EA044991B1 - ANTIBODY FORMATION EX VIVO - Google Patents

Description

The invention relates to the field of medicine, molecular biology and immunology.

Ex vivo B cell cultures are important tools for the production of antibodies, preferably monoclonal antibodies. Monoclonal antibodies (MAbs) are multiple identical copies of a single antibody molecule that bind to antigens with the same affinity and perform the same effector functions. Among the advantages of MAbs is their specificity with respect to the same epitope on the antigen. This specificity provides certain clinical advantages of MAT compared to more traditional treatments, while offering patients an effective, well-tolerated treatment option with low overall side effects. Moreover, MAbs are preferred for biological and medical research.

The traditional approach for producing mAbs is hybridoma technology, in which a B cell is fused with a myeloma cell to form a hybrid antibody, forming cell lines (hybridomas). However, hybridoma technology with human B cells has not been very successful because the resulting hybridomas are unstable. At the same time, an improved technology was developed where ex vivo B cell cultures with a long replicative life cycle are obtained (WO 2007/067046). This technology involves ex vivo human cultures where Bcl-6 is co-expressed with Blimp-1 and/or anti-apoptotic nucleic acid in B cells. This improves the replicative life cycle of these B cells. Typically, human B cells are cultured to produce human mAbs. Human mAbs are preferred for therapeutic administration into the human body due to their lower immunogenicity compared to antibodies of other types. Using the technology according to WO 2007/067046, ex vivo human B cell cultures are obtained with an average doubling time of approximately 25-36 hours.

For the commercial production of targeted mAbs, such as therapeutic mAbs, it is advantageous to use B cell cultures, and B cells have a short doubling time. A short doubling time is also very important in therapeutic approaches such as cancer therapy, for example, when a non-human mammal is immunized with the patient's cancer cells, then cancer-specific B cells are harvested from the animal and used to generate antibodies ex vivo. Since such antibodies are drugs specifically prepared for a particular patient, they should be generated as quickly as possible so that the patient can begin his/her AT therapy as soon as possible. Such antibodies, which are specific for an individual's tumor, cannot be generated in advance.

One object of the present invention is to provide means and methods for generating improved ex vivo B cell cultures with shorter doubling times.

In the present invention, it has surprisingly been found that B cell cultures with a shorter doubling time compared to the B cell cultures described in WO 2007/067046 are obtained by using rabbit B cells. While commonly used B cells such as human B cells, mouse B cells and llama B cells typically have a doubling time of 25-36 hours, the inventors of the present invention have unexpectedly discovered that rabbit B cell cultures can be obtained over time doubling time 20 hours or less. The discovery of this fact allows for significantly faster production of target antibodies, leading to higher yields over a period of time, which is especially valuable for commercial antibody production and therapeutic applications.

Accordingly, the present invention provides the use of a rabbit B cell to produce an ex vivo B cell culture with an average doubling time of 20 hours or less. According to the present invention, ex vivo rabbit B cell cultures are typically prepared by expressing Bcl-6 or its rabbit homolog and an anti-apoptotic nucleic acid molecule in a rabbit B cell. In this regard, there is also provided a method for producing an ex vivo B cell culture with an average doubling time of 20 hours or less, a method comprising:

inducing, improving and/or maintaining the expression of Bcl-6 or its rabbit homolog in a B cell and inducing, improving and/or maintaining the expression of an anti-apoptotic nucleic acid molecule in said B cell, characterized in that said B cell is a rabbit B cell .

It is preferable to generate ex vivo rabbit B cell cultures with an average doubling time of less than 20 hours. More preferably, said average doubling time is less than 19 hours or even less than 18 hours. A shorter doubling time allows for faster and higher antibody production, which improves the time - and efficiency - of conducting research and screening of the desired antibody and isolating and/or identifying the target antibodies. Moreover, if a patient-specific mAb needs to be developed, the shorter doubling time of rabbit B cells allows for faster initiation of patient-specific mAb therapy.

Thus, the method of the present invention using rabbit B cells provides the advantage that the antibody can be obtained, tested, identified, isolated and/or produced ex vivo in a shorter period of time compared to currently known cultures of human, mouse or llama B cells.

The fact that the present invention provides a B cell culture with a short doubling time provides the advantage that sufficient amounts of antibody can be produced in a shorter period of time compared to existing methods. For example, in the method disclosed in WO 2007/067046, a collection of B cells obtained from a human individual is stabilized using Bcl-6 and an anti-apoptotic nucleic acid (or compounds that enhance the expression of such nucleic acids) and then cultured. This results in stabilized human cells that are capable of both proliferating and producing antibody. During culture, the stabilized B cells produce antibody, which is secreted into the culture medium. Subsequently, these antibodies are preferably tested for the desired specificity (and/or affinity). Current assay techniques typically require an antibody concentration of at least 100 ng/ml culture medium. This minimum antibody concentration is obtained after 15-20 days of culturing stabilized human B cells. Thus, when using human B cell cultures, the antibody is harvested at least 15-20 days after the culture begins to grow, typically around day 20. Llama B cells have a similar growth rate to human B cells, so if a llama B cell culture is used, antibody collection is also typically done at least 15 to 20 days after the culture begins to grow. With mouse B cells having a longer doubling time, antibodies with a minimum concentration of 100 ng/ml are typically obtained after more than 20 days.

Following antibody testing, appropriate target B cells are often selected and isolated for further use. Given that antibody testing typically takes approximately three days, targeted human or llama B cells are typically selected and isolated after 18 to 23 days of B cell culture growth, while targeted mouse B cells are typically selected and isolated after more than 23 days. The isolated B cells are further cultured. Thus, a B cell culture with the target human, llama, or mouse B cells is typically obtained approximately three weeks after the B cell culture begins to grow. However, with the method according to the present invention, an antibody concentration of at least 100 ng/ml is already obtained after 11-12 days. Thus, it is possible to collect the antibody as early as 11-12 days after the B cell culture begins to grow, taking into account that it is necessary to maintain the human (or llama) B cell culture for at least 15-20 days before collecting the antibody . If the assay procedure takes three days, the target rabbit B cells are thus selected and isolated within 14-15 days from the start of B cell culture growth, which is significantly faster compared to the situation where human or mouse B cells are cultured. To summarize, while obtaining a human, llama, or mouse B cell culture that produces a sufficient concentration of antibody typically takes approximately three weeks, it has been found that according to the present invention, a B cell culture with rabbit B cells producing a sufficient concentration AT, already received after two weeks. This is an important advantage compared to existing methods. In one aspect of the present invention, there is provided a method for producing antibodies, preferably for use in one or more testing assays requiring a minimum antibody concentration of at least 100 ng/ml, the method comprising:

inducing, improving and/or maintaining Bcl-6 expression in a rabbit B cell;

inducing, improving and/or maintaining expression of an anti-apoptotic nucleic acid molecule in said B cell;

culturing said B cell ex vivo; and collecting antibodies produced by said B cell for 10-14 days, preferably 11-12 days. These collected antibodies are preferably tested using one or more assays requiring a minimum antibody concentration of at least 100 ng/ml.

As described above, the resulting antibodies are typically used for testing for the desired specificity and/or affinity. Modern testing methods usually require a minimum antibody concentration of 100 ng/ml, but if more sensitive detection methods are used, antibody collection can be performed earlier. Regardless of the sensitivity of the test method, using rabbit B cells with the method according to the present invention, the required minimum concentration of antibody is obtained earlier compared to using currently known human, llama or mouse B cells due to the significantly faster doubling time of B cells. rabbit cells. For example, if a minimum antibody concentration of only 30 ng/ml is required instead of 100 ng/ml, this concentration is typically achieved using human B cells after 1318 days from the start of growth of the B cell culture, while the B cell A rabbit culture would require only 9-10 days to obtain this minimum concentration of antibody. Thus, again, antibody testing and isolation of target B cells can be performed earlier. Currently, in practice, the inventors obtain and study rabbit antibodies within 7-14 days from the beginning of growth of the B-cell culture. Prior to the present invention, ex vivo B cell cultures allowing antibody testing at significantly earlier stages than ex vivo human B cell cultures were not available. In this regard, a method for producing antibodies is also proposed, a method comprising:

inducing, improving and/or maintaining the expression of Bcl-6 or its rabbit homolog in a rabbit B cell;

inducing, improving and/or maintaining expression of an anti-apoptotic nucleic acid molecule in said B cell;

culturing said B cell ex vivo; and collecting antibodies produced by said B cell for 7-14 days, preferably 9-12 or 9-10 days. These collected antibodies are preferably tested using one or more assays requiring a minimum antibody concentration of about 30 ng/ml.

As used herein, the term rabbit B cell means a B cell that is derived from a rabbit or a B cell that is derived from a rabbit B cell.

An example of a rabbit B cell-derived B cell is the progeny of a rabbit B cell formed after one or more rounds of cell division. Such progeny, for example, contain an ex vivo culture of rabbit B cells.

An ex vivo rabbit B cell culture is a culture that contains rabbit B cells and/or their progeny. Other types of cells may also be present in the culture. For example, B cell mitogen cells, such as CD40 positive L cells and/or EL4B5 cells, are typically also present in the B cell culture of the present invention. In addition, other types of cells that were also present in the sample containing B cells could still be present in the B cell culture. If present under B cell culture conditions, such non-B cells are generally less able to proliferate than B cells, so the number of such contaminating cells will generally decrease over time. Preferably, at least 70% of the cells in the rabbit B cell cultures are rabbit B cells. More preferably, at least 75, 80, 85, 90 or 95% of the cells of said rabbit B cell culture are rabbit B cells. In a particularly preferred embodiment, rabbit cells and B cell mitogen cells, such as CD40 positive L cells and/or EL4B5 cells, are substantially the only cell types present in a rabbit B cell culture.

Preferably, the B cells of a rabbit B cell culture according to the present invention are the progeny of one original rabbit B cell, such that monoclonal antibodies are produced by the B cell culture.

As used herein, the term average doubling time is defined as the required average time, starting from a culture with a certain initial number of B cells, to obtain a culture with a number of B cells that is twice the said initial number of B cells. Since not every B cell will proliferate at exactly the same rate, average values for the B cell culture as a whole are generally used.

Bcl-6 encodes a transcriptional repressor that is required for normal development and maturation of B cells and T cells and which is required for germinal center formation. In the germinal center of B cells, there is strong expression of Bcl-6, while in plasma cells there is almost no expression. Bcl-6 inhibits the differentiation of activated B cells into plasma cells. In the method of the present invention, the expression product of Bcl-6 or the expression product of its rabbit homologue remains present in the ex vivo cultured rabbit B cells. The presence of Bcl-6 or its rabbit homologue, together with the presence of an anti-apoptotic nucleic acid, prolongs the replicative life cycle of B cells. Expression of Bcl-6 or its rabbit homolog is preferably induced, improved or maintained by introducing into the rabbit B cell(s) used for culture a compound that promotes expression of Bcl-6 or a compound that stimulates expression of the rabbit homolog Bcl-6, or by culturing rabbit B cells in the presence of such a compound.

In this regard, there is also provided a method according to the present invention, comprising:

providing said rabbit B cells with a compound capable of directly or indirectly increasing the expression of Bcl-6 or the expression of a rabbit homologue of Bcl-6; and/or culturing said rabbit B cell in the presence of a compound capable of directly or indirectly increasing the expression of Bcl-6 or the expression of a rabbit homologue of Bcl-6.

Various compounds are known in the art that are capable of directly or indirectly increasing the expression of Bcl-6 or the expression of the rabbit homologue of Bcl-6. For example, such a compound contains signal transducer and transcription activation protein 5 (STAT5) or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and/or a nucleic acid coding sequence thereof. STAT5 is a signal transducer that can improve Bcl-6 expression. There are two known forms of STAT5, STAT5a and STAT5b, which

- 3 044991 are encoded by two different sequentially linked genes. Introduction and/or activation of STAT5 or its rabbit homolog results in increased Bcl-6 or increased rabbit Bcl-6 homologue. Therefore, STAT5 or its rabbit homolog, or a functional part or functional derivative thereof, is capable of directly increasing the expression of Bcl-6 or the expression of the rabbit homolog of Bcl-6. In this regard, a method according to the present invention is provided, comprising providing said rabbit B cell with STAT5 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, or providing said rabbit B cell with a nucleic acid molecule encoding STAT5 or a rabbit homologue thereof, or a functional derivative thereof. part or functional derivative, or culturing said rabbit B cell in the presence of STAT5 or in the presence of a rabbit homologue thereof, or a functional part or functional derivative thereof.

The presence of STAT5 or its rabbit homologue directly increases the amount of Bcl-6 or the amount of the rabbit homolog of Bcl-6. In addition, it is possible to indirectly increase the expression of Bcl-6 or the expression of its rabbit homologue. For example, this is accomplished by regulating the amount of a particular compound, which in turn is capable of directly or indirectly activating STAT5 or its rabbit homologue and/or increasing the expression of STAT5 or the expression of its rabbit homolog. Therefore, in one embodiment, the expression and/or activity of endogenous and/or exogenous STAT5 or the expression of its rabbit homologue is increased. For example, it is possible to indirectly improve the expression of Bcl-6 or the expression of its rabbit homologue by culturing a rabbit B cell in the presence of interleukin (IL) 2 and/or IL4, which are capable of activating STAT5 or activating the rabbit homolog of STAT5, which in turn increases expression of Bcl-6 or expression of the rabbit homolog of Bcl-6.

As used herein, the term rabbit homolog, for example, Bcl-6 or STAT5, means a rabbit protein similar to Bcl-6 or STAT5, which means that in rabbit B cells it performs a similar function to that of Bcl-6 or STAT5 in human B cells.

However, it is preferable to provide a rabbit B cell with a nucleic acid molecule encoding Bcl-6 or encoding a rabbit homologue thereof, or a functional portion or functional derivative thereof. Thus, it is possible to directly regulate the concentration of Bcl-6 or the concentration of its rabbit homologue in the specified rabbit B cell. In this regard, also provided is a method according to the present invention, comprising providing said rabbit B cell with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue thereof, or a functional portion or functional derivative thereof. In one embodiment, said nucleic acid molecule is constitutively active, meaning that there is continuous expression of Bcl-6 or a rabbit homolog thereof, or a functional portion or functional derivative thereof, regardless of the presence of a regulator. In another embodiment, said nucleic acid molecule is inducible, meaning that its expression is regulated by at least one inducer and/or repressor. Thus, the expression of said nucleic acid molecule is controlled as desired. For example, Tet-On and Tet-Off expression systems (eg, Tet-on® and Tet-Off® enhanced inducible gene expression systems, Clontech) can be used to inducibly express a target nucleic acid sequence. In these systems, transcriptional activator (tTA) expression is regulated by the presence (Tet-On) or absence (Tet-Off) of tetracycline (TC) or a doxycycline-like derivative (dox). In principle, tTA consists of the Escherichia coli Tet repressor protein (TetR) and the transactivating domain of herpes simplex virus VP16. tTA regulates the transcription of a target nucleic acid sequence under the control of a tetracycline response element (TRE) containing a Tet operator (TetO) DNA sequence and a promoter sequence, for example, the human cytomegalovirus (hCMV) promoter. A nucleic acid sequence encoding, for example, Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, can be placed downstream of the promoter.

In the Tet-off system, tTA binds to the TRE in the absence of TC or dox and transcription of the target nucleic acid sequence is activated, while in the presence of TC or dox tTA fails to bind to the TRE and expression of the target nucleic acid sequence is inhibited. In contrast, the Tet-On system uses reverse tTA (rtTA), which can only bind to the TRE in the presence of dox. Transcription of the target nucleic acid sequence is inhibited in the absence of dox and activated in the presence of dox.

In another embodiment, inducible expression is accomplished using a hormonal inducible gene expression system, for example, such as an ecdysone-inducible gene expression system (eg, RheoSwitch®, New England Biolabs) (Christopherson, K. S. et al. PNAS 89, 6314-8 ( 1992)). Ecdysone is an insect steroid hormone, such as from Drosophila melanogaster. In cells transfected with the ecdysone receptor, a heterodimer consisting of the ecdysone receptor (Ecr) and the retinoid X receptor (RXR) is formed in the presence of an ecdysone agonist selected from ecdysone, one of its analogs such as muristerone A and ponasterone. Oh, and the non-steroidal agonist ecdysone. In the presence of an agonist, Ecr and RXR interact and bind to the ecdysone response element, which is present on the expression cassette. Expression of a target nucleic acid sequence that is placed in an expression cassette downstream of the ecdysone response element is thus induced by exposing rabbit B cells to the ecdysone agonist.

In yet another embodiment of the present invention, inducible expression is accomplished using an arabinose-inducible gene expression system (eg, pBAD/gIII kit, Invitrogen) (Guzman, L.M. et al. Bacteriol 177, 4121-4130 (1995)). Arabinose is a monosaccharide containing five carbon atoms. In cells transfected with the arabinose-inducible PBAD promoter, expression of the target nucleic acid sequence placed downstream of PBAD can be further induced in the presence of arabinose.

In addition, (a nucleic acid molecule encoding) a Bcl-6 protein or a rabbit homologue thereof, or a functional portion or a functional derivative thereof, can also be used, wherein the activity of the above Bcl-6 or a rabbit homolog or a functional portion or a functional derivative thereof is controlled at least one inducer and/or repressor. A non-limiting example is a fusion protein, wherein the regulatory element is fused to a sequence encoding at least a portion of Bcl-6 or its rabbit homologue. For example, the estrogen receptor (ER) is fused to Bcl-6, resulting in an ER-Bcl-6 fusion protein. This fusion protein is inactive because it forms a complex with heat shock proteins in the cytosol. Following administration of the exogenous inducer 4-hydroxy-tamoxifen (4HT), the ER-Bcl6 fusion protein is dissociated from the heat shock proteins so that the Bcl-6 portion of the fusion protein becomes active.

As used herein, the term anti-apoptotic nucleic acid molecule refers to a nucleic acid molecule that is capable of delaying and/or preventing apoptosis in a rabbit B cell. Preferably, said anti-apoptotic nucleic acid molecule is capable of delaying and/or preventing apoptosis in a rabbit plasmablast-like B cell that is capable of both proliferating and producing antibody. It is preferable to use an anti-apoptotic nucleic acid molecule that contains an exogenous nucleic acid molecule. This means that either a nucleic acid sequence is used that is not naturally expressed in rabbit B cells, or an additional copy of a naturally occurring nucleic acid sequence is used such that expression in the resulting rabbit B cells is improved compared to natural rabbit B cells.

Various anti-apoptotic nucleic acid molecules are known in the art, so various embodiments are available. It is preferable to use an anti-apoptotic nucleic acid molecule that is an anti-apoptotic member of the Bcl-2 family because Bcl-2 anti-apoptotic proteins are good inhibitors of apoptosis in B cells. Many of the processes controlled by the Bcl-2 family (whose family contains both pro- and anti-apoptotic proteins) belong to the mitochondrial apoptotic pathway. Use of antiapoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, Bcl-2-related protein A1 (also called Bcl-2-A1 or A1), Bcl-2 like 10 (Bcl2L10) and Mcl-1, or their rabbit homologues, or a functional portion or functional derivative thereof, is preferred since Bcl-2, Bcl-xL, Bcl-w, A1, Bcl2L10 and Mcl-1 are generally integrated with the outer mitochondrial membrane. They directly bind to and inhibit proapoptotic proteins that belong to the Bcl-2 family to protect the integrity of the mitochondrial membrane.

Accordingly, in a preferred embodiment, a method according to the present invention is provided, wherein said anti-apoptotic nucleic acid molecule comprises an anti-apoptotic gene of the Bcl-2 family, preferably Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w , or Bcl2L10, or a rabbit homologue thereof, or a functional part or functional derivative thereof.

In one embodiment, the expression of Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10 or a rabbit homologue thereof is induced, improved or maintained by administration to the B cell(s) rabbit with at least one compound capable of stimulating the expression of any of these anti-apoptotic genes, or by culturing rabbit B cells in the presence of such compound(s). In this regard, a method according to the present invention is also provided, comprising:

providing said rabbit B cell with a compound capable of directly or indirectly improving the expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10 or their rabbit homologue; and/or culturing said rabbit B cell in the presence of a compound capable of directly or indirectly improving the expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/ or Bcl2L10, or their rabbit homologue.

However, it is preferable to provide a rabbit B cell with at least one molecule

- 5 044991 nucleic acid encoding an anti-apoptotic gene of the Bcl-2 family, preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10 and their rabbit homologues, and their functional parts and functional derivatives. In this way, it is also possible to directly increase the amount of the expression product in said rabbit B cell. In this regard, a method according to the present invention is also provided, comprising providing said rabbit B cells with at least one nucleic acid molecule encoding an anti-apoptotic gene of the Bcl-2 family, preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl -2, A1, Bcl-w, Bcl2L10 and their rabbit homologues, and their functional parts and functional derivatives. In one embodiment, said nucleic acid molecule is constitutively active, which means that said nucleic acid molecule is continuously expressed. In another embodiment, said nucleic acid molecule is inducible, meaning that its expression is regulated by at least one inducer and/or repressor. Non-limiting examples of inducible nucleic acid expression systems known in the art are previously presented herein.

In a particularly preferred embodiment, said anti-apoptotic nucleic acid molecule encodes Bcl-xL or Mcl-1, or a rabbit homologue thereof, or a functional portion or functional derivative thereof. According to the present invention, the combination of Bcl-6 and Bcl-xL is particularly capable of extending the replicative life cycle of rabbit B cells, thereby generating long-lasting cultures of the resulting rabbit B cell-like lymphoblasts. The same is true for the combination of Bcl-6 and Mcl-1. Most preferably, said anti-apoptotic nucleic acid molecule encodes Bcl-xL or a functional portion or functional derivative thereof.

The functional portion of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10 or their rabbit homolog is a protein molecule that has the same ability - in essence, not necessarily in quantity - to increase the replicative life cycle of B- rabbit cells compared with native Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-W or Bcl2L10, or their rabbit homolog, respectively. Such a functional part, for example, is devoid of amino acids that are not involved, or only very little, in this ability.

For example, functional portions of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10 or their rabbit homologue are defined herein as Bcl-xL, Mcl-1, Bcl-2, A1, Bcl fragments -w, Bcl2L10, respectively, or their rabbit homologue, which retained the same type of anti-apoptotic characteristics as full-length Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, respectively, or their rabbit homolog (by essence, not necessarily in quantity). The functional parts of Bcl-xL, Mcl-1, Bcl2, A1, Bcl-w, Bcl2L10 or their rabbit homolog are generally shorter fragments of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, respectively, or their rabbit homologue, which are capable of delaying and/or preventing apoptosis in rabbit B cells. Such functional parts, for example, lack sequences that do not significantly contribute to the antiapoptotic activity of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10. A functional portion of Bcl-6 or its rabbit homolog, for example, is a shorter fragment of Bcl-6 or a shorter fragment of its rabbit homologue that is capable of extending the replicative life cycle of a rabbit B cell.

A functional derivative of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or their rabbit homolog, is defined herein as Bcl-6, Bcl-xL, Mcl-1, Bcl protein -2, A1, Bcl-w or Bcl2L10, respectively, or their rabbit homolog, which was changed, but retained its ability (in essence, not necessarily in quantity) to increase the replicative life cycle of the rabbit B cell. A functional derivative is made in many ways, for example, by replacing a conserved amino acid, where one amino acid is replaced by another amino acid with generally similar properties (size, hydrophobicity, etc.) in such a way as not to seriously affect the overall functioning. Alternatively, the functional derivative, for example, contains a fusion protein with a detectable tag or an inducible compound.

Another aspect of the present invention solves the problem of efficiently introducing a target nucleic acid molecule into rabbit B cells. Contrary to expectations, the present inventors discovered that a widely used amphoretroviral vector, which is suitable for infecting rodent cells such as mouse cells, and which therefore should also be capable of transducing rabbit B cells, was unable to effectively transduce rabbit B cells. ; The transduction efficiency of amphovector 4 days after transduction in rabbit B cells was below 1%. Thus, the inventors of the present invention were forced to look for other gene delivery vehicles. Surprisingly, the present inventors have discovered that a gene delivery vehicle that contains the extracellular domain of the gibbon leukemia virus (GALV) envelope protein is capable of transducing rabbit B cells with high efficiency, typically 80-90% within 35 days of transduction. This property was quite unexpected, since the gibbon leukemia virus does not infect rabbits under natural conditions. Therefore, it was not expected that rabbit cells

- 6 044991 will contain a receptor for the GALV envelope protein; the current finding was mere coincidence. It should be noted that the transduction efficiency of human B cells with a vector containing the extracellular domain of the GALV envelope protein is typically 60-70% 4 days after transduction, which is generally lower than the transduction efficiency of rabbit B cells with this vector, despite that human B cells are primate cells. This is surprising because since the monkey is also a primate, it was expected that a vector containing the GALV-based coat protein would be better able to infect primate cells compared to rabbit cells. It is noteworthy that the use of a vector containing the extracellular domain of the GALV envelope protein does not effectively transduce mouse B cells, which is consistent with the fact that gibbon leukemia virus does not infect mice.

Now that the discovery of the present invention has been presented that rabbit B cells can be efficiently transduced using at least a functional portion of the extracellular domain of the GALV envelope protein, and that the transduction efficiency is even higher than that of human B cells, new areas of application have become available. . It is now possible to introduce a target nucleic acid molecule into rabbit B cells with high efficiency, which is particularly advantageous for the formation of a rabbit B cell culture according to the present invention. Accordingly, in a preferred embodiment of the present invention there is provided a method for increasing the replicative life cycle of a rabbit B cell, the method comprising:

inducing, improving and/or maintaining the expression of Bcl-6 or its rabbit homolog in a rabbit B cell and inducing, improving and/or maintaining the expression of at least one anti-apoptotic nucleic acid in said B cell, characterized in that said rabbit B cell is provided a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homolog thereof, or encoding a functional portion or functional derivative thereof, and/or at least one anti-apoptotic nucleic acid molecule through transduction with a gene delivery vehicle that contains the extracellular domain of a leukemia virus envelope protein gibbons (GALV), or at least a functional portion of said extracellular domain, or through transduction with a gene delivery vehicle containing a protein having at least 70% sequence identity with the extracellular domain of the GALV coat protein, or through transduction with a gene delivery vehicle containing a protein having at least 70% sequence identity with at least a functional portion of the extracellular domain of the GALV envelope protein. In one preferred embodiment, said extracellular domain is the GALV envelope protein of the SEATO strain. Said extracellular domain preferably contains the sequence

MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQ

VLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDS

DYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWD CETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKD WITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPR EAPPPSLPDSNSTALATSAQTPTV RKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGAT ESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIGKVPFTH QHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPRIYY YPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIK GPIDLQQGLTSLQI AIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSG AVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL.

Preferably, said sequence identity is at least 75%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84% , more preferably at least 85%, more preferably at least 86%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%.

As will be appreciated by one skilled in the art, said extracellular domain is located on the surface (envelope) of wild-type gibbon leukemia virus such that it can bind

- 7 044991 to be called with the host cell, is also preferably located on the surface (shell) of the gene delivery carrier for transduction of rabbit B cells. In one particularly preferred embodiment, a vector or other gene delivery vehicle is used that comprises an envelope protein comprising an extracellular domain and a transmembrane domain of the GALV envelope protein or a functional portion thereof, which is fused to the cytoplasmic domain of the amphooshell protein. This allows for specific efficient transduction of rabbit B cells, as shown in the examples.

As used herein, the term functional portion of the extracellular domain of a GALV envelope protein means a portion of said extracellular domain that is still capable of binding to rabbit B cells, thereby promoting infection and/or transduction of rabbit B cells. Such a functional portion may lack one or more amino acid residues that are not essential for rabbit B cell binding, infection and/or transduction.

Of course, now that the discovery of the present invention has been proposed, it is possible to transduce rabbit B cells with any target nucleic acid molecule using at least a functional portion of the extracellular domain of the GALV envelope protein. In this regard, an isolated or recombinant rabbit B cell is also provided, linked to an extracellular domain of the GALV envelope protein, or linked to at least a functional portion of said extracellular domain, or linked to a protein having at least 70% sequence identity with at least functional part of the extracellular domain of the GALV envelope protein. Also provided is an isolated or recombinant rabbit B cell that is linked through at least a functional portion of the extracellular domain of a GALV envelope protein, or through a protein having at least 70% sequence identity with at least a functional portion of an extracellular domain of a GALV envelope protein, with gene delivery carrier. In one preferred embodiment, said extracellular domain is the extracellular domain of the GALV envelope protein of the SEATO strain. Said extracellular domain preferably contains the sequence

MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQ VLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDS DYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWD CETTGTGYWLSKSSKDLFrVKWDQNSEW TQKFQQCHQTGWCNPLKIDFTDKGKLSKD WITGKTWGLRFYVSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPR EAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGAT ESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGH GLCIGKVPFTH QHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPRIYY YPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQI AIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSG AVRDSMK KLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL.

Again, said sequence identity is preferably at least 75%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95% , more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably 100%. Said gene delivery vehicle preferably contains a target nucleic acid molecule, preferably a nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and/or an anti-apoptotic nucleic acid sequence. With such a gene delivery vehicle, it is possible to produce a stable rabbit B cell culture according to the present invention. Said anti-apoptotic nucleic acid sequence is preferably an anti-apoptotic gene of the Bcl2 family, most preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10 and their rabbit homologues, and functional parts thereof and functional derivatives.

It is also proposed to use the extracellular domain of the GALV envelope protein, or at least its functional part, which is capable of binding to a rabbit B cell, to introduce

- 8 044991 introduction of a target nucleic acid molecule into a rabbit B cell, as well as the use of a protein having at least 70% sequence identity with at least a functional portion of the extracellular domain of the GALV coat protein to introduce the target nucleic acid molecule into a B cell a rabbit. Also proposed is the use of a gene delivery carrier containing at least a functional part of the extracellular domain of the GALV envelope protein, or a gene delivery carrier containing a protein having at least 70% sequence identity with at least a functional part of the extracellular domain of the GALV envelope protein, said delivery carrier a gene further comprising a nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and at least one anti-apoptotic nucleic acid sequence for enhancing the replicative life cycle of a rabbit B cell. In one preferred embodiment, said extracellular domain is the extracellular domain of the GALV envelope protein of the SEATO strain. Said extracellular domain preferably contains the sequence

MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQ

VLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDS DYTAAYKQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWD CETTGTGYWLSKSSKDLITVKWDQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKD WITGKTWGLRFYVSGHPGVQFT IRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPR EAPPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGAT ESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIGKVPFTH QHLCNQTLSINSSGDHQYLLPSNHSWWACSTGLTPCLSTSVFNQTRDFCI QVQLIPRIYY YPEEVLLQAYDNSHPRTKREAVSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQI AIDADLRALQDSVSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSG AVRDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL.

A preferred chimeric coat protein, which is also used in the Examples, is shown in FIG. 9. This chimeric envelope protein contains the extracellular domain of the GALV envelope protein (with the sequence

MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQ

Vlsqtgdvvdvdtkvtwtwptlkpdvcalaswadipgtdvsskrvrppds dytaaykqitwgatespramsstfyvcprdgleslyCGLYCGLYCGLYCHECHECHECHESSKDLSKDLISKDLITIT VKWDQNSEWTQKFQCHQCHQCHQNPLKIDFTDDKLSKD WitgktwglrfyvshpgvqvqvqtirlkitnMpavlvvlvlvlvlPPPPSLPPSLPDSNSTALATSAQtvrktivtivtivtivtlPPPSTIVTIVTLENTLN TPPPPTGDRLFDLVQGAFLNATNATNATNATNPGATEASGESGESGESGESGESSGESTLDRCRWGTQKLTLCIGHGKVPFTH QHLCNQTLLPSNHSHSTPCLSTSVFNFN Qtrdfciqvqliplipriyy YpeevlqaydnshprtkrevslvlglglglglglgtgpidlqlqgltslqdsvsklesevvvvlplflflcAalaalkecaaccapf YIDHSG AVRDSMKKLKEKLDKLERQKNWYEGWFNNSPWFTLL, highlighted with fat font for FIG. 9) and the transmembrane domain of the GALV envelope protein (with the sequence STIAGPLLLLLLLLILGPCII, indicated by underlining in Fig. 9), fused with the cytoplasmic domain of the amphooshell protein (with the sequence NRLVQFVKDRISVVQALVLTQQYIIQLKPIEYEP, indicated in italics and underlined by a dotted line in Fig. 9). A vector or other gene delivery vehicle containing this preferred chimeric envelope protein is particularly capable of introducing the target nucleic acid molecule into rabbit B cells.

In this regard, an isolated or recombinant rabbit B cell coupled to a chimeric envelope protein as shown in FIG. 9 or with a protein containing a chimeric coat protein as shown in FIG. 9, or with a protein having at least 70% sequence identity with the chimeric coat protein as shown in FIG. 9. Also provided is an isolated or recombinant rabbit B cell, which is linked via a chimeric envelope protein as shown in FIG. 9 or through a protein containing a chimeric coat protein as shown in FIG. 9, or through a protein having at least 70% sequence identity with the chimeric coat protein as shown in FIG. 9, with a vector or other gene delivery vehicle.

Such a vector or other gene delivery vehicle is particularly suitable for transducing a rabbit B cell with a target nucleic acid molecule. In this regard, it is also proposed

- 9 044991 use of a chimeric coat protein as shown in FIG. 9 or a protein containing a chimeric coat protein as shown in FIG. 9, or a protein having at least 70% sequence identity with the chimeric coat protein as shown in FIG. 9, to introduce a target nucleic acid molecule into a rabbit B cell.

Such a vector or other gene delivery vehicle is particularly suitable for extending the replicative life cycle of a rabbit B cell. In this regard, a method is also provided for increasing the replicative life cycle of a rabbit B cell, a method comprising:

inducing, improving and/or maintaining the expression of Bcl-6 or its rabbit homolog in a rabbit B cell and inducing, improving and/or maintaining the expression of at least one anti-apoptotic nucleic acid in said B cell, characterized in that said rabbit B cell is provided a nucleic acid molecule encoding Bcl-6 or encoding a rabbit homolog thereof, or encoding a functional portion or functional derivative thereof, and/or at least one anti-apoptotic nucleic acid molecule through transduction with a vector or other gene delivery vehicle containing a chimeric envelope protein as indicated in fig. 9 or a protein containing a chimeric coat protein as shown in FIG. 9, or a protein having at least 70% sequence identity with the chimeric coat protein as shown in FIG. 9.

Also proposed is the use of a gene delivery vehicle containing a chimeric coat protein as shown in FIG. 9 or a protein containing a chimeric coat protein as shown in FIG. 9, or a protein having at least 70% sequence identity with the chimeric coat protein as shown in FIG. 9, said gene delivery vehicle further comprising a nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and at least one anti-apoptotic nucleic acid sequence for enhancing the replicative life cycle of a rabbit B cell. It should be emphasized that although a GALV-based gene delivery vehicle is very suitable for efficiently transducing rabbit B cells with one or more target nucleic acid molecule(s), such as Bcl-6 and molecule anti-apoptotic nucleic acid, the use of a GALV-based gene delivery vehicle is not necessary to obtain rabbit B cells with a short doubling time of 20 hours or less. Other gene delivery vehicles can be used to introduce Bcl-6 and the anti-apoptotic nucleic acid molecule into rabbit B cells (although the efficiency will often be lower) to produce rabbit B cells with a doubling time of 20 hours or less. As Bcl-6 and the anti-apoptotic nucleic acid molecule are introduced into rabbit B cells, a rapidly growing B cell culture can be obtained, although it may take longer to grow the smaller number of initially transduced rabbit B cells. An advantage of using a gene delivery vehicle capable of efficiently transducing rabbit B cells, such as a GALV-based gene delivery vehicle as described herein, is that an increased ratio of the initially isolated B cells will be transduced, such that the B cells , obtained from them, will be present in the resulting B-cell culture. This results in a higher diversity of B cells in a B cell culture compared to a situation where a gene delivery vehicle with a lower transduction efficiency is used because in the latter case a low ratio of the original B cells is transduced. The presence of higher B cell diversity in the resulting B cell culture increases the likelihood of isolating one or more B cells with the desired property. Therefore, in principle, the higher the transduction efficiency of the gene delivery vehicle, the higher the B cell diversity in the resulting B cell culture.

In order to induce expression in rabbit B cells, the target nucleic acid molecule is preferably operably linked to a promoter. Non-limiting examples include the CMV promoter and the CAG promoter. In one aspect, such a promoter is inducible, meaning that its activity is dependent on at least one compound, such as a transcription factor.

As used herein, the term gene delivery vehicle means any compound capable of transferring a nucleic acid molecule into a host cell. Non-limiting examples of gene delivery vehicles include (viral) vectors and plasmids. The term gene delivery vehicle containing at least a functional portion of the extracellular domain of a GALV envelope protein means a gene delivery vehicle comprising at least a portion of an extracellular domain of a GALV envelope protein, wherein said extracellular domain or said portion thereof is capable of binding to a rabbit B cell, so that the nucleic acid can be introduced into said rabbit B cell. As presented above in the present description, the specified extracellular domain or part thereof is preferably placed on the surface of the gene delivery vehicle so that it can bind to the receptor on the rabbit B cell. Likewise, if the gene delivery vehicle of the present invention contains a protein having at least 70% sequence identity of at least

- 10 044991 At least with the functional part of the extracellular domain of the GALV envelope protein, the protein is preferably located on the surface of the gene delivery vehicle so that it can bind to the receptor on the rabbit B cell.

Percent identity of an amino acid or nucleic acid sequence, or the term % sequence identity, is defined herein as the percentage of residues in a potentially matching amino acid or nucleic acid sequence that matches residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve maximum percentage of identity. Methods and computer programs for alignment are well known in the art, for example, Align 2.

The GALV envelope protein is a protein that is naturally present in the viral envelope of gibbon leukemia virus and which is involved in infection of host cells. Target specificity is usually determined by the coat protein. In one embodiment, said envelope protein is the GALV envelope protein of the SEATO strain. Retroviral vectors containing the GALV envelope protein are known in the art and can be prepared using techniques that are widely used in the field of molecular biology, for example, see Lam et al., 1996.

The term operably linked to a promoter means that the target nucleic acid sequence is located sufficiently close to the promoter such that the promoter can influence its expression. Typically, such a promoter will induce or enhance the expression of said target nucleic acid. The term expression activity refers to such induction or enhancement of expression.

As mentioned earlier in the present description, the present invention provides the discovery that a rabbit B cell culture can be obtained with a shorter average doubling time compared to currently known human or mouse B cell cultures. This is all the more surprising since non-rabbit compounds, such as human Bcl-6, mouse IL21 and human CD40L nucleic acid sequences, were used in the present Examples. As shown in the examples, the present inventors transduced rabbit B cells with a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL or Mcl-1 sequence. Even when human sequences were used and rabbit cells were cultured in the presence of mouse IL21 and human CD40L, rabbit B cells were surprisingly able to proliferate faster and produce more antibody compared to human and mouse B cells.

Therefore, according to the present invention, rabbit B cells proliferate very well when using human and mouse compounds. Under these reaction conditions, rabbit B cells proliferate even better than human and mouse B cells. Among other things, this has the advantage that the currently used reaction conditions for human B cells do not have to be the same for rabbit B cells. It is not necessary to obtain rabbit IL21, rabbit CD40, or rabbit nucleic acid sequences encoding Bcl-6 or an anti-apoptotic gene. Instead, currently available human or mouse compounds can be used. Accordingly, in one aspect of the present invention there is provided a method of increasing the replicative life cycle of a rabbit B cell, the method comprising:

inducing, improving and/or maintaining the expression of Bcl-6 or its rabbit homologue in a rabbit B cell and inducing, improving and/or maintaining the expression of an anti-apoptotic nucleic acid molecule in said B cell, characterized in that said rabbit B cell is provided with at least one nucleic acid molecule selected from the group consisting of:

nucleic acid molecules encoding non-rabbit Bcl-6 or a functional portion or functional derivative thereof; and non-rabbit anti-apoptotic nucleic acid molecules.

Preferably, said non-rabbit nucleic acid molecule is a human nucleic acid molecule, since human Bcl-6 and human anti-apoptotic sequences appear to perform particularly well in rabbit B cells. In a particularly preferred embodiment, the rabbit B cell is provided with a nucleic acid molecule encoding human Bcl-6 and a human anti-apoptotic nucleic acid molecule, preferably encoding human Bcl-xL or human Mcl-1, or human Bcl-2, or human A1, or Human Bcl-w, or human Bcl2L10.

In addition, a method according to the present invention is provided, further comprising providing said rabbit B cell with IL21 and CD40L. Preferably, non-rabbit IL21 and/or non-rabbit CD40L are used. Preferably, said IL21 is mouse or human IL21, most preferably mouse IL21. In another preferred embodiment, said CD40L is mouse or human CD40L, most preferably human CD40L.

- 11 044991

In addition to increasing the expression of Bcl-6 and the expression of an anti-apoptotic nucleic acid molecule, it is also preferable to induce, improve and/or maintain the expression of Blimp-1 or its rabbit homologue in a rabbit B cell. This improves antibody production by said B cell. Thus, in one aspect, a method of the present invention is provided, the method further comprising inducing, improving and/or maintaining expression of Blimp-1 or its rabbit homologue in said rabbit B cell.

The extent of expression of Blimp-1 or its rabbit homologue in a rabbit B cell is regulated in various ways. In one embodiment, a rabbit B cell is provided with a compound capable of directly or indirectly increasing the expression of Blimp-1, or the expression of its rabbit homolog. Additionally or alternatively, a rabbit B cell is cultured in the presence of a compound capable of directly or indirectly increasing the expression of Blimp-1 or the expression of its rabbit homolog. In this regard, a method according to the present invention is also provided, further comprising:

providing said rabbit B cell with a compound capable of directly or indirectly increasing the expression of Blimp-1 or the expression of its rabbit homologue; and/or culturing said rabbit B cell in the presence of a compound capable of directly or indirectly increasing the expression of Blimp-1 or the expression of its rabbit homolog.

Said compound capable of increasing the expression of Blimp-1 or its rabbit homolog most preferably contains IL21. Therefore, in one preferred embodiment of the present invention, the B cells are cultured from a rabbit in the presence of IL21 for at least a portion of the culture time.

In another embodiment, said compound capable of increasing Blimp-1 expression comprises signal transporter and activator of transcription 3 (STAT3) protein or a functional portion or functional derivative thereof, and/or a nucleic acid molecule encoding the same. STAT3 is a signal transporter that is involved in B cell development and differentiation. STAT3 is capable of up-regulating Blimp-1 expression. In one preferred embodiment, a rabbit B cell is provided with a nucleic acid molecule encoding STAT3 or a functional portion or functional derivative thereof, wherein the expression of said nucleic acid molecule is controlled by an exogenous repressor inducer such that the amount of STAT3 expression is controlled as desired. For example, one of the previously mentioned inducible expression systems is used. In one embodiment, a fusion product comprising STAT3 or a functional moiety or a functional derivative and ER is used. For example, a rabbit B cell is provided with a nucleic acid molecule encoding estrogen receptor (ER) and STAT3 as an ER-STAT3 fusion protein. This fusion protein is inactive because it forms a complex with heat shock proteins in the cytosol. Thus, STAT3 is unable to reach the nucleus and Blimp-1 expression is not improved. Upon administration of the exogenous inducer 4-hydroxy-tamoxifen (4HT), the ER-STAT3 fusion protein is dissociated from heat shock proteins so that STAT3 is able to enter the nucleus and activate Blimp-1 expression.

As used herein, a functional portion of STAT3 is defined as a fragment of STAT3 that has the same ability - in essence, not necessarily in quantity - to increase the expression of Blimp-1 or its rabbit homolog as native STAT3. Such a functional part, for example, is devoid of amino acids, which do not participate or only participate very little in this ability.

A functional derivative of STAT3 is defined as a STAT3 protein that has been altered but retains its ability (in essence, not necessarily in quantity) to increase the expression of Blimp-1 or its rabbit homolog. A functional derivative is obtained in many ways, for example, through the replacement of a conserved amino acid, whereby one amino acid is replaced by another amino acid with generally similar properties (size, hydrophobicity, etc.), such that the overall functioning is not seriously altered. Alternatively, the functional derivative, for example, contains a fusion protein with a detectable tag or an inducible compound.

Since STAT3 is capable of increasing the expression of Blimp-1 or increasing the expression of its rabbit homologue, it is also possible to indirectly increase the expression of Blimp-1 or its rabbit homologue by administering a compound capable of increasing the activity and/or expression of STAT3. Accordingly, in one embodiment, a rabbit B cell is provided with a compound capable of improving STAT3 activity such that the expression of Blimp-1 or its rabbit homologue is indirectly improved.

STAT3 is activated in various ways. Preferably, STAT3 is activated by providing the rabbit B cell with a cytokine. Cytokines naturally involved in B cell differentiation are very effective in regulating STAT proteins. IL21 and IL6 are very effective activators of STAT3, but IL2, IL7, IL10, IL15, and IL27 are also known to activate STAT3. In addition, Toll-like receptors (TLRs), which are involved in innate immunity, are also capable of activating STAT3. Accordingly, in one embodiment, a method according to the present invention is provided, wherein said rabbit B cell culture is cultured in

- 12 044991 presence of IL21, IL2, IL6, IL7, IL10, IL15 and/or IL27. It is most preferred to use IL21 since IL21 is particularly suitable for improving antibody production by rabbit B cell cultures of the present invention. IL21 is capable of up-regulating expression

Blimp-1 even if Blimp-1 expression is neutralized by BCL6.

Additionally or alternatively, mutated Janus kinase (JAK) or a mutated rabbit JAK homolog is used to activate STAT3. Naturally, JAK is capable of phosphorylating STAT3 after it itself has been activated by at least one cytokine. A mutated Janus kinase or a mutated rabbit JAK homolog capable of activating STAT3 independent of the presence of cytokines is particularly suitable for use in the method of the present invention.

In yet another embodiment, the expression of Blimp-1 or a rabbit homologue thereof is increased by providing a rabbit B cell with suppressor of cytokine signaling (SOCS) protein or a rabbit homologue thereof, and/or by activating the SOCS protein or a rabbit homologue thereof in the cell. Alternatively, or additionally, at least one of the E proteins E47, E12, E2-2 and HEB is used to increase the expression of Blimp-1 or its rabbit homologue. E47 is a transcription factor that belongs to a family of helix-loop-helix proteins called E proteins. There are four E proteins, E12, E47, E2-2 and HEB, which are involved in lymphocyte development. E12 and E47 are encoded by the same gene, called E2A, which is spliced differently. E proteins have been described as tumor suppressors. One of the specific targets of E47 is the Socs1 and Socs3 genes.

Thus, in one aspect, there is provided a method of the present invention further increasing the expression of Blimp-1 or a rabbit homologue thereof in a rabbit B cell by providing said B cell with a compound capable of directly or indirectly increasing the expression of Blimp-1 or a rabbit homologue thereof, and/ or culturing said B cell in the presence of a compound capable of directly or indirectly increasing the expression of Blimp-1 or its rabbit homologue, wherein said compound contains:

STAT3 or a functional part or functional derivative thereof, and/or a compound capable of activating STAT3, and/or a compound capable of increasing STAT3 expression, and/or

IL21, IL2, IL6, IL7, IL10, IL15, IL27, SOCS protein, one of the E proteins E47, E12, E2-2 or HEB, mutated Janus kinase and/or nucleic acid sequence encoding STAT3, or its rabbit homolog or a functional part, or a functional derivative.

Most preferably, said compound is IL21.

In addition, the invention provides isolated or recombinant rabbit B cells obtained using the method of the present invention. Such isolated or recombinant rabbit B cells preferably contain an exogenous anti-apoptotic nucleic acid sequence and an exogenous nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof. In this regard, an isolated or recombinant rabbit B cell is also provided comprising an exogenous nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and an exogenous anti-apoptotic nucleic acid sequence. As explained above, the exogenous nucleic acid molecule contains either a nucleic acid sequence that does not naturally occur in rabbit B cells or an additional copy of a nucleic acid sequence from natural rabbit B cells. Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, and their rabbit homologues are the preferred anti-apoptotic nucleic acid molecules. Accordingly, in one preferred aspect, an isolated or recombinant rabbit B cell is provided that comprises an exogenous nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and an exogenous nucleic acid sequence encoding Bcl-6. xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10 or a rabbit homologue thereof, or a functional part or functional derivative thereof.

Said nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and said exogenous anti-apoptotic nucleic acid sequence may be present on a single nucleic acid molecule. Alternatively, the sequences are present on at least two different nucleic acid molecules.

Preferably, non-rabbit sequences are used, as explained previously. Accordingly, a preferred embodiment provides an isolated or recombinant rabbit B cell comprising a non-rabbit anti-apoptotic nucleic acid sequence and a non-rabbit nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof. Said non-rabbit nucleic acid sequence preferably contains human sequences.

- 13 044991

In a particularly preferred embodiment, an isolated or recombinant

Rabbit B cell, which contains:

an anti-apoptotic nucleic acid sequence encoding human Bcl-6 or a functional part or functional derivative thereof, and a human anti-apoptotic nucleic acid sequence preferably encoding human Bcl-xL or human Mcl-1 or human Bcl-2 or human A1 or Bcl- human w, or human Bcl2L10, or a functional part or functional derivative thereof. In this same case, said nucleic acid sequence encoding Bcl-6 or a functional portion or functional derivative thereof and said anti-apoptotic nucleic acid sequence may be present on a single nucleic acid molecule, or, alternatively, these sequences may be present on at least one two different nucleic acid molecules.

The present invention also provides ex vivo rabbit B cell cultures produced by the methods of the present invention. An important advantage is the fact that ex vivo rabbit B cell cultures are now produced with a short average doubling time. In this regard, an ex vivo rabbit B cell culture has been proposed that has an average doubling time of 20 hours or less. In a further preferred embodiment, an ex vivo rabbit B cell culture is provided containing rabbit B cells according to the present invention. Said rabbit B cells preferably contain a nucleic acid sequence encoding human Bcl-6 or a functional portion or functional derivative thereof, and an anti-apoptotic nucleic acid sequence. In addition, an ex vivo rabbit B cell culture is proposed, containing rabbit B cells in the presence of non-rabbit IL21 and/or non-rabbit CD40L. Preferably, said IL21 is mouse or human IL21, most preferably mouse IL21. In another preferred embodiment, said CD40L is mouse or human CD40L, most preferably human CD40L.

Also provided is an antibody produced by the method of the present invention, as well as an antibody produced by a rabbit B cell according to the present invention or by an ex vivo rabbit B cell culture according to the present invention. Such an antibody is particularly preferred for therapeutic or diagnostic applications. Preferably, said antibody is a monoclonal antibody.

The present invention is also described in the following examples. These examples do not limit the scope of the present invention, but serve only to clarify the invention.

Examples

Example 1.

B cell transduction.

Gene transfer into lymphocytes by traditional methods such as calcium phosphate precipitation, liposome formation or electroporation is inefficient, but more importantly, stable gene integration overall is lacking. However, viral transduction directly results in stable gene integration into the target cell genome and can be very effective if the appropriate viral envelope is selected. Both retroviral and lentiviral transduction are suitable for efficient gene transfer. While retroviral integration depends on cytokinesis, lentiviral transduction can be applied to nondividing cells such as plasma B cells. Large-scale preparation of recombinant retrovirus can be easily produced through the use of stable production cell lines such as the Phoenix expression platform (Kinsella and Nolan, 1996). Production of high titer lentiviruses tends to be more cumbersome, mainly due to the toxicity of the expressed proteins and viral envelopes.

For the present examples, we used a Moloney murine leukemia virus (MMLV) platform using producer cells expressing either the amphotropic envelope or the gibbon leukemia virus (GALV) envelope (Wilson et al., 1995). In the case of the GALV-based vector, the transmembrane domain of the GALV envelope protein of the SEATO strain was fused with the cytoplasmic domain of the amphooshell protein (Fig. 9).

The transfer vector is set up such that Bcl-6, Bcl-xL and green fluorescent protein (GFP) marker are simultaneously translated from the same viral RNA (Fig. 8). This multicistronic approach is achieved by placing a self-cleaving 2A peptide sequence (Szymczak et al., 2004) between the BCL-6 and BCL-xl coding regions and the internal ribosome entry site (IRES) upstream of the GFP reporter gene. Manifestations of viral transduction are high and objective.

Generation of immortalized rabbit B cells.

Human memory B cells were immortalized using the BCL-6/Bcl-xL technology described by Kwakkenbos et al., 2010 and patent application WO 2007/067046.

Briefly, PBMCs from rabbit blood were isolated using a Ficoll density gradient and stained for Ig expression using an antibody that recognizes Ig (IgG H+L: heavy chain and light

- 14 044991 kappa and lambda IgG chains), sometimes in combination with a specific IgM antibody. B cells were isolated (Ig positive or Ig positive + IgM negative) using a FACS sorter and stimulated on γ-irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L L cells, 105 cells ml ^-1 ). together with recombinant mouse interleukin (IL)-21 for 36-48 hours. Cells were collected and washed with medium without fetal calf serum (FCS), and the cells were then transferred to Retronectin® (Takara, Shiga, Japan) tissue culture-coated plates, where they were transduced with a retroviral vector containing BCL-6, Bcl-xL and GFP as a reporter protein. Alternatively, B cells were transduced with a retroviral vector containing BCL-6, Mcl-1 and GFP. Transduced B cells were maintained in culture with CD40 ligand expressing L cells and IL-21. In fig. 1 compares the transduction efficiency for GALV and amphotropic retroviruses on day 4 after transduction. Four days after transduction with an amphotropic type retrovirus, 0.8% of cells were transduced, compared with 80% of cells after transduction with a GALV type retrovirus. It is clear that the GALV type retrovirus is superior to the amphotropic type retrovirus in transducing rabbit B cells.

Example 2.

Cell cultures.

We maintained B cells (2x105 cells ml ^-1 ) in Iskov's modified Dulbecco's medium (Gibco) containing 8% FBS and penicillin-streptomycin (Roche) supplemented with recombinant mouse interleukin 21 (IL-21) (50 ng ml ^-1 ), and cultured them on γ-irradiated (50 Gy) mouse L-cell fibroblasts stably expressing CD40L (CD40L L-cells, 105 cells ml ^-1 ). To determine cellular doubling time, cells were cultured in 24-well plates at 50-100,000 cells per well together with CD40L and IL-21 L cells. Every 3-4 days, cells were counted and 50-100,000 cells were transferred to a new well. In fig. 2 shows growth curves for B cells from two human donors (89 and 93), one sample of a llama B cell (Llama), and one sample of a rabbit B cell transduced with a GALV-type retrovirus carrying a nucleic acid molecule containing the sequence Human Bcl-6, and human Bcl-xL (Rb 6XL). In addition, a growth curve is shown for one rabbit sample transduced with a GALV-type retrovirus carrying a nucleic acid molecule containing the sequence of human Bcl-6 and human Mcl-1 (Rb 6M). Transduced rabbit B cells have an average doubling time of 19 hours and thus grow faster than human or llama B cells, which have an average doubling time of 26 to 32 hours. These average doubling times were originally calculated by determining the increase in B cells. cells over several 3-4 day time intervals and averaging the results obtained. Subsequently, the overall average doubling time over the entire culture period was calculated. This resulted in an average doubling time of 18 hours for transduced rabbit B cells, an average doubling time of 25-29 hours for transduced human B cells, and an average doubling time of 27 hours for transduced llama B cells. This confirms the observations of the present inventors that their methods yield rabbit B cell cultures with an average doubling time of 20 hours or less, while human, mouse, and llama B cells typically have doubling times of 25 to 36 hours.

Example 3.

B cell receptor expression and antigen-specific staining.

Immortalized human B cells express the B cell receptor. This quality allows for antigen-specific staining and sorting of B cells. To determine whether B cell receptor expression also existed on transduced rabbit B cells, B cell clones were stained with fluorescently labeled antibodies specifically reactive with either rabbit IgG, rabbit IgM, or rabbit IgA. B cells were washed in cold (4°C) cell culture medium and incubated on ice in the dark in cell culture medium containing immunofluorescently labeled antibodies that are specific for either rabbit IgG, IgM, IgA, or labeled antigen. Excess labeled antibody or antigen was then washed away and B cell receptor expression analysis was performed on a FACS analyzer; Guava easycyte (Millipore) or FACS Aria3 (BD).

In fig. 3, three different B cell clones of different isotypes are stained with fluorescently labeled antibodies that specifically recognize the rabbit antibody isotype IgG, IgA, or IgM. It is apparent that the B cell receptor can be efficiently stained for different rabbit antibody isotypes. In this regard, the present inventors conclude that immortalized rabbit B cells also express the B cell receptor.

In addition, fluorescently labeled influenza proteins were also used to stain influenza-specific B cells from rabbits that were immunized with human influenza vaccine or untreated control rabbits (Fig. 4). Rabbit B cells were fluorescently stained with H1, H3, or influenza B and sorted on a 1 cell/well basis using a FACS sorter.

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Example 4.

Development of clonal rabbit B-cell cultures derived from a single cell.

Transduced B cells were sorted on a single cell/well basis using a FACS sorter and cultured in the presence of γ-irradiated (50 Gy) mouse L cell fibroblasts stably expressing CD40L (CD40L L cells, 105 cells ml ^-1 ) together with recombinant mouse IL-21. Fresh CD40L and IL-21 L cells were added every 3–4 days. Beginning at day 9 after cell seeding (one cell per well), supernatants were assayed by ELISA for rabbit immunoglobulin G (IgG) production. For comparison, an assay for human IgG in the supernatant of human B cell clones was also performed in parallel.

In fig. Figure 7 shows the antibody concentration in the supernatant over time, starting from day 9 after the initiation of single-cell cultures. The antibody concentration was determined for two human donors and one rabbit B-cell sample, which were transduced with a GALV-type retrovirus carrying a nucleic acid molecule containing the sequence of human Bcl-6 and human Bcl-xL, and for one rabbit B-cell sample. transduction of which was carried out with a GALV-type retrovirus carrying a nucleic acid molecule containing the sequence of human Bcl-6 and human Mcl-1. B cell clones from rabbits produced IgG at concentrations of 30 ng/ml and 100 ng/ml for a shorter period of time (9-10 days and 11-12 days, respectively) than human B cell clones (13-18 and 15-20 days respectively). This fact provides the important advantage that it allows for earlier screening of rabbit B cell clones for target antibodies compared to human B cell clones.

Example 5.

Immunization of rabbits.

New Zealand White rabbits were immunized with a human influenza vaccine containing 15 μg H1N1, 15 μg H3N2 and 15 μg influenza B in Freund's complete adjuvants. After 3 weeks, rabbits were re-immunized with the same vaccine in Freund's incomplete adjuvants. Five days after the boost, rabbits were bled, B cells were isolated from the blood, and transduction was carried out with a GALV-type retrovirus (containing the extracellular domain and transmembrane domain of the GALV envelope protein of the SEATO strain, fused with the cytoplasmic domain of the amphooshell protein), carrying a nucleic acid molecule containing sequence of human Bcl-6, and human Bcl-xL. Transduced B cells were seeded at various cell densities in culture plates and cultured as described in Example 4. In addition, transduced B cells were labeled with fluorescently labeled vaccine components; H1, H3 or influenza B and sorted on a 1 cell/well basis using a FACS sorter and cultured as described in Example 4. Supernatants of cultured cells were analyzed for binding to the whole vaccine or its individual components. The results presented in Fig. 4-6 show that antigen-specific B cells can be identified in the B cell pool from vaccinated rabbits by plating cells at different densities (Fig. 5) and also very efficiently by cell sorting using labeled antigens (Fig. 4 and 6).

Example 6.

Rabbit B cells immortalized by introducing the Bcl-6 and Bcl-xl genes using an amphotropic retrovirus.

Immortalization of rabbit B cells by introducing the Bcl-6 and Bcl-xl genes can be achieved through the use of various types of vectors, for example, retroviruses such as GALV and amphotropic type, as shown in example 1. Growth of B cells transduced with a retrovirus amphotropic type was further carried out to confirm that introduction of Bcl-6 and Bcl-xl by amphotropic retrovirus also leads to immortalization of rabbit B cells. Four days after transduction with the amphotropic type retrovirus, 0.8% of the cells were transduced, compared with 80% of the cells after transduction with the GALV type retrovirus (Figs. 1 and 10). Ten days after transduction, 94% of the cell population transduced with the amphotropic retrovirus was GFP positive, demonstrating that transduced cells grow faster than non-transduced cells (Fig. 10).

In order to determine cell doubling time, cells were cultured as in Example 2 in 24-well plates at 50-100,000 cells per well together with CD40L and IL-21 L cells. Every 3-4 days, cells were counted and 50-100,000 cells were transferred to a new well. In fig. 11 shows a growth curve for rabbit B cells transduced with an amphotropic virus. The estimated doubling time is 19 hours, which is comparable to rabbit B cells transduced with a GALV-type retrovirus (18 hours). In conclusion, introduction of Bcl-6 and Bcl-xl into rabbit B cells via an amphotropic retrovirus also resulted in the immortalization of rabbit B cells, although the transduction efficiency was significantly lower compared to the GALV-based vector.

Brief description of drawings

Fig. 1. Transduction of rabbit memory B cells. Rabbit B cells were isolated from peripheral blood mononuclear cells (PBMCs) based on Ig expression. Cells were activated for

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36-40 h on CD40L L cells with rm-IL-21. Transduction of cells was carried out with a retroviral vector containing BCL6 and Bcl-xL. Retroviruses of both the GALV type and the amphotropic type were studied. The 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 by the results of GFP expression. The GALV type retrovirus demonstrated a higher transduction efficiency (80%) compared to the amphotropic type retrovirus (0.8%).

Fig. 2. 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 analyzed in parallel for B cells from llama cells and human cells from two different donors, which were transduced with a similar retroviral vector containing BCL6 and Bcl-xL.

Fig. 3. The expression of surface immunoglobulins IgG, IgM and IgA was determined using FACS on three different Bcl-6, Bcl-xL-transduced rabbit B cell clones.

Fig. 4. Identification of antigen-specific rabbit B cells in a pool of rabbit B cells with different specificities.

Fig. 5. Rabbit antigen-specific antibodies were raised against various components of a human influenza vaccine containing 15 μg H1N1, 15 μg H3N2, and 15 μg influenza B. Rabbits were primary and secondary immunized with human influenza vaccine. B cells were immortalized and seeded at various densities in 384-well plates on CD40L L cells in the presence of recombinant mouse IL-21. Antibodies present in the rabbit cell culture supernatants were screened by ELISA for influenza specificity. Antigen-specific antibodies were observed for all vaccine components.

Fig. 6. Immortalized B cells from rabbits immunized with a human influenza vaccine containing 15 μg H1N1, 15 μg H3N2, and 15 μg influenza B were stained with fluorescently labeled influenza proteins. B cells exhibiting influenza protein binding were sorted on a 1 cell/well basis in 384-well plates for CD40L L cells in the presence of recombinant mouse IL21 using a FACSAria sorter. Supernatants were screened by ELISA for influenza-specific antibodies. Antigen-specific antibodies were detected at high frequencies for vaccine components used for antigen-specific sorting.

Fig. 7. Antibody concentration in the supernatant of clonal B cells at various time points. Transduced human, llama and mouse B cells were seeded on a 1 cell/well basis in the presence of irradiated CD40L L cells and supplemented with mouse IL-21. CD40L and IL-21 L cells were renewed every 3-4 days. The IgG concentration was analyzed by ELISA for individual wells at various time points during culture. Each measurement was carried out on different wells. B cell transduction was performed with either 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.

Fig. 8. Schematic illustration of the vector used to transduce rabbit and human B cells.

Fig. 9. Sequence of the extracellular domain of the GALV SEATO coat protein (in bold) and the transmembrane domain of the GALV SEATO coat protein (underlined) fused to the cytoplasmic domain of the amphotecoat protein (in italics and dotted underlining).

Fig. 10. Transduction of rabbit memory B cells and rapid growth of rabbit B cells transduced with an amphotropic type retrovirus. Rabbit B cells were isolated from peripheral blood mononuclear cells (PBMCs) based on Ig expression. Cells were activated for 36-40 hours on CD40L L cells with rm-IL-21. Transduction of cells was carried out with a retroviral vector containing BCL6 and Bcl-xL. Retroviruses of both the GALV type and the amphotropic type were studied. The 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 by the results of GFP expression. The GALV type retrovirus demonstrated a higher (80%) transduction efficiency compared to the amphotropic type retrovirus (0.8%). After 10 days, 94% of rabbit B cells transduced with amphotropic retrovirus were immortalized by GFP expression, demonstrating vigorous growth of transduced cells over nontransduced cells.

Fig. 11. Growth curve was analyzed for rabbit B cells transduced with an amphotropic type retroviral vector containing BCL6 and Bcl-xL

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Claims (23)

Links Christopherson, K.S. et al. PNAS 89, 6314-8 (1992) Guzman, L. M. et al. Bacteriol 177, 4121-4130 (1995) T.M. Kinsella, G.P. Nolan, Hum Gene Ther 7 (1996) 1405. Kwakkenbos et al. Generation of stable monoclonal antibody-producing B cell receptor positive human memory B cells by genetic programming. Nature Medicine (2010) vol. 16 (1) pp. 123-8 Lam et al. Improved gene transfer into human lymphocytes using retroviruses with the gibbon ape leukemia virus envelope. Human gene therapy 7 (1996) 1415-1422 A.L. Szymczak, C.J. Workman, Y. Wang, K.M. Vignali, S. Dilioglou, E.F. Vanin, D.A.A. Vignali, Nat Biotechnol 22 (2004) 589. C.A. Wilson, M.V. Eiden, W.B. Anderson, C. Lehel, Z. Olah, J Virol 69 (1995) 534. WO 2007/067046 CLAIM 1. Use of a rabbit B cell expressing an exogenous nucleic acid molecule encoding the Bcl-6 protein or its rabbit homologue and expressing an exogenous anti-apoptotic nucleic acid molecule to produce an ex vivo B cell culture with an average doubling time of 20 hours or less. 2. A method of obtaining a B cell culture ex vivo with an average doubling time of 20 hours or less, comprising: causing expression of a nucleic acid molecule encoding a Bcl-6 protein or a rabbit homolog thereof and at least one anti-apoptotic nucleic acid molecule in a rabbit B cell. 3. The method according to claim 2, additionally including: culturing said B cell ex vivo; and collecting antibodies produced by said B cell for 7-14 days. 4. The method according to claim 3, where the collection of antibodies is carried out within 9-12 days. 5. A method for increasing the replicative life cycle of a rabbit B cell, comprising: introducing into said rabbit B cell a nucleic acid molecule encoding the Bcl-6 protein or its rabbit homologue; and at least one anti-apoptotic nucleic acid, by transducing a rabbit B cell with a gene delivery vehicle comprising at least a functional portion of the extracellular domain of the gibbon leukemia virus (GALV) envelope protein or a sequence that is at least 70% identical to the functional portion of the extracellular domain GALV coat protein. 6. The method according to claim 5, wherein said extracellular domain is the extracellular domain of the envelope protein of the GALV strain SEATO virus. 7. The method of claim 5, wherein said gene delivery vehicle comprises a chimeric envelope protein, or a protein containing said chimeric envelope protein, or a protein that has at least 70% sequence identity with said chimeric envelope protein. 8. The method according to claim 7, where the chimeric envelope protein contains an extracellular domain and a transmembrane domain fused to a cytoplasmic domain, and has the sequence: - 18 044991 MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNPHQPMTLTWQVLS QTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLESWDIPGTDVSSSKRVRPPDSDYTAAY KQITWGAIGCSYPRARTRMASSTFYVCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYW LSKSSKDLITVKWDQNSEWT QKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFT1RLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREAPPPSLPDSNSTALA TSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTLNATNPGATESCWLCLAMGPPYYEAI ASSGEVAYSTDLDRCRWGTQGKLTLTEVSGHGLCIG KVPFTHQHLCNQTLSINSSGDHQYL LPSNHSWWACSTGLTPCLSTSVFNQTRDFCIQVQLIPR1YYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAG1GTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDSVSKLEDSLTSLSE VVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAVRDSMKK LKEKLDKRQLERQKSQN WYEGWFNNSPWFTTLLSTIAGPLLLLLLLLLILGPCIINRLVQFVKDRISVVQALVLTQQYHQL KP1EYEP. 9. Method according to any one of claims 2-8, characterized in that: the nucleic acid encodes non-rabbit Bcl-6 or a functional portion or functional derivative thereof; and/or the at least one anti-apoptotic nucleic acid molecule is a non-rabbit anti-apoptotic nucleic acid molecule, wherein the functional portion and functional derivative of Bcl-6 retains the ability to extend the replicative life cycle of a rabbit B cell. 10. The method of claim 9, wherein said non-rabbit nucleic acid molecules are human nucleic acid molecules. 11. The method according to any one of claims 2 to 10, wherein at least one anti-apoptotic nucleic acid molecule contains a Bcl2 family gene. 12. The method according to claim 11, in which the Bcl2 family gene is selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl-2L10 and their rabbit homologues, functional parts and functional derivatives . 13. The method according to any one of claims 2 to 12, further comprising inducing, improving and/or maintaining the expression of Blimp-1 or its rabbit homologue in said rabbit B cell. 14. The method of claim 13, wherein: improving the expression of Bcl-6 or its rabbit homologue is accomplished by culturing said rabbit B cell in the presence of a compound capable of directly or indirectly improving the expression of Bcl-6 or the expression of its rabbit homologue. 15. The method according to any one of claims 2 to 14, further comprising culturing said rabbit B cell in the presence of at least one compound capable of directly or indirectly improving the expression of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl-2L10 and/or their rabbit homolog. 16. The method according to claim 15 wherein said rabbit B cell is cultured in the presence of IL21 and CD40L. 17. The method of claim 16, wherein said IL21 is mouse or human IL21, and/or said CD40L is mouse or human CD40L. 18. The method according to any one of claims 2 to 17, further comprising culturing said rabbit B-cell in the presence of at least one compound capable of directly or indirectly improving the expression of Blimp-1 or the expression of a rabbit homolog of Blimp-1. 19. A recombinant rabbit B cell comprising a nucleic acid sequence encoding Bcl-6 or a rabbit homologue thereof, or a functional portion or functional derivative thereof, and an antiapoptotic nucleic acid sequence transduced with at least a functional portion of said extracellular domain of the GALV envelope protein or sequence having at least 70% sequence identity with at least a functional portion of the extracellular domain of the GALV envelope protein. 20. The rabbit B cell according to claim 19, wherein said anti-apoptotic nucleic acid sequence is a nucleic acid sequence encoding a protein selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w , Bcl-2L10, and their rabbit homolog, a functional moiety thereof, a functional derivative thereof, and any combination thereof, wherein the functional moiety or functional derivative is Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl-2L10, or their rabbit homologue retains the ability to extend the replicative life cycle of rabbit B cells. 21. Recombinant rabbit B cell containing: an exogenous non-rabbit anti-apoptotic nucleic acid molecule encoding Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl-2L10 or a functional part or functional derivative thereof, and an exogenous non-rabbit nucleic acid molecule encoding Bcl-6 or its a functional moiety or functional derivative, wherein the functional moiety or functional derivative of Bcl-6 and Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl-2L10 retains the ability to extend the replicative life cycle of a rabbit B cell. 22. The rabbit B cell of claim 21, wherein said non-rabbit nucleic acid sequence is a human nucleic acid sequence. 23. An ex vivo rabbit B cell culture that has an average doubling time of 20 hours or less, wherein the B cells of said ex vivo rabbit B cell culture contain an exogenous molecule -