Abstract
The invention provides a method for influencing the stability of an antibody producing cell, comprising directly or indirectly influencing the amount of BCL6 and/or Blimp 1 expression product within said antibody producing cell. Stable antibody producing cells and cell lines are also provided, as well as methods for producing antibodies using such cells and/or cell lines.
Claims
1. An antibody producing B cell that is capable of replicating for at least nine weeks, wherein the B cell comprises an exogenous nucleic acid sequence encoding a peptide comprising Bcl6, or a functional part thereof, capable of increasing the replicative life span of an antibody producing B cell, and wherein Bcl6 and Blimp-1 are co-expressed in the B cell.
2. The antibody producing B cell according to claim 1, comprising: an exogenous nucleic acid sequence encoding a peptide comprising Bcl6, or a functional part thereof, capable of increasing the replicative life span of an antibody producing B cell, and an exogenous compound that increases or maintains the amount of Blimp-1 expression product.
3. The antibody producing B cell according to claim 2, comprising: an exogenous nucleic acid sequence encoding a peptide comprising Bcl6, or a functional part thereof, capable of increasing the replicative life span of an antibody producing B cell, and an exogenous nucleic acid sequence encoding a peptide comprising STAT3, or a functional part thereof, capable of upregulating Blimp 1 expression.
4. The antibody producing B cell according to claim 3, wherein the STAT3 is constitutively active.
5. The antibody producing B cell according to claim 3, wherein expression of the nucleic acid sequence encoding STAT3 or a functional part of STAT3 is regulated by an activator and/or repressor that is inducible by an exogenous compound.
6. The antibody producing B cell according to claim 1, further comprising an exogenous anti-apoptotic nucleic acid sequence.
7. The antibody producing B cell according to claim 6, wherein the exogenous anti-apoptotic nucleic acid sequence encodes a member of the anti-apoptotic Bcl 2 family.
8. The antibody producing B cell according to claim 6, wherein the exogenous anti-apoptotic nucleic acid sequence encodes a peptide comprising Bcl-xL, or a functional part thereof, capable of increasing the replicative life span of an antibody producing B cell.
9. An antibody producing B cell that is capable of replicating for at least nine weeks, comprising: an exogenous nucleic acid sequence encoding a peptide comprising Bcl6, or a functional part thereof, capable of increasing the replicative life span of an antibody producing B cell, and an exogenous anti-apoptotic nucleic acid sequence.
10. The antibody producing B cell according to claim 9, wherein the exogenous anti-apoptotic nucleic acid sequence encodes a member of the anti-apoptotic Bcl 2 family.
11. The antibody producing B cell according to claim 9, wherein the exogenous anti-apoptotic nucleic acid sequence encodes a peptide comprising Bcl-xL, or a functional part thereof capable of increasing the replicative life span of an antibody producing B cell.
12. The antibody producing B cell according to claim 8, wherein expression of the nucleic acid sequence encoding a peptide comprising Bcl6, Bcl-xL or a functional part of Bcl6 or Bcl-xL, is regulated by at least one of an activator and/or a repressor that is inducible by an exogenous compound.
13. A B cell culture, comprising antibody producing B cells according to claim 1.
14. The B cell culture according to claim 13, wherein the culture further comprises at least one of IL 21 and/or Il 10.
15. A B cell culture, comprising antibody producing B cells according to claim 9.
16. The B cell culture according to claim 15, wherein the culture further comprises at least one of IL 21 and/or Il 10.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0193] FIG. 1 illustrates enhanced growth of BCL6.sup.+ cells cultured with IL-21. 100% pure BCL6.sup.+ memory B cells were cultured in the presence of IL-2 and IL-4 (conventional culture conditions), or with IL-21 alone. The total expansion of live cells over 17 days of culture with IL-21 is shown.
[0194] FIG. 2 illustrates: plasmablast immortalization of BCL6-positive cells with IL-21. Memory B cells were transduced With a retrovirus expressing BCL6-GFP and cultured with IL-2 and IL-4 (to prevent differentiation) or with IL-21 for 14 days. The surface staining for CD38 and CD20 of GFP.sup.+ (that is, BCL6.sup.+) cells is shown. IL-21 induces an 8-fold increase in the amount of B cells with a plasmablast phenotype.
[0195] FIG. 3 illustrates IL-21 upregulates BLIMP1 in BCL6.sup.+ B cells. 100% pure BCL6-.DELTA.NGFR.sup.+ were cultured with IL-2 and IL-4 or IL-21 for 24 days. cDNA was generated from total RNA and mRNA levels of BLIMP1 and HPRT (loading control) were determined by reverse transcriptase polymerase chain reaction.
[0196] FIG. 4 illustrates: non-limiting model of one embodiment according to the present invention.
[0197] FIG. 5 illustrates a general overview of ideal culture scheme, see for more details the material and methods section of Example 3.
[0198] FIG. 6A. illustrates growth dynamics of IL-2 and IL-4 vs. IL-21 stimulated B cell. Peripheral blood (PB) memory B cells derived from two donors (B18 and B19) were stimulated either with IL-21 or IL-2 and IL-4. Cells were transduced with caSTAT5b-ER-IRES-NGFR at day 2 for the IL-21 and at day 5 for IL-2 and IL-4 treated cultures; 4HT was added at day 13.
[0199] FIG. 6B illustrates of 4 donors Tetanus Toxoid specific B cells were sorted from PB (cell numbers ranged from 1000-10.000). Cells were cultured in 96 well with IL-21 and transduced with caSTAT5b-ER-IRES-NGFR on day 2. 4HT was added on day 4 and IL-21 was replaced with IL-2, IL-4 and 4HT after 7 days (B14 and B15) or was replaced after 20 days (B16 and B17). Cells were counted by hand and dead cells were excluded.
[0200] FIG. 7 illustrates percentage caSTAT5b-ER-IRES-NGFR transduced cells was determined using the LSR II (BD). Of two donors (B18 and B19) IL-2 and IL-4 vs. IL-21 time series experiment were performed. Of each donor ¼ of the cells were transduced using the IL-2 and IL-4 protocol, the remaining ¾ was transduced using IL-21. Directly after the IL-21 transduction (36 h) one third of the IL-21 culture was switched to IL-2 and IL-4. This was repeated on day 5, 10 and 20 of the IL-21 culture.
[0201] FIG. 8A illustrates total human IgG and IgM antibody production by caSTAT5b-ER-IRES-NGFR transduced PB derived memory B cells, as described in FIGS. 6 and 7. Mean IgG production of donor B18 and B19 is shown. B cells were transduced using the IL-2 and IL-4 vs. the IL-21 protocol. The IgG production indicated with the open symbols represent all cultures that had been treated with IL-21, irrespective when they were switched to IL-2 and IL-4.
[0202] FIG. 8B illustrates IgM production in samples as described above, note that the time scale is different.
[0203] FIG. 9 illustrates antibody production of B cell clones derived from memory B cells of donors B18 and B19 transduced with caSTAT5b-ER-IRES-NGFR. Ten-day-old cultures that were derived from IL-21 stimulated B cells (stimulated for 36 h) were used for LD culture. Twelve clones were obtained; 5 from and 7 from B19. IgG production is the mean of three time points; IgM production is the mean of two time points.
[0204] FIG. 10A. illustrates IgG Tetanus Toxoid ELISA on supernatant of polyclonal, 100% caSTA5b-ER-IRES-NGFR positive, Tetanus Toxoid sorted human B cells. Of 7 donors rapidly proliferating clonal cultures were derived. Shown is the average TT antibody production of at least 3 different measurements per donor. Each time the relative OD was determined (generally a relative increase of >2 to 3 times the background is assumed positive).
[0205] FIG. 10B illustrates that to determine if TT IgG ELISA negative cultures could be producing IgM, the same 7 donor samples were tested in a total IgM ELISA.
[0206] FIG. 11 illustrates anti-Tetanus Toxoid ELISA. The binding of IgG and IgM .alpha.-TT specific antibodies by ELISA was determined. Supernatants of 100% NGFR positive clonal B cell cultures derived from donors B15, B16, B18 and B19 were tested. Two times the background was set as positive.
[0207] FIG. 12A illustrates total IgG and IgM production after restimulation of clonal B cell cultures donor B16 which produces IgG.
[0208] FIG. 12B illustrates restimulation of clonal B cell donor B19 which produces IgM. Production was measured in supernatant of cultures that were either cultured with IL-2, IL-4 and in the presence or absence of 4HT or with IL-21 and in the presence or absence of 4HT. Cultures containing IL-2 and IL-4 did not show an increase in antibody secretion (not shown). Only cultures that responded to the restimulation are shown (10 out of 14 IgG and 8 out of 9 IgM clones responded)
[0209] FIG. 13A illustrates antibodies secreted by IL-21 restimulated and 4HT deprived cultures, as described in the legend of FIG. 8 were tested for their antigen specificity. The supernatants derived from restimulated donor B16 clonal TT cultures were tested in the .alpha.-TT IgG ELISA.
[0210] FIG. 13B illustrates the supernatants derived from donor B19 cultures were tested in the IgM ELISA (b). Shown is the relative increase in antibody binding compared to the negative control, samples B19-10B7 and 10E1 were cut off at 30 for visibility; values were 96 and 121, respectively.
[0211] FIG. 14 illustrates LMP1 RT-PCR was performed on RNA isolated from frozen cell pellets of indicated cultures. Shown are 15 cultures which were randomly selected and tested for EBV infection. Sample coding: B indicates donor B29, C indicates donor B30 and both were cultured at 1000 c/w (e3) or 100 c/w (e2). All cultures were transduced with BCL6 except for B28 UTD (untransduced) and B29 UTD; JY cells were used as positive controls.
[0212] FIG. 15 illustrates IgG antibody production (ng/ml) by BCL6 and EBV cultures as described in table 5. For each condition the average of two samples is shown each of which consist of a longitudinal follow up of 6 to 10 time points. An asterisks indicates samples are significantly different (p<0.05, unpaired student t-test). The samples cultured with IL-2 and IL-4 are a combination of EBV and BCL6 positive and negative cultures longitudinal followed.
[0213] FIG. 16 illustrates Kappa-FITC and Lambda-PE staining on CD19, and NGFR positive cells. One B cell is either positive for Kappa or Lambda. Samples were measured using a LSRII (BD) and analysed using FlowJo software.
[0214] FIG. 17 illustrates a representative LMP1 PCR performed on DNA isolated from frozen cell pellets of donor B25 1000 c/w MBC transduced with caSTAT5b and suspected to be EBV positive based on the color of the culture medium, growth kinetics and phenotype as observed by light microscopy. All other cultures were EBV negative.
[0215] FIG. 18 illustrates average IgG production (ng/ml) in caSTAT5b-ER B cells cultured without 4HT, with IL-2 IL-4 or IL-21 and with or without EBV. The increase in antibody production in the presence of IL-21 was significant compared to cultures with IL-2 and IL-4 (p<0.05). The increase in antibody production in IL-21 containing, EBV infected cultures was significant compared to cultures without EBV (p<0.05), nonparametric Mann-Whitney)
[0216] FIG. 19 (A-C) illustrate BCL6-IRES-YFP.sup.+ cells were transduced with STAT3ER-IRES-GFP and expanded on CD40L L cells with IL-2 and IL-4. BCL6/STAT3ER positive cells were sorted by FACS and equal numbers were cultured in the absence of cytokines, but in the presence or absence of 4HT (1 .mu.M) for 4 days. (A) shows live cell numbers after 4 days. (B) shows semi-quantitative RT-PCR for BLIMP1 and HPRT1 expression in BCL6-YFP.sup.+/STAT3ER-GFP.sup.+ cells. (C) shows IgG production in BCL6-YFP.sup.+STAT3ER-GFP.sup.+ treated for 4 days.+.4HT.
[0217] FIG. 20 (A-B) illustrates: (A). CD19.sup.+ B cells were transduced with control YFP-IRES-YFP (cYFP); BCL6-IRES-YFP (BCL6-YFP) or BcIXL-GFP (Bcl-xL-GFP). Cells were then maintained on CD40L and IL-4 and the percent YFP or GFP positive was determined over time by FACS. All data represented in A and B are derived from CD19+CD3.sup.− gating. (B). Unsorted bulk cultures of Bcl-xL-IRES-GFP and BCL6-IRES-YFP double transduced B cells on CD40L and IL-4. Individual GFP.sup.+, YFP.sup.+, and GFP/YFP double positive cells was determined by FACS
[0218] FIG. 21(A-B) illustrates: IL-21 increased proliferation of B cells transduced with Bcl-xL, BCL6, or Bcl-xL+BCL6 double transduced cells. At day 17 after transduction and maintainence on CD40L and IL-4, cultures were split and cultured on CD40L in the presence of IL-4, or IL-21. Absolute number of transduced cells was determined and the cumulative expansion was calculated in single transduced cells (A) or in double transduced cells (B).
[0219] FIG. 22 illustrates Long-Term Cultures are EBV.sup.−
[0220] RT-PCR analysis of Bcl-xL, BCL6, LMP1, and EBNA1 mRNA expression in Day 66 cultures of BCL6-transduced cells (Lane 1) and BCL6/Bcl-xL-double transduced bulk cultures (Lane 2). 1 .mu.l of a cDNA reaction performed in the absence of reverse transcriptase (−RT) reaction was used as a negative control for genomic DNA contamination. Positive controls: Bcl-xL, STATS-ER transduced B cells cultured with 4-HT; BCL6, LMP1, and EBNA1, human Raji B cells.
[0221] FIG. 23 illustrates doubling time of Bcl-xL, BCL6, and Bcl-xL−BCL6 double transduced cells. Based on the number of transduced (GFP.sup.+,YFP.sup.+) B cells the doubling time between days 51-59 of culture was calculated in Bcl-xL−, and BCL6-transduced cells in single transductions as well as in Bcl-xL+BCL6 double-transduced bulk cultures.
[0222] FIG. 24 illustrates L591 a Hodgkin cell line, was transduced by lentivirus containing E47-IRES-GFP. GFP positive cells were sorted and cultured independent of L cells (CD40 stimulation) and cytokines. Cell numbers were determined in time.
REFERENCES
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