B-cell cultivation method

Abstract

Herein is reported a method for co-cultivating B-cells in the presence of phorbol myristate acetate, IL-1beta, TNFalpha, IL-2, IL-10 and IL-6.

Claims

1. A method for co-cultivating a single deposited B-cell comprising the step of co-cultivating the B-cell with feeder cells in the presence of 1.5-7.25 ng/ml phorbol myristate acetate, wherein neither of a thymocyte cultivation supernatant, a macrophage cultivation supernatant, or a T-cell cultivation supernatant is added to the cultivation.

2. A method for co-cultivating a B-cell comprising the step of co-cultivating the B-cell with feeder cells in the presence of phorbol myristate acetate, IL-1beta, TNFalpha, IL-2, IL-10 and IL-6, wherein neither of a thymocyte cultivation supernatant, a macrophage cultivation supernatant, or a T-cell cultivation supernatant is added to the cultivation.

3. The method according to claim 2, wherein the co-cultivating is in the presence of 0.3-3 ng/ml phorbol myristate acetate, 0.02-0.2 ng/ml IL-1beta, 0.02-0.2 ng/ml TNFalpha, 0.5-5 ng/ml IL-2, 0.1-1 ng/ml IL-10, and 0.1-1 ng/ml IL-6.

4. The method according to claim 2, wherein the co-cultivating is in the presence of about 0.05-0.7 ng/ml phorbol myristate acetate, about 0.2 ng/ml IL-1beta, about 0.2 ng/ml TNFalpha, about 5 ng/ml IL-2, about 1 ng/ml IL-10, and about 1 ng/ml IL-6.

5. The method according to claim 2, wherein the co-cultivating is in the presence of about 0.2-0.35 ng/ml phorbol myristate acetate, about 0.064 ng/ml IL-1beta, about 0.064 ng/ml TNFalpha, about 1.6 ng/ml IL-2, about 0.32 ng/ml IL-10, and about 0.32 ng/ml IL-6.

6. The method according to claim 2, wherein the co-cultivating is in the presence of about 1.3 ng/ml phorbol myristate acetate, about 0.064 ng/ml IL-1beta, about 0.064 ng/ml TNFalpha, about 1.6 ng/ml IL-2, about 0.32 ng/ml IL-10, and about 0.32 ng/ml IL-6.

7. The method according to claim 3, wherein the IL-1beta has an activity of 5.5-14*10.sup.8 IU/mg, the TNFalpha has an activity of 2.3-2.9*10.sup.8 U/mg, the IL-2 has an activity of 6-7*10.sup.6 IU/mg, the IL-10 has an activity of 6-7.5*10.sup.5 IU/mg, and the IL-6 has an activity of 9.2-16.1*10.sup.8 U/mg.

8. The method according to claim 1, wherein the feeder cells are EL4-B5 cells.

9. The method according to claim 1, wherein the co-cultivating is further in the presence of SAC.

10. A method for producing an antibody comprising the co-cultivation method according to claim 1.

11. A feeder mix comprising phorbol myristate acetate, IL-1beta, TNFalpha, IL-2, IL-10 and IL-6.

12. The feeder mix according to claim 11 comprising: 0.3-3 ng/ml phorbol myristate acetate, 0.02-0.2 ng/ml IL-1beta, 0.02-0.2 ng/ml TNFalpha, 0.5-5 ng/ml IL-2, 0.1-1 ng/ml IL-10, and 0.1-1 ng/ml IL-6.

13. The feeder mix according to claim 11 comprising: 0.05-0.7 ng/ml phorbol myristate acetate, about 0.2 ng/ml IL-1beta, about 0.2 ng/ml TNFalpha, about 5 ng/ml IL-2, about 1 ng/ml IL-10, and about 1 ng/ml IL-6.

14. The feeder mix according to claim 11 comprising: 0.2-0.35 ng/ml phorbol myristate acetate, about 0.064 ng/ml IL-1beta, about 0.064 ng/ml TNFalpha, about 1.6 ng/ml IL-2, about 0.32 ng/ml IL-10, and about 0.32 ng/ml IL-6.

15. The feeder mix according to claim 11 comprising: about 1.3 ng/ml phorbol myristate acetate, about 0.064 ng/ml IL-1beta, about 0.064 ng/ml TNFalpha, about 1.6 ng/ml IL-2, about 0.32 ng/ml IL-10, and about 0.32 ng/ml IL-6.

16. The feeder mix according to claim 11, wherein the IL-1beta has an activity of 5.5-14*10.sup.8 IU/mg, the TNFalpha has an activity of 2.3-2.9*10.sup.8 U/mg, the IL-2 has an activity of 6-7*10.sup.6 IU/mg, the IL-10 has an activity of 6-7.5*10.sup.5 IU/mg, and the IL-6 has an activity of 9.2-16.1*10.sup.8 U/mg.

17. Use of a feeder mix according to claim 11 in the co-cultivation of B-cells and feeder cells.

18. The method according to claim 2, wherein the feeder cells are EL4-B5 cells.

19. The method according to claim 2, wherein the co-cultivating is further in the presence of SAC.

20. A method for producing an antibody comprising the co-cultivation method according to claim 2.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0553] The invention is based at least in part on the finding that phorbol myristate acetate alone or a combination of phorbol myristate acetate (PMA) with synthetic cytokines and synthetic interleukins can replace the commonly employed macrophage/T-cell cultivation supernatant used in the co-cultivation of B-cells and feeder cells, such as murine EL4-B5 feeder cells.

[0554] With the elimination of the need to use non-defined macrophage/T-cell cultivation supernatants in the co-cultivation of B-cells and feeder cells the co-cultivation process is made, amongst other things, more robust, less expensive and easily adaptable to B-cell derived from different species.

[0555] The invention is further based at least in part on the finding that when employing phorbol myristate acetate in combination with synthetic cytokines and synthetic interleukins the concentrations of each of these components has to be within certain ranges in order to ensure a positive effect.

[0556] Immunization

[0557] 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.

[0558] 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.

[0559] In the method as reported herein B-cells obtained from any source e.g. human, mouse, hamster or rabbit, can be used. Depending on the source of the B-cell the feeder cells and the feeder mix are adjusted/chosen.

[0560] In case of a rabbit B-cell the feeder cell is either an EL4-B5 cell or a mammalian cell, such as a CHO cell or a BHK cell or a HEK cell, expressing rabbit CD40L. In one embodiment the rabbit is selected from 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.

[0561] In case of a human B-cell the feeder cell is either an EL4-B5 cell or a mammalian cell, such as a CHO cell or a BHK cell or a HEK cell, expressing human CD40L.

[0562] In case of a murine B-cell the feeder cell is either an EL4-B5 cell or a mammalian cell, such as a CHO cell or a BHK cell or a HEK cell, expressing mouse CD40L. In one embodiment the mouse is an NMRI-mouse or a balb/c-mouse.

[0563] In case of a hamster B-cell the feeder cell is either an EL4-B5 cell or a mammalian cell, such as a CHO cell or a BHK cell or a HEK cell, expressing hamster CD40L. In one embodiment the hamster is selected from Armenian hamster (Cricetulus migratorius), Chinese hamster (Cricetulus griseus), and Syrian hamster (Mesocricetulus auratus). In one embodiment the hamster is the Armenia hamster.

[0564] Source and Isolation of B-Cells

[0565] The 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.

[0566] In one embodiment B-cells, e.g. from the blood or the spleen, are obtained of from 4 days after immunization until at most 14 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).

[0567] 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 divides and grows 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.

[0568] Selection Steps Prior to Co-Cultivation

[0569] B-cells producing antibodies that specifically bind an antigen can be enriched 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).

[0570] 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.

[0571] Macrophages can be depleted from PBMCs by adhesion to the surface of the cell culture plate (see pre-incubation step).

[0572] Incubating the population of B-cells in co-cultivation medium prior to the single cell depositing increases the total number of antibody secreting cells obtained after the single cell depositing compared to a single cell depositing directly after the isolation and optional enrichment of the population of B-cells from the blood of a non-human animal (in one embodiment the non-human animal is a rabbit). Specifically the incubating is at about 37 C. for about one hour in EL-4 B5 medium, e.g. using a cell culture incubator.

[0573] In one embodiment of the methods as reported herein the cells are from a protein-immunized animal and are depleted of macrophages prior to the labeling.

[0574] 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. Therein the respective antigen is presented attached to a surface and cells binding thereto are selectively enriched in the cell population in case the bound cells are processed further, or reduced in the cell population in case the cells remaining in solution are processed further.

[0575] 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.

[0576] With the labeling of non-target cell populations and non-specifically binding lymphocytes it is possible to selectively deplete these cells. In this 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 can be reduced or even minimized. By a single cell depositing of mature B-cells (memory B-cells, affinity matured plasmablasts and plasma cells) by fluorescence activated cell sorting using the labeling a higher number of IgG.sup.+-wells/cell clones can be obtained in the co-cultivation step.

[0577] Different cell populations can be labeled by using different surface markers such as CD3.sup.+-cells (T-cells), CD119.sup.+-cells (B-cells), IgM.sup.+-cells (mature naive B-cells), IgG.sup.+-cells (mature B-cells), LC.sup.+-cells, CD38.sup.+-cells (e.g. plasmablasts), and IgG.sup.+CD38.sup.+-cells (pre-plasma cells).

[0578] Immuno-fluorescence labeling for selection of mature IgG.sup.+-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 required is a labeling that results in about 0.1% to 2.5% of labeled cells of the total cell population.

[0579] 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.

TABLE-US-00001 TABLE Immuno-fluorescence labeling for the determination of mature mouse-, hamster- and rabbit-B-cells. B-cell origin sorting of B-cells with fraction of all viable cells (%) mouse IgG.sup.+CD19.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

[0580] 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.

[0581] Single Cell Depositing

[0582] 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.

[0583] An additional centrifugation step after the single cell depositing and prior to the co-cultivation increases the number of antibody secreting cells and increases the amount of the secreted IgG.

[0584] 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. In one preferred embodiment the centrifuging is for 5 min. at 300g.

[0585] Co-Cultivation

[0586] The single deposited B-cells are co-cultivated with feeder cells in the presence of a synthetic feeder mix as reported herein.

[0587] In one embodiment the B-cells are co-cultivated with murine EL-4 B5 cells as feeder cells.

[0588] As outlined above an increase in the yield in the co-cultivation step (number of IgG.sup.+-wells/cell clones as well as IgG-concentration) and also an enrichment or isolation of mature IgG.sup.+-B-cell from PBMCs can be achieved by suitable immuno fluorescence labeling.

[0589] The single cell depositing of IgG.sup.+IgM.sup.-cells can be used for B-cells of immunized and non-immunized non-human animals.

[0590] The single cell depositing of IgG.sup.+-, and/or IgG.sup.+CD138.sup.+-, and/or CD138.sup.+- and/or IgG.sup.+IgM.sup.-B-cells, and/or IgG.sup.+LC.sup.+-, and/or IgG.sup.+CD138.sup.+LC.sup.+-, and/or CD138.sup.+LC.sup.+- and/or IgG.sup.+IgM.sup.LC.sup.+-B-cells can be used for rabbit-B-cells.

[0591] The immuno-fluorescence labeling used for B-cells obtained from the blood of an experimental non-human animal can also be used for the labeling of B-cells obtained from the spleen and other immunological organs of an experimental non-human animal, such as mouse, hamster and rabbit. For rabbit-blood derived B-cells 0.2% of IgG.sup.+-cells were found after depletion of macrophages. Peyer'sche plaques from rabbit showed 0.4% of IgG.sup.+-cells and spleen showed 0.3% of IgG.sup.+-cells after depletion of macrophages.

[0592] With the methods 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 sufficient antibody concentrations 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.

[0593] The co-cultivation step with feeder cells can be preceded and also succeeded by a number of additional steps.

[0594] Due to the origin of the feeder mix as cultivation supernatant, i.e. its production from the supernatant of cultivated thymocytes (thymocyte cultivation supernatantTSN), considerable batch to batch variations occur.

[0595] TSN is needed in combination with feeder cells to stimulate the (single deposited) B-cell, thereby inducing proliferation and antibody secretion.

[0596] In order to overcome this variability a synthetic feeder mix having the same or comparable stimulation properties can be employed.

[0597] The B-cell-species-specific additives for the synthetic feeder mix result in increased amounts of secreted antibody by the respective B-cell clone. Concomitantly highly producing cells contain more mRNA which in turn facilitates the reverse transcription and sequencing of the encoding nucleic acid, e.g. with a redundant, non-specific primer set.

[0598] The co-cultivation is in one embodiment of all methods as reported herein in polystyrene multi well plates with wells with a round bottom. The working volume of the wells is in one embodiment of all methods as reported herein of 50 l to 250 l. In one embodiment the wells are coated at least partially with a non-fibrous substrate prepared from a blend of polymer plastic resin and amphipathic molecules, wherein the amphipathic molecule comprises a hydrophilic moiety and a hydrophobic region, wherein the hydrophobic regions are anchored within the substrate and the hydrophilic moieties are exposed on the substrate. In one embodiment the amphipathic molecules are chosen from alkylamine ethoxylated, poly (ethylene imine), octyldecamine or mixtures thereof (see e.g. EP 1 860 181).

[0599] Characterization of Co-Cultivated Cells

[0600] 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.

[0601] 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.

[0602] Isolation of mRNA, Cloning and Sequencing

[0603] From the B-cells the total mRNA can be isolated and transcribed in cDNA. With specific primers the cognate VH- and VL-region encoding nucleic acid can be amplified. Almost no identical sequences are obtained. The method provides for highly diverse antibodies binding to the same antigen.

[0604] The primers used for the amplification of the VH-encoding nucleic acid can be used for cDNA obtained from cells from the NMRI-mouse, the Armenian Hamster, the Balb/c-mouse as well as the Syrian hamster and the rabbit.

[0605] In one embodiment of all methods as reported herein the amino acid sequence is derived from the amplified VH-encoding nucleic acid and the exact start and end point is identified by locating the amino acid sequences of EVQL/QVQL to VSS (VH-region) and DIVM/DIQM to KLEIK (VL-region).

[0606] Also reported herein is a method for producing an antibody comprising the following steps: [0607] a) providing a population of (mature) B-cells (obtained from the blood of an experimental non-human animal), [0608] b) staining the cells of the population of B-cells with at least one fluorescence dye (in one embodiment with one to three, or two to three fluorescence dyes), [0609] c) depositing single cells of the stained population of B-cells in individual containers (in one embodiment is the container a well of a multi well plate), [0610] d) cultivating the deposited individual B-cells in the presence of feeder cells and a feeder mix (in one embodiment the feeder cells are EL-4 B5 cells, in one embodiment the feeder mix is the synthetic feeder mix as reported herein), [0611] e) determining the binding specificity of the antibodies secreted in the cultivation of the individual B-cells, [0612] f) determining the amino acid sequence of the variable light and heavy chain domain of specifically binding antibodies by a reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining a monoclonal antibody variable light and heavy chain domain encoding nucleic acid, [0613] g) introducing the monoclonal antibody light and heavy chain variable domain encoding nucleic acid in an expression cassette for the expression of an antibody, [0614] h) introducing the nucleic acid in a cell, [0615] i) cultivating the cell and recovering the antibody from the cell or the cell culture supernatant and thereby producing an antibody.

[0616] In one embodiment the non-human animal is selected from rat, mouse, hamster, rabbit, non-human primates, sheep, dog, cow, chicken, amphibians, and reptiles.

[0617] The Method as Reported Herein

[0618] Herein is reported a method for the co-cultivation of single deposited B-cells, which can be of any source, with feeder cells in a suitable co-cultivation medium, wherein all cell growth stimulating additives are of synthetic origin.

[0619] The invention is based at least in part on the finding that phorbol myristate acetate alone or a combination of phorbol myristate acetate (PMA) with synthetic cytokines and synthetic interleukins can replace the commonly employed macrophage/T-cell cultivation supernatant used in the co-cultivation of B-cells and feeder cells, such as murine EL4-B5 feeder cells.

[0620] The invention is further based at least in part on the finding that when employing phorbol myristate acetate in combination with synthetic cytokines and synthetic interleukins the concentrations of each of these components has to be within certain ranges in order to ensure a positive effect.

[0621] 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 [0622] co-cultivating the one or more B-cells with feeder cells in the presence of phorbol myristate acetate (PMA) (and thereby producing immunoglobulin).

[0623] In one embodiment the co-cultivating is in the presence of 1.5-7.25 ng/ml phorbol myristate acetate.

[0624] 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 [0625] co-cultivating the one or more B-cells with feeder cells in the presence of phorbol myristate acetate (PMA), IL-1beta, TNFalpha, IL-2, IL-10 and IL-6 (and thereby producing immunoglobulin).

[0626] In one embodiment the co-cultivating is in the presence of [0627] about 0.3-3 ng/ml phorbol myristate acetate, [0628] about 0.02-0.2 ng/ml IL-1beta, [0629] about 0.02-0.2 ng/ml TNFalpha, [0630] about 0.5-5 ng/ml IL-2, [0631] about 0.1-1 ng/ml IL-10, and [0632] about 0.1-1 ng/ml IL-6.

[0633] In one embodiment the co-cultivating is in the presence of [0634] about 0.3-3 ng/ml phorbol myristate acetate, [0635] about 0.064 ng/ml IL-1beta, [0636] about 0.064 ng/ml TNFalpha, [0637] about 1.6 ng/ml IL-2, [0638] about 0.32 ng/ml IL-10, and [0639] about 0.32 ng/ml IL-6.

[0640] In one embodiment the co-cultivating is in the presence of [0641] about 0.9-3 ng/ml phorbol myristate acetate, [0642] about 0.02-0.2 ng/ml IL-1beta, [0643] about 0.02-0.2 ng/ml TNFalpha, [0644] about 0.5-5 ng/ml IL-2, [0645] about 0.1-1 ng/ml IL-10, and [0646] about 0.1-1 ng/ml IL-6.

[0647] In one embodiment the co-cultivating is in the presence of [0648] about 0.9-3 ng/ml phorbol myristate acetate, [0649] about 0.064 ng/ml IL-1beta, [0650] about 0.064 ng/ml TNFalpha, [0651] about 1.6 ng/ml IL-2, [0652] about 0.32 ng/ml IL-10, and [0653] about 0.32 ng/ml IL-6.

[0654] In one embodiment the co-cultivating is in the presence of [0655] about 0.9-3 ng/ml phorbol myristate acetate, [0656] about 0.02 ng/ml IL-1beta, [0657] about 0.02 ng/ml TNFalpha, [0658] about 0.5 ng/ml IL-2, [0659] about 0.1 ng/ml IL-10, and [0660] about 0.1 ng/ml IL-6.

[0661] In one embodiment the co-cultivating is in the presence of [0662] about 0.5-2.5 ng/ml phorbol myristate acetate, [0663] about 0.02-0.2 ng/ml IL-1beta, [0664] about 0.02-0.2 ng/ml TNFalpha, [0665] about 0.5-5 ng/ml IL-2, [0666] about 0.1-1 ng/ml IL-10, and [0667] about 0.1-1 ng/ml IL-6.

[0668] In one embodiment the co-cultivating is in the presence of [0669] about 0.5-2.5 ng/ml phorbol myristate acetate, [0670] about 0.064 ng/ml IL-1beta, [0671] about 0.064 ng/ml TNFalpha, [0672] about 1.6 ng/ml IL-2, [0673] about 0.32 ng/ml IL-10, and [0674] about 0.32 ng/ml IL-6.

[0675] In one embodiment the co-cultivating is in the presence of [0676] about 0.2-0.35 ng/ml phorbol myristate acetate, [0677] about 0.02-0.2 ng/ml IL-1beta, [0678] about 0.02-0.2 ng/ml TNFalpha, [0679] about 0.5-5 ng/ml IL-2, [0680] about 0.1-1 ng/ml IL-10, and [0681] about 0.1-1 ng/ml IL-6.

[0682] In one embodiment the co-cultivating is in the presence of [0683] about 0.2-0.35 ng/ml phorbol myristate acetate, [0684] about 0.064 ng/ml IL-1beta, [0685] about 0.064 ng/ml TNFalpha, [0686] about 1.6 ng/ml IL-2, [0687] about 0.32 ng/ml IL-10, and [0688] about 0.32 ng/ml IL-6.

[0689] In one embodiment the co-cultivating is in the presence of [0690] about 0.05-0.7 ng/ml phorbol myristate acetate, [0691] about 0.02-0.2 ng/ml IL-1beta, [0692] about 0.02-0.2 ng/ml TNFalpha, [0693] about 0.5-5 ng/ml IL-2, [0694] about 0.1-1 ng/ml IL-10, and [0695] about 0.1-1 ng/ml IL-6.

[0696] In one embodiment the co-cultivating is in the presence of [0697] about 0.05-0.7 ng/ml phorbol myristate acetate, [0698] about 0.2 ng/ml IL-1beta, [0699] about 0.2 ng/ml TNFalpha, [0700] about 5 ng/ml IL-2, [0701] about 1 ng/ml IL-10, and [0702] about 1 ng/ml IL-6.

[0703] In one embodiment the co-cultivating is in the presence of [0704] about 0.05-0.7 ng/ml phorbol myristate acetate, [0705] about 0.064 ng/ml IL-1beta, [0706] about 0.064 ng/ml TNFalpha, [0707] about 1.6 ng/ml IL-2, [0708] about 0.32 ng/ml IL-10, and [0709] about 0.32 ng/ml IL-6.

[0710] In one preferred embodiment the co-cultivating is in the presence of [0711] about 1.3 ng/ml phorbol myristate acetate, [0712] about 0.064 ng/ml IL-1beta, [0713] about 0.064 ng/ml TNFalpha, [0714] about 1.6 ng/ml IL-2, [0715] about 0.32 ng/ml IL-10, and [0716] about 0.32 ng/ml IL-6.

[0717] In one preferred embodiment the co-cultivating is in the presence of [0718] about 0.9 ng/ml phorbol myristate acetate, [0719] about 0.02 ng/ml IL-1beta, [0720] about 0.02 ng/ml TNFalpha, [0721] about 0.5 ng/ml IL-2, [0722] about 0.1 ng/ml IL-10, and [0723] about 0.1 ng/ml IL-6.

[0724] In one embodiment the amount of phorbol myristate acetate (PMA) used in the co-cultivation is given by weight and the amount of IL-1beta, TNFalpha, IL-2, IL-10 and IL-6 used in the co-cultivation is given by activity.

[0725] In one embodiment the co-cultivating is in the presence of [0726] about 0.3-3 ng/ml phorbol myristate acetate, [0727] about 0.02-0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0728] about 0.02-0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0729] about 0.5-5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0730] about 0.1-1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0731] about 0.1-1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0732] In one embodiment the co-cultivating is in the presence of [0733] about 0.3-3 ng/ml phorbol myristate acetate, [0734] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0735] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0736] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0737] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0738] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0739] In one embodiment the co-cultivating is in the presence of [0740] about 0.9-3 ng/ml phorbol myristate acetate, [0741] about 0.02-0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0742] about 0.02-0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0743] about 0.5-5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0744] about 0.1-1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0745] about 0.1-1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0746] In one embodiment the co-cultivating is in the presence of [0747] about 0.9-3 ng/ml phorbol myristate acetate, [0748] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0749] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0750] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0751] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0752] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0753] In one embodiment the co-cultivating is in the presence of [0754] about 0.9-3 ng/ml phorbol myristate acetate, [0755] about 0.02 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0756] about 0.02 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0757] about 0.5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0758] about 0.1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0759] about 0.1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0760] In one embodiment the co-cultivating is in the presence of [0761] about 0.5-2.5 ng/ml phorbol myristate acetate, [0762] about 0.02-0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0763] about 0.02-0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0764] about 0.5-5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0765] about 0.1-1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0766] about 0.1-1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0767] In one embodiment the co-cultivating is in the presence of [0768] about 0.5-2.5 ng/ml phorbol myristate acetate, [0769] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0770] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0771] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0772] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0773] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0774] In one embodiment the co-cultivating is in the presence of [0775] about 0.2-0.35 ng/ml phorbol myristate acetate, [0776] about 0.02-0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0777] about 0.02-0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0778] about 0.5-5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0779] about 0.1-1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0780] about 0.1-1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0781] In one embodiment the co-cultivating is in the presence of [0782] about 0.2-0.35 ng/ml phorbol myristate acetate, [0783] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta [0784] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0785] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0786] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0787] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0788] In one embodiment the co-cultivating is in the presence of [0789] about 0.05-0.7 ng/ml phorbol myristate acetate, [0790] about 0.02-0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0791] about 0.02-0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0792] about 0.5-5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0793] about 0.1-1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0794] about 0.1-1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0795] In one embodiment the co-cultivating is in the presence of [0796] about 0.05-0.7 ng/ml phorbol myristate acetate, [0797] about 0.2 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0798] about 0.2 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0799] about 5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, about 1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0800] about 1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0801] In one embodiment the co-cultivating is in the presence of [0802] about 0.05-0.7 ng/ml phorbol myristate acetate, [0803] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0804] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0805] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0806] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0807] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0808] In one preferred embodiment the co-cultivating is in the presence of [0809] about 1.3 ng/ml phorbol myristate acetate, [0810] about 0.064 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0811] about 0.064 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0812] about 1.6 ng/ml with 6-7 (preferably 6.3)*10.sup.6 U/mg (murine) IL-2, [0813] about 0.32 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0814] about 0.32 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0815] In one preferred embodiment the co-cultivating is in the presence of [0816] about 0.9 ng/ml phorbol myristate acetate, [0817] about 0.02 ng/ml with 5.5-14*10.sup.8 IU/mg (murine) IL-1beta, [0818] about 0.02 ng/ml with 2.3-2.9*10.sup.8 U/mg (murine) TNFalpha, [0819] about 0.5 ng/ml with 6-7 (preferably 6.3)*10.sup.6 IU/mg (murine) IL-2, [0820] about 0.1 ng/ml with 6-7.5*10.sup.5 IU/mg (murine) IL-10, and [0821] about 0.1 ng/ml with 9.2-16.1*10.sup.8 U/mg (murine) IL-6.

[0822] In one embodiment the feeder cells are EL-4 B5 cells.

[0823] 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.

[0824] In one embodiment the co-cultivating is further in the presence of Staphylococcus aureus strain Cowans cells (SAC).

[0825] In one embodiment the incubating is for 5 to 14 days.

[0826] In all methods as reported herein the co-cultivation is in the absence of a thymocyte cultivation supernatant. Thus, the added feeder mix is not a cultivation supernatant.

[0827] In one embodiment neither of a thymocyte cultivation supernatant, a macrophage cultivation supernatant, or a T-cell cultivation supernatant is added to the cultivation.

[0828] One additional aspect as reported herein is a feeder mix comprising (consisting of) about 2 ng/ml (murine) IL-1beta, about 2 ng/ml (murine) TNFalpha, about 50 ng/ml (murine) IL-2, about 10 ng/ml (murine) IL-10 and about 10 ng/ml (murine) IL-6.

[0829] One additional aspect as reported herein is the use of a (synthetic) feeder mix comprising (consisting of) about 2 ng/ml (murine) IL-1beta, about 2 ng/ml (murine) TNFalpha, about 50 ng/ml (murine) IL-2, about 10 ng/ml (murine) IL-10 and about 10 ng/ml (murine) IL-6 in the co-cultivation of (murine) EL-4 B5 cells with (single deposited) human, murine or rabbit B-cells.

[0830] When using a synthetic feeder mix comprising about 2 ng/ml (murine) IL-1beta, about 2 ng/ml (murine) TNFalpha, about 50 ng/ml (murine) IL-2, about 10 ng/ml (murine) IL-10 and about 10 ng/ml (murine) IL-6 in the co-cultivation of (murine) EL4-B5 cells with (single deposited) (rabbit) B-cells the frequency of IgG positive wells is comparable with that when TSN is used.

TABLE-US-00002 TABLE 2 ng/ml IL-1beta 2 ng/ml TNFalpha 50 ng/ml mIL-2 10 ng/ml mIL-10 TSN 10 ng/ml mlL-6 IgG positive wells 64.9 58.3 [% of total wells]

[0831] Also the average productivity is comparable with that when TSN is used (see following Table).

TABLE-US-00003 TABLE 2 ng/ml IL-1beta 2 ng/ml TNFalpha 50 ng/ml mIL-2 10 ng/ml mIL-10 TSN 10 ng/ml mIL-6 average IgG 3.2 2.6 productivity [g/ml]

[0832] When adding PMA (3 ng/ml) in addition to the synthetic feeder mix then the results are significantly different (see following Table).

TABLE-US-00004 TABLE 2 ng/ml IL-1beta 2 ng/ml IL-1beta 2 ng/ml TNFalpha 2 ng/ml TNFalpha 50 ng/ml mIL-2 50 ng/ml mIL-2 10 ng/ml mIL-10 10 ng/ml mIL-10 10 ng/ml mIL-6 10 ng/ml mIL-6 SAC SAC 3 ng/ml PMA IgG positive wells 63.1 2.7 [% of total wells] average IgG 2.1 0.07 productivity [g/ml]

[0833] It has been found that when employing phorbol myristate acetate in combination with synthetic cytokines and synthetic interleukins the concentrations of each of these components has to be within certain ranges in order to ensure a synergistic effect.

[0834] The synthetic feeder mix comprising 2 ng/ml (murine) IL-1beta, 2 ng/ml (murine) TNFalpha, 50 ng/ml (murine) IL-2, 10 ng/ml (murine) IL-10 and 10 ng/ml (murine) IL-6 is termed in the following Table 1CM.

[0835] Thus, a 0.1CM feeder mix as in the following Table corresponds to a synthetic feeder mix comprising 0.2 ng/ml (murine) IL-1beta, 0.2 ng/ml (murine) TNFalpha, 5 ng/ml (murine) IL-2, 1 ng/ml (murine) IL-10 and 1 ng/ml (murine) IL-6.

[0836] It has surprisingly been found that in the presence of PMA the concentration of the synthetic feeder mix can be reduced without impairing with the increased number of IgG positive wells as well as increased productivity. At the same time the cells are not over-stimulated and thereby abrogating the positive effect.

[0837] It has also surprisingly been found that by using PMA alone, i.e. in the absence of cytokines and interleukins, comparable numbers of IgG positive wells and even increased productivity can be achieved.

[0838] Number of IgG positive wells [% of total]:

TABLE-US-00005 CM PMA 2x 1.1x 1x 0.32x 0.1x 0.032x 0.01x 0x 3 ng/ml n.d. n.d. 13.1 13.1 35.7 72.6 69.0 n.d. 0.95 ng/ml n.d. n.d. 8.3 23.8 50.0 67.9 69.0 28.6 0.3 ng/ml n.d. n.d. 17.9 57.1 67.9 70.2 69.0 n.d. 0.095 ng/ml n.d. n.d. 38.1 n.d. n.d. n.d. n.d. n.d. 0 ng/ml 23.8 36.9 27.4 46.4 50.0 36.9 41.7 n.d. n.d. = not determined

[0839] Average IgG productivity [g/ml]:

TABLE-US-00006 CM PMA 2x 1.1x 1x 0.32x 0.1x 0.032x 0.01x 0x 3 ng/ml n.d. n.d. 0.351 0.125 0.363 3.480 3.893 n.d. 0.95 ng/ml n.d. n.d. 0.100 0.134 1.623 5.340 4.235 1.650 0.3 ng/ml n.d. n.d. 0.720 2.662 4.450 3.476 1.931 n.d. 0.095 ng/ml n.d. n.d. 1.390 n.d. n.d. n.d. n.d. n.d. 0 ng/ml 0.549 1.496 0.675 2.233 1.817 0.928 0.375 n.d. n.d. = not determined

[0840] The number of antigen specific clones is not affected (shown in the following for B-cells obtained from a rabbit immunized with human serum albumin (HSA)).

[0841] Number of anti-HSA antibody positive wells [% of total]:

TABLE-US-00007 CM PMA 2x 1.1x 1x 0.32x 0.1x 0.032x 0.01x 0x 3 ng/ml n.d. n.d. 6.0 3.6 7.1 34.5 33.3 n.d. 0.95 ng./ml n.d. n.d. 3.6 7.1 27.4 31.0 27.4 19.0 0.3 ng/ml n.d. n.d. 3.6 22.6 28.6 27.4 28.6 n.d. 0.095 ng/ml n.d. n.d. 23.8 n.d. n.d. n.d. n.d. n.d. 0 ng/ml 6.0 20.2 3.6 23.8 26.2 16.7 17.9 n.d. n.d. = not determined

[0842] Number of anti-HSA antibody positive wells [% of IgG positive wells]:

TABLE-US-00008 CM PMA 2x 1.1x 1x 0.32x 0.1x 0.032x 0.01x 0x 3 ng/ml n.d. n.d. 45.5 27.3 20.0 47.5 48.3 n.d. 0.95 ng/ml n.d. n.d. 42.9 30.0 54.8 45.6 39.7 66.7 0.3 ng/ml n.d. n.d. 20.0 39.6 42.1 39.0 41.4 n.d. 0.095 ng/ml n.d. n.d. 62.5 n.d. n.d. n.d. n.d. n.d. 0 ng/ml 25.0 54.8 13.0 51.3 52.4 45.2 42.9 n.d. n.d. = not determined

[0843] For the conditions 0.1CM, 0.032CM and without CM the influence of the PMA concentration was evaluated further (relative weighting from o to +++++).

TABLE-US-00009 IgG+ productivity HSA+ HSA+ PMA wells IgG+ wells wells [ng/ml] [% total] [g/ml] [% total] [% IgG+] 0.949 + 0.712 +++ ++ +++ ++++ 0.534 +++++ +++ +++ +++ 0.3 +++++ +++++ 0.225 +++++ +++++ +++++ ++++ 0.127 ++ ++++ + ++ 0.054 ++++ +++ +++ +++ 0.011 + + +++ +++++ 0.002 + + ++

TABLE-US-00010 IgG+ productivity HSA+ HSA+ PMA wells IgG+ wells wells [ng/ml] [% total] [g/ml] [% total] [% IgG+] 2.25 ++++ ++++ ++ 1.688 +++++ +++++ +++++ + 1.266 +++ +++++ +++++ ++++ 0.949 ++++ +++++ +++++ +++ 0.712 ++ +++ + 0.534 ++ +++ ++ +++ 0.3 ++++ +++ ++++ +++ 0.127 + ++ 0.011 +++ +++++

TABLE-US-00011 IgG+ productivity HSA+ HSA+ PMA wells IgG+ wells wells [ng/ml] [% total] [g/ml] [% total] [% IgG+] 7.106 +++++ ++ ++++ + 5.331 ++++ +++++ +++ ++ 3.999 +++ +++ + 2.251 +++++ +++++ +++++ +++ 1.688 ++ +++ +++++ +++++ 0.95 + + +++ 0.535 + +++++

[0844] Without being bound by this theory it is assumed that in order to have a robust method values within an overlapping range resulting in the best overall number of IgG-positive wells, productivity and number of antigen-specific wells are to be used.

[0845] The following examples and sequences 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.

EXAMPLES

[0846] Materials and Methods

[0847] Recombinant DNA Techniques

[0848] 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.

[0849] Media and Buffers

[0850] Blocking buffer for ELISA comprises 1PBS and 1% BSA.

[0851] Coating buffer for ELISA comprises 4.29 g Na2CO3*10 H2O and 2.93 g NaHCO3 add water to a final volume of 1 liter, pH 9.6 adjusted with 2 N HCl.

[0852] Ethanol-solution for RNA isolation comprises 70% Ethanol or 80% Ethanol.

[0853] FACS-buffer for immuno fluorescence staining comprises 1PBS and 0.1% BSA.

[0854] IMDM-buffer for ELISA comprises 1PBS, 5% IMDM and 0.5% BSA.

[0855] Incubation buffer 1 for ELISA comprises 1PBS, 0.5% CroteinC.

[0856] Incubation buffer 2 for ELISA comprises 1PBS, 0.5% CroteinC and 0.02% Tween 20.

[0857] Incubation buffer 3 for ELISA comprises 1PBS, 0.1% BSA.

[0858] 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.

[0859] Wash buffer 1 for ELISA comprises 1PBS, 0.05% Tween 20.

[0860] Wash buffer 2 for ELISA comprises 1PBS, 0.1% Tween 20.

[0861] Wash buffer 3 for ELISA comprises water, 0.9% NaCl, 0.05% Tween 20.

[0862] EL-4 B5 medium comprises RPMI 1640 supplemented with 10% FCS, 2 mM Glutamine, 1% penicillin/streptomycin solution, 2 mM sodium pyruvate, 10 mM HEPES and 0.05 mM -mercaptoethanol.

[0863] Animal Care and Immunization

[0864] The experimental animals were held according to the German animal protection law (TierSCHG) as well as according to the respective European guidelines.

[0865] NZW rabbits obtained from Charles River Laboratories International, Inc. were used for immunization. The animals were housed according to the Appendix A Guidelines for accommodation and care of animals in an AAALACi accredited animal facility. All animal immunization protocols and experiments were approved by the Government of Upper Bavaria (permit number 55.2-1-54-2532-90-14) and performed according to the German Animal Welfare Act and the Directive 2010/63 of the European Parliament and Council.

[0866] Generally, 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.

[0867] 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.

[0868] In more detail, NZW rabbits, 12-16 weeks old, were immunized with 400 g recombinant human serum albumin protein (HSA; CAS RN 70024-90-7; Sigma), emulsified with complete Freund's adjuvant, at day 0 by intradermal application, followed by 200 g HSA emulsified with complete or incomplete Freund's adjuvant at weeks 1, 2, 6, 10 and 23, by alternating intramuscular and subcutaneous injections.

[0869] Removal of Organs, Blood and Macrophages

[0870] Generally, 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.

[0871] Specifically, blood (10% of estimated total blood volume) was taken at days 4, 5 and 6 post immunizations, starting from the 3rd immunization onwards. Serum was prepared for immunogen-specific IgG titer determination by ELISA, and peripheral mononuclear cells were isolated, which were used as a source of antigen-specific B cells in the B cell cloning process.

[0872] Macrophages were isolated from the obtained blood by attachment to cell culture plastic.

[0873] EDTA containing whole blood was diluted twofold with 1PBS before density centrifugation on lymphocyte 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.

[0874] Density Gradient Centrifugation

[0875] 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).

[0876] Withdrawn blood was diluted 2:1 with phosphate buffered saline (PBS). 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. The vial was centrifuged for 20 min. at 800g without braking. The lymphocytes were obtained from the white interim layer. The removed cells were supplemented with 10 ml PBS and centrifuged at 800g for 10 min. The supernatant was discarded and the pellet was resuspended, washed, centrifuged. The final pellet was resuspended in PBS or medium.

[0877] Depletion of Macrophages

[0878] Sterile 6-well plates (cell culture grade) were used to deplete macrophages and monocytes through unspecific adhesion. Wells were either uncoated, coated with KLH (keyhole limpet haemocyanine) or with streptavidin. 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 or medium.

[0879] Enrichment of Antigen-Specific B-Cells

[0880] 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 or thrice 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 5 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 800g. The cells were kept on ice until the immune fluorescence staining. The pellet was optionally resuspended in PBS.

[0881] Cultivation of T-Cells

[0882] The T-cells were isolated from 4-5 week old rabbits. The cells were centrifuged and immediately cultivated or frozen in aliquots of 310.sup.7 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 48 hours at 37 C.

[0883] Cultivation of Macrophages

[0884] 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 48 hours in 35 ml medium.

[0885] Co-Cultivation of T-Cells and Macrophages

[0886] T-cells and macrophages were cultivated for 48 hours in separate flasks. Prior to combining both cell populations, the T-cells were centrifuged for 10 min. at 800g. The supernatant was discarded and the cell pellet was resuspended in 10 ml medium. The T-cells were adjusted to a minimal cell density of 510.sup.5 cells/ml and 10 pg phorbol-12-myristate-13-acetate (PMA) and 5 ng or 50 ng Phytohemagglutinin M (PHA-M) per ml of medium were added. The cultivation medium was removed from macrophages and the T-cell suspension was added to the flasks containing macrophages. 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.

[0887] Immunofluorescence Staining

[0888] Protocol 1:

[0889] Depending on the number of cells to be stained the cells were provided in 100 l medium (less than 10.sup.6 cells) or 200 l medium (more than 10.sup.6 cells), respectively. The fluorescent labeled antibody was diluted with 5% serum of the experimental animal and FACS buffer to a final volume of 100 l or 200 l, respectively. The reaction mixture was incubated on a roller rack for 40 min. at 4 C. in the dark. After the incubation the cells were washed twice at 300g for 5 min. The pellet was resuspended in 400 l PBS and filtered through a 70 m sieve. The filtered solution was transferred to a FACS-vial and directly before the FACS experiment dead cells were stained by addition of propidium iodide (6.25 g/ml). If the antibody was conjugated to biotin the antibody was detected in a second step with streptavidin labeled fluorophore (e.g. Alexa Flour(R) 647 (antibody 197)).

[0890] Protocol 2:

[0891] Anti-rabbit IgG FITC used for single cell sorting was from AbD Serotec (STAR121F, Dsseldorf, Germany).

[0892] For surface staining, cells were incubated with the optimally diluted anti-rabbit IgG FITC antibody in PBS for 30 min. with rolling at 4 C. in the dark. Following centrifugation, the supernatants were removed by aspiration. The PBMCs were subjected to two cycles of centrifugation and washing with ice cold PBS. Finally the PBMCs were resuspended in ice cold PBS and immediately subjected to the FACS analyses. Propidium iodide in a concentration of 5 g/ml (BD Pharmingen, San Diego, Calif., USA) was added prior to the FACS analyses to discriminate between dead and live cells. In other experiments the stained cells were single deposited by FACS.

[0893] A Becton Dickinson FACSAria equipped with a computer and the FACSDiva software (BD Biosciences, USA) were used to collect and analyze the data.

[0894] Proliferation Assays

[0895] a) Cell Titer Glo (CTG) Viability Assay [0896] The CTG viability assay (Promega; # G7571) was used according to the instructions of the manufacturer.

[0897] b) .sup.3H Thymidine Assay [0898] 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).

[0899] c) Microscopic Analysis [0900] 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.

[0901] d) Analysis of B-Cell Activation Via CFSE-Labeling. [0902] 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.

[0903] Quantification of IgG

[0904] Generally, 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. 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.

[0905] Specifically, a mixture of 0.5 g/ml of biotinylated mouse anti-rabbit IgG antibody (Sigma-Aldrich) and 0.35 g/ml anti-rabbit IgG HRP conjugate (Sigma-Aldrich) was transferred to 384 well streptavidin coated microtiter plates (MicroCoat Biotechnologie GmbH). Dilutions of B-cell supernatants in PBS supplemented with 0.5% BSA and 0.05% Tween-20 were added and incubated for 90 min at RT. After repeated washing (6) with PBST (phosphate buffered saline with 0.2% Tween-20) buffer the plates were developed with BM Blue HRP substrate solution and color formation was measured by absorbance at 370 nm. A commercial rabbit IgG (Sigma-Aldrich) was used as a calibration standard.

[0906] Detection of Antigen-Specific IgG

[0907] Generally, antibodies produced by single deposited and co-cultivated B-cells or from B-cells obtained from an immunized experimental animal can be characterized with respect to specific antigen binding. The ELISA was performed at room temperature and the ELISA-solution was incubated between the individual steps on a shaker at 20g. In the first step the antigen was bound to the wells of a 96-well multi well plate. If the antigen was a protein it had been diluted in coating buffer and applied directly to the plate. Peptide antigens were bound via the specific binding pair biotin/streptavidin. The wells of the multi well plate can be already coated with soluble CroteinC (CrC) by the manufacturer. If not, the wells were incubated after the immobilization of the antigen with 200 l blocking buffer. After the incubation with 100 l antigen solution per well (pre-coated multi well plate) or 200 l blocking buffer, respectively, non-bound antigen or blocking buffer was removed by washing with wash buffer. The diluted B-cell supernatants were added in a volume of 100 l per well and incubated. After the incubation the wells were washed. Afterwards the detection antibody was added in a volume of 100 l per well. The antibody can be either conjugated to horseradish peroxidase or labeled with biotin. The detection antibody was determined with a streptavidin-horseradish peroxidase conjugate. After the incubation the multi well plate was washed and afterwards 50 l of a substrate solution containing 3,3,5,5 tetramethyl benzidine (TMB) were added per well and incubated for a period as given in Table X. The enzymatic reaction was stopped by the addition of 50 l sulfuric acid and the optical density was determined at 450 nm and 680 nm with a photometer (Rainbow Thermo ELISA Reader) and the Xread plus-software.

[0908] Specifically, the assay was performed at room temperature (RT) on 384-well Maxisorp microtiter plates (Thermo Scientific) with PBS (phosphate buffered saline) buffer supplemented with 0.5% Gelatine and 0.025% Tween V-20. The plates were coated with 0.5 g/ml of human serum albumin (HSA, Sigma-Aldrich) for at least 2 hours to overnight. After washing (3) with PBST (PBS with 0.1% Tween-20) buffer the wells were blocked with PBS with 0.5% Gelatine and 0.1% Tween-20. Again, the plates were washed 3 and afterwards dilutions of B-cell supernatants were added. After an incubation of 60 min and 3 washing steps with PBST a 1:4000 dilution of a HRP-conjugated anti-rabbit IgG antibody (Amersham) was transferred to the wells and incubated for 60 min. Finally, the plates were repeatedly washed (6) with PBST and developed with BM Blue HRP substrate solution for 30 min. Absorbance was measured at 392-405 nm.

[0909] Panning on Antigen

[0910] a) Coating of Plates

[0911] 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.

[0912] 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.

[0913] b) Panning of B-cells on Peptides

[0914] 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.

Example 1

[0915] Cultivation of EL-4 B5 Cells

[0916] 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.

[0917] 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/mi. The cells have a doubling time of approximately 18 hours.

[0918] Cells were harvested and adjusted to a cell density of 110.sup.6 cells/ml before -irradiation at 50 Gy.

Example 2

[0919] Co-Cultivation of B-Cells and EL-4 B5 Cells

[0920] Single cell sorted B-cells were cultured in 96-well plates with 200 l/well EL-4 B5 medium with Pansorbin Cells (SAC) (Calbiochem (Merck), Darmstadt, Deutschland), EL-4 B5 cells (510.sup.4/well) and rabbit thymocyte supernatant (TSN) or cytokine mix (CM) with PMA, respectively, 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).