EX VIVO ANTIBODY PRODUCTION
The present invention provides means and methods for producing improved ex vivo B cell cultures with a short doubling time.
1 . (canceled)
2 . A method for obtaining an ex vivo B cell culture 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,
wherein said B cell is a rabbit B cell; and
wherein said ex vivo B cell culture has a mean doubling time of 20 hours or less.
3 . A method for increasing the replicative life span of a rabbit B cell, 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,
wherein said rabbit B cell is provided with:
a nucleic acid molecule encoding Bcl-6, or
a rabbit homologue of said nucleic acid molecule encoding Bcl-6, 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 . A method of introducing a nucleic acid molecule of interest into a rabbit B cell comprising:
transduction of said rabbit B cell with 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.
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.
6 . The method according to claim 2 , wherein 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 . The method according to claim 6 , wherein said non-rabbit nucleic acid molecule is a human nucleic acid molecule.
8 . The method according to claim 2 , said at least one anti-apoptotic nucleic acid molecule comprising a gene of the Bcl2 family, 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 . The method according to claim 2 , further comprising: inducing, enhancing and/or maintaining expression of Blimp-1, or a rabbit homologue thereof, in said rabbit B-cell.
10 . The method according to claim 2 , further comprising providing said rabbit B cell with IL21 and CD40L.
11 . The method according to claim 10 , wherein said IL21 is mouse or human IL21 and/or wherein said CD40L is mouse or human CD40L.
12 . The method according to claim 2 , 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 . The method according to claim 2 , 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 . The method according to claim 2 , 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: wherein said cell is 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 . The rabbit B cell according to claim 15 , wherein said cell 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 comprising 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 . The 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, encoding Bcl-xL, Mcl, Bcl-2, A1, Bcl-w, 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 . The 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, wherein said ex vivo rabbit B cell culture has a mean doubling time of 20 hours or less.
21 . The ex vivo rabbit B cell culture comprising rabbit B cells according to claim 18 .
22 . An ex vivo rabbit B cell culture when obtained by a method of claim 2 .
23 . An antibody obtained by a method according to claim 2 .
24 . An antibody produced by a rabbit B cell according to claim 18 .
25 . (canceled)
26 . The method according to claim 3 , wherein said extracellular domain is of an envelope protein of GALV strain SEATO.
27 . The method according to claim 3 , said gene delivery vehicle comprising a chimeric envelope protein, or a protein comprising a chimeric envelope protein, or a protein that has at least 70% sequence identity with a chimeric envelope protein.