HK1212731B - Single b-cell cultivation method - Google Patents

Description

Single B cell culture method

This application is a divisional application of PCT application PCT/EP2011/058616, filed on May 26, 2011, with the invention name “Single B Cell Culture Method”. The date on which the PCT application entered the Chinese national phase is November 28, 2012, with application number 201180026559.6.

Herein is reported a method for obtaining the amino acid sequence of at least the variable domain of a monoclonal antibody secreted by a single B cell obtained from a population of B cells from an experimental animal by single cell sedimentation and co-cultivation with feeder cells in the presence of a feeder mix.

Background of the Invention

To obtain cells that secrete monoclonal antibodies, the hybridoma technique developed by Koehler and Milstein is widely used. However, in hybridoma technology, only a subset of B cells obtained from immunized experimental animals can be fused and proliferated. The source of B cells is usually an organ of the immunized experimental animal, such as the spleen.

Zubler et al. developed different methods for obtaining cells that secrete monoclonal antibodies starting in 1984 (see, for example, Eur. J. Immunol. 14 (1984) 357-63, J. Exp. Med. 160 (1984) 1170-1183). These methods involved obtaining B cells from the blood of immunized experimental animals and co-culturing them with murine EL-4 B5 feeder cells in the presence of a feeder mixture containing cytokines. Using this method, up to 50 ng/ml of antibody could be obtained after 10-12 days of co-culture.

Weitkamp, J-H., et al. (J. Immunol. Meth. 275 (2003) 223-237) reported the generation of recombinant human monoclonal antibodies against rotavirus from single antigen-specific B cells selected using fluorescent virus-like particles. US 2006/0051348 reported a method for producing multiple isolated antibodies against multiple homologous antigens. WO 2008/144763 and WO 2008/045140 reported antibodies against IL-6 and their uses, as well as methods for culturing clonal populations of antigen-specific B cells. US 2007/0269868 reported a method for culturing clonal populations of antigen-specific B cells. Masri et al. (Mol. Immunol. 44 (2007) 2101-2106) reported the cloning and expression of functional Fab fragments in Escherichia coli (E. coli) obtained from single human lymphocytes against anthrax toxin. A method for preparing an immunoglobulin library is reported in WO2007/031550.

SUMMARY OF THE INVENTION

This article reports a method for isolating B cells from a B cell population with special properties. First, within 4 weeks after the first immunization of the experimental animal, induced antibody-producing cells can be isolated and the binding specificity of the antibody can be determined. Second, the quantity and/or quality (e.g., antibody production/secretion capacity) of the antibody-producing cells can be improved by any of the following steps: i) a preincubation step, and/or ii) a centrifugation step, and/or iii) a panning step. Third, the feeder mixture for co-culturing B cells and feeder cells can be improved by adding IL-21, or IL-6, or SAC, or BAFF.

Thus, in one aspect herein is reported a method for selecting B cells, said method comprising the following steps:

a) optionally labeling B cells of the B cell population,

b) individually co-culturing each B cell of the B cell population that has been deposited as a single cell with feeder cells,

c) selecting B cells that proliferated and secreted antibodies in step b).

In one aspect, also reported herein is a method for obtaining a B cell clone, said method comprising the steps of:

a) Obtain B cells from experimental animals,

b) labeling of B cells,

c) depositing the labeled B cells as single cells,

d) co-culturing the single-cell deposited B cells and feeder cells alone,

e) selecting the B cells that proliferated and secreted the antibody in step d) and thereby obtaining B cell clones.

In another aspect, herein is reported a method for producing an antibody that specifically binds to a target antigen, the method comprising the steps of:

a) optionally labeling cells of the B-cell population with at least one fluorescent dye,

b) culturing each B cell of the B cell population that has been deposited as a single cell in a separate container in the presence of feeder cells and a feeder mix to obtain a separate B cell clone and a culture supernatant,

c) selecting B cell clones that produce antibodies that specifically bind to the target antigen,

d) culturing cells containing a nucleic acid encoding an antibody that specifically binds to a target antigen, the antibody being produced by the B cell clone selected in step c), or a humanized variant thereof, and recovering the antibody from the cells or the culture supernatant, thereby producing the antibody.

In one embodiment, the method comprises one or more of the following steps:

After step c): c1) determining the nucleic acid sequences encoding the variable light chain domain and the variable heavy chain domain of the antibody by reverse transcriptase PCR,

After step c1): c2) transfecting the cells with a nucleic acid comprising nucleic acid sequences encoding the antibody variable light chain domain and variable heavy chain domain.

In one aspect, also reported herein is a method for producing an antibody, said method comprising the steps of:

a) providing a (mature) B cell population (obtained from the blood of experimental animals),

b) labeling cells of the B-cell population with at least one fluorescent dye (in one embodiment with 1 to 3, or 2 to 3 fluorescent dyes),

c) depositing single cells of the labeled B cell population in separate containers (in one embodiment, the containers are wells of a multi-well plate),

d) culturing 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 natural TSN, in one embodiment, the feeder mix is a synthetic feeder mix),

e) determining the binding specificity of the antibodies secreted in the culture medium of the respective B cells,

f) determining the amino acid sequences of the variable light and heavy chain domains of the specific binding antibody by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining nucleic acids encoding the variable light and heavy chain domains of the monoclonal antibody,

g) introducing into an expression cassette the nucleic acids encoding the variable light and heavy chain variable domains of the monoclonal antibody for antibody expression,

h) introducing the nucleic acid into the cell,

i) culturing cells and recovering antibodies from the cells or cell culture supernatant, thereby producing the antibodies.

In one embodiment of all aspects as reported herein, the method comprises the step of incubating the B cell population in co-culture medium prior to single cell deposition. In one embodiment, the incubation is performed at about 37°C. In one embodiment, the incubation is performed for 0.5 to 2 hours. In a specific embodiment, the incubation is performed for about 1 hour. In one embodiment, the incubation is performed at about 37°C for about 1 hour.

In one embodiment of all aspects as reported herein, the method comprises the step of centrifuging the single cell deposited B cells prior to co-culturing. In one embodiment, the centrifugation is performed for about 1 minute to about 30 minutes. In a specific embodiment, the centrifugation is performed for about 5 minutes. In one embodiment, the centrifugation is performed at about 100 x g to about 1,000 x g. In a specific embodiment, the centrifugation is performed at about 300 x g. In one embodiment, the centrifugation is performed at about 300 x g for about 5 minutes.

In one embodiment of all aspects as reported herein the method comprises, directly before the labelling step, the step of panning B cells with immobilised antigen.

In one embodiment of all aspects as reported herein the B-cell population is obtained from the blood of the animal by density gradient centrifugation.

In one embodiment of all aspects as reported herein, the B cell population is obtained from the blood of an experimental animal 4 days after immunization. In another embodiment, the B cell population is obtained from the blood of an experimental animal from 4 days to at least 9 days after immunization. In a further embodiment, the B cell population is obtained from the blood of an experimental animal from 4 days to 9 days after immunization.

In one embodiment of all aspects as reported herein the B-cell population is isolated by density gradient centrifugation.

In one embodiment of all aspects as reported herein the B-cell is a mature B-cell.

In one embodiment of all aspects as reported herein the labeling is performed with 1 to 3 fluorescent dyes. In a specific embodiment the labeling is performed with 2 to 3 fluorescent dyes.

In one embodiment of all aspects as reported herein the labelling of B cells results in labelling of 0.1 to 2.5% of the cells of the total B cell population.

In one embodiment of all aspects as reported herein the B-cell is a mouse B-cell, or a hamster B-cell, or a rabbit B-cell.

In one embodiment of all aspects as reported herein the single cell deposition is performed in wells of a multiwell plate.

In one embodiment of all aspects as reported herein the feeder cells are murine EL-4 B5 cells.

In one embodiment of all aspects as reported herein said antibody is a monoclonal antibody.

In one embodiment of all aspects as reported herein the marker is IgG ⁺ CD19 ⁺ -B cells, IgG ⁺ CD38 ⁺ -B cells, IgG ⁺ CD268 ⁺ -B cells, IgG ⁻ CD138 ⁺ -B cells, CD27 ⁺ CD138 ⁺ -B cells, or CD3 ⁻ CD27 ⁺ -B cells.

In one embodiment of all aspects as reported herein the B-cells are of mouse origin and the marker is IgG ⁺ CD19 ⁺ -B cells, and/or IgG ⁻ CD138 ⁺ -B cells.

In one embodiment of all aspects as reported herein the B-cells are of hamster origin and the marker is IgG ⁺ IgM ⁻ B-cells.

In one embodiment of all aspects as reported herein the B-cells are of rabbit origin and the marker is IgG ⁺ -B cells and/or CD138 ⁺ -B cells, or CD138 ⁺ IgG ⁺ -B cells and/or IgG ⁺ IgM ⁻ -B cells.

In one embodiment of all aspects as reported herein the co-cultivation is in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1% (w/v) 200 mM glutamine solution comprising penicillin and streptomycin, 2% (v/v) 100 mM sodium pyruvate solution, and 1% (v/v) 1 M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethanesulfonic acid (HEPES) buffer. In another embodiment the co-cultivation medium further comprises 0.05 mM β-mercaptoethanol.

In one embodiment of all aspects as reported herein the B-cells are co-cultured with feeder cells and a feeder mix.In one embodiment the feeder mix is natural thymocyte culture supernatant (TSN) or a synthetic feeder mix.

In a specific embodiment, the feeder mixture is a synthetic feeder mixture. In one embodiment, the synthetic feeder mixture comprises interleukin-1β and tumor necrosis factor alpha. In one embodiment, the synthetic feeder mixture comprises interleukin-2 (IL-2) and/or interleukin-10 (IL-10). In one embodiment, the synthetic feeder mixture further comprises Staphylococcus aureus (Staphylococcus aureus) Cowans strain cells (SAC). In one embodiment, the synthetic feeder mixture comprises interleukin-21 (IL-21). In one embodiment, the synthetic feeder mixture comprises B cell activating factor (BAFF) of the tumor necrosis factor family. In one embodiment, the synthetic feeder mixture comprises interleukin-6 (IL-6). In one embodiment, the synthetic feeder mixture comprises interleukin-4 (IL-4).

In one embodiment, the co-cultivation is performed in the presence of thymocyte culture supernatant as a feeder mix.In a specific embodiment, the thymocyte culture supernatant is obtained from thymocytes of the thymus of a young animal.

In one embodiment, the method for obtaining a B cell clone further comprises the following steps:

f) determining the amino acid sequences of the variable light and heavy chain domains of the antibodies produced by the selected B cell clones in step e) by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining the monoclonal antibody amino acid variable domain sequence.

In one embodiment, the experimental animal is selected from the group consisting of mice, hamsters and rabbits.

Detailed Description of the Invention

The method reported herein allows for the rapid characterization of the binding specificity of monoclonal antibodies obtained from individual B-cell clones, i.e., within 4 weeks of the first immunization of an experimental animal, induced antibody-producing cells can be isolated and the binding specificity of the antibodies produced therefrom can be determined, wherein at least 4 different experiments can be performed depending on the amount/concentration of the antibody in the supernatant of the B-cell co-culture.

immunity:

Non-human animals, such as mice, rabbits, hamsters and rats are generally used as animal models for evaluating antibody-based therapies. Therefore, it is often necessary to provide cross-reactive antibodies that bind to non-human animal antigens and human antigens. The method reported herein can be used to provide cross-reactive antibodies. In the method reported herein, B cells obtained from, for example, mice, hamsters and rabbits can be used. In one embodiment, the mouse is an NMRI-mouse or a baIb/c-mouse. In another embodiment, the hamster is selected from the group consisting of Armenian hamsters (gray hamsters (Cricetulus migratorius)), Chinese hamsters (black-lined hamsters (Cricetulusgriseus)) and Syrian hamsters (golden hamsters (Mesocricetulus auratus)). In a specific embodiment, the hamster is an Armenian hamster. In one embodiment, the rabbit is selected from the group consisting of New Zealand white (NZW) rabbits, Zimmermann rabbits (ZIKA), Alicia mutant rabbits, basilea mutant rabbits, transgenic rabbits with human immunoglobulin loci, rbIgM knockout rabbits, and hybrids thereof.

In one embodiment, the experimental animals, such as mice, hamsters, and rabbits, selected for immunization are no older than 12 weeks.

Source and isolation of B cells:

The blood of experimental animals provides a high diversity of antibody-producing B cells. The antibodies secreted by the B cells obtained from these cells have almost no identical or overlapping amino acid sequences within the CDRs, thus showing high diversity.

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

The B cells in the blood of experimental animals can be obtained using any method known to those skilled in the art. For example, density gradient centrifugation (DGC) or red blood cell lysis (lysis) can be used. Density gradient centrifugation provides a higher total yield, i.e., B cell clone number, than hypotonic lysis. In addition, in the cells obtained by density gradient centrifugation, more cells divide and grow in the co-culture step. The secreted antibody concentration is also higher than the cells obtained by different methods. Therefore, in one embodiment, a B cell population is provided by density gradient centrifugation.

Table 1: Number of IgG producing wells/cell clones when cells were obtained by density gradient centrifugation (DGC) or hypotonic lysis of erythrocytes

Co-culture prior to selection steps:

B cells producing antibodies that specifically bind to 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).

The term "specifically binds" and grammatical equivalents thereof indicate that the antibody binds its target with a dissociation constant (Kd) of ^10-7 M or less, in one embodiment, from ^10-8 M to ^10-13 M, and in another embodiment, from ^10-9 M to ^10-13 M. The term is also used to indicate that the antibody does not specifically bind to other biomolecules present, i.e., it binds to other biomolecules with a dissociation constant (Kd) of ^10-6 M or greater, in one embodiment, from ^10-6 M to 1 M.

In one embodiment of all methods as reported herein the PBMCs are depleted of macrophages. As described below this can be advantageous, e.g. in one embodiment of rabbit-derived B cells, for the co-cultivation step.

Macrophages can be removed by adherence to the cell culture plate surface (see Pre-incubation step).

In one embodiment of the methods as reported herein the cells are from an animal immunized with the protein and macrophages are depleted prior to labelling.

It has been found that incubating the B cell population in co-culture medium prior to single cell deposition increases the total number of antibody-secreting cells obtained after single cell deposition compared to direct single cell deposition after isolation and optional enrichment of the B cell population from the blood of experimental animals (using rabbits as an example, see Tables 2a and 2b). Specifically, incubation in EL-4 B5 medium at about 37°C for about 1 hour, for example, using a cell culture incubator.

Table 2a: IgG positive wells/cell clones (rb = rabbit) incubated or not in EL-4 B5 medium for 1 hour before single cell deposition of all cells.

＊Remove macrophages

cells and monocytes

Table 2b: IgG positive wells/cell clones that were or were not incubated in EL-4 B5 medium for 1 hour before B cell monolayer deposition

In one embodiment of the methods as reported herein cells are obtained from an animal immunized with the protein and macrophages are depleted.

By using panning methods, cells that do not produce antibodies that bind to the antigen can be reduced or similarly enriched for cells that do produce antibodies that bind to the antigen, respectively. Where a binding partner is present that is bound to the surface, the cells that bind thereto are selectively enriched in the cell population if the bound cells are further processed, or the cells that bind thereto are reduced in the cell population if the cells remaining in the solution are further processed.

Table 3: Enrichment of B cells secreting antigen-specific antibodies by panning with the respective antigens

In one embodiment, the method reported herein includes a selection step before single cell deposition, wherein B cells that produce 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. In order to select B cells from different experimental animal species, different cell surface markers can be used. It has been found that many available cell surface markers cannot provide suitable labels, whether used alone or in combination.

By marking non-target cell populations and non-specifically bound lymphocytes, these cells can be selectively removed. In this removal step, only incomplete removal can be achieved. Although removal is not quantitative, its subsequent fluorescent labeling of the remaining cells provides advantages because the number of interfering cells can be reduced or even minimized. By using the following markers outlined below, single cell deposition of mature B cells (memory B cells, affinity-matured plasmablasts and plasma cells) is carried out by fluorescence-activated cell sorting, and a higher number of IgG ⁺ -wells/cell clones can be obtained in the co-cultivation step.

The term "label" refers to the presence or absence of a surface marker that can be detected by adding a specifically binding and labeled anti-surface marker antibody. Thus, after incubation with the respective specifically binding and labeled anti-surface marker antibody, the presence of the surface marker is determined by the appearance of fluorescence, while the absence of the surface marker is determined by the absence of fluorescence, for example, in the case of a fluorescent marker.

Different cell populations can be marked by using different surface markers, such as CD3 ⁺ -cells (T cells), CD19 ⁺ -cells (B cells), IgM ⁺ -cells (mature naive B cells), IgG ⁺ -cells (mature B cells), CD38 ⁺ -cells (e.g. plasmablasts), and IgG ⁺ CD38 ⁺ -cells (proplasmablasts).

As reported herein, developed for selecting mature IgG ⁺ -B cell (such as memory B cell, plasmablast and plasma cell) immunofluorescence labeling.In order to select or enrich B cell, single labeling or double labeling or triple labeling cell.Also require that labeling causes about 0.1% to 2.5% of the labeled cells of total cell population.In one embodiment, by on 0.1% to 2.5% B cell in cell population, in another embodiment, by on 0.3% to 1.5% B cell in cell population, in another embodiment, by the labeling of the surface molecule existing on 0.5% to 1% B cell in cell population selects the B cell that is deposited as unicellular.

0.5-1% of IgG ⁺ -B cells in a PBMC population can be doubly labeled as IgG ⁺ CD19 ⁺ -cells, IgG ⁺ CD38 ⁺ -cells and IgG ⁺ CD268 ⁺ -cells. Thus, in one embodiment of all methods as reported herein, IgG ⁺ CD19 ⁺ -B cells, IgG ⁺ CD38 ⁺ -B cells or IgG ⁺ CD268 ⁺ -B cells are deposited as single cells.

0.5-1% of IgG ⁻ -B cells in a PBMC population can be doubly labeled as IgG ⁻ CD138 ⁺ -cells.Thus, in one embodiment of all methods as reported herein the IgG ⁻ CD138 ⁺ -B cells are deposited as single cells.

Labelling of CD27 ⁺ CD138 ⁺ - or CD3 ^" CD27 ⁺ - cells results in approximately 1.5% of the cells of the cell population being labeled, respectively. Thus, in one embodiment of all methods as reported herein the CD27 ⁺ CD138 ⁺ - or CD3 ^" CD27 ⁺ - B cells are deposited as single cells.

0.6%±0.1% of the IgG ⁺ -hamster B cells in the PBMC population could be double labeled as IgG ⁺ IgM ⁻ -hamster B cells.Thus, in one embodiment of all methods as reported herein the IgG ⁺ IgM ⁻ -hamster B cells are deposited as single cells.

In one embodiment, IgG ^- CD138 ⁺ -B cells are deposited as single cells from B cells obtained from immunized animals. In one embodiment of all methods reported herein, IgG ⁺ CD19 ⁺ -B cells are deposited as single cells from B cells obtained from non-immunized animals. In another embodiment of all methods reported herein, IgG ⁺ IgM ^-- B cells are deposited as single cells from B cells obtained from non-immunized or immunized animals. In one embodiment of all methods reported herein, IgG ⁺ CD19 ⁺ -mouse B cells are deposited as single cells. This selection step results in an increase or even the highest yield of IgG ⁺ -wells in the subsequent co-culture step. In another embodiment of all methods reported herein, IgG ^- CD138 ⁺ -mouse B cells are deposited as single cells. Therewith, cells that first produce the highest amount of B cell clones are selected, and secondly, cells that produce the highest IgG concentration are selected (see Table 5). In another embodiment of all methods reported herein, IgG ⁺ CD19 ⁺ -mouse B cells and IgG ^- CD138 ⁺ -mouse B cells are deposited as single cells. In a specific embodiment, the method has the proviso that, if the cells are of rabbit origin, the marker is not IgG ⁺ -B cells and/or CD138 ⁺ -B cells.

IgG ⁺ -mouse B cells can be labeled with anti-mouse-IgG-antibody 227 (Ab 227), IgG ⁺ -hamster B cells can be labeled with anti-hamster-IgG-antibody 213 (AB 213) and/or anti-hamster-IgG-antibody 225 (AB 225), and rabbit B cells can be labeled with anti-IgG-antibody 184 (see Table 4).

Table 4: Immunofluorescence labeling of B cells - This table shows the average labeled proportion of mouse B cell (A-E), hamster B cell (F-H) and rabbit B cell (I-J) populations.

PE: phycoerythrin

APC: Allophycocyanin

FITC: fluorescein isothiocyanate

It must be noted that not all commercially available antibodies can be used for labeling due to their low or non-existent specificity.

Mouse B cells can be labeled with anti-IgG antibody 227, and hamster B cells can be labeled with anti-IgG antibody 213.

IgG ⁺ CD19 ⁺ -murine B cells can be labeled with antibody 227 and antibody 218,

IgG ⁺ IgM ^- - Mouse B cells can be labeled with antibodies 227 and 219.

IgG ⁺ IgM ^- - Hamster B cells can be labeled with antibodies 213 and 224,

IgG ⁺ -rabbit B cells can be labeled with antibody 184,

IgG ⁺ IgM ^- - rabbit B cells can be labeled with Antibody 184, Antibody 254 and SA 263,

IgG ⁺ CD138 ⁺ -rabbit B cells can be labeled with Antibody 259 and Antibody 256.

Murine B cells can be labeled with anti-CD27 antibody 235 or 236 (AB 235, AB 236), anti-CD38 antibody 192 (AB 192), anti-CD138 antibody 233 (AB 233), and anti-CD268 antibody 246 (AB 246).

Table 5: Immunofluorescence labeling for mature mouse (A-J), hamster (K) and rabbit (L-N) B cells

A647:Alexa647

FITC: fluorescein isothiocyanate

In one embodiment, the method includes the steps of depleting the B cell population of macrophages and enriching the B cell population for B cells that secrete antibodies that specifically bind to the target antigen.

Single cell deposition:

The method reported herein includes the step of depositing the B cells of the B cell population as single cells. In one embodiment of all methods reported herein, single cells are deposited by fluorescence activated cell sorting (FACS). The required labeling for FACS single cell deposition can be performed as reported in the previous sections.

In one embodiment of all methods reported herein, specifically labeled B cells are deposited as single cells. In another embodiment of all methods reported herein, the labeling is labeling a cell surface marker with a fluorescently labeled antibody. In another embodiment, the methods reported herein provide monoclonal antibodies. In one embodiment of all methods reported herein, mature B cells are deposited as single cells.

It has also been found that an additional centrifugation step after single cell sedimentation and before co-culture provides an increased number of antibody secreting cells and increases the amount of secreted IgG (for experimental animals with human immunoglobulin loci, see Table 6).

Table 6: IgG positive wells/cell clones with or without centrifugation step after single cell sedimentation

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 a specific embodiment the centrifugation is performed at 300 x g for 5 min.

Co-culture:

There may be a number of additional steps before and after the co-cultivation step with feeder cells.

In one embodiment of all methods reported herein, single deposited B cells and feeder cells are co-cultured in the presence of a feeder mix. In a specific embodiment, B cells are co-cultured with murine EL-4 B5 feeder cells. Improvements in yield (IgG ⁺ -wells/cell clone number and IgG concentration) during the co-cultivation step, as well as the enrichment or isolation of mature IgG ⁺ -B cells from PBMCs, can be achieved by suitable immunofluorescence labeling as described above.

The highest number of IgG ⁺ -wells/cell clones can be obtained by single cell deposition of IgG ⁺ CD19 ⁺ -and/or IgG ⁺ CD38 ⁺ -B cells from freshly isolated PBMCs. Good results can be obtained by single cell deposition of IgG ⁺ CD19 ⁺ -, IgG ⁺ CD38 ⁺ -and/or IgG ^- CD138 ⁺ -B cells after removal of macrophages or KLH-specific cells (keyhole limpet hemocyanin). Improved results can be obtained by single cell deposition of IgG ⁺ CD19 ⁺ -, IgG ⁺ CD38 ⁺ -and/or IgG ^- CD138 ⁺ -B cells after removal of antigen-specific B cells. Thus, in one embodiment of all methods reported herein, IgG ⁺ CD19 ⁺ -, IgG ⁺ CD38 ⁺ -and/or IgG ^- CD138 ⁺ -B cells are deposited as single cells.

It has been found that single cell deposition based on the markers as described above results in the highest proportion of IgG ⁺ -wells/cell clones and wells/cell clones with the highest IgG concentration in the supernatant. Thus, in one embodiment of all methods reported herein, IgG ⁺ CD19 ⁺ - and/or IgG ^- CD138 ⁺ -murine B cells are deposited as single cells. In one embodiment of all methods reported herein, IgG ⁺ IgM ^- -hamster B cells are deposited as single cells. In one embodiment of all methods reported herein, IgG ⁺ -, and/or IgG ⁺ CD138 ⁺ -, and/or CD138 ⁺ - and/or IgG ⁺ IgM ^- -rabbit B cells are deposited as single cells.

Table 7: Yield in co-culture depending on immunofluorescence labeling

For murine B cells, the highest number of IgG ⁺ -wells/cell clones can be obtained after co-culture by single cell deposition of IgG ⁺ CD19 ⁺ -cells after each enrichment (enr.) and/or depletion (depl.) step. Alternatively, the optimal IgG concentration in the supernatant can be obtained by single cell deposition of IgG ^- CD138 ⁺ -cell wells/cell clones. Single cell deposition of IgG ^- CD138 ⁺ -cells can be used for B cells from immunized animals. Single cell deposition of IgG ⁺ CD19 ⁺ -cells can be used for B cells from non-immunized animals. Single cell deposition of IgG ⁺ IgM ^- -cells can be used for hamster B cells from immunized and non-immunized animals. Single cell deposition of IgG ⁺ -, and/or IgG ⁺ CD138 ⁺ , and/or CD138 ⁺ -, and/or IgG ⁺ IgM ^- -B cells can be used for rabbit B cells.

The immunofluorescence labeling used for B cells obtained from the blood of experimental animals can also be used to label B cells obtained from the spleen and other immune organs of experimental animals (such as mice, hamsters and rabbits). For mouse B cells, the proportion of IgG ⁺ -B cells from the spleen is about 0.8%, compared to 0.4% IgG ⁺ CD19 ⁺ -cells. For hamster B cells, the corresponding values are 1.9% and 0.5% IgG ⁺ IgM ^- -cells. For rabbit blood-derived B cells, 0.2% IgG ⁺ -cells were found after removal of macrophages. After removal of macrophages, Peyer'sche spots from rabbits showed 0.4% IgG ⁺ -cells and spleen showed 0.3% IgG ⁺ -cells.

Using the method reported herein, after about 7 days of co-cultivation, i.e., after 5, 6, 7 or 8 days, in particular after 7 or 8 days, an antibody concentration of from about 30 ng/ml to as high as 15 μg/ml or higher (average value of about 500 ng/ml) can be obtained. Using the amount of antibody thus provided, a variety of different analyses can be performed to characterize the antibody in more detail, such as regarding binding specificity. By characterizing the improvement of the antibody in the screening/selection process at this early stage, the number of nucleic acid separations required and the sequencing reactions that must be performed can be reduced. Additionally, the B cell clone provides some amount of mRNA encoding monoclonal light and heavy chain variable regions that allows the use of degenerate PCR primers, and avoids the need for highly specific primers. The number of PCR cycles required is also reduced. Therefore, in one embodiment, reverse transcriptase PCR uses degenerate PCR primers of the light and heavy chain variable domains.

In one embodiment of all methods reported herein, the feeder mixture is a thymocyte culture supernatant. In a specific embodiment, the thymocyte culture supernatant is obtained from thymocytes of the thymus of a corresponding young animal. Compared to isolating thymocytes from the blood of adult animals, the thymus of young animals is particularly suitable for use. The term "young animal" refers to an animal before sexual maturity has occurred. For example, a young hamster is less than 6 weeks old, in particular less than 4 weeks old. For example, a young mouse is less than 8 weeks old, in particular less than 5 weeks old.

Because feeder mixes are derived from supernatant derived from cultured thymocytes ( thymocyte cultivation supernatant — TSN), there is considerable batch-to-batch variation. To overcome this variability, synthetic feeder mixes have been developed, consisting of synthetic ingredients. A feeder mix consisting of IL-1β (interleukin-1β), TNFα (tumor necrosis factor α), IL-2 (interleukin-2), and IL-10 (interleukin-10) is known from Tucci, A., et al., J. Immunol. 148 (1992) 2778-2784.

This article reports a synthetic feeder mix for co-culturing single deposited B cells and feeder cells. Also reported herein are B cell species-specific additives for the synthetic feeder mix that are used to increase the amount of antibody secreted by the corresponding B cell clone. High-producing cells also contain more mRNA, which in turn facilitates reverse transcription and sequencing of encoding nucleic acids, e.g., using redundant, nonspecific primer sets.

By adding SAC (Staphylococcus aureus Cowans strain cells, using a single SAC batch), the number of antibody-secreting B cells and the average IgG concentration in the supernatant after co-culture can be increased. It has been found that a concentration range of SAC added to the co-culture can be defined, as decreasing as well as increasing SAC concentrations reduce the amount of secreted antibody.

Table 8a: Results of huCk ELISA (huCk = human CK) or rbIgG ELISA of cell culture supernatants of B cells obtained from experimental animals with the human IgG locus or wild-type rabbits (NZW) co-cultured with EL-4 B5 feeder cells, using TSN as feeder mix with or without the addition of SAC.

As can be seen, SAC ratios from 1:20,000 to 1:150,000 provide increasing numbers of IgG ⁺ -wells/cell clones, with the highest numbers shown at ratios from 1:50,000 to 1:100,000. In one embodiment, the amount of SAC added to the culture medium is determined by providing a dilution series and determining at which dilution the added SAC provides the highest number of IgG positive wells/cell clones.

It has been observed that by adding SAC to the feeder mix, the co-culture of B cells is surprisingly altered in such a way that only single deposited B cells have a growth advantage, whereas when a mixture of PBLs (e.g., B cells and endogenous T cells) is used for the co-culture, B cell growth is inhibited.

Table 8b: huCk ELISA or rbIgG ELISA results of cell culture supernatants of PBL and single deposited B cells co-cultured with EL-4 B5 feeder cells, using TSN as feeder mix, and adding SAC

＊Remove macrophages

cell

Additional data obtained using different feeding mixtures are shown in Tables 9 and 10 below.

In one embodiment of all methods reported herein, the feed mixture for the synthesis of B cell co-cultivation comprises IL-1β, TNFα, IL-2, IL-10 and IL-21 (interleukin-21). In one embodiment of all methods reported herein, the feed mixture for the synthesis of B cell co-cultivation comprises IL-1β, TNFα, IL-2, IL-10 and SAC. In a specific embodiment, IL-1β, TNFα, IL-2, IL-10 and IL-21 are recombinant mouse IL-1β, mouse TNFα, mouse IL-2, mouse IL-10, and mouse IL-21.

In one embodiment of all methods as reported herein the synthetic feeder mix for co-cultivation of murine B cells comprises IL-1β, IL-2, IL-10, TNF-α and BAFF. In a specific embodiment BAFF is added at a concentration of 5 ng/ml.

In one embodiment of all methods as reported herein, the synthetic feeder mix for the co-cultivation of hamster B cells comprises IL-1β, IL-2, IL-10, TNF-α, IL-6 and SAC. In a specific embodiment, IL-6 is added at a concentration of 10 ng/ml. In a specific embodiment, SAC is added at a ratio of 1:75,000.

Table 9: rblgG ELISA results of cell culture supernatants of rabbit B cells co-cultured with EL-4 B5 feeder cells and different synthetic feeder mixes containing different combinations of recombinant murine substances

Table 10: IgG+-wells of cell culture supernatants of rabbit B cells co-cultured with EL-4 B5 feeder cells and TSN or feeder mix containing recombinant murine material and SAC (rb = rabbit, m = mouse)

Co-culture of feeder cells and murine B cells in the absence of IL-2, IL-10, and both IL-2 and IL-10 resulted in increased IgG ⁺ -well yields but decreased IgG concentrations. The absence of TNFα also decreased IgG concentrations. In the absence of IL-1β, no IgG was detected in the supernatant.

Co-culture of hamster B cells without IL-2 or IL-10 resulted in IgG ⁺ -wells with detectable IgG concentrations. In contrast, B cell growth was barely detectable in co-cultures without IL-2 and IL-10. In the absence of TNF-α or IL-1β, no IgG secretion was detected.

Co-culture of mouse, hamster, and rabbit B cells in the presence of EL-4 B5 feeder cells requires at least IL-1β and TNFα. For co-culture of mouse cells, IL-2 and IL-10 can be omitted. Hamster B cells can be cultured in the absence of IL-2 or IL-10. Rabbit B cells can be cultured in the absence of IL-2, IL-10, or IL-6.

For murine and hamster B cells, addition of IL-4 to the feeder mix increased the number of IgG ⁺ -wells/cell clones as well as the IgG concentration in the supernatant. Thus, in one embodiment of all methods as reported herein, the feeder mix for the co-cultivation of murine or hamster B cells comprises IL-4.

Adding IL-6 to the feeder mixture for the co-cultivation of mouse B cells or hamster B cells results in an increased IgG ⁺ -well/cell clone number or an increased IgG concentration, respectively. Therefore, in one embodiment of all methods reported herein, the feeder mixture for the co-cultivation of mouse B cells or hamster B cells comprises IL-6. In a specific embodiment, IL-6 is added at a concentration of 50 ng/ml. In a specific embodiment, if high IgG concentration is required, IL-6 is added at a concentration of 10 ng/ml. In a specific embodiment, IL-6 is added after the B cells of selection and EL-4 B5 cells are co-cultivated for 3 days.

One aspect as reported herein is a synthetic feeder mix for the co-culturing of B cells and feeder cells comprising IL-1β, TNFα, IL-10, and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4, and IL-6.

One aspect as reported herein is a synthetic feeder mix for the co-culturing of B cells and feeder cells comprising IL-1β, TNFα, IL-2, IL-10 and SAC.

One aspect as reported herein is a synthetic feeder mix for the co-culture of murine B cells and feeder cells consisting of IL-1β, TNFα and optionally comprising IL-21 and/or SAC and/or BAFF and/or IL-6.

One aspect as reported herein is a synthetic feeder mix for the co-culture of murine B cells and feeder cells comprising IL-1β, IL-2, IL-10, TNF-α and BAFF.

One aspect as reported herein is a synthetic feeder mix for the co-culturing of murine or hamster B cells and feeder cells comprising IL-1β, TNFα, IL-2, IL-10 and IL-6.

One aspect as reported herein is a synthetic feeder mix for the co-culture of hamster B cells and feeder cells consisting of IL-1β, TNFα, and IL-2 or IL-10, and optionally comprising IL-21, and/or SAC, and/or BAFF.

One aspect as reported herein is a synthetic feeder mix for the co-culture of hamster B cells and feeder cells comprising IL-1β, IL-2, IL-10, TNF-α, IL-6 and SAC.

One aspect as reported herein is a synthetic feeder mix for the co-culture of rabbit B cells and feeder cells comprising IL-1β, TNFα, IL-10 and IL-6.

One aspect as reported herein is a synthetic feeder mix for the co-culture of rabbit B cells and feeder cells comprising IL-1β, TNFα, IL-10, IL-6 or IL-2, and SAC.

In a specific embodiment, the IL-1β, TNFα, IL-2, IL-10, and IL-21 are recombinant murine IL-1β, murine TNFα, murine IL-2, murine IL-10, and murine IL-21.

In a specific embodiment, BAFF is added at a concentration of 5 ng/ml.

In a specific embodiment, IL-6 is added at a concentration of 10 ng/ml.

In a particular embodiment, SAC is added at a ratio of 1:75,000.

In a specific embodiment, the feeder cells are murine EL-4 B5 cells.

Addition of a potassium channel inhibitor (PAP-1, 5-(4-phenoxybutoxy)psoralen) surprisingly increased B cell rbIgG secretion in a concentration-dependent manner without reducing the number of B cell clones. Typically, cytokines that induce rbIgG production are associated with a decrease in the total number of B cell clones. This was not the case with PAP-1.

Table 11: rbIgG ELISA results of cell culture supernatants of B cells co-cultured with EL-4 B5 feeder cells in the presence of TSN and SAC (=w/o) and different concentrations of PAP-1. DMSO: solvent for PAP-1 (1 μM)

The highest IgG concentration in the supernatant was obtained using a TSN concentration of 7.5%.

Table 12: Effect of TSN on co-culture. A TSN concentration of 7.5% resulted in improved B cell growth and productivity.

The highest combination of IgG ⁺ -well number and IgG concentration in the supernatant can be obtained by using 30,000 feeder cells per well of a 96-well plate.In one embodiment of all methods as reported herein the number of feeder cells per single deposited B-cell is about 30,000.

Table 13: Effect of the amount of EL-4 B5 feeder cells on co-culture

In one embodiment of all methods as reported herein, the co-cultivation is performed in a polystyrene multiwell plate with round-bottomed wells. In one embodiment of all methods as reported herein, the working volume of the wells is 50 μl to 250 μl. In a specific embodiment, the wells are at least partially coated with a non-fibrous substrate prepared from a blend of a polymeric plastic resin and an amphiphilic molecule, wherein the amphiphilic molecule comprises a hydrophilic part and a hydrophobic region, wherein the hydrophobic region is anchored within the substrate and the hydrophilic part is exposed on the substrate. In a specific embodiment, the amphiphilic molecule is selected from ethoxylated alkylamines, poly(ethyleneimine), octyldecamine or mixtures thereof (see for example EP 1 860 181).

Characterization of co-cultured cells:

For the (qualitative and quantitative) determination of IgG secreted after co-culture, all methods known to the person skilled in the art can generally be used, such as ELISA. In one embodiment of all methods reported herein, ELISA is used. In a specific embodiment for the determination of IgG secreted by murine B cells, an ELISA is used in which the anti-IgG antibodies AB216 (capture antibody) and AB 215 (tracer antibody) are used. In a specific embodiment for the determination of IgG secreted by hamster B cells, an ELISA is used in which the monoclonal antibodies AB 220 (capture antibody) and AB 213 (tracer antibody) are used.

Depending on the characterization results, a B cell clone can be obtained, i.e., selected. The term "clone" refers to a population of B cells that have been divided and secreted antibodies from a single B cell. Thus, a B cell clone produces a monoclonal antibody.

Isolation, cloning and sequencing of mRNA:

Total mRNA can be isolated from B cells and transcribed into cDNA. Using specific primers, nucleic acids encoding the cognate VH and VL regions can be amplified. Sequencing of the nucleic acids thus obtained demonstrates that the majority (71-95%) of the antibodies obtained are monoclonal. Sequencing of individual B cells also reveals that virtually no identical sequences are obtained. Thus, the method provides a high diversity of antibodies that bind to the same antigen.

Primers for amplifying VH-encoding nucleic acids can be used with cDNA obtained from cells derived from NMRI-mice, Armenian hamsters, Balb/c-mice, and Syrian hamsters and rabbits.

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

The term "antibody" refers to a protein consisting of one or more polypeptide chains substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include different constant region genes and numerous immunoglobulin variable region genes. Immunoglobulins can exist in a variety of forms, including, for example, Fv, Fab and F(ab) ₂ and single chain (scFv), diabodies, monovalent, bivalent, trivalent or tetravalent forms, as well as bispecific, trispecific or tetraspecific forms (e.g., Huston, JS, et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5879-5883; Bird, RE, et al., Science 242 (1988) 423-426; generally, Hood et al., Immunology, Benjamin NY, 2nd ed. (1984); and Hunkapiller, T. and Hood, L., Nature 323 (1986) 15-16).

Also reported herein is a method for producing an antibody, said method comprising the steps of:

a) providing a (mature) B cell population (obtained from the blood of experimental animals),

b) staining the cells of the B-cell population with at least one fluorescent dye (in one embodiment with 1 to 3, or 2 to 3 fluorescent dyes),

c) depositing single cells of the stained B cell population in separate containers (in one embodiment, the containers are wells of a multi-well plate),

d) culturing 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 natural TSN, in one embodiment, the feeder mix is a synthetic feeder mix),

e) determining the binding specificity of the antibodies secreted in the respective B cell cultures,

f) determining the amino acid sequences of the variable light and heavy chain domains of the specifically binding antibody by reverse transcriptase PCR and nucleotide sequencing, and thereby obtaining nucleic acids encoding the variable light and heavy chain domains of the monoclonal antibody,

g) introducing nucleic acids encoding the light and heavy chain variable domains of the monoclonal antibody into an expression cassette for expressing the antibody,

h) introducing the nucleic acid into the cell,

i) culturing cells and recovering antibodies from the cells or cell culture supernatant, thereby producing the antibodies.

"Expression cassette" refers to a construct containing the necessary regulatory elements (eg, a promoter and polyadenylation site) for expressing at least a nucleic acid contained therein in a cell.

"Laboratory animal" means a non-human mammal. In one embodiment, the laboratory animal is selected from rats, mice, hamsters, rabbits, non-human primates, sheep, dogs, cows, chickens, amphibians and reptiles.

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

Example

Example 1

Culture media and buffers:

The blocking buffer for ELISA contained 1X PBS and 1% BSA.

The coating buffer for ELISA contained 4.29 g of Na ₂ CO ₃ * 10 _{H 2} O and 2.93 g of NaHCO ₃ , which was made up to a final volume of 1 liter with water and adjusted to pH 9.6 with 2N HCl.

The ethanol solution used for RNA isolation contained 70% ethanol or 80% ethanol.

FACS buffer for immunofluorescence staining contained 1X PBS and 0.1% BSA.

IMDM buffer for ELISA contains 1X PBS, 5% IMDM and 0.5% BSA.

Incubation buffer 1 for ELISA contains 1X PBS, 0.5% Crotein C.

Incubation buffer 2 for ELISA contains 1X PBS, 0.5% Crotein C, and 0.02% Tween 20.

Incubation buffer 3 for ELISA contains 1X PBS, 0.1% BSA.

Incubation buffer 4 for ELISA contained 1X PBS, 0.5% BSA, 0.05% Tween, PBS (10X), 0.01M _{KH 2} PO ₄ , 0.1M Na ₂ HPO ₄ , 1.37M NaCl, 0.027M KCl, pH 7.0.

PCR buffer contained 500 mM KCl, 15 mM MgCl ₂ , 100 mM Tris/HCl, pH 9.0.

Wash buffer 1 for ELISA contained 1X PBS, 0.05% Tween 20.

Wash buffer 2 for ELISA contained 1X PBS, 0.1% Tween 20.

Wash buffer 3 for ELISA contained water, 0.9% NaCl, 0.05% Tween 20.

EL-4 B5 medium contains RPMI 1640, 10% FCS, 1% glutamine/penicillin/streptomycin mixture, 2% 100 mM sodium pyruvate, 1% 1 M HEPES buffer.

Example 2

Animal care and immunization

The experimental animals were maintained in accordance with the German Animal Protection Act (TierSCHG) and according to the corresponding European guidelines.

Mice and hamsters were immunized between 6 and 8 weeks of age and up to 12 weeks of age. The antigen was first administered with complete Freund's adjuvant (CFA). Further applications used incomplete Freund's adjuvant (IFA). An emulsion containing the antigen was administered subcutaneously, with the emulsion containing 50 to 100 μg of antigen, depending on the weight of the animal.

NZW rabbits (Charles River Laboratories International, Inc.) were used for immunization. The antigen was dissolved in _K₃PO₄ buffer (pH 7.0 ₎ at a concentration of 1 mg/ml and mixed with complete Freund's adjuvant (CFA) (1:1) until a stable emulsion was formed. Rabbits received an intradermal (id) injection of 2 ml of the emulsion, followed by a second intramuscular (im) and a third subcutaneous (sc) injection of 1 ml each at 1-week intervals. A fourth intramuscular injection of 1 ml was administered 2 weeks later, followed by two additional subcutaneous injections at 4-week intervals.

During the immunization period, serum antibody titers were determined using an antigen-specific assay. Blood or spleen was removed from immunized animals at an _IC50 antibody titer of 1:10,000. To reactivate antigen-specific B cells, 30 μg to 50 μg of antigen were administered intravenously to the experimental animals 3 days prior to blood or spleen removal.

Example 3

Removal of organs, blood, and macrophages

Blood was obtained from mice and hamsters by retroorbital venipuncture. Blood was obtained from rabbits by puncture of the ear vein or ear artery (for larger volumes). Whole blood (10 ml) was collected from rabbits 4-6 days after the 3rd, 4th, 5th, and 6th immunizations and used for single cell sorting by FACS.

Isolate macrophages by attachment to cell culture plastic. From mice and hamsters, approximately 3*10 ⁵ macrophages can be obtained per animal using this method.

If larger numbers of mouse or hamster macrophages are needed, isolate peritoneal macrophages. To do this, the animal must be at least 3 months old. To remove peritoneal macrophages, sacrifice the animal and immediately inject 5 ml of EL-4 B5 medium at 37°C into the peritoneal cavity. After massaging the animal's abdomen for 5 minutes, remove the cell-containing solution.

Example 4

Culture of EL-4 B5 cells

Thaw frozen EL-4 B5 cells quickly in a 37°C water bath and dilute with 10 ml of EL-4 B5 medium. Centrifuge at 300 x g for 10 minutes, discard the supernatant, and resuspend the pellet in medium. After another centrifugation step, discard the supernatant again and resuspend the pellet in 1 ml of medium.

EL-4 B5 cells were seeded in 175 ^cm flasks at a cell density of 3 x ¹⁰ cells/ml. The cell density was determined every 2 days and adjusted to 3 x ¹⁰ cells/ml. The cells have a doubling time of approximately 12 hours and must be cultured at a cell density below 5 x ¹⁰ cells/ml because the cells lose their stimulatory properties at higher cell densities.

When the total cell count reached approximately 1.5 x ¹⁰⁹ cells, the culture medium was removed by centrifugation. The cells were then irradiated with 50 gray (5000 rad). After viable cell counts were determined by trypan blue staining, the cells were aliquoted into 5 x ¹⁰⁶ to 1 x ¹⁰⁷ aliquots and frozen at -80°C.

For co-culture, cells were thawed and washed twice with EL-4 B5 medium. To determine the number of viable cells, the cell suspension was diluted 1:10 with 0.4% (w/v) trypan blue solution, 10 μl of the mixture was transferred to a Neubauer counting chamber, and the cells were counted.

Example 5

Density gradient centrifugation

Isolation of peripheral blood mononuclear cells (PBMCs) was achieved by density gradient separation using BITRATE® A (-mammalian, cedarlane) according to the manufacturer's instructions.

Dilute the drawn blood 2:1 with phosphate-buffered saline (PBS). Place an equal volume of density separation medium in a centrifuge tube and carefully add the diluted blood through the tube wall. Centrifuge the tube at 800 x g for 20 minutes without braking. Recover lymphocytes from the white interlayer. Replenish the drawn cells with 10 ml of PBS and centrifuge at 800 x g for 10 minutes. Discard the supernatant and resuspend the pellet, wash, and centrifuge. Resuspend the final pellet in PBS.

Example 6

Hypotonic lysis of red blood cells

To disrupt red blood cells by hypotonic lysis, ammonium chloride solution (BD Lyse ^™ ) was diluted 1:10 with water and added to whole blood at a ratio of 1:16. To lyse the red blood cells, the mixture was incubated in the dark for 15 minutes. To separate cell debris from intact cells, the solution was centrifuged at 800 x g for 10 minutes. The supernatant was discarded, and the pellet was resuspended in PBS, washed again, centrifuged, and the pellet was resuspended in PBS.

Example 7

Preparation of cells from internal organs of experimental animals

To prepare spleen and thymocytes, dissect the corresponding organs in a culture dish and extract the cells in PBS. To remove excess tissue, filter the cell suspension through a 100 μm sieve. To obtain lymphocytes from spleen cells, use density gradient centrifugation. No additional enrichment step is required for thymocytes.

Example 8

Removal of macrophages

Use sterile 6-well plates (cell culture grade) to remove macrophages and monocytes by nonspecific adhesion. Coat the wells with KLH (keyhole limpet hemocyanin) or with streptavidin and a control peptide. Add 3 ml to a maximum of 4 ml of culture medium and up to 6 x 10 ⁶ peripheral blood mononuclear cells from immunized rabbits to each well and allow binding in an incubator at 37°C for 60 to 90 minutes. Then, transfer the supernatant containing the lymphocytes to a centrifuge tube and centrifuge at 800 x g for 10 minutes. Resuspend the pellet in PBS.

50% of the cells in the supernatant were used for the panning step; the remaining 50% of the cells were directly subjected to immunofluorescence staining and single-cell sorting.

Example 9

Depletion of KLH-specific B cells

Incubate 4 ml of a solution containing 2 μg/ml keyhole limpet hemocyanin (KLH) in the wells of a multiwell plate with coating buffer overnight at room temperature. Remove the supernatant and wash the wells twice with PBS before the removal step. Adjust the blood cells to a cell density of 2 x 10 ⁶ cells/ml and add 3 ml to each well of the multiwell plate. Incubate the multiwell plate at 37°C for 60 to 90 minutes. Transfer the supernatant to a centrifuge tube, wash the wells twice with PBS, and combine the supernatants in the centrifuge tube. Pellet the cells by centrifugation at 800 x g for 10 minutes and resuspend the pellet in PBS.

Example 10

Enrichment of antigen-specific B cells

Dilute the corresponding antigen to a final concentration of 2ug/ml with coating buffer. Add 3ml of this solution to the wells of a 6-well multi-well plate and incubate overnight at room temperature. Remove the supernatant before use and wash the wells twice with PBS. Adjust the B cell solution concentration to 2x ¹⁰⁶ cells/ml and add 3ml to each well of the 6-well multi-well plate. Incubate the plate at 37°C for 60 to 90 minutes. Remove the supernatant and wash the wells 2 to 4 times with PBS. To recover antigen-specific B cells, add 1ml of trypsin/EDTA solution to the wells of the multi-well plate and incubate at 37°C for 10 to 15 minutes. Terminate the incubation by adding culture medium and transfer the supernatant to a centrifuge tube. Wash the wells twice with PBS and combine the supernatant with other supernatants. Precipitate the cells by centrifugation at 800x g for 10 minutes. Resuspend the pellet in PBS.

Example 11

Co-culture of B cells and EL-4 B5 cells

a) Co-culture was performed in a 96-well multi-well plate with a round bottom. A basal solution containing EL-4 B5 cells (1.6 x ¹⁰ cells/15.2 ml) and cytokines was prepared in EL-4 B5 culture medium. 200 μl of basal solution was added to each well of the multi-well plate. Single B cells were added to each well by fluorescence-activated cell sorting. After the addition of B cells, the plate was centrifuged at 300 x g for 5 minutes. The plate was incubated at 37°C for 7 days.

b) Individually sorted B cells and Pansorbin cells (1:20,000) (Calbiochem (Merck), Darmstadt, Germany), 5% _rabbit thymocyte supernatant, and λ-irradiated EL-4-B5 mouse thymoma cells (2×10 4 /well) were cultured in 96-well plates in an incubator at 37°C and 5% CO 2 in an atmosphere of 5% ^CO 2 for 7 days. The B cell culture supernatant was removed for screening, and the cells were immediately harvested for variable region gene cloning or frozen at −80°C in 100 μl of RLT buffer (Qiagen, Hilden, Germany).

Example 12

T-cell culture

Isolate T cells from the thymus of 3-4 week-old mice and hamsters, or 4-5 week-old rabbits. Centrifuge the cells and culture immediately or freeze in aliquots of 3 x ¹⁰⁷ cells. Seed thymocytes in 175 ^cm2 culture flasks at a minimum cell density of 5 x ¹⁰⁵ cells/ml in EL-4 B5 medium and incubate at 37°C for 48 hours.

Example 13

Macrophage culture

Macrophages were isolated from the peritoneal cavity of mice and hamsters at least 3 months old. Peritoneal macrophages from mice or hamsters, or blood mononuclear cells from rabbits, were cultured in 175 ^cm flasks at 37°C for 1.5 hours at a cell density of at least 1 x ¹⁰ cells/ml in EL-4 B5 medium. The culture medium was then removed and non-attached cells were removed from attached macrophages by washing with warm EL-4 B5 medium, followed by culturing in 35 ml of culture medium for 48 hours.

Example 14

Co-culture of T-cells and macrophages

T cells and macrophages were cultured in separate bottles for 48 hours. Before combining the two cell populations, the T cells were centrifuged at 800 x g for 10 minutes. The supernatant was discarded and the cell pellet was resuspended in 10 ml of culture medium. The T cells were adjusted to a minimum cell density of 5 x ¹⁰ cells/ml, and 10 pg of phorbol 12-myristate-13-acetate (PMA) and 5 ng or 50 ng of phytohemagglutinin M (PHA-M) were added to each ml of culture medium. The culture medium of the macrophages was removed and the T cell suspension was added to the bottle containing the macrophages. After 36 hours of co-culture, the culture medium was removed and referred to as TSN solution. To remove the remaining cells, the TSN solution was filtered through a 0.22 μm filter. The TSN solution was frozen at -80°C in 4 ml aliquots.

Example 15

Immunofluorescence staining

Depending on the number of cells to be stained, cells were provided in 100 μl of culture medium (less than 10 ⁶ cells) or 200 μl of culture medium (more than 10 ⁶ cells). Fluorescently labeled antibodies were diluted to a final volume of 100 μl or 200 μl, respectively, with 5% laboratory animal serum and FACS buffer. The reaction mixture was incubated on a roller rack in the dark at 4°C for 40 minutes. After incubation, the cells were washed twice at 300 x g for 5 minutes. The pellet was resuspended in 400 μl of PBS and filtered through a 70 μm sieve. The filtered solution was transferred to a FACS tube and dead cells were stained directly by adding propidium iodide (6.25 μg/ml) before the start of the FACS experiment. If the labeled antibody was labeled with biotin, the antibody was detected in a second step using streptavidin-labeled AlexaFlour(R) 647 (antibody 197).

Example 16

Quantification of IgG

After 7 days of co-cultivation, the co-cultivated 96-well multiwell plate was centrifuged at 300 xg for 5 minutes. 150 μl of the supernatant was removed and diluted with PBS in a 2:1 ratio in a second 96-well multiwell plate.

ELISA was performed as outlined in Example 17.

Antibodies were used at a concentration of 50 ng/ml. If the OD after 5 minutes of incubation was 1 or above 1, a dilution series from 0.8 to 108 ng/ml IgG was tested.

Example 17

Detection of antigen-specific IgG

Antibodies produced by single deposited and co-cultured B cells or B cells obtained from immunized animals can be characterized with respect to specific antigen binding. ELISAs are performed at room temperature, and the ELISA solution is incubated at 20 x g on a shaker between steps. In the first step, the antigen is bound to the wells of a 96-well multiwell plate. If the antigen is a protein, it is diluted in coating buffer and applied directly to the plate. Peptide antigens bind to biotin/streptavidin via a specific binding pair. The wells of the multiwell plate may have been coated with soluble Crotein C (CrC) by the manufacturer. If not, the wells are incubated with 200 μl of blocking buffer after antigen immobilization. After incubation with 100 μl of antigen solution/well (pre-coated multiwell plate) or 200 μl of blocking buffer, unbound antigen or blocking buffer is removed by washing with wash buffer. Diluted B cell supernatant is added to each well and incubated at a volume of 100 μl. After incubation, the wells are washed. Detection antibody is then added at a volume of 100 μl per well. The antibody can be conjugated to horseradish peroxidase or labeled with biotin. The detection antibody is detected using a streptavidin-horseradish peroxidase conjugate. After incubation, the multiwell plate is washed and 50 μl of a substrate solution containing 3,3',5,5'-tetramethylbenzidine (TMB) is added to each well and incubated for the time provided in Table X. The enzymatic reaction is stopped by adding 50 μl of sulfuric acid and the optical density is measured at 450 nm and 680 nm using a photometer (Rainbow Thermo ELISA Reader) and Xread plus software.

Example 18

Isolation of ribonucleic acid (RNA)

First, pellet the cells from which RNA is to be isolated by centrifugation. Lyse the cell pellet by adding 100 μl of RLT buffer containing 10 μl/ml β-mercaptoethanol. Resuspend the cells by mixing several times with a pipette. Transfer the solution to the wells of a multiwell plate. Shake the plate briefly at 200 x g and freeze at -20°C.

RNA isolation was performed using a kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Example 19

Reverse transcription polymerase chain reaction

Reverse transcription is carried out in the volume of 20 μ l. Each reaction is carried out with and without the control of reverse transcriptase. Each reaction pre-mixes 1 μ l dNTP (each 10mM), 0.4 μ l oligo (dT) _12-18 (0.2 μ g) and 0.6 μ l random hexamer primer (0.03 μ g), then is added to 8.5 μ l at H The RNA in O. 65 ℃ of incubation reaction mixtures 5 minutes, then are directly transferred onto ice. After this pre-mix 2 μ l RT-buffer (10x), 4 μ l MgCl (25mM), 2 μ l DTT (0.1M) and 1 μ l RNAse Out ^™ (40 units) are also added to ice-cold reaction mixtures. After incubation at room temperature for 2 minutes, 0.5 μ l Superscript ^™ II reverse transcriptase (25 units) is added. Incubation reaction mixtures at room temperature for 10 minutes.

Reverse transcription was performed at 42° C. for 50 minutes. After reverse transcription, the reverse transcriptase was inactivated by incubation at 70° C. for 15 minutes. The cDNA was stored at −20° C.

Example 20

polymerase chain reaction

The polymerase chain reaction was performed using the Taq PCR Core Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. PCR was performed in a volume of 20 μl. The samples were transferred to a

Example 21

Sequencing

All sequences were determined by SequiServe (Vaterstetten, Germany).

Example 22

Panning antigen

a) Plate coating

Biotin/Streptavidin: Incubate sterile streptavidin-coated 6-well plates (cell culture grade) with biotinylated antigen at a concentration of 0.5-2 μg/ml in PBS for 1 hour at room temperature. Wash the plates three times with sterile PBS before use.

Covalently bound protein: Coat 6-well cell culture plates with 2 μg/ml of protein in carbonate buffer (0.1 M sodium bicarbonate, 34 mM sodium bicarbonate, pH 9.55) overnight at 4° C. Wash plates three times with sterile PBS before use.

b) Panning of B cells on peptides

Seed up to ⁶ x 10 cells/4 ml of culture medium in a 6-well tissue culture plate coated with the corresponding peptide and allow to bind for 1 hour at 37°C in an incubator. Remove non-adherent cells by carefully washing the wells 1-2 times with 1x PBS. Detach remaining adherent cells by incubating with trypsin in an incubator at 37°C for 10 minutes, followed by two washes in culture medium. Keep cells on ice until immunofluorescence staining.

Claims (14)

1. A method for selecting B cells, the method comprising the following steps: a) Each B cell in a B cell population co-cultured with mouse EL-4B5 cells used as feeder cells, wherein each B cell has been deposited as a single cell. b) Select the B cell clones that proliferate and secrete antibodies in step a). The co-culture was carried out in the presence of a synthetic feeding mixture comprising IL-1β, TNFα, IL-10 and one or more selected from IL-21, SAC, BAFF, IL-2, IL-4 and IL-6. 2. A method for producing an antibody that binds to a target antigen, the method comprising the following steps: a) Each B cell in a population of B cells was co-cultured in the presence of mouse EL-4B5 cells as feeder cells and one or more of IL-1β, TNFα, IL-10, and IL-21, SAC, BAFF, IL-2, IL-4, and IL-6 as a feeder mixture, wherein each B cell had been deposited as a single cell in a separate container. b) Select B cell clones that produce antibodies that specifically bind to the target antigen. c) Culture cells containing nucleic acids encoding antibodies produced by the B cell clone selected in step b), or humanized variants thereof, and recover the antibodies from the cells or culture supernatant, thereby producing antibodies. 3. The method according to any one of the preceding claims, the method further comprising the step of incubating a population of B cells in a co-culture medium prior to single-cell deposition. 4. The method according to claim 3, characterized in that the incubation is carried out at about 37°C for about 1 hour. 5. The method of claim 1 or 2, further comprising the step of centrifuging the single-cell deposited B cells prior to co-culturing. 6. The method of claim 5, characterized in that the centrifugation is performed at about 300 x g for about 5 minutes. 7. The method according to claim 1 or 2, wherein the B cells are mature B cells. 8. The method according to claim 1 or 2, characterized in that, for single-cell deposition, B cells are labeled with 2 or 3 fluorescent dyes. 9. The method of claim 8, characterized in that, for deposition as a single cell, 0.1% to 2.5% of the total B cell population is labeled. 10. The method according to claim 1 or 2, wherein the B cells are mouse B cells, hamster B cells, or rabbit B cells. 11. The method according to claim 1 or 2, characterized in that co-culture is carried out in RPMI 1640 medium supplemented with 10% (v/v) FCS, 1% (w/v) 200 mM glutamine solution containing penicillin and streptomycin, 2% (v/v) 100 mM sodium pyruvate solution, and 1% (v/v) 1M 2-(4-(2-hydroxyethyl)-1-piperazine)-ethanesulfonic acid (HEPES) buffer. 12. The method of claim 8, characterized in that the B cells are mouse-derived and labeled as IgG ⁺ CD19 ⁺ -B cells, and/or IgG ^- CD138 ⁺ -B cells, and IgG ⁺ CD19 ⁺ -B cells, and/or IgG ^- CD138 ⁺ -B cells are deposited as single cells. 13. The method of claim 8, characterized in that the B cells are hamster-derived and labeled as IgG ⁺ IgM ^- B cells, and the IgG ⁺ IgM ^- B cells are deposited as single cells. 14. The method of claim 8, characterized in that the B cells are rabbit-derived and labeled as IgG ⁺ -B cells and/or CD138 ⁺ -B cells, or CD138 ⁺ IgG ⁺ -B cells and/or IgG ⁺ IgM ^-- B cells, and that IgG ⁺ -B cells and/or CD138 ⁺ -B cells, or CD138 ⁺ IgG ⁺ -B cells and/or IgG ⁺ IgM ^-- B cells are deposited as single cells.