NZ722156B2 - Ex vivo antibody production - Google Patents

Abstract

The present invention provides means and methods for producing improved ex vivo rabbit B cell cultures with a short doubling time of less than 20 hours or less. The methods comprise inducing, enhancing or maintaining expression of Bcl-6 and at least one anti-apoptotic nucleic acid comprising a gene of the Bcl-2 family in a rabbit B cell. The invention further relates to the use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein for introducing a nucleic acid molecule encoding Bcl-6 and the anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell. The invention further relates to rabbit B cells and ex vivo rabbit B cell cultures obtained by such methods. of the Bcl-2 family in a rabbit B cell. The invention further relates to the use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein for introducing a nucleic acid molecule encoding Bcl-6 and the anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell. The invention further relates to rabbit B cells and ex vivo rabbit B cell cultures obtained by such methods.

Description

(12) Granted patent caon (19) NZ (11) 722156 (13) B2 (47) Publicaon date: 2021.12.24 (54) EX VIVO ANTIBODY PRODUCTION (51) Internaonal Patent Classificaon(s): C12N 5/0781 C07K 16/00 C12N 15/867 (22) Filing date: (73) Owner(s): 2014.12.24 KLING BIOTHERAPEUTICS B.V. (23) Complete specificaon filing date: (74) Contact: 2014.12.24 AJ PARK (30) aonal Priority Data: (72) Inventor(s): EP 13199584.7 2013.12.24 VAN HELDEN, Pauline Maria Wilhelmina KWAKKENBOS, Mark Jeroen (86) Internaonal Applicaon No.: SPITS, Hergen BEAUMONT, Tim (87) Internaonal Publicaon number: /099534 (57) Abstract: The present invenon provides means and methods for producing improved ex vivo rabbit B cell es with a short doubling me of less than 20 hours or less. The methods comprise inducing, enhancing or maintaining sion of Bcl-6 and at least one optoc nucleic acid comprising a gene of the Bcl-2 family in a rabbit B cell. The invenon further relates to the use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence identy with the extracellular domain of a GALV envelope protein for introducing a nucleic acid molecule encoding Bcl-6 and the an-apoptoc nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell. The invenon further s to rabbit B cells and ex vivo rabbit B cell cultures obtained by such methods.

NZ 722156 B2 Title: Ex vivo antibody production The invention relates to the fields of medicine, molecular biology and logy.

Ex vivo B cell cultures are important tools for producing antibodies, preferably monoclonal dies. onal antibodies (mAbs) represent le identical copies of a single antibody molecule, which copies bind to antigens with the same affinity and e the same effector functions. Amongst the benefits of mAbs is their specificity for the same epitope on an antigen. This specificity confers certain clinical advantages on mAbs over more conventional treatments while offering patients an effective, well-tolerated therapy option with generally low side effects. Moreover mAbs are useful for ical and medical research.

A conventional approach for obtaining mAbs is hybridoma technology, wherein a B cell is fused with a myeloma cell in order to form hybrid antibody producing cell lines (hybridomas). However, hybridoma technology with human B cells has not been very successful because the resulting omas are unstable.

Meanwhile, an improved technology has been developed wherein ex vivo B cell cultures are produced with a prolonged replicative life span ().

This technology involves human ex vivo cultures wherein Bcl-6, together with Blimp-1 and/or an anti-apoptotic nucleic acid, are sed in the B cells. This improves the replicative life span of these B cells. Typically, human B cells are ed in order to obtain human mAbs. Human mAbs are preferred for therapeutic applications in humans due to the lower genicity as compared to antibodies of other s. Using the technology of , ex vivo human B cell cultures with a mean doubling time of about 25-36 hours are obtained.

In order to commercially produce mAbs of interest, such as therapeutic mAbs, it is advantageous to use B cell cultures wherein the B cells have a short doubling time. A short doubling time is also very important in therapeutic approaches like cancer therapy, for instance when a non-human mammal is immunized with cancer cells of a patient, where after cancer-specific B cells are harvested from the animal and used for ex vivo antibody production. Since such antibodies are a tailor-made medicine for the individual patient, they should be produced as fast as possible so that the patient can start his/her Ab y as soon as le. Such antibodies that are specific for an individual’s tumor cannot be produced beforehand.

It is one of the objects of the present invention to e means and methods for producing improved ex vivo B cell cultures with a shorter doubling time; and or to at least e the public with a useful choice.

The t invention provides the insight that B cell cultures with a shorter doubling time, as compared to the B cell cultures disclosed in , are ed when rabbit B cells are used. Whereas commonly used B cells such as human B cells, murine B cells and llama B cells typically have a doubling time of 25-36 hours, the present inventors have surprisingly found that rabbit B cell cultures can be obtained with a doubling time of 20 hours or less. This insight allows significant faster production of antibodies of interest, ing in a higher yield within a given time frame, which is particularly valuable for commercial antibody production and therapeutic applications.

Summary of the invention In a first aspect the present invention es a method for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method comprising: - inducing, enhancing or ining expression of Bcl-6 in a B cell, - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl-2 family in said B cell, characterized in that said B cell is a rabbit B cell.

In a second aspect the present invention provides a method for increasing the replicative life span of a rabbit B cell, the method comprising: - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B cell, and - inducing, enhancing or maintaining sion of at least one antiapoptotic nucleic acid comprising a gene of the Bcl-2 family in said B cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, with at least one anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family, or with a combination f, via uction with a gene delivery vehicle that comprises the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a n that has at least 70% sequence identity with the extracellular domain of a GALV envelope n.

In a third aspect the present invention provides a use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, for introducing a nucleic acid le encoding Bcl-6 and at least one anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell.

In a fourth aspect the present invention es a method for obtaining antibodies, comprising: - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B cell; - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid le comprising a gene of the Bcl2- family in said B cell; - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 7-14 days.

In a fifth aspect the present invention es a rabbit B cell, which is bound via the ellular domain of a GALV envelope protein, or via a protein that has at least 70% sequence ty with the extracellular domain of a GALV envelope protein, to a gene delivery vehicle that comprises a nucleic acid sequence encoding Bcl-6 and an anti-apoptotic nucleic acid sequence comprising a gene of the Bcl-2 family.

In a sixth aspect the present invention provides an isolated or recombinant rabbit B cell comprising: - a non-rabbit anti-apoptotic c acid molecule comprising a gene of the Bcl-2 family, and - a non-rabbit nucleic acid molecule encoding Bcl-6.

In a h aspect the present invention es an ex vivo rabbit B cell culture comprising the rabbit B cells according to the fifth or sixth aspects, which has a mean doubling time of 20 hours or less.

In an eighth aspect the present invention provides an ex vivo rabbit B cell culture when obtained by a method according to the first aspect.

In a ninth aspect the present invention provides a use of a gene delivery vehicle comprising the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% ce identity with the extracellular domain of a GALV envelope protein, and a nucleic acid sequence encoding Bcl-6 and at least one antiapoptotic nucleic acid sequence sing a gene of the Bcl-2 family, for increasing the replicative life span of a rabbit B cell.

Also described is a use of a rabbit B cell for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less. Ex vivo rabbit B cell cultures described herein are typically obtained by expression of Bcl-6, or a rabbit homologue thereof, and an anti-apoptotic nucleic acid molecule in a rabbit B-cell.

Further described is therefore a method for obtaining an ex vivo B cell culture with a mean doubling time of 20 hours or less, the method comprising: - inducing, enhancing and/or ining expression of Bcl-6, or a rabbit gue thereof, in a B-cell and - inducing, enhancing and/or ining expression of an anti-apoptotic nucleic acid molecule in said B-cell, characterized in that said B cell is a rabbit B cell. ably, ex vivo rabbit B cell cultures are produced with a mean doubling time of less than 20 hours. More preferably, said mean doubling time is less than 19 hours or even less than 18 hours. A shorter doubling time allows faster and higher antibody production, which enhances the time to - and efficacy of - testing and screening for a desired antibody and ion and/or identification of antibodies of interest. Moreover, if mAbs need to be developed for an individual patient, the shorter doubling time of rabbit B cells allows for a quicker start of the patient’s ic mAb therapy.

A method described herein, using rabbit B cells, thus provides the advantage that antibody can be obtained, , identified, isolated and/or produced ex vivo within a shorter time frame as compared to tly known human, murine or llama B cell cultures.

The fact that the present disclosure describes a B cell culture with a short doubling time es the advantage that a sufficient quantity of antibody can be obtained within a shorter period of time as compared to existing methods. For instance, in a method as disclosed in a collection of B cells obtained from a human individual is stabilized using Bcl-6 and an anti-apoptotic nucleic acid (or compounds increasing the expression of such nucleic acids) and uently ed. This results in stabilized human B cells, which are capable of both proliferating and producing antibody. During culturing, the stabilized B cells produce antibody, which is secreted into the culture medium. uently, these antibodies are preferably tested for a desired specificity (and/or affinity). For current test procedures, an antibody concentration of at least 100 ng/ml culture medium is typically ed. After 15-20 days of culturing stabilized human B cells, such l antibody concentration is obtained. Therefore, using human B cell cultures, antibody is harvested at least 15-20 days after starting the culture, typically around day 20. Llama B cells have a similar growth rate as human B cells, so that if a llama B cell culture is used, antibody is also typically harvested at least 15-20 days after starting the culture. With murine B cells, which have a longer doubling time, antibodies with a minimal concentration of 100 ng/ml are typically obtained after more than 20 days.

After g the antibodies, the corresponding B cells of st are often selected and isolated for further use. Given the fact that antibody testing normally takes about three days, human or llama B cells of interested are typically selected and ed after 18-23 days from the start of the B cell culture, whereas murine B cells of interest are typically selected and isolated after more than 23 days. The isolated B cells are then further cultured. A B cell e with human, llama or murine B cells of interest is thus typically obtained after about three weeks from the start of the B cell culture. With a method described herein, however, an antibody concentration of at least 100 ng/ml is already obtained after 11-12 days.

Hence, antibody can now already be harvested 11-12 days after starting the B cell culture, whereas one had to maintain a human (or llama) B cell culture for at least -20 days before harvesting antibody. If the testing procedure takes three days, rabbit B cells of interest are thus selected and isolated within 14-15 days from the start of the B cell culture, which is significantly faster as compared to the situation wherein human or murine B cells are cultured. In conclusion, whereas it typically takes about three weeks for obtaining a human, llama or murine B cell culture which es a sufficient tration of antibody, with the insight of the present disclosure a B cell culture with rabbit B cells producing a ient Ab concentration is already obtained after two weeks. This is a major advantage over existing methods. One embodiment bed herein relates to a method for ing antibodies, preferably for use in one or more testing assays requiring a minimal antibody concentration of at least 100 ng/ml, the method comprising: - inducing, enhancing and/or ining expression of Bcl-6 in a rabbit B-cell; - inducing, enhancing and/or ining expression of an anti-apoptotic nucleic acid molecule in said B-cell; - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 10-14 days, preferably within 11-12 days. Said harvested antibodies are ably tested using one or more assays requiring a minimal antibody concentration of at least 100 ng/ml.

As described above, the obtained antibodies are typically used for testing for a desired specificity and/or affinity. Current test methods often require a l antibody concentration of 100 ng/ml, but if more sensitive detection s are used, the antibodies can be harvested earlier. Whatever the sensitivity of the test method, using rabbit B cells with a method according to the present disclosure, the required minimal antibody concentration is obtained earlier as compared to the use of currently known human, llama or murine B cells, due to the significant faster ng time of rabbit B cells. For instance, if a minimal antibody concentration of only 30 ng/ml is required, instead of 100 ng/ml, this concentration is typically reached using human B cells after 13-18 days from the start of the B cell culture, s a rabbit B cell culture would only need 9-10 days to obtain this l antibody concentration. Thus, again, antibody testing and isolation of B cells of interest can be performed earlier. In practice, the current ors obtain and test the rabbit antibodies within 7-14 days from the start of a B cell culture. Before the present disclosure, ex vivo B cell cultures allowing antibody testing at significant earlier stages as compared to ex vivo human B cell cultures were not ble.

Further described is therefore a method for obtaining antibodies, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a rabbit B-cell; - inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid molecule in said rabbit ; - culturing said B cell ex vivo; and - ting antibodies produced by said B cell within 7-14 days, preferably within 9-12 or 9-10 days. Said harvested antibodies are ably tested using one or more assays requiring a minimal antibody concentration of about 30 ng/ml.

As used herein, the term “rabbit B cell” means a B cell that has been obtained from a rabbit, or a B cell that originates from a rabbit B cell. An example of B cells ating from a rabbit B cell is the progeny of a rabbit B cell that is formed after one or more cell division cycles. Such progeny for instance includes an ex vivo culture of rabbit B cells.

An ex vivo rabbit B cell culture is a culture that ns rabbit B cells and/or progeny thereof. Other kinds of cells may also be present in the culture. For instance, B cell stimulator cells such as CD40 positive L cells and/or EL4B5 cells are typically also present in a B cell culture described herein. onally, other kinds of cells, which were also present in a B cell-containing sample, could still be present in a B cell culture. When t in B cell culturing conditions, such non- B cells are typically less capable of proliferating as compared to B cells, so that the number of such contaminating cells will typically decline in time. Preferably, at least 70% of the cells of a rabbit B cell culture 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 B cells and B cell stimulator cells such as CD40 positive L cells and/or EL4B5 cells are essentially the only kinds of cell t in a rabbit B cell culture.

Preferably, the B cells of a rabbit B cell culture described herein are y of one al rabbit B cell, so that monoclonal antibodies are produced by the B cell culture.

The term “mean doubling time” is defined herein as the mean time required, starting from a culture with a certain original amount of B cells, to obtain a culture with a number of B cells that is two times said original B cell number. Since not every B cell will proliferate at exactly the same rate, mean values are typically used for a B cell culture as a whole.

Bcl-6 encodes a transcriptional repressor which is required for normal B cell and T cell development and tion and which is required for the formation of germinal centers. Bcl-6 is highly expressed in germinal center B cells whereas it is hardly expressed in plasma cells. Bcl-6 inhibits differentiation of activated B cells into plasma cells. In a method described herein, Bcl-6 expression product, or the expression product of a rabbit homologue f, remains present in the rabbit B cells of an ex vivo culture. The presence of Bcl-6, or a rabbit homologue thereof, together with the ce of an anti-apoptotic nucleic acid, prolongs the replicative life span of the B cells. Expression of Bcl-6, or a rabbit homologue thereof, is preferably induced, ed or maintained by administering a Bcl-6 expression-promoting compound, or a compound that promotes sion of a rabbit homologue of Bcl-6, to the rabbit B cell(s) used for culturing, or by culturing rabbit B cells in the presence of such compound.

Further bed is therefore a method according to the present disclosure, comprising: - ing said rabbit B cell with a compound capable of ly or indirectly enhancing expression of Bcl-6, or 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 enhancing expression of Bcl-6, or expression of a rabbit homologue of Bcl- Various compounds e of directly or indirectly enhancing expression of Bcl-6, or expression of a rabbit homologue of Bcl-6, are known in the art. Such compound for instance comprises a Signal Transducer of Activation and Transcription 5 (STAT5) protein, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and/or a nucleic acid ce coding ore. STAT5 is a signal transducer capable of enhancing Bcl-6 expression.

There are two known forms of STAT5, STAT5a and STAT5b, which are encoded by two different, tandemly linked genes. Administration and/or activation of STAT5, or a rabbit homologue thereof, results in enhanced levels of Bcl-6, or enhanced levels of a rabbit homologue of Bcl-6. Hence, STAT5, or a rabbit gue thereof, or a functional part or a functional derivative thereof is capable of directly increasing expression of Bcl-6, or expression of a rabbit homologue of Bcl-6. bed is ore a method according to the present disclosure comprising providing said rabbit B cell with STAT5, or with a rabbit homologue thereof, or with a functional part or a functional derivative thereof, or providing said rabbit B cell with a nucleic acid molecule encoding STAT5, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, 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 a functional derivative thereof.

The presence of STAT5, or a rabbit homologue thereof, directly increases the amount of Bcl-6, or the amount of a rabbit homologue of Bcl-6. It is also possible to indirectly increase expression of Bcl-6, or expression of a rabbit homologue thereof.

This is for instance done by regulating the amount of a certain compound, which in turn is capable of directly or ctly activating STAT5, or a rabbit homologue thereof, and/or increasing expression of STAT5, or sion of a rabbit homologue thereof. Hence, in one ment the expression and/or activity of endogenous and/or exogenous STAT5, or the expression of a rabbit homologue thereof, is increased. It is for instance le to indirectly e expression of Bcl-6, or expression of a rabbit homologue thereof, by culturing a rabbit B cell in the presence of interleukin (IL) 2 and/or IL4 which are e of activating STAT5, or activating a rabbit homologue of STAT5, which in turn increases expression of Bcl-6, or expression of a rabbit homologue of Bcl-6.

As used herein, the term t homologue” of, for instance, Bcl-6 or STAT5 means a rabbit protein corresponding to Bcl-6 or STAT5, which means that it has a corresponding, similar function in rabbit B cells as compared to the function of Bcl- 6 or STAT5 in human B cells.

It is, however, preferred to provide a rabbit B cell with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue thereof, or a functional part or a functional derivative thereof. This way, it is possible to directly te the concentration of Bcl-6, or the concentration of a rabbit homologue thereof, in said rabbit B cell. Also described is therefore a method according to the present disclosure comprising providing said rabbit B cell with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue of Bcl-6, or a functional part or a functional derivative thereof. In one embodiment, said nucleic acid le is constitutively active, meaning that Bcl-6, or a rabbit homologue f, or a functional part or a functional derivative thereof, is continuously expressed, independent of the presence of a regulator. In another embodiment, said nucleic acid molecule is inducible, meaning that the expression thereof is regulated by at least one inducer and/or repressor. This way, expression of said nucleic acid molecule is ted at will. For instance, Tet-On and Tet-Off expression systems (for example Tet-On® and Tet-Off® Advanced Inducible Gene Expression s, Clontech) can be used for inducible expression of a nucleic acid sequence of interest. In these systems expression of the transcriptional activator (tTA) is regulated by the presence (Tet-On) or absence (Tet-Off) of tetracycline (TC) or a derivative like doxycycline (dox). In principle, tTA is composed of the Escherichia coli Tet repressor protein (TetR) and the Herpes simplex virus transactivating domain VP16. tTA regulates transcription of a nucleic acid sequence of interest under the control of a tetracycline-responsive element (TRE) comprising the Tet operator (TetO) DNA sequence and a promoter sequence, for ce the human galovirus (hCMV) promoter. A nucleic acid sequence encoding, for instance, Bcl6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof, can be placed downstream of this er.

In the Tet-off system, tTA binds to TRE in the absence of TC or dox and ription of a nucleic acid sequence of interest is activated, whereas in the presence of TC or dox tTA cannot bind TRE and expression of a c acid sequence of st is inhibited. In contrast, the Tet-on system uses a e tTA (rtTA) that can only bind the TRE in the presence of dox. Transcription of a nucleic acid sequence of interest is inhibited in the absence of dox and activated in the presence of dox.

In another embodiment, inducible expression is executed using a hormone inducible gene expression system such as for instance an ecdysone inducible gene expression system (for e RheoSwitch®, New England Biolabs) (Christopherson, K.S. et al. PNAS 89, 6314-8 (1992)). Ecdysone is an insect steroid hormone from for example Drosophila melanogaster. In cells transfected with the ecdysone receptor, a heterodimer consisting of the ecdysone or (Ecr) and id X receptor (RXR) is formed in the presence of an ecdyson agonist selected from ecdysone, one of its analogues such as erone A and ponasterone A, and a non-steroid ecdysone agonist. In the presence of an agonist, Ecr and RXR interact and bind to an ecdysone response element that is present on an expression cassette. Exaperssion of a nucleic acid sequence of interest that is placed in an expression cassette downstream of the ecdysone response t is thus induced by exposing a rabbit B-cell to an ecdyson agonist.

In yet another embodiment of the present disclosure inducible expression is executed using an arabinose-inducible gene sion system (for example pBAD/gIII kit, Invitrogen) (Guzman, L. M. et al. iol 177, 4121–4130 ).

Arabinose is a monosaccharide containing five carbon atoms. In cells transfected with the arabinose-inducible promoter PBAD sion of a nucleic acid sequence of interest placed downstream of PBAD can then be induced in the presence of arabinose.

It is also possible to use (a nucleic acid le encoding) a Bcl-6 protein, or a rabbit homologue thereof, or a functional part or functional derivative thereof, wherein the activity of said Bcl-6 or rabbit homologue or functional part or functional derivative is regulated by at least one inducer and/or repressor. A nonlimiting example is a fusion n n a regulatory element is fused to a sequence encoding at least part of Bcl-6 or a rabbit homologue thereof. For instance, an estrogen receptor (ER) is fused to Bcl-6, resulting in fusion protein ERBcl-6.

This fusion protein is inactive because it forms a complex with heat shock proteins in the cytosol. Upon administration of the exogenous inducer 4 hydroxytamoxifen (4HT), the fusion protein ER-Bcl-6 dissociates from the heat shock proteins, so that the Bcl-6 part of the fusion protein becomes .

As used herein, the term “anti-apoptotic nucleic acid molecule” refers to a c acid molecule, which is capable of delaying and/or preventing sis in a rabbit B cell. Preferably, said anti-apoptotic nucleic acid molecule is capable of ng and/or preventing apoptosis in a plasmablast-like rabbit B cell, which is capable of both proliferating and producing antibody. Preferably, an anti-apoptotic nucleic acid molecule is used which ses an exogenous nucleic acid molecule.

This means that either a nucleic acid sequence is used which is not naturally expressed in rabbit B cells, or that an additional copy of a naturally occurring c acid sequence is used, so that expression in the resulting rabbit B cells is enhanced as compared to natural rabbit B cells. Various poptotic nucleic acid molecules are known in the art, so that various embodiments are available.

Preferably, an anti-apoptotic nucleic acid molecule is used which is an anti- apoptotic member of the Bcl-2 family because anti-apoptotic Bcl-2 proteins are good apoptosis inhibiters in B cells. Many processes that are controlled by the Bcl-2 family (which family includes both pro- and anti-apoptotic proteins) relate to the mitochondrial pathway of apoptosis. The use of anti-apoptotic Bcl-2 family members Bcl-2, Bcl-xL, Bcl-w, Bclrelated protein A1 (also named Bcl2-A1 or A1), Bcl-2 like 10 (Bcl2L10) and Mcl-1, or a rabbit homologue thereof, or a onal part or functional tive thereof, is preferred because Bcl-2, Bcl-xL, Bcl-w, A1, Bcl2L10 and Mcl-1 are generally integrated with the outer mitochondrial membrane. They ly bind and inhibit the pro-apoptotic proteins that belong to the Bcl-2 family to t ondrial membrane integrity.

A preferred embodiment therefore describes a method according to the present disclosure, wherein said anti-apoptotic nucleic acid molecule comprises an anti-apoptotic gene of the Bcl2 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 a functional derivative thereof.

In one embodiment, expression of Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a rabbit homologue thereof, is induced, ed or maintained by administering at least one compound, e of promoting expression of any of these anti-apoptotic genes, to rabbit B cell(s), or by culturing rabbit B cells in the presence of such compound(s). Further described is therefore a method according to the present disclosure, comprising: - providing said rabbit B cell with a compound capable of directly or indirectly ing expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10, or a rabbit homologue thereof; and/or - culturing said rabbit B cell in the ce of a compound capable of directly or indirectly enhancing expression of Bcl-xL and/or Mcl-1 and/or Bcl-2 and/or A1 and/or Bcl-w and/or Bcl2L10, or a rabbit homologue thereof.

Preferably, however, a rabbit B cell is described herein with at least one nucleic acid molecule encoding an anti-apoptotic gene of the Bcl2 family, preferably selected from the group ting of , Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof, and onal parts and functional derivatives thereof.

This way, it is possible to directly enhance the amount of sion product in said rabbit B cell. Also described is therefore a method according to the present disclosure, comprising providing said rabbit B cell with at least one nucleic acid molecule encoding an anti-apoptotic gene of the Bcl2 family, preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof, and onal parts and functional derivatives thereof. In one embodiment, said nucleic acid molecule is constitutively active, meaning that said nucleic acid molecule is continuously expressed. In another embodiment, said nucleic acid le is inducible, meaning that the expression thereof is regulated by at least one inducer and/or repressor. miting examples of inducible nucleic acid expression systems known in the art are described herein before.

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 part or a functional derivative thereof. According to the present disclosure, a combination of Bcl-6 and Bcl-xL is particularly well capable of increasing the replicative life span of rabbit B-cells, thereby forming long term es of the resulting plasmablast-like B-cells. The same holds true for a combination of Bcl-6 and Mcl-1. Most ably, said anti-apoptotic nucleic acid encodes Bcl-xL or a functional part or a onal derivative thereof.

A functional part of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or of a rabbit homologue thereof, is a proteinaceous molecule that has the same capability - in kind, not necessarily in amount - of increasing the replicative life span of a rabbit B cell as compared to natural Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or a rabbit homologue thereof, respectively. Such onal part is for instance devoid of amino acids that are not, or only very little, involved in said capability.

For instance, functional parts of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, or of a rabbit homologue f, are defined herein as fragments of BclxL , Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, respectively, or of a rabbit homologue thereof, which have retained the same kind of anti-apoptotic characteristics as full length Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, tively, or a rabbit homologue thereof (in kind, but not necessarily in amount). Functional parts of BclxL , Mcl-1, Bcl-2, A1, Bcl-w or 0, or of a rabbit homologue f, are typically shorter fragments of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, respectively, or of a rabbit homologue thereof, which are capable of delaying and/or preventing apoptosis in a rabbit B-cell. Such functional parts are for instance devoid of sequences which do not significantly contribute to the anti-apoptotic activity of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10. A functional part of Bcl-6, or of a rabbit gue thereof, is typically a shorter fragment of Bcl-6, or a shorter fragment of a rabbit homologue f, which is capable of increasing the replicative life span of a rabbit B cell.

A functional derivative of Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, or of a rabbit gue thereof, is defined as a Bcl-6, Bcl-xL, Mcl-1, Bcl-2, A1, Bclw or Bcl2L10 protein, respectively, or a rabbit homologue thereof, which has been altered but has maintained its capability (in kind, not necessarily in amount) of increasing the replicative life span of a rabbit B cell. A onal derivative is ed in many ways, for instance through conservative amino acid substitution wherein one amino acid is substituted by another amino acid with generally similar properties (size, hydrophobicity, etc), such that the l oning is not seriously affected. Alternatively, a functional derivative for instance ses a fusion protein with a detectable label or with an inducible compound.

Another aspect of the t disclosure solves the problem of efficiently introducing a nucleic acid molecule of interest into rabbit B cells. Contrary to expectations, the inventors found that the commonly used ampho retroviral vector, which is suitable for infecting rodent cells such as murine cells and which was therefore expected to be also capable of transducing rabbit B cells, appeared not to transduce rabbit B cells efficiently; the transduction efficiency of an ampho vector at 4 days after transduction appeared lower than 1% in rabbit B cells. Therefore, the present inventors had to search for other gene delivery vehicles. Surprisingly, the inventors discovered that a gene delivery vehicle which comprises the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein is capable of transducing rabbit B cells with a high efficiency, typically of 80-90% at 3-5 days after transduction. This property was quite unexpected, since a gibbon ape leukemia virus does not naturally infect s. Rabbit cells were ore not expected to contain a or for a GALV pe protein; the current finding was mere coincidence. Of note, transduction efficiency of human B cells with a vector containing the extracellular domain of a GALV envelope protein is lly 60-70% at 4 days after transduction, which is often lower than the transduction efficiency of rabbit B cells with this vector, despite the fact that human B cells are primate cells. This is surprising because, since an ape is also a primate, a vector containing a GALV-based envelope protein was expected to be better capable of infecting primate cells as compared to rabbit cells. Of note, murine B cells are indeed not efficiently transduced using a vector that comprises the extracellular domain of a GALV envelope n, consistent with the fact that a gibbon ape leukemia virus does not infect mice.

Now that the insight of the present disclosure has been provided that it is possible to efficiently transduce rabbit B cells using at least a functional part of the extracellular domain of a GALV envelope protein, and that the transduction efficiency is even higher than the transduction efficiency of human B cells, new applications have become available. It has now become possible to introduce a nucleic acid molecule of st into rabbit B cells with high efficiency, which is particularly advantageous for producing an ex vivo rabbit B cell e described herein. A preferred embodiment described herein is therefore a method for increasing the replicative life span of a rabbit B cell, the method comprising: - ng, ing and/or maintaining expression of Bcl-6, or of a rabbit homologue thereof, in a rabbit B-cell and - inducing, enhancing and/or ining sion of at least one anti-apoptotic nucleic acid in said B-cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, or encoding a rabbit homologue thereof, or encoding a functional part or a functional derivative thereof, and/or with at least one anti-apoptotic nucleic acid molecule, via transduction with a gene delivery vehicle that ses the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or at least a functional part of said extracellular domain, or via uction with a gene delivery vehicle that comprises a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, or via transduction with a gene delivery vehicle that comprises a protein that has at least 70% sequence ty with at least a onal part of the extracellular domain of a GALV envelope protein. In one preferred embodiment, said extracellular domain is of an envelope n of GALV strain SEATO. Said extracellular domain preferably ses the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA DSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL. 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 understood by the skilled person, said extracellular domain, which is located at the surface (envelope) of a wild type gibbon ape leukemia virus so that it can bind a host cell, is ably also located at the surface (envelope) of a gene ry vehicle for transducing rabbit B cells. In one particularly preferred ment, a vector or other gene delivery vehicle is used that comprises an envelope protein which contains the extracellular domain and transmembrane domain of a GALV envelope protein, or a functional part f, which is fused to the cytoplasmic domain of an ampho envelope protein. This allows particular efficient transduction of rabbit B cells, as shown in the Examples.

As used herein, the term “a functional part of the extracellular domain of a GALV envelope protein” means a part of said extracellular domain which is still capable of binding rabbit B cells, thereby mediating infection and/or transduction of the rabbit B cells. Such functional part may lack one, or multiple, amino acid residues which are not essential for g, infection and/or transduction of rabbit B cells.

Of course, now that the insight of the present sure has been provided, rabbit B cells can be transduced with any nucleic acid molecule of interest using at least a functional part of the ellular domain of a GALV envelope protein. r described is therefore an isolated or recombinant rabbit B cell bound to the extracellular domain of a GALV envelope protein, or bound to at least a functional part of said extracellular domain, or bound to a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein. An isolated or inant rabbit B cell that is bound via at least a functional part of the extracellular domain of a GALV envelope protein, or via a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV pe protein, to a gene delivery vehicle is also described herewith. In one red embodiment, said extracellular domain is of an envelope protein of GALV strain SEATO. Said extracellular domain preferably ses the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL ATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT HGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL. Again, said sequence ty 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 ably 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 ably comprises a nucleic acid molecule of st, preferably a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or functional derivative thereof, and/or an anti-apoptotic nucleic acid sequence. With such gene delivery vehicle, a stabile rabbit B cell culture described herein can be produced. Said anti-apoptotic nucleic acid sequence is preferably an poptotic gene of the Bcl2 family, most preferably selected from the group consisting of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w, Bcl2L10, and rabbit homologues thereof, and functional parts and functional derivatives thereof.

A use of the extracellular domain of a GALV pe n, or at least a functional part thereof that is capable of binding a rabbit B cell, for introducing a nucleic acid molecule of interest into a rabbit B cell is also herewith described, as well as a use of a protein that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein for introducing a nucleic acid molecule of interest into a rabbit B cell. Further described is a use of a gene ry e comprising at least a functional part of the extracellular domain of a GALV envelope protein, or a gene delivery vehicle comprising a protein that has at least 70% sequence ty with at least a functional part of the extracellular domain of a GALV pe protein, said gene delivery vehicle further comprising a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a onal derivative f, and at least one anti-apoptotic nucleic acid sequence, for increasing the replicative life span of a rabbit B cell. In one preferred embodiment, said extracellular domain is of an envelope protein of GALV strain SEATO. Said extracellular domain preferably comprises the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES WDIPGTDVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW DQNSEWTQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL.

A preferred chimeric envelope protein, which is also used in the Examples, is shown in Figure 9. This chimeric pe protein contains the extracellular domain of a GALV envelope protein (with the sequence MVLLPGSMLLTSNLHHLRHQMSPGSWKRLIILLSCVFGGGGTSLQNKNP HQPMTLTWQVLSQTGDVVWDTKAVQPPWTWWPTLKPDVCALAASLES DVSSSKRVRPPDSDYTAAYKQITWGAIGCSYPRARTRMASSTFY VCPRDGRTLSEARRCGGLESLYCKEWDCETTGTGYWLSKSSKDLITVKW TQKFQQCHQTGWCNPLKIDFTDKGKLSKDWITGKTWGLRFY VSGHPGVQFTIRLKITNMPAVAVGPDLVLVEQGPPRTSLALPPPLPPREA PPPSLPDSNSTALATSAQTPTVRKTIVTLNTPPPTTGDRLFDLVQGAFLTL NATNPGATESCWLCLAMGPPYYEAIASSGEVAYSTDLDRCRWGTQGKLT LTEVSGHGLCIGKVPFTHQHLCNQTLSINSSGDHQYLLPSNHSWWACST GLTPCLSTSVFNQTRDFCIQVQLIPRIYYYPEEVLLQAYDNSHPRTKREA VSLTLAVLLGLGITAGIGTGSTALIKGPIDLQQGLTSLQIAIDADLRALQDS VSKLEDSLTSLSEVVLQNRRGLDLLFLKEGGLCAALKEECCFYIDHSGAV RDSMKKLKEKLDKRQLERQKSQNWYEGWFNNSPWFTTLL, indicated in bold in Figure 9) and the transmembrane domain of a GALV pe protein (with the sequence STIAGPLLLLLLLLILGPCII, indicated underlined in Figure 9), fused to the cytoplasmic domain of an ampho envelope n (with the sequence NRLVQFVKDRISVVQALVLTQQYHQLKPIEYEP, indicated in italics and -underlined in Figure 9). A vector or other gene delivery vehicle that comprises this preferred chimeric envelope protein is particularly well capable of introducing a c acid molecule of interest into rabbit B cells.

Further described is therefore an isolated or recombinant rabbit B cell bound to a chimeric envelope protein as depicted in Figure 9, or to a n comprising a chimeric envelope protein as depicted in Figure 9 or to a protein that has at least 70% sequence identity with a chimeric envelope protein as depicted in Figure 9. An isolated or recombinant rabbit B cell that is bound via a chimeric pe protein as depicted in Figure 9, or via a protein comprising a chimeric envelope protein as depicted in Figure 9 or via a protein that has at least 70% sequence identity with a chimeric envelope protein as depicted in Figure 9, to a vector or other gene delivery vehicle is also described herewith.

Such vector or other gene delivery vehicle is particularly suitable for transducing rabbit B cells with a nucleic acid molecule of interest. Further described is therefore a use of a chimeric envelope protein as depicted in Figure 9, or a protein comprising a chimeric envelope protein as depicted in Figure 9 or a protein that has at least 70% sequence identity with a ic pe protein as depicted in Figure 9, for introducing a nucleic acid molecule of interest into a rabbit B cell.

Such vector or other gene delivery vehicle is particularly suitable for increasing the replicative life span of rabbit B cells. Further described is ore a method for sing the replicative life span of a rabbit B cell, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6, or of a rabbit homologue thereof, in a rabbit B-cell and - inducing, enhancing and/or maintaining expression of at least one anti-apoptotic nucleic acid in said B-cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule ng Bcl-6, or encoding a rabbit homologue thereof, or encoding a functional part or a functional derivative thereof, and/or with at least one anti-apoptotic nucleic acid molecule, via transduction with a vector or other gene delivery e that comprises a chimeric pe protein as depicted in Figure 9, or a protein comprising a chimeric envelope protein as depicted in Figure 9, or a protein that has at least 70% ce identity with a chimeric envelope protein as depicted in Figure 9.

Also bed is a use of a gene delivery vehicle comprising a chimeric envelope protein as depicted in Figure 9, or a protein sing a chimeric envelope protein as ed in Figure 9, or a protein that has at least 70% sequence identity with a chimeric envelope protein as depicted in Figure 9, said gene delivery vehicle further comprising a nucleic acid sequence encoding Bcl-6 , or a rabbit homologue f, or a functional part or a onal derivative f, and at least one anti-apoptotic nucleic acid sequence, for increasing the replicative life span of a rabbit B cell.

It is emphasized that, although a GALV-based gene delivery e is very suitable for efficient transduction of rabbit B cells with one or more nucleic acid molecule(s) of interest, such as Bcl-6 and an anti-apoptotic nucleic acid molecule, the use of a GALV-based gene delivery vehicle is not mandatory for obtaining rabbit B cells with a short doubling time of 20 hours or less. Other gene delivery vehicles can also be used for introducing Bcl-6 and an anti-apoptotic nucleic acid molecule into rabbit B cells (although the efficiency will often be lower), in order to produce rabbit B cells with a doubling time of 20 hours or less. As long as Bcl-6 and an anti-apoptotic nucleic acid molecule are introduced into rabbit B cells, a fastgrowing B cell e can be obtained, although it may take longer for the lower amount of originally transduced rabbit B cells to grow out. An advantage of the use of a gene delivery e that is able to efficiently transduce rabbit B cells, such as a GALV-based gene delivery e as described , is that a higher proportion of the originally isolated B cells will be transduced, so that B cells derived therefrom will be present in the resulting B cell culture. This results in a higher ity of B cells within the B cell culture as ed to a situation wherein a gene delivery vehicle with a lower transduction efficiency is used, because in the latter case a lower proportion of the original B cells are transduced. The presence of a higher diversity of B cells within the resulting B cell culture improves the chance of isolating one or more B cells with a desired property. Hence, in principle, the higher the transduction efficiency of the gene delivery vehicle, the higher the diversity of B cells within the resulting B cell culture.

In order to induce expression in rabbit B cells, a nucleic acid molecule of interest is preferably operably linked to a er. Non-limiting examples include a CMV promoter and a CAG promoter. In one aspect, such promoter is inducible, meaning that its activity is influenced by at least one compound, such as for instance a ription factor.

As used herein, the term “gene delivery vehicle” means any compound capable of transferring a c acid molecule into a host cell. Non-limiting examples of gene delivery vehicles include (viral) vectors and plasmids. The term “gene delivery vehicle comprising at least a functional part of the extracellular domain of a GALV envelope protein” means a gene ry vehicle comprising at least part of the extracellular domain of a GALV envelope protein, wherein said extracellular domain, or said part thereof, is capable of binding a rabbit B cell so that nucleic acid can be introduced into said rabbit B cell. As described herein before, said extracellular domain, or part thereof, is preferably located at the surface of the gene delivery vehicle, so that it can bind a receptor on a rabbit B cell.

Likewise, if a gene ry vehicle described herein comprises a n that has at least 70% sequence identity with at least a functional part of the extracellular domain of a GALV envelope protein, said protein is preferably located at the surface of the gene delivery vehicle, so that it can bind a receptor on a rabbit B cell.

The percentage of identity of an amino acid or nucleic acid sequence, or the term “% sequence identity”, is defined herein as the percentage of residues in a candidate amino acid or c acid sequence that is identical with the residues in a reference sequence after aligning the two sequences and introducing gaps, if necessary, to achieve the maximum percent ty. Methods and computer programs for the alignment are well known in the art, for example "Align 2".

A GALV envelope protein is a protein that is naturally present in the viral pe of gibbon ape leukemia virus and that is involved in infection of host cells.

The target specificity is typically determined by the envelope protein. In one embodiment, said pe protein is of GALV strain SEATO. iral vectors containing the GALV envelope protein are known in the art and can be produced using procedures that are commonly used in the art of molecular biology, see for instance Lam et al., 1996.

The term “operably linked to a promoter” means that a nucleic acid sequence of interest is located sufficiently close to a promoter so that the promoter can influence expression thereof. Typically, such promoter will induce or increase expression of said nucleic acid of interest. The term “expression ty” refers to such induction or enhancement of expression.

As ned herein before, the present disclosure provides the insight that an ex vivo rabbit B cell culture can be ed with a r mean ng time as compared to currently known human or murine B cell cultures. This is all the more surprising because bbit compounds, such as a human Bcl-6 nucleic acid sequence, murine IL21 and human CD40L, were used in the current 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 ce. Even though human sequences were used, and the rabbit cells were ed in the presence of murine IL21 and human CD40L, the rabbit B cells surprisingly appeared to proliferate faster and to produce more antibody as compared to human and murine B cells.

Hence, according to the present dislosure, rabbit B cells proliferate very well using human and murine compounds. Under these reaction conditions, the rabbit B cells even proliferate better than human and murine B cells. This has amongst other things the age that currently used reaction conditions for human B cells do not have to be adjusted for rabbit B cells. There is no need to obtain rabbit IL21, rabbit CD40 or rabbit c acid sequences encoding Bcl-6 or an anti- apoptotic gene. Instead, currently ble human or murine nds can be used. One embodiment described herein is a method for increasing the replicative life span of a rabbit B cell, the method comprising: - inducing, enhancing and/or maintaining expression of Bcl-6, or a rabbit homologue thereof, in a rabbit B-cell and - inducing, enhancing and/or maintaining expression of 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: * a nucleic acid molecule encoding a non-rabbit Bcl-6 or a functional part or a functional derivative thereof; and * a non-rabbit anti-apoptotic nucleic acid molecule.

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

Furthermore, a method described herein further sing ing said rabbit B cell with IL21 and CD40L. Preferably, non-rabbit IL21 and/or non-rabbit CD40L is used. Preferably, said IL21 is murine or human IL21, most preferably murine IL21. In another preferred embodiment, said CD40L is murine or human CD40L, most preferably human CD40L.

Besides increasing Bcl-6 expression and the expression of an anti-apoptotic c acid molecule, it is also advantageous to , enhance and/or maintain expression of Blimp-1, or a rabbit homologue thereof, in a rabbit B-cell. This enhances antibody production of said B cell. Also described is a method according to the disclosure, wherein the method further comprises inducing, ing and/or maintaining expression of Blimp-1, or a rabbit homologue thereof, in said rabbit B-cell.

The extent of expression of Blimp-1, or of a rabbit homologue thereof, in a rabbit B cell is regulated in a variety of ways. In one embodiment a rabbit B cell is provided with a compound, which is capable of directly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue thereof. Additionally, or alternatively, a rabbit B cell is cultured in the presence of a compound capable of ly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue thereof. Further described is therefore a method described herein, further comprising: - providing said rabbit B cell with a compound capable of directly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue thereof; and/or - culturing said rabbit B cell in the presence of a compound capable of ly or indirectly increasing expression of Blimp-1, or expression of a rabbit homologue thereof.

Said compound capable of sing expression of Blimp-1, or of a rabbit homologue thereof, most preferably comprises IL21. Hence, in one preferred ment described , rabbit B cells are cultured in the ce of IL21, at least during part of the culture time.

In another embodiment said compound capable of increasing Blimp-1 sion ses a Signal Transducer of Activation and Transcription 3 (STAT3) protein or a functional part or a functional derivative thereof, and/or a nucleic acid molecule coding therefore. STAT3 is a signal transducer, which is ed in B cell pment and differentiation. STAT3 is capable of upregulating 1 expression. In one preferred embodiment, a rabbit B cell is provided with a nucleic acid molecule encoding STAT3 or a functional part or a onal derivative thereof, wherein the expression of said nucleic acid molecule is regulated by an ous inducer of repressor, so that the extent of STAT3 sion is regulated at will. For instance, one of the earlier mentioned inducible expression systems is used. In one embodiment a fusion product comprising STAT3, or a functional part or a functional derivative, and ER is used. For instance, a rabbit B cell is provided with a c acid molecule encoding an estrogen or (ER) and STAT3 as a fusion n ER-STAT3. This fusion protein is ve because it forms a complex with heat shock proteins in the cytosol. This way, STAT3 is unable to reach the nucleus and Blimp-1 expression is not enhanced. Upon administration of the exogenous inducer 4 hydroxy-tamoxifen (4HT), the fusion protein ER-STAT3 dissociates from the heat shock proteins, so that STAT3 is capable of entering the nucleus and activating Blimp-1 sion.

As used herein, a functional part of STAT3 is defined as a fragment of STAT3 that has the same capability - in kind, not necessarily in amount - of increasing sion of Blimp-1, or of a rabbit homologue thereof, as compared to natural STAT3. Such functional part is for instance devoid of amino acids that are not, or only very little, involved in said capability.

A functional derivative of STAT3 is defined as a STAT3 protein, which has been altered but has maintained its capability (in kind, not necessarily in amount) of increasing expression of Blimp-1, or of a rabbit homologue thereof. A functional tive is provided in many ways, for instance through conservative amino acid substitution wherein one amino acid is substituted by another amino acid with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is not seriously affected. Alternatively, a functional derivative for instance comprises a fusion protein with a detectable label or with an inducible Since STAT3 is capable of increasing expression of 1, or increasing expression of a rabbit homologue thereof, it is also possible to indirectly increase sion of Blimp-1, or of a rabbit homologue thereof, by administering a compound capable of increasing the activity and/or expression of STAT3. In one embodiment, a rabbit B cell is therefore ed with a compound that is capable of enhancing the activity of STAT3, so that expression of Blimp-1, or of a rabbit homologue thereof, is indirectly enhanced.

STAT3 is activated in a variety of ways. Preferably, STAT3 is activated by ing a rabbit B cell with a ne. Cytokines, being naturally involved in B cell differentiation, are very effective in regulating STAT proteins. Very effective activators of STAT3 are IL21 and IL6, but also IL2, IL7, IL10, IL15 and IL27 are known to activate STAT3. er, Toll-like receptors (TLRs), which are involved in innate immunity, are also capable of ting STAT3. One embodiment s to a method described herein, wherein said rabbit B cell is cultured in the presence of IL21, IL2, IL6, IL7, IL10, IL15 and/or IL27. Most ably IL21 is used, since IL21 is particularly suitable for enhancing antibody production of rabbit B cell cultures described herein. IL21 is capable of upregulating Blimp-1 expression, even when Blimp-1 sion is counteracted by BCL6.

Additionally, or alternatively a mutated Janus kinase (JAK) , or a mutated rabbit homologue of a JAK, is used in order to activate STAT3. Naturally, a JAK is e of orylating STAT3 after it has itself been activated by at least one cytokine. A mutated Janus kinase, or a mutated rabbit homologue of a JAK, capable of activating STAT3 independently of the presence of cytokines, is particularly suitable in a method described herein.

In yet another embodiment, expression of Blimp-1, or of a rabbit homologue thereof, is increased by providing a rabbit B cell with a suppressor of cytokine signalling (SOCS) protein, or a rabbit homologue thereof, and/or by activating a SOCS protein or a rabbit homologue thereof within said cell. Alternatively, or additionally, at least one of the E-proteins E47, E12, E2-2 and HEB is used in order to increase expression of Blimp-1, or of a rabbit homologue thereof. E47 is a transcription factor that belongs to a family of helix-loop-helix proteins, named 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 one gene, named E2A, which is d differently. E proteins have been described as tumor suppressors.

One of the specific targets of E47 are the Socs1 and Socs3 genes.

Also described is a method according to the present disclosure, further increasing expression of Blimp-1, or of a rabbit homologue thereof, in a rabbit B cell by ing said B cell with a compound capable of directly or indirectly increasing expression of Blimp-1, or of a rabbit homologue thereof, and/or culturing said B cell in the presence of a compound capable of directly or indirectly increasing sion of Blimp-1, or of a rabbit homologue thereof, wherein said compound comprises: - STAT3 or a onal part or a functional derivative thereof, and/or - a compound capable of activating STAT3, and/or - a compound capable of enhancing sion of STAT3, and/or - IL21, IL2, IL6, IL7, IL10, IL15, IL27, a SOCS protein, one of the E-proteins E47, E12, E2-2 or HEB, a mutated Janus kinase and/or a nucleic acid sequence encoding STAT3 , or a rabbit homologue or a functional part or a functional derivative thereof.

Most preferably, said compound is IL21.

Also bed is isolated or recombinant rabbit B cells obtainable with a method described herein. Such isolated or recombinant rabbit B cells preferably comprise an exogenous anti-apoptotic nucleic acid sequence and an exogenous nucleic acid sequence encoding Bcl-6 , or a rabbit gue thereof, or a functional part or a functional derivative f. Further bed is therefore an isolated or inant rabbit B cell, comprising an exogenous nucleic acid sequence encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and an exogenous anti-apoptotic nucleic acid ce. As explained before, said exogenous nucleic acid molecule either contains a nucleic acid sequence that does not naturally occur in rabbit B cells, or an additional copy of a natural rabbit B cell nucleic acid sequence. Bcl-xL, Mcl-1, Bcl- 2, A1, Bcl-w and Bcl2L10, and rabbit homologues thereof, are preferred antiapoptotic nucleic acid molecules. One preferred aspect therefore describes an isolated or recombinant rabbit B cell, which comprises an ous nucleic acid ce encoding Bcl-6 , or a rabbit homologue thereof, or a functional part or a functional derivative thereof, and an exogenous nucleic acid sequence encoding BclxL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a rabbit homologue thereof, or a onal part or a functional derivative thereof.

Said nucleic acid sequence ng Bcl-6 , or a rabbit gue thereof, or a functional part or a functional derivative thereof, and said exogenous antiapoptotic nucleic acid sequence may be present on one nucleic acid molecule.

Alternatively, these ces are present on at least two different nucleic acid molecules.

Preferably, non-rabbit sequences are used, as explained before. A preferred embodiment therefore describes an isolated or recombinant rabbit B cell comprising a bbit poptotic c acid sequence and a non-rabbit nucleic acid sequence encoding Bcl-6, or a rabbit homologue thereof, or a functional part or a functional derivative thereof. Said bbit nucleic acid sequence preferably contain human sequences.

In a particularly preferred embodiment, an ed or recombinant rabbit B cell is provided which comprises: - a nucleic acid sequence encoding human Bcl-6 or a functional part or a 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 human Bcl-w or human Bcl2L10, or a functional part or a functional derivative thereof. Again, said nucleic acid sequence encoding Bcl-6 or a functional part or a functional derivative thereof, and said anti-apoptotic nucleic acid sequence, may be present on one nucleic acid molecule, or, alternatively, these sequences may be present on at least two ent nucleic acid molecules.

Also described are ex vivo rabbit B cell cultures obtainable by the s described . An important advantage is the fact that ex vivo B cell cultures are now obtained with a short mean doubling time. Described is therefore an ex vivo rabbit B cell culture which has a mean doubling time of 20 hours or less. A further preferred embodiment describes an ex vivo rabbit B cell culture comprising rabbit B cells bed herein. Said rabbit B cells preferably comprise a nucleic acid sequence encoding human Bcl-6 or a functional part or a functional derivative thereof, and an anti-apoptotic nucleic acid sequence. Also described is an ex vivo rabbit B cell culture comprising rabbit B cells in the presence of non-rabbit IL21 and/or non-rabbit CD40L. Preferably, said IL21 is murine or human IL21, most preferably murine IL21. In another preferred ment, said CD40L is murine or human CD40L, most preferably human CD40L.

An antibody when obtained by a method described herein is also described herewith, as well as an antibody produced by a rabbit B cell described herein or by an ex vivo rabbit B cell culture described herein. Such antibody is particularly useful for therapeutic or stic applications. Preferably, said antibody is a monoclonal antibody.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting ents in this specification, and claims which include the term “comprising”, it is to be understood that other features that are additional to the features prefaced by this term in each statement or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in r manner.

In this ication where reference has been made to patent specifications, other external documents, or other sources of information, this is lly for the purpose of providing a context for discussing the features of the invention. Unless specifically stated ise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

In the description in this specification reference may be made to subject matter that is not within the scope of the claims of the current application. That subject matter should be y identifiable by a person skilled in the art and may assist in putting into practice the invention as defined in the claims of this application.

The invention is r explained in the following examples. These examples do not limit the scope of the invention, but merely serve to clarify the invention.

Examples Transduction of B cells Gene transfer into lymphocytes by traditional methods like calcium phosphate precipitation, liposome formation or electroporation is inefficient but more importantly stable gene integration is generally absent. Viral transduction however leads directly to stable gene integration into the genome of the target cell and can be very efficient if the proper virus envelope is chosen. Both retroviral and lentiviral transductions are le for efficient gene transfer. While iral integration is dependent on cell division, lentiviral transduction can also be applied to non-dividing cells like plasma B cells. Large-scale preparation of inant retrovirus can easily be achieved by using stable producer cell lines such as the Phoenix expression platform (Kinsella and Nolan, 1996). tion of high titer lentivirus tends to be more cumbersome mainly because of the toxicity of the sed virus proteins and envelopes.

For the current es, we used a Moloney Murine Leukemia Virus (MMLV) based platform, using either amphotropic or Gibbon Ape Leukemia Virus (GALV) envelope expressing producer cells (Wilson et al., 1995). In our GALV-based vector, the transmembrane domain of the GALV strain SEATO envelope protein was fused to the asmic domain of an ampho envelope protein (Figure 9).

The transfer vector is set-up such that Bcl-6, Bcl-xL and the green fluorescent protein (GFP) marker protein are simultaneously translated from the same viral RNA (Figure 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 an Internal mal Entry Sequence (IRES) upstream of the GFP reporter gene. Viral transduction encies are high and unbiased.

Generation of alized rabbit B cells Human memory B cells were immortalized using the BCL-6 / Bcl-xL technology described by nbos et al., 2010 and patent application . In brief, PBMC’s from rabbit blood were isolated using a ficoll density gradient and d for Ig sion using an antibody that recognizes Ig (IgG H+L: IgG heavy chain and kappa and lambda light chains) mes in combination with an IgM specific 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 harvested and washed with medium without FCS and cells were then transferred to Retronectin® (Takara, Shiga, Japan)- coated tissue culture plates where they were transduced with a retroviral vector containing BCL-6, , and GFP as a reporter protein.

Alternatively cells were transduced with a retroviral vector containing BCL-6, Mcl- 1 and GFP. Transduced B cells were maintained in culture with CD40 Ligand sing L-cells and IL-21. In Figure 1 the transduction efficiency is compared for GALV and amphotropic type retroviruses at 4 days after transduction. Four days after transduction with the amphotropic type retrovirus 0.8% of the cells was transduced compared to 80% of cells after transduction with a GALV type retrovirus. Clearly the GALV type retrovirus is superior to the amphotropic type retrovirus for ucing rabbit B cells.

Example 2 Cell culture.

We maintained B cells (2 × 105 cells ml−1) in Iscove’s modified co’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 ine cell doubling time cells were cultured in 24-well plates at 50-100.000 cells/well together with CD40L- L cells and IL-21. Every 3-4 days cell were d and 50-100.000 cells transferred to a new well. In Figure 2 growth curves are depicted for B cells from two human donors (89 and 93), one llama B-cell sample (Llama) and one rabbit B-cell sample which was transduced with a GALV type retrovirus carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL (Rb 6XL). Also a growth curve is depicted for one rabbit sample that was transduced with a GALV type retrovirus carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Mcl-1 (Rb 6M). The transduced rabbit B cells have an average doubling time of 19 hours and thus grow faster than the human or llama B cells that have doubling times between 26 and 32 hours. These average doubling times were originally calculated by determining the increase of B cells during l 3-4 days time intervals, and averaging the obtained results. Subsequently, the overall average doubling time during the whole culturing period was calculated. This resulted in an e doubling time of the transduced rabbit B cells of 18 hours, an e doubling time of the transduced human B cells of 25-29 hours and an average doubling time of the transduced llama B cells of 27 hours. This ms our observations that our methods yield rabbit B-cell cultures with a mean doubling time of 20 hours or less, whereas human, murine and llama B cells typically have a doubling time of between 25 and 36 hours.

Example 3 B-cell receptor sion and antigen-specific staining Immortalized human B cells express the B-cell receptor. This quality enables antigen-specific staining and sorting of B cells. To determine r the B-cell receptor is also expressed on uced rabbit B cells, B-cell clones are stained with fluorescently labeled antibodies reacting ically 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 with cell culture medium containing immunofluorescently labelled antibodies that are specific for either rabbit IgG, IgM, IgA or labelled antigen. Afterwards excess of labelled antibodies or antigen was washed away and B-cell receptor expression analysed on a FACS analyser; the Guava easycyte pore) or FACS Aria3 (BD).

In Figure 3 three different B-cell clones of different isotypes were d with fluorescently labelled antibodies specifically recognizing rabbit antibody isotype IgG, IgA or IgM. Clearly the B-cell receptor can be efficiently stained for the different rabbit antibody isotypes. We therefore conclude that immortalized rabbit B cells also express the B-cell receptor.

In addition, also fluorescently labeled influenza proteins were used to stain for influenza-specific B-cells from rabbits that had been immunized with a human influenza vaccine or untreated control s e 4). Rabbit B cells were stained with fluorescently labeled H1, H3 or nza B and sorted 1 cell per well using a FACS sorter. e 4 Development of single-cell derived, clonal rabbit B cell cultures.

Transduced B cells were sorted one cell per well using a FACS sorter and ed 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.

Every 3-4 days fresh CD40L- L cells and IL-21 were added. Starting 9 days after seeding the cells (one cell per well), the supernatants were analyzed in ELISA for the production of rabbit immunoglobulin G (IgG). For comparison also the human IgG in the supernatant of human B-cell clones was analyzed in parallel.

In Figure 7 the antibody concentration in the supernatant is depicted over time starting at 9 days after the initiation of the single cell cultures. The antibody concentration was determined for two human donors and one rabbit B-cell sample that were transduced with a GALV type retrovirus carrying a nucleic acid le containing a human Bcl-6 sequence and a human Bcl-xL and for one rabbit B-cell sample that was uced with a GALV type retrovirus carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Mcl-1. B cell clones from rabbits produce IgG concentrations of 30 ng/ml and 100 ng/ml within a r time period (9-10 days and 11-12 days, respectively) than do the human B- cell clones (13-18 and 15-20 days, respectively). This provides the important advantage that it allows for earlier screening for dies of interest of rabbit B cell clones, compared to human B cell clones.

Immunization of rabbits. 2 New Zealand White rabbits were immunized with a human influenza vaccine containing 15ug H1N1, 15ug H3N2 and 15ug infl B in complete Freunds adjuvans.

After 3 weeks rabbits were boosted with the same vaccine in lete Freunds adjuvans. Five days after the boost rabbits were bled, B-cells were isolated from the blood and transduced with a GALV type irus (containing the extracellular domain and transmembrane domain of the GALV strain SEATO envelope protein, fused to the cytoplasmic domain of an ampho envelope protein) carrying a nucleic acid molecule containing a human Bcl-6 sequence and a human Bcl-xL. Transduced B cells were seeded at different cell ies into culture plates and cultured as described in Example 4. Also, transduced B cells were labeled with scently labeled components of the vaccine; H1, H3 or influenza B and sorted 1 cell per well using a FACS sorter and cultured as described in Example 4. The atants of the cultured cells were analyzed for g to the complete vaccine or to its individual components. The results are depicted in Figures 4-6 and show that antigen-specific B cells can be identified in the B-cell pool from vaccinated rabbits by seeding cells at different density (Figure 5) and also very efficiently by sorting cells using the labeled antigens (Figure 4 and Figure Example 6 Rabbit B cells are immortalized by the introduction of the genes Bcl-6 and Bcl-xl using an amphotropic type retrovirus.

Immortalization of rabbit B cells by introduction of the genes Bcl-6 and Bcl-xl can be achieved by using different types of vectors, such as for instance GALV and amphotrophic type retroviruses as is shown in Example 1. The growth of B cells transduced with the amphotrophic type retrovirus was further pursued to confirm that introduction of Bcl-6 and Bcl-xl by amphotrophic retrovirus also leads to immortalization of rabbit B cells. Four days after transduction with the amphotropic type retrovirus 0.8% of the cells was uced compared to 80% of cells after transduction with a GALV type irus (Figure 1 and Figure 10). Ten days after uction 94% of the cell population transduced with the amphotrophic retrovirus was GFP ve demonstrating that the transduced cells overgrow the non-transduced cells (Figure 10).

To ine cell doubling time cells were cultured as done in Example 2 in 24- well plates at 50-100.000 cells/well together with CD40L- L cells and IL-21. Every 3-4 days cell were counted and 50-100.000 cells transferred to a new well. In Figure 11 the growth curve is depicted for rabbit B cells transduced with amphotrophic virus. The calculated doubling time is 19 hours, which is comparable to rabbit B cells transduced with GALV type retrovirus (18 hours). In conclusion, introduction of Bcl-6 and Bcl-xl into rabbit B cells by rophic retrovirus also results in immortalization of rabbit B cells, gh the transduction efficiency is much lower as compared to a GALV based vector.

Brief description of the drawings Figure 1.

Transduction of rabbit memory B cells. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for rs on CD40L L-cells with rm-IL-21. Cells were transduced with a retroviral vector containing BCL6 and Bcl- xL. Both GALV and amphotropic type iruses were . Transduced cells are then cultured on CD40L-L cells in the presence of recombinant mouse IL-21.

After four days of culture the transduction efficiency was determined based on GFP expression. GALV typed retrovirus showed superior (80%) transduction efficiency compared to amphotropic (0.8%) typed retrovirus.

Figure 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 analysed in parallel B cells from llama cells and human cells from two different donors that were transduced with an cal retroviral vector containing BCL6 and Bcl-xL. Figure 2 also shows transduced rabbit B cells grow rapidly.

Doubling time: Human B cells: 25-29 hrs Rabbit B cells 6XL: 18hrs Rabbit B cells 6M: 18hrs Llama: 27 hrs.

Figure 3.

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

Figure 4. fication of antigen-specific rabbit B-cells within a pool of rabbit B cells with ent specificities.

Figure 5.

Antigen-specific rabbit antibodies were obtained against the different components of a human influenza vaccine containing 15ug H1N1, 15ug H3N2 and 15ug infl B .

Rabbits were immunized and boosted with the human influenza vaccine. B cells were immortalized and seeded at different densities in 384-well plates on CD40L-L cells in the ce of recombinant mouse IL-21. Antibodies present in the rabbit B cell e supernatants were screened in ELISA for influenza-specificity.

Antigen-specific antibodies were observed for all the components of the vaccine.

Figure 6.

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

Figure 7.

Antibody concentration in the supernatant of clonal B cells at different time points.

Human, llama and rabbit transduced B cells were seeded 1 cell per well in the ce of irradiated CD40L- L cells and mented with mouse IL-21. Every 3-4 days CD40L- L cells and IL-21 were replenished. The IgG concentration was analyzed in ELISA for individual wells at different time points during culture.

Each measurement was done on different wells. The rabbit B cells were either transduced with a retroviral vector containing BCL6 and Bcl-xL or a retroviral vector containing BCL6 and Mcl-1. All other cells (human and llama) were transduced with BCL6 and Bcl-xL.

Figure 8.

Schematic representation of the vector used to transduce the rabbit and human B cells Figure 9.

Sequence of the extracellular domain of GALV SEATO envelope protein (bold) and the transmembrane domain of the GALV SEATO envelope protein (underlined), fused to the cytoplasmic domain of ampho envelope protein (italics + dottedunderlined Figure 10.

Transduction of rabbit memory B cells and wth of rabbit B cells transduced with amphotrophic type retrovirus. Rabbit B cells were isolated from PBMCs based on Ig expression. Cells were activated for 36-40hrs on CD40L L-cells with rm-IL-21. Cells were transduced with a retroviral vector containing BCL6 and Bcl- xL. Both GALV and amphotropic type iruses were tested. Transduced cells were then cultured on CD40L-L cells in the presence of recombinant mouse IL-21.

After four days of e the transduction efficiency was determined based on GFP expression. GALV typed irus showed superior (80%) transduction efficiency compared to amphotropic (0.8%) typed irus. After 10 days 94% of rabbit B cells transduced with amphotrophic type retrovirus were immortalized based on GFP expression showing outgrowth of transduced cells over non-transduced cells.

Figure 11.

A growth curve was analyzed for rabbit B cells transduced with a amphotrophic type retroviral vector containing BCL6 and Bcl-xL. Figure 11 also shows Rabbit cells transduced with ampho-coated virus grow rly fast as cells transduced by GALV-coated virus.

Doubling time: Rabbit B cells Ampho 6XL: 19 hrs.

References Christopherson, K.S. et al. PNAS 89, 6314-8 (1992) Guzman, L. M. et al. Bacteriol 177, 4121–4130 (1995) T.M. la, G.P. Nolan, Hum Gene Ther 7 (1996) 1405.

Kwakkenbos et al. tion 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. i, Nat Biotechnol 22 (2004) 589.

C.A. Wilson, M.V. Eiden, W.B. Anderson, C. Lehel, Z. Olah, J Virol 69 (1995) 534.

Claims (22)

Claims

1. A method for obtaining an ex vivo B cell culture with a mean ng time of 20 hours or less, the method comprising: 5 - inducing, enhancing or maintaining sion of Bcl-6 in a B cell, - inducing, enhancing or maintaining expression of at least one antiapoptotic nucleic acid molecule comprising a gene of the Bcl-2 family in said B cell, 10 characterized in that said B cell is a rabbit B cell.

2. A method for increasing the ative life span of a rabbit B cell, the method comprising: - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B 15 cell, and - ng, enhancing or ining expression of at least one antiapoptotic nucleic acid comprising a gene of the Bcl-2 family in said B cell, characterized in that said rabbit B cell is provided with a nucleic acid molecule encoding Bcl-6, with at least one anti-apoptotic nucleic acid 20 molecule comprising a gene of the Bcl-2 family, or with a ation thereof, via transduction with a gene delivery vehicle that comprises the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein or a protein that has at least 70% sequence ty with the extracellular domain of a GALV envelope protein.

3. Use of the extracellular domain of a gibbon ape leukemia virus (GALV) envelope protein, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, for introducing a nucleic acid molecule encoding Bcl-6 and at least one anti- tic nucleic acid molecule comprising a gene of the Bcl-2 family into a rabbit B cell.

4. A method for obtaining antibodies, sing: 5 - inducing, enhancing or maintaining expression of Bcl-6 in a rabbit B cell; - inducing, enhancing or maintaining sion of at least one optotic nucleic acid molecule comprising a gene of the Bcl2- family in said B cell; 10 - culturing said B cell ex vivo; and - harvesting antibodies produced by said B cell within 7-14 days.

5. The method of claim 4, wherein antibodies produced by said B cell are harvested within 9-12 days.

6. The method according to any one of claims 1-2, and 4-5, characterized in that said rabbit B cell is provided with: * a nucleic acid molecule encoding a non-rabbit Bcl-6, or * at least one non-rabbit anti-apoptotic nucleic acid molecule 20 comprising a gene of the Bcl-2 family, or a combination thereof.

7. The method according to claim 6, wherein said bbit nucleic acid molecule is an anti-apoptotic nucleic acid molecule comprising a human 25 gene of the Bcl-2 family or a nucleic acid molecule encoding a human Bcl-6.

8. The method ing to any one of claims 1-7, wherein said gene of the Bcl-2 family is selected from the group consisting of Bcl-xL, Mcl 1, Bcl-2, 30 A1, Bcl-w and Bcl2L10.

9. The method ing to any one of claims 1-2 and 4-8, wherein the method also comprises ng, enhancing or maintaining expression of Blimp 1 in said rabbit B cell.

10. The method according to any one of claims 1-2 and 4-9, further comprising providing said rabbit B cell with IL21 and CD40L.

11. The method according to claim 10, wherein said IL21 is mouse or 10 human IL21, or wherein said CD40L is mouse or human CD40L, or a combination thereof.

12. The method according to any one of claims 1-2 and 4-11, further comprising: 15 - providing said rabbit B cell with a nucleic acid molecule encoding STAT3; or- culturing said rabbit B cell in the presence of IL21, or a combination thereof.

13. A rabbit B cell, which is bound via the extracellular domain of a 20 GALV pe protein, or via a protein that has at least 70% sequence identity with the extracellular domain of a GALV pe protein, to a gene delivery vehicle that comprises a nucleic acid sequence encoding Bcl-6 and an anti-apoptotic nucleic acid sequence comprising a gene of the Bcl-2 family.

14. The rabbit B cell ing to claim 13, 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, 0, and any combination thereof.

15. An isolated or recombinant rabbit B cell comprising: - a non-rabbit anti-apoptotic nucleic acid molecule comprising a gene of the Bcl-2 family, and - a non-rabbit c acid molecule encoding Bcl-6.

16 The isolated or recombinant rabbit B cell according to claim 15, wherein the gene of the Bcl-2 family encodes Bcl-xL, Mcl-1, Bcl-2, A1, Bcl w, or 0. 10

17. The rabbit B cell according to claim 15 or 16, wherein said non-rabbit nucleic acid molecule comprises a human gene of the Bcl-2 family, or s human Bcl-6, or a combination thereof.

18. An ex vivo rabbit B cell e comprising the rabbit B cells 15 according to any one of claims 13-17, which has a mean doubling time of 20 hours or less.

19. An ex vivo rabbit B cell culture when obtained by a method according to any one of claims 1 and 6-12.

20. Use of a gene delivery vehicle comprising the extracellular domain of a gibbon ape leukemia virus (GALV) envelope n, or a protein that has at least 70% sequence identity with the extracellular domain of a GALV envelope protein, and a nucleic acid sequence encoding Bcl-6 and at least 25 one anti-apoptotic nucleic acid sequence comprising a gene of the Bcl-2 family, for increasing the ative life span of a rabbit B cell.

21. The method according to claim 2, the use according to claim 3, or the rabbit B cell according to claim 13 or 14, or a use according to claim 20, wherein said extracellular domain is of an envelope protein of GALV strain SEATO.

22. The method according to claim 2, or the rabbit B cell according to 5 claim 13 or 14, or the use according to claim 20, n said gene delivery vehicle comprises a chimeric pe protein as depicted in