectors for the expression of an (human or humanized) antibody, [0198] j) introducing the expression vector(s) in a cell, [0199] k) cultivating the cell and recovering the antibody from the cell or the cell culture supernatant and thereby producing the antibody.

[0200] In one embodiment the obtaining one or more nucleic acids encoding the secreted antibody's variable domains from the B-cell clone comprises the following steps [0201] extracting total RNA from the antibody-producing B-cell clone, [0202] performing a single stranded cDNA synthesis/reverse transcription of the extracted polyA.sup.+ mRNA, [0203] performing a PCR with a set of species specific primer, [0204] optionally removal of the PCR primer/purification of the PCR product, [0205] optionally sequencing of the PCR product.

[0206] In one embodiment the introducing the monoclonal antibody variable light and/or heavy chain variable domain encoding nucleic acid in an expression vector for the expression of an (human or humanized) antibody comprises the following steps [0207] T4 polymerase incubation of the variable light and heavy chain variable domain, [0208] linearization and amplification of the expression vector, [0209] T4 polymerase incubation of the amplified expression vector, [0210] sequence and ligation independent cloning of the variable domain encoding nucleic acid into the amplified expression vector, and [0211] preparation of the vector(s) from pool of vector transformed E. coli cells.

[0212] In one embodiment the methods above comprise immediately prior to the labeling step the following step [0213] incubating the population of B-cells with (target) antigen, which is immobilized on a solid surface, and recovering (only) B-cells bound to the immobilized antigen.

[0214] In one embodiment the population of B-cells is a non-human animal B-cell population. In one embodiment the B-cell population is a mouse B-cell population, or a hamster B-cell population, or a rabbit B-cell population. In one preferred embodiment the B-cell population is a rabbit B-cell population.

[0215] In one embodiment the population of B-cells is obtained from the blood of a non-human animal 4 days after the immunization. In one embodiment the population of B-cells is obtained from the blood of a non-human animal of from 4 days up to at most 9 days after immunization.

[0216] In one embodiment the B-cell population is a human B-cell population.

[0217] In one embodiment the population of B-cells is obtained from blood by density gradient centrifugation.

[0218] In one embodiment the B-cells are mature B-cells.

[0219] In one embodiment of the single cells are deposited (individually) into the wells of a multi-well plate.

[0220] In one embodiment the antibody is a monoclonal antibody.

[0221] In one embodiment the deposited cells are labeled with one or three fluorescence dyes and the incubation is with two to four fluorescently labeled antibodies.

[0222] In one embodiment the labeling of the B-cells of the population of B-cells results in labeling of 0.1% to 2.5% of the cells of the (total) B-cell population.

[0223] In one embodiment the labeling is of B-cell surface IgG.

[0224] In one embodiment the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG.sup.+IgM.sup.-B-cells).

[0225] In one embodiment of all aspects the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-light chain antibody (the labeling is of cell surface IgG and cell surface antibody light chain) and the selection is of cells positive for cell surface IgG and positive for cell surface antibody light chain (results in single cell deposition of IgG.sup.+LC.sup.+-B-cells).

[0226] In one embodiment the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG.sup.+IgM.sup.-B-cells), whereby the population of B-cells has been incubated with (target) antigen, which is immobilized on a solid surface, and (only) B-cells bound to the immobilized antigen have been recovered and subjected to the incubation with the fluorescently labeled antibodies.

[0227] In one embodiment the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-CD19 antibody (the labeling is of cell surface IgG and cell surface CD19) and the selection is of cells positive for cell surface IgG and also positive for cell surface CD19 (results in single cell deposition of IgG.sup.+CD19.sup.+-B-cells).

[0228] In one embodiment the incubation is in addition with a fluorescently labeled anti-light chain antibody (the labeling is of cell surface antibody light chain in addition to the other two labels) and the selection is of cells positive for cell surface antibody light chain (results in single cell deposition of LC.sup.+-B-cells).

[0229] In one embodiment the B-cell population is a rabbit B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody (the labeling is of cell surface IgG) and the selection is of cells positive for cell surface IgG (results in single cell deposition of IgG.sup.+-B-cells).

[0230] In one preferred embodiment the B-cell population is a rabbit B-cell population and the incubation is with a fluorescently labeled anti-IgG antibody and a fluorescently labeled anti-IgM antibody (the labeling is of cell surface IgG and cell surface IgM) and the selection is of cells positive for cell surface IgG and negative for cell surface IgM (results in single cell deposition of IgG.sup.+IgM.sup.-B-cells).

[0231] In one embodiment the incubation of the rabbit B-cells is in addition with a fluorescently labeled anti-light chain antibody (the labeling is of cell surface antibody light chain in addition to the other two labels) and the selection is of cells positive for cell surface antibody light chain (results in single cell deposition of LC-B-cells).

[0232] In one embodiment the co-cultivating is in a co-cultivation medium comprising RPMI (1640) medium supplemented with 10% (v/v) FCS, 1% (w/v) of a 200 mM glutamine solution that comprises penicillin and streptomycin, 2% (v/v) of a 100 mM sodium pyruvate solution, and 1% (v/v) of a 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethane sulfonic acid (HEPES) buffer. In one embodiment the co-cultivation medium further comprises 0.05 mM beta-mercaptoethanol.

[0233] In one embodiment the animal is an experimental animal. In one embodiment the experimental animal is selected from mouse, hamster, and rabbit. In one embodiment the experimental animal is a rabbit.

[0234] The following FIGURE and examples are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

DESCRIPTION OF THE FIGURE

[0235] The FIGURE shows the lot-to-lot variability of TSN lots produced with a method known from the art (left part) and with the inventive method as reported herein (right part). Solid square: ratio of the number IgG-positive wells of the respective TSN lot to the number of IgG-positive wells obtained with a reference TSN lot; dash-point-dash line: average of the ratio of the number of IgG positive wells; solid triangles: ratio of the average IgG concentration in the wells obtained with the respective TSN lot to the average IgG concentration in the wells obtained with a reference TSN lot; dashed line: average of the IgG concentration ratios.

EXAMPLES

Materials and Methods

Recombinant DNA Techniques

[0236] Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The molecular biological reagents were used according to the manufacturer's instructions.

Media and Buffers

[0237] Blocking buffer for ELISA comprises 1PBS and 1% BSA.

[0238] Coating buffer for ELISA comprises 4.29 g Na2CO3* 10 H2O and 2.93 g NaHCO3add water to a final volume of 1 liter, pH 9.6 adjusted with 2 N HCl.

[0239] Ethanol-solution for RNA isolation comprises 70% Ethanol or 80% Ethanol.

[0240] FACS-buffer for immuno fluorescence staining comprises 1PBS and 0.1% BSA.

[0241] IMDM-buffer for ELISA comprises 1PBS, 5% IMDM and 0.5% BSA.

[0242] Incubation buffer 1 for ELISA comprises 1PBS, 0.5 CroteinC.

[0243] Incubation buffer 2 for ELISA comprises 1PBS, 0.5 CroteinC and 0.02% Tween 20.

[0244] Incubation buffer 3 for ELISA comprises 1PBS, 0.1% BSA.

[0245] Incubation buffer 4 for ELISA comprises 1PBS, 0.5% BSA, 0.05% Tween, PBS (10), 0.01 M KH2PO4, 0.1 M Na2HPO4, 1.37 M NaCl, 0.027 M KCl, pH 7.0.

[0246] PCR-buffer comprises 500 mM KCl, 15 mM MgCl2, 100 mM Tris/HCl, pH 9.0.

[0247] Wash buffer 1 for ELISA comprises 1PBS, 0.05% Tween 20.

[0248] Wash buffer 2 for ELISA comprises 1PBS, 0.1% Tween 20.

[0249] Wash buffer 3 for ELISA comprises water, 0.9% NaCl, 0.05 Tween 20.

[0250] EL-4 B5 medium comprises RPMI 1640 (Pan Biotech, Aidenbach, Germany) supplemented with 10% FCS (Hyclone, Logan, Utah, USA), 2 mM Glutamine, 1% penicillin/streptomycin solution (PAA, Pasching, Austria), 2 mM sodium pyruvate, 10 mM HEPES (PAN Biotech, Aidenbach, Germany) and 0.05 mM -mercaptoethanol (Gibco, Paisley, Scotland).

Animal Care

[0251] The experimental animals were held according to the German animal protection law (TierSCHG) as well as according to the respective European guidelines.

[0252] Mice and hamster were received at an age of from 6 to 8 weeks and were immunized prior to an age of 12 weeks. The antigen was at first applied together with complete Freud's adjuvant (CFA). Further applications were with incomplete Freud's adjuvant (IFA). The antigen containing emulsion was applied subcutaneously whereby the emulsion comprised an amount of from 50 to 100 g antigen depending on the weight of the receiving experimental animal.

Proliferation Assays

[0253] a) Cell Titer Glo (CTG) viability assay [0254] The CTG viability assay (Promega; # G7571) was used according to the instructions of the manufacturer. [0255] b).sup.3H Thymidine Assay [0256] After 6 days of incubation .sup.3H-Thymidin was added (0.5 Ci/well) and incubated for further 16 hours. The incorporation of .sup.3H-Thymidine during cell proliferation was determined with a microplate scintillation counter (Wallac). [0257] c) Microscopic analysis [0258] For the acquisition of microscopic images, a phase contrast microscope from Leica (Leica DM IL) combined with a high resolution camera (Leica DFC290 HD) was used. [0259] d) Analysis of B-cell activation via CF SE-labeling. [0260] Isolated B-cells were washed with sterile phosphate buffer saline solution (PBS). Up to 110.sup.7 cells were resuspended in 1 ml protein-free PBS and incubated with CFSE (# C34554, Invitrogen/Molecular Probes) for 3 to 10 minutes at a final concentration of 2.5 M at 37 C. CFSE loading was stopped by addition of an excess of FCS-supplemented medium. After extensive washing with FCS-containing medium, B-cells were used in co-culture experiments. Proliferation of CD19.sup.+ gated (B-)cells as a consequence of CFSE dilution was confirmed by flow cytometric analysis (FL-1 channel) after indicated time points.

Quantification of IgG

[0261] The 96-well multi well plate in which the co-cultivation was performed was centrifuged after seven days of co-cultivation at 300g for 5 min. 150 l supernatant was removed and diluted at a ratio of 2:1 with PBS in a second 96-well multi well plate.

[0262] The antibody was used at a concentration of 50 ng/ml. If the OD was or exceeded 1 after an incubation time of 5 min. a dilution series of from 0.8 to 108 ng/ml IgG was tested.

Panning on Antigen

a) Coating of Plates

[0263] Biotin/Streptavidin: Sterile streptavidin-coated 6-well plates (cell culture grade) were incubated with biotinylated antigen at a concentration of 0.5-1(2) g/ml in PBS at room temperature for one hour. Plates were washed in sterile PBS three times before use.

[0264] Covalently bound protein: Sterile cell culture 6-well plates were coated with 2 g/ml protein in carbonate buffer (0.1 M sodium bicarbonate, 34 mM disodium hydrogen carbonate, pH 9.55) over night at 4 C. Plates were washed in sterile PBS three times before use.

b) Panning of B-Cells on Peptides

[0265] 6-well tissue culture plates coated with the respective antigen were seeded with up to 610.sup.6 cells per 4 ml medium and allowed to bind for one hour at 37 C. in the incubator. Non-adherent cells were removed by carefully washing the wells 1-2 times with 1PBS. The remaining sticky cells were detached by trypsin for 10 min. at 37 C. in the incubator and then washed twice in media. The cells were kept on ice until the immune fluorescence staining.

Gene Synthesis

[0266] Desired gene segments encoding cDNA were prepared by Geneart GmbH (Regensburg, Germany). The gene segments are flanked by singular restriction endonuclease cleavage sites to facilitate expression construct cloning as described below. The DNA sequence of the subcloned gene fragments were confirmed by DNA sequencing.

Rabbit B-Cell Medium/EL4-B5 Medium:

[0267]

TABLE-US-00006 500 ml RPMI 1640 #P04-17500 PAN Biotech 50 ml FCS #A15-512 PAA 5 ml L-glutamine #25030-024 Invitrogen 5 ml potassium pyruvate #P04-43100 PAN Biotech 5 ml HEPES #15630-056 Invitrogen 500 l -mercaptoethanol #31350010 Invitrogen 1 ml Pen/Strep #11074440001 Roche Di a. GmbH

Additives to Rabbit B-Cell Medium

[0268]

TABLE-US-00007 SAC #507858 Calbiochem

Multi-Well-Plates

[0269]

TABLE-US-00008 96erU-plate #3799 Corning

Phenotyping/Sorting of Antibodies

[0270]

TABLE-US-00009 goat anti-rabbit IgG Fc-antibody AbDSerotec STAR121F anti-human/murine (rabbit cross- reactive) anti CD40L antibody: anti-muCD40L antibody R&D systems AF1163 anti-huCD40L antibody &D systems AF617R donkey anti-goat IgG antibody Molecular Probes A11055 Alexa 488

Miscellaneous

[0271]

TABLE-US-00010 anti-FITC antibody-coupled Miltenyi Biotec #130-048-701 microbeads human B-cell negative isolation kit Invitrogen #113.13D Nucleofector Kit T Lonza VCA-1002 CBA for total IgG BD Biosciences #558679

Example 1

Immunization of Rabbits

[0272] NZW rabbits (Charles River Laboratories International, Inc.) were used for immunization. The antigen was solved in K.sub.3PO.sub.4 buffer pH 7.0 at a concentration of 1 mg/ml and mixed (1:1) with complete Freud's adjuvant (CFA) till generation of stabile emulsion. The rabbits received an intra-dermal (i.d.) injection of 2 ml of emulsion followed by a second intra muscular (i.m.) and third subcutaneous (s.c.) injection each with 1 ml in one week interval. The fourth i.m. injection of 1 ml was performed two weeks later followed by two further s.c. injections of 1 ml in four weeks interval.

[0273] During the immunization serum antibody titer was determined with an antigen specific assay. At an antibody titer with an IC.sub.50 of 1:10000 the blood or the spleen of the immunized animal was removed. For reactivation of antigen specific B-cells 30 g to 50 g of the antigen was applied intravenously to the experimental animal three days prior to the removal of the blood or the spleen.

Example 2

Removal of Organs, Blood and Macrophages

[0274] Blood from rabbits was obtained by punctuation of the ear vein or, for larger volumes, of the ear artery. Whole blood (10 ml) was collected from rabbits 4-6 days after the third, fourth, fifth and sixth immunization and used for single cell sorting by FACS.

[0275] Macrophages were isolated from the obtained blood by attachment to cell culture plastic.

[0276] If a larger amount of was required, peritoneal macrophages were isolated. For this the animals have to be at least 3 months of age. For the removal of peritoneal macrophages, animals were sacrificed and 5 ml of EL-4 B5 medium with a temperature of 37 C. was immediately injected into the peritoneal cavity. After kneading the animal's belly for 5 minutes, the solution containing the cells was removed.

[0277] EDTA containing whole blood was diluted twofold with 1PBS before density centrifugation on lympholyte mammal (Cedarlane Laboratories) or Ficoll Paque Plus (GE Healthcare, cat. #17-1440-03), which was performed to isolate rabbit PBMC. PBMCs were washed twice before staining with antibodies.

Example 3

Density Gradient Centrifugation

[0278] The isolation of peripheral blood mononuclear cells (PBMCs) was effected by density gradient separation with Lympholyte according to manufacturer's instructions A (Lympholyte-mammal, Cedarlane).

[0279] Withdrawn blood (optionally supplemented with EDTA) was diluted 1:1 with phosphate buffered saline (PBS), e.g. 60 ml blood plus 60 ml buffer. In a centrifuge vial the same volume of density separation medium was provided and the diluted blood is carefully added via the wall of the vial on top of the density separation medium. The ratio of the density separation medium to the PBS-diluted blood is about 1:1.5. Depending on the total volume of diluted blood a certain number of vials will be required. The vials were centrifuged for 20 min. at 800g without braking. Each individual white interim layer was added to 25 ml PBS, supplemented with PBS to a total of 50 ml and centrifuged at 800g for 10 min. The supernatants were discarded, the pellet were resuspended in 1/120 of the final volume of PBS, pellets were combined on a 1:1 basis and PBS was added to a final volume of 50 ml. Thereafter the vials were centrifuged again. Depending on the number of blood samples the samples are combined on a 1:1 ratio after each centrifugation step until only one sample is left. The final pellet was resuspended in PBS.

Example 4

Hypotonic Lysis of Red Blood Cells

[0280] For disruption of red blood cells by hypotonic lysis an ammonium chloride solution (BD Lys) was diluted 1:10 with water and added at a ratio of 1:16 to whole blood. For lysis of the red blood cells the mixture was incubated for 15 min. in the dark. For separation of cell debris from intact cells the solution was centrifuged for 10 min. at 800g. The supernatant was discarded, the pellet was resuspended in PBS, washed again, centrifuged and the pellet was resuspended in PBS. Example 8

Example 5

Depletion of Macrophages

[0281] Sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes through unspecific adhesion. Wells were either coated with KLH (keyhole limpet haemocyanine) or with streptavidin and the control peptides. Each well was filled with 3 ml to (at maximum) 4 ml medium and up to 610.sup.6 peripheral blood mononuclear cells from the immunized rabbit and allowed to bind for 60 to 90 min. at 37 C. in the incubator. Thereafter the lymphocyte containing supernatant was transferred to a centrifugation vial and centrifuged at 800g for 10 min. The pellet was resuspended in PBS.

Example 6

Enrichment of Antigen-Specific B-Cells

[0282] The respective antigen was diluted with coating buffer to a final concentration of 2 g/ml. 3 ml of this solution were added to the well of a 6-well multi well plate and incubated over night at room temperature. Prior to use the supernatant was removed and the wells were washed twice with PBS. The B-cell solution was adjusted to a cell density of 210.sup.6 cells/ml and 3 ml are added to each well (up to 610.sup.6 cells per 3-4 ml medium) of a 6-well multi well plate. The plate was incubated for 60 to 90 min. at 37 C. The supernatant was removed and non-adherent cells were removed by carefully washing the wells 1-4 times with 1PBS. For recovery of the sticky antigen-specific B-cells 1 ml of a trypsin/EDTA-solution was added to the wells of the multi well plate and incubated for 10 to 15 min. at 37 C. The incubation was stopped by addition of medium and the supernatant was transferred to a centrifugation vial. The wells were washed twice with PBS and the supernatants were combined with the other supernatants. The cells were pelleted by centrifugation for 10 min. at 800 x g. The cells were kept on ice until the immune fluorescence staining. The pellet was optionally resuspended in PBS.

Example 7

Cultivation of T-Cells

[0283] The T-cells were isolated from the thymus of 3-4 week old mice and hamsters, or of 4-5 week old rabbits, respectively. The cells were centrifuged and immediately cultivated or frozen in aliquots of 4-510 cells. The thymocytes were seeded with a minimum cell density of 510.sup.5 cells/ml of EL-4 B5 medium in 175 cm.sup.2 culture flasks and incubated for up to 48 hours (40-48 hours depending on the TSN production method the macrophages will be used in; see Examples 9 and 10) at 37 C.

Example 8

Cultivation of Macrophages

[0284] Macrophages were isolated from the peritoneal cavity of mice and hamsters, respectively, of an age of at least three months. Peritoneal macrophages from mice or hamsters, or blood mononuclear cells from rabbits were cultivated in EL-4 B5 medium at a cell density of at least 110.sup.5 cells/ml in 175 cm.sup.2 culture flasks for 1.5 hours at 37 C. Afterwards the medium was removed and non-attached cells were removed from the attached macrophages by washing with warm EL-4 B5 medium, followed by cultivation for about 48 hours in 35 ml medium.

Example 9

Co-Cultivation of T-Cells and Macrophages According to the Current Invention

[0285] T-cells (see Example 7, 40 hours cultivation) and macrophages (see Example 8) were cultivated in separate flasks. Prior to combining both cell populations, the T-cells were centrifuged for 10 min. at 800 x g. The supernatant was discarded and the cell pellet was resuspended in 10 ml EL-4 B5 medium. The final cultivation medium contained T-cells adjusted to a cell density of 510.sup.5 cells/ml, 10 ng phorbol-12-myristate-13-acetate (PMA) per ml of medium, and 5 g phytohemagglutinin M (PHA-M) per ml of medium (=T-cell suspension). Thereafter, the cultivation medium was removed from the macrophages (=medium-depleted macrophages). An amount/volume of the T-cell suspension was added to the flasks containing the medium-depleted macrophages to obtain a final but defined macrophage cell density of from 1.25-210.sup.6 macrophages/ml. After 30-46 hours of co-cultivation, the cultivation medium was removed and was termed TSN solution. For removal of remaining cells the TSN solution was filtered through a 0.22 m filter. The TSN solution was frozen at 80 C. in aliquots (of 4.2 ml).

Example 10Comparative Example

Co-Cultivation of T-Cells and Macrophages According to the State of the Art

[0286] T-cells (see Example 7, 48 hours cultivation) and macrophages (see Example 8) were cultivated in separate flasks. Prior to combining both cell populations, the T-cells were centrifuged for 10 min. at 800 x g. The supernatant was discarded and the cell pellet was resuspended in 10 ml EL-4 B5 medium. The final cultivation medium contained T-cells adjusted to a cell density of 510.sup.5 cells/ml, 10 ng phorbol-12-myristate-13-acetate (PMA) per ml of medium, and 5 g phytohemagglutinin M (PHA-M) per ml of medium (=T-cell suspension). Thereafter, the cultivation medium was removed from the macrophages (=medium-depleted macrophages). An amount/volume of the T-cell suspension was added to the flasks containing the medium-depleted macrophages to obtain a final macrophage cell density of 110.sup.6 macrophages/ml. After 36 hours of co-cultivation, the cultivation medium was removed and was termed TSN solution. For removal of remaining cells the TSN solution was filtered through a 0.22 m filter. The TSN solution was frozen at 80 C. in aliquots (of 4 ml).

Example 11

Cultivation of EL-4 B5 Cells

[0287] The frozen EL-4 B5 cells were thawed rapidly in a water bath at 37 C. and diluted with 10 ml EL-4 B5 medium. After centrifugation at 300g for 10 minutes the supernatant was discarded and the pellet resuspended in 1 ml medium.

[0288] The EL-4 B5 cells were inoculated at a cell density of 810 cells/ml in T175 cultivation flasks. Cell density was determined every second day and adjusted to 810.sup.4 cells/ml. The cells have a doubling time of approximately 10 hours.

[0289] Cells were harvested and adjusted to a cell density of 110.sup.6 cells/ml before -irradiation at 50 Gy.

Example 12

Co-Cultivation of B-Cells and EL-4 B5 Cells

[0290] Single sorted B-cells were cultured in 96-well plates with 210 l/well EL-4 B5 medium with Pansorbin Cells (1:20000) (Calbiochem (Merck), Darmstadt, Deutschland), 5% thymocyte supernatant produced according to Example 9 or 10 and gamma-irradiated EL-4-B5 murine thymoma cells (210.sup.4/well) for 7 days at 37 C. in an atmosphere of 5% CO.sub.2 in the incubator. B-cell culture supernatants were removed for screening and the cells harvested immediately for variable region gene cloning or frozen at 80 C. in 100 l RLT buffer (Qiagen, Hilden, Germany).

[0291] It has been found that depending on the amount of macrophages present in the co-cultivation of thymocytes (T-cells) with mononuclear cells (macrophages), i.e. depending on the ratio of said cells, the produced TSN shows different properties. The respective results are shown in the following Table (total wells=4*84). The reference value was obtained with the comparative state of the art method according to Example 10.

TABLE-US-00011 TABLE average ratio frequency of average [*10.sup.6 cells/ml] IgG positive productivity of thymocytes: average IgG wells all IgG positive mononuclear positive wells [% of total wells cells [n] wells] [g/ml] example 0.5:1 (ref.) 33.3 2.3 39.6 2.7 0.35 0.05 10 0.5:1.25 38.8 4.8 46.1 5.7 0.81 0.10 9 0.5:1.5 34.5 5.5 41.1 6.6 1.22 0.12 9 0.5:2 37.3 1.8 44.4 2.1 1.32 0.23 9

Example 13

[0292] Co-Cultivation of B-Cells and EL-4 B5 Cells with Different Lots Produced According to Examples 9 and 10

[0293] To show the robustness of the herein reported TSN production method compared to the method known from the art multiple lots have been prepared with the methods according to Examples 9 and 10. The results are presented in the following Tables.

According to Example 10:

[0294]

TABLE-US-00012 ratio IgG positive wells ratio average productivity wells TSN lot to reference to reference reference 1.00 1.00 1 0.80 0.75 2 0.83 0.89 3 0.84 0.65 4 0.78 0.42 5 1.04 0.49 6 0.88 0.56 7 0.96 0.64 8 0.94 0.69 9 1.11 0.79 10 0.83 0.66 11 1.11 0.39 12 1.04 0.78 13 0.95 0.48 average 0.93 0.63
According to example 9:

TABLE-US-00013 ratio IgG positive wells ratio average productivity wells TSN lot to reference to reference reference 1.00 1.00 14 0.83 0.52 15 0.96 0.96 16 0.99 1.55 17 1.04 1.15 18 0.98 1.21 19 1.33 1.53 20 0.81 1.37 21 1.05 1.54 22 1.01 1.09 23 1.14 1.26 24 1.23 1.02 25 1.03 1.38 26 1.03 0.96 27 1.04 1.31 28 1.17 1.52 29 1.10 1.46 30 1.00 1.95 31 0.97 2.60 32 0.94 1.27 33 0.94 1.15 34 0.85 1.05 35 0.96 1.32 average 1.02 1.33