EX VIVO ANTIBODY PRODUCTION

Abstract

The present invention provides means and methods for producing improved ex vivo B cell cultures with a short doubling time.

Claims

1. Use of a rabbit B cell for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less.

2. A method for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method comprising: inducing, enhancing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a B-cell and inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid molecule in said B-cell, characterized in that said B cell is a rabbit B cell.

3. A method for increasing the replicative life span of a rabbit B cell, the method comprising: inducing, enhancing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a rabbit B-cell and inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid in said B-cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and/or with at least one anti-apoptotic nucleic acid molecule, via transduction with a gene delivery vehicle that comprises at least a functional part of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein.

4. Use of at least a functional part of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein, for introducing a nucleic acid molecule of interest into a rabbit B cell.

5. A method for obtaining antibodies, comprising: inducing, enhancing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a rabbit B-cell; inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid molecule in said B-cell; culturing said B cell ex vivo; and harvesting antibodies produced by said B cell within 7-14 days, preferably within 9-12 days.

6. A method according to any one of claims 2, 3 or 5, characterized in that said rabbit B cell is provided with: a nucleic acid molecule encoding a non-rabbit Bcl-6 or a functional part or a functional derivative thereof, and/or at least one non-rabbit anti-apoptotic nucleic acid molecule.

7. A method according to claim 6, wherein said non-rabbit nucleic acid molecule is a human nucleic acid molecule.

8. A method according to any one of claims 2-3 or 5-7, wherein said at least one anti-apoptotic nucleic acid molecule comprises a gene of the Bcl2 family, preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof and functional parts thereof and functional derivatives thereof.

9. A method according to any one of claims 2-3 or 5-8, wherein the method also comprises inducing, enhancing and/or maintaining expression of Blimp-1, or a rabbit homologue thereof, in said rabbit B-cell.

10. A method according to any one of claims 2-3 or 5-9, further comprising providing said rabbit B cell with IL21 and CD40L.

11. A method according to claim 10, wherein said IL21 is mouse or human IL21 and/or wherein said CD40L is mouse or human CD40L.

12. A method according to any one of claims 2-3 or 5-11, comprising: providing said rabbit B cell with a compound capable of directly or indirectly enhancing expression of Bcl-6, or expression of a rabbit homologue thereof; and/or culturing said rabbit B cell in the presence of a compound capable of directly or indirectly enhancing expression of Bcl-6, or expression of a rabbit homologue thereof.

13. A method according to any one of claims 2-3 or 5-12, comprising: providing said rabbit B cell with at least one compound capable of directly or indirectly enhancing expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10 and/or or a rabbit homologue thereof; and/or culturing said rabbit B cell in the presence of at least one compound capable of directly or indirectly enhancing expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10 and/or or a rabbit homologue thereof.

14. A method according to any one of claims 2-3 or 5-13, further comprising: providing said rabbit B cell with at least one compound capable of directly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue of Blimp-1; and/or culturing said rabbit B cell in the presence of at least one compound capable of directly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue of Blimp-1.

15. An isolated or recombinant rabbit B cell bound to at least a functional part of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or to a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein.

16. A rabbit B cell according to claim 15, which is bound via at least a functional part of the extracellular domain of a GALV envelope protein, or via a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein, to a gene delivery vehicle that comprises a nucleic acid sequence encoding Bcl-6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and/or an anti-apoptotic nucleic acid sequence.

17. A rabbit B cell according to claim 16, wherein said anti-apoptotic nucleic acid sequence is a nucleic acid sequence encoding a protein selected from the group consisting of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, a rabbit homologue thereof, a functional part thereof, a functional derivative thereof, and any combination thereof.

18. An isolated or recombinant rabbit B cell comprising: a non-rabbit anti-apoptotic nucleic acid molecule, preferably encoding Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10 or a functional part or a functional derivative thereof, and a non-rabbit nucleic acid molecule encoding Bcl-6 or a functional part or a functional derivative thereof.

19. A rabbit B cell according to claim 18, wherein said non-rabbit nucleic acid sequence is a human nucleic acid sequence.

20. An ex vivo rabbit B cell culture which has a mean doubling time of 20 hours or less.

21. An ex vivo rabbit B cell culture comprising rabbit B cells according to claim 18 or 19.

22. An ex vivo rabbit B cell culture when obtained by a method according to any one of claims 2-3 or 5-14.

23. An antibody when obtained by a method according to any one of claims 2-3 or 5-14.

24. An antibody produced by a rabbit B cell according to claim 18 or 19 or by an ex vivo rabbit B cell culture according to any one of claims 20-22.

25. Use of a gene delivery vehicle comprising at least a functional part of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein, and a nucleic acid sequence encoding Bcl-6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and/or at least one anti-apoptotic nucleic acid sequence, for increasing the replicative life span of a rabbit B cell.

26. A method according to claim 3, or a use according to claim 4, or a rabbit B cell according to claim 15 or 16, or a use according to claim 25, wherein said extracellular domain is of an envelope protein of GALV strain SEATO.

27. A method according to claim 3 or a rabbit B cell according to claim 16 or a use according to claim 25, wherein said gene delivery vehicle comprises a chimeric envelope protein as depicted in FIG. 9, or a protein comprising a chimeric envelope protein as depicted in FIG. 9, or a protein that has at least 70% sequence identity with a chimeric envelope protein as depicted in FIG. 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0123] FIG. 1.

[0124] Transduction of rabbit memory B cells. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for 36-40 hrs on CD40L L-cells with rm-IL-21. Cells were transduced with a retroviral vector containing BCL6 and Bcl-xL. Both GALV and amphotropic type retroviruses were tested. Transduced cells are then cultured on CD40L-L cells in the presence of recombinant mouse IL-21. After four days of culture the transduction efficiency was determined based on GFP expression. GALV typed retrovirus showed superior (80%) transduction efficiency compared to amphotropic (0.8%) typed retrovirus.

[0125] FIG. 2.

[0126] Growth curves were analyzed for rabbit B cells transduced with a retroviral vector containing BCL6 and Bcl-xL or a retroviral vector containing BCL6 and Mcl-1. For comparison growth curves were analysed in parallel B cells from llama cells and human cells from two different donors that were transduced with an identical retroviral vector containing BCL6 and Bcl-xL.

[0127] FIG. 3.

[0128] IgG, IgM and IgA surface immunoglobulin expression was detected using FACS on three different Bcl-6 Bcl-xL transduced rabbit B-cell clones.

[0129] FIG. 4.

[0130] Identification of antigen-specific rabbit B-cells within a pool of rabbit B cells with different specificities.

[0131] FIG. 5.

[0132] Antigen-specific rabbit antibodies were obtained against the different components of a human influenza vaccine containing 15 ug H1N1, 15 ug H3N2 and 15 ug infl B. Rabbits were immunized and boosted with the human influenza vaccine. B cells were immortalized and seeded at different densities in 384-well plates on CD40L-L cells in the presence of recombinant mouse IL-21. Antibodies present in the rabbit B cell culture supernatants were screened in ELISA for influenza-specificity. Antigen-specific antibodies were observed for all the components of the vaccine.

[0133] FIG. 6.

[0134] Immortalized B cells from rabbits immunized with a human influenza vaccine containing 15 ug H1N1, 15 ug H3N2 and 15 ug infl B were stained with fluorescently labelled influenza proteins. B cells showing binding to the influenza proteins were sorted 1 cell per well in 384-well plates on CD40L-L cells in the presence of recombinant mouse IL-21 using a FACSAria sorter. Supernatants were screened in ELISA for influenza-specific antibodies. Antigen-specific antibodies were observed with a high frequency for the components of the vaccine that were used for antigen-specific sorting.

[0135] FIG. 7.

[0136] Antibody concentration in the supernatant of clonal B cells at different time points. Human, llama and rabbit transduced B cells were seeded 1 cell per well in the presence of irradiated CD40L-L cells and supplemented with mouse IL-21. Every 3-4 days CD40L-L cells and IL-21 were replenished. The IgG concentration was analyzed in ELISA for individual wells at different time points during culture. Each measurement was done on different wells. The rabbit B cells were either transduced with a retroviral vector containing BCL6 and Bcl-xL or a retroviral vector containing BCL6 and Mcl-1. All other cells (human and llama) were transduced with BCL6 and Bcl-xL.

[0137] FIG. 8.

[0138] Schematic representation of the vector used to transduce the rabbit and human B cells

[0139] FIG. 9.

[0140] Sequence of the extracellular domain of GALV SEATO envelope protein (bold) and the transmembrane domain of the GALV SEATO envelope protein (underlined), fused to the cytoplasmic domain of ampho envelope protein (italics+dotted-underlined).

[0141] FIG. 10.

[0142] Transduction of rabbit memory B cells and outgrowth of rabbit B cells transduced with amphotropic type retrovirus. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for 36-40 hrs on CD40L L-cells with rm-IL-21. Cells were transduced with a retroviral vector containing BCL6 and Bcl-xL. Both GALV and amphotropic type retroviruses were tested. Transduced cells were then cultured on CD40L-L cells in the presence of recombinant mouse IL-21. After four days of culture the transduction efficiency was determined based on GFP expression. GALV typed retrovirus showed superior (80%) transduction efficiency compared to amphotropic (0.8%) typed retrovirus. After 10 days 94% of rabbit B cells transduced with amphotropic type retrovirus were immortalized based on GFP expression showing outgrowth of transduced cells over non-transduced cells.

[0143] FIG. 11.

[0144] A growth curve was analyzed for rabbit B cells transduced with a amphotropic type retroviral vector containing BCL6 and Bcl-xL

REFERENCES

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