Method for producing antigen-specific B cells and their use for the production of hybridoma cells and monoclonal antibodies

Abstract

The invention refers to a non-therapeutic method for producing antigen-specific B cells by using the adoptive cell transfer of primed B cells, especially of spleen cells including B cells of a previously immunized non-human animal and by administering an antigen of interest to a naïve non-human animal.

Claims

1. A non-therapeutic method for producing antigen-specific B cells by using adoptive cell transfer, wherein the method comprises the steps of: a) providing primed B cells isolated from non-human animals which have been immunized with an antigen of interest; b) administration of the primed B cells and the antigen of interest to a naïve non-human animal in order to induce a humoral immune response against the antigen of interest, c) isolation of antigen-specific B cells from the non-human animal of step b).

2. The method according to claim 1, wherein the primed B cells are spleen cells including B cells isolated from non-human animals which have been previously immunized with the antigen of interest.

3. The method according to claim 1, wherein the primed B cells to be administered to the naïve non-human animal are pre-treated by washing in serum-free cell culture medium and by lysis of the red blood cells.

4. The method according to claim 1, wherein the primed B cells to be administered to the naïve non-human animal are pre-treated by: washing the primed B cells in serum-free cell culture medium; pelleting the primed B cells by centrifugation; re-suspending the obtained cell pellet using red blood cell lysis buffer; adding serum-free culture medium; pelleting the primed B cells by centrifugation; and re-suspending the final cell pellet in physiological saline.

5. The method according to claim 1, wherein the primed B cells are administered to the naive non-human animal prior to the administration of the antigen of interest.

6. The method according to claim 1, wherein the administration of the primed B cells and/or of the antigen of interest is performed by injection, preferably by intraperitoneally, intravenously, subcutaneous or intralymphatic injection.

7. The method according to claim 1, wherein the developing immune response in the naïve non-human animal administered with the primed B cells and the antigen of interest is monitored, preferably the serum antibody titres in the blood are determined at predetermined time points.

8. The method according to claim 1, wherein the non-human animal administered with the primed B cells and the antigen of interest is boosted by additional administration of antigen of interest, preferably the time point for boosting is determined depending on the extent of the observed immune response.

9. The method according to claim 1, wherein the non-human animal to be administered with the primed B cells and the antigen of interest is a mammal, preferably selected from the group consisting of rodent, mouse, rat, guinea pig, rabbit, bovine, horse, dog, cat, goat, sheep, and pig, more preferably the non-human animal is a mouse or a rat.

10. The method according to claim 1, wherein the antigen of interest is one selected from the group consisting of peptides or peptide conjugates, proteins or fragments thereof; carbohydrates; organic and inorganic molecules; receptors produced recombinantly or by animal cells, bacterial cells, and viruses; enzymes; agonists and antagonists of biological pathways; hormones; nucleic acids, lipids, nanoparticles and cytokines.

11. The method according to claim 1, wherein the primed B cells and/or the B cells of the non-human animal administered with the primed B cells and the antigen of interest are isolated from spleen, blood or any other primary or secondary lymphoid organ.

12. A method for producing a hybridoma cell, comprising the steps of: production of antigen-specific B cells as defined in any one of claims 1 to 11; and fusing an antigen-specific B cell and a myeloma cell, so as to prepare a hybridoma cell producing an antibody specific for the antigen of interest.

13. A hybridoma produced according to a method of claim 12.

14. A method for producing a monoclonal antibody, comprising the steps of: culturing the hybridoma cell produced by the method as defined in claim 12; and obtaining a monoclonal antibody against the antigen of interest from the culture of the hybridoma cell or the recombinant expression of the antibody genes.

15. A monoclonal antibody produced according to a method of claim 14.

Description

EXAMPLES

Materials

[0028] BALB/c (8 to 12 weeks old) purchased from JANVIER LABS [0029] Antigen 4, 5 and 6 being peptides coupled to bovine thyroglobulin, the antigen solutions are used in physiological saline at 1 mg/ml, (insoluble antigens are injected as suspension) [0030] Freund's Complete Adjuvant, (CFA or FCA) and Freund's incomplete Adjuvant (IFA or FIA) [0031] Red blood cell (RBC) lysis buffer [0032] Myeloma cells SP 2/0-Ag-14 purchased from the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, DE)

Example 1

Preparation of Antigen-Specific B Cells Using Adoptive Cell Transfer of Spleen Cells of Previously Immunized Mice and Administration of an Antigen of Interest to Naïve Mice

[0033] The cell transfer is performed with freshly isolated spleen cells containing spleen cell populations including primed B cells from previously immunized mice or spleen cells resurrected from frozen spleen cell stocks, i.e. backup spleen cells previously isolated from immunized mice. [0034] a) Immunization of mice with either antigen 4, 5 or 6 (see Table 1) for hybridoma generation using the standard protocol and the provision of primed B cells to be used for adoptive cell transfer.

[0035] For each antigen, 5 Mice are immunized with the antigen. The animals are given weekly injections of the antigen of interest emulsified in equal volumes of antigen and Freund's Adjuvants. Complete Freund's adjuvant is only used for the first immunization. Subsequent immunizations are performed with Incomplete Freund's adjuvant. The last immunization is performed in plain phosphate-buffered saline (PBS) or physiological saline. Alternative adjuvants such as Ribi, TiterMax, or Alum can also be used. Over the course of the standard 6-week immunization schedule, each mouse usually receives a total of four to five injections. The titer of the immune response is typically measured after the third injection and every subsequent immunization. Fresh spleen cells are isolated and aliquots are immediately used for hybridoma production using the standard technique or processed for adoptive cell transfer. Any additional surplus cells are frozen as backup cells for later processing if necessary. [0036] b) Transfer of freshly prepared spleen cells into naïve mice and their subsequent immunization with either antigen 4 or 6 to generate hybridomas by the adoptive transfer method.

[0037] Immediately following the standard cell fusion, an aliquot of 150×10.sup.6 cells of the same spleen cell population used in step (a) is washed in a 50-ml centrifuge tube by centrifugation in 50 ml of serum-free cell culture medium at 200×g for 8 min at room temperature. The cell pellet is re-suspended in 3.5 ml of red blood cell lysis buffer, and after 1 min of incubation serum-free culture medium is added, up to a final volume of 50 ml. An aliquot of 200 μl of the resulting cell suspension is taken for cell counting using a haemocytometer and the cell suspension is centrifuged again. The final cell pellet is re-suspended in physiological saline and the cell number is adjusted to 50×10.sup.6/200 μl. A group of naïve mice (typically two mice) is injected intraperitoneally with 200 μl/animal of cell suspension, followed by 25-100 μg/animal of antigen 4 or 6, also in physiological saline, 1 hour later. The injections are carried out intraperitoneally. In order to monitor the developing immune response, the recipient mice are bled after 1 week and their serum antibody titers are determined by ELISA. Depending on the extent of the observed immune response, the mice can be boosted again in antigen-dependent interval(s) before finally using them for hybridoma production according to standard protocols. Typically, after a resting period of 7-14 days, the mice are ready to receiving their final boost for hybridoma generation. However, in case of insufficient serum antibody titers, the recipient mice can be rested (2-4 weeks) and boosted multiple times before measuring their serum antibody titers again. [0038] c) Transfer of resurrected spleen cells into naïve mice and their subsequent immunization with antigen 5 to generate hybridomas by the adoptive transfer method using frozen spleen cell stock.

[0039] Frozen spleen cells are thawed and then prepared according to the procedure as described above under (b). For spleen cell resurrection, a cryo vial is retrieved from liquid nitrogen storage and the cells are rapidly thawed in a 37° C. water bath. The vial is transferred from the water bath to the laminar flow hood before its content is completely thawed. The right point in time for transfer of the cryo vial to the hood would be when the frozen cell pellet in the inverted cryo vial can be readily released from the bottom of the vial, i.e. drops down inside the vial after a forceful sudden downward movement. In the hood, the surface of the vial is disinfected with 70% isopropanol and the vial is opened. The cells in the still thawing cell pellet are step by step (0.5 ml volumes) re-suspended by multiply adding and withdrawing serum-free cell culture medium. The resulting 0.5 ml aliquots of thawing cells are transferred to 10 ml of serum-free cell culture medium and the cell suspension is then centrifuged at 200×g for 8 min at room temperature. The final cell pellet is re-suspended in 50 ml of serum-free cell culture medium and the cells are counted using a haemocytometer. After red blood cell lysis, the cells are processed for adoptive transfer as described above under b).

Example 2

Hybridoma and Monoclonal Antibody Bulk Production

[0040] Following hybridoma generation by adoptive transfer, monoclonal antibodies were bulk produced using standard in vitro tissue culture techniques and the antibodies were purified using standard affinity chromatography techniques before measuring the antibody concentrations spectrophotometrically. [0041] The following production rates were determined: [0042] Antigen 4: Hybridom a: 17.4 μg/ml IgG1/κ [0043] Hybridom b: 34.6 μg/ml IgG1/κ [0044] Hybridom c: 51.4 μg/ml IgG1/κ [0045] Antigen 5: Hybridom a: 25.7 μg/ml IgG1/κ [0046] Hybridom b: 20.6 μg/ml IgG1/κ [0047] Antigen 6: Hybridom a: 31.2 μg/ml IgG2b/κ [0048] Hybridom b: 64.0 μg/ml IgG1/κ

[0049] Table 1 compares the numbers of hybridomas generated by the standard technique and the adoptive cell transfer technique.

TABLE-US-00001 TABLE 1 Number of antigen-positive primary hybridomas according to the method of generation Number of primary cultures generated through: Standard technique Adoptive cell transfer Antigen No. 4 3 21 Antigen No. 5 20 30 Antigen No. 6 5 34