NZ723331B2

NZ723331B2 - Means and methods for producing stable antibodies

Info

Publication number: NZ723331B2
Application number: NZ723331A
Authority: NZ
Inventors: Adrianus Quirinus Bakker; Mark Jeroen Kwakkenbos; Koen Wagner
Original assignee: Kling Biotherapeutics Bv
Priority date: 2014-01-31
Filing date: 2015-01-30
Publication date: 2021-11-30

Abstract

The present invention provides methods for obtaining, from an ex vivo B cell culture, B cells that are capable of producing antibodies with a higher stability and with the same or a lower binding affinity, as compared to an initial parent antibody. These B cells are obtained by selecting B cells with a higher binding avidity than the average binding avidity of the ex vivo B cell culture. Antibodies produced by these high avidity B cells are tested in order to determine their affinity and their thermal stability and/or their resistance to aggregation and/or their chemical stability. h a higher binding avidity than the average binding avidity of the ex vivo B cell culture. Antibodies produced by these high avidity B cells are tested in order to determine their affinity and their thermal stability and/or their resistance to aggregation and/or their chemical stability.

Description

(12) Granted patent speciﬁcaon (19) NZ (11) 723331 (13) B2 (47) Publicaon date: 2021.12.24 (54) MEANS AND METHODS FOR ING STABLE DIES (51) aonal Patent ﬁcaon(s): C07K 16/00 C07K 16/10 C12N 5/0781 (22) Filing date: (73) s): 2015.01.30 KLING BIOTHERAPEUTICS B.V. (23) Complete speciﬁcaon ﬁling date: (74) Contact: 2015.01.30 AJ PARK (30) Internaonal Priority Data: (72) Inventor(s): EP 14153480.0 2014.01.31 KWAKKENBOS, Mark Jeroen BAKKER, Adrianus Quirinus (86) Internaonal Applicaon No.: WAGNER, Koen (87) Internaonal Publicaon number: WO/2015/115892 (57) Abstract: The present invenon provides methods for obtaining, from an ex vivo B cell culture, B cells that are capable of producing anbodies with a higher ity and with the same or a lower binding aﬃnity, as compared to an inial parent anbody. These B cells are obtained by selecng B cells with a higher binding avidity than the average binding avidity of the ex vivo B cell culture.

Anbodies produced by these high avidity B cells are tested in order to determine their aﬃnity and their thermal stability and/or their resistance to aggregaon and/or their chemical stability. 723331 B2 Title: Means and methods for ing stable dies The invention relates to the fields of medicine, molecular biology and immunology. More specifically, the invention relates to the field of antibodies.

Ex vivo B cell cultures are important tools for producing antibodies, preferably monoclonal antibodies. Monoclonal antibodies (mAbs) represent multiple cal copies of a single dy molecule, which copies bind to antigens with the same affinity and promote the same effector functions. Amongst the ts of mAbs is their specificity for the same e on an antigen. This specificity confers n 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 biological and medical research.

A conventional approach for obtaining mAbs is hybridoma technology, wherein a B cell is fused with a a 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 hybridomas are unstable.

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

This technology involves human ex vivo cultures wherein BCL6, together with Blimp-1 and/or an anti-apoptotic nucleic acid, are expressed in the B cells. This improves the replicative life span of these B cells. Typically, human B cells are cultured in order to obtain human mAbs. Human mAbs are red for therapeutic applications in humans due to the lower immunogenicity as compared to antibodies of other species.

One of the problems faced when commercially producing antibodies, e.g. for pharmaceutical or research applications, is to obtain antibodies that are stable enough for instance for production in large quantities, for stration to ts and/or for long-term storage. Considerable effort is put in increasing the stability of in particular therapeutic antibodies. In the early phases of research and development of antibodies with a specificity and high affinity for an antigen of interest, stability is typically not a property that is taken into account. Instead, stability of antibodies is altered by introducing mutations in the encoding nucleic acid and testing the resulting antibodies for their stability once dies with the desired specificity and affinity are identified. Given the cost of producing these mutated antibodies and the time involved, alternative methods for obtaining stable antibodies are desired.

It is an object of the present invention to provide means and s for selecting B cells capable of producing stable antibodies and for producing such stable antibodies, as well as for producing B cells capable of producing such stable antibodies; and/or to at least provide the public with a useful choice. In particular, the method of the present invention allow for the ion of stable antibodies at an early stage in the development process.

Summary of the invention In a first aspect the present invention provides an ex vivo method for producing a B cell capable of producing antibody for an antigen of interest comprising: a) selecting at least one B cell capable of producing antibody specific for said n of interest or at least one B cell capable of developing into a B cell capable of ing antibody specific for said antigen of interest; b) providing said at least one B cell with a c acid encoding BCL6 and with an anti-apoptotic nucleic acid of the BCL2 family; c) allowing expansion of said at least one B cell into a first B cell e; d) selecting at least one B cell from said first B cell culture with a binding avidity for said antigen of interest that is higher than the average g y for said antigen of interest of B cells in said first B cell culture; e) optionally allowing expansion of said at least one B cell selected in step d) into a second B cell culture; f) ining the thermal stability and/or the resistance to aggregation and/or the chemical stability, and the affinity for said antigen of interest, of antibodies produced by said at least one B cell ed in step d) or by said second B cell culture; and g) selecting at least one B cell capable of producing antibodies with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by B cells of said first B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average ty for said antigen of interest of antibodies produced by said first B cell culture.

In a second aspect the present invention provides a method for isolating from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by said ex vivo B cell culture, and with an affinity for said antigen of st that is the same as, or less than, the average affinity for said antigen of st of antibodies ed by said ex vivo B cell e the method comprising: a) ing an ex vivo B cell culture e of producing antibody specific for an n of interest; b) selecting at least one B cell from said ex vivo B cell culture with a binding avidity for said antigen of interest that is higher than the e binding avidity of B cells of said ex vivo B cell e for said antigen of interest; c) determining the thermal stability and/or the resistance to aggregation and/or the chemical stability, and the affinity for said n of interest, of antibodies produced by said at least one B cell selected in step b); and d) isolating at least one B cell capable of producing dies with a higher thermal stability and/or ance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by B cells of said ex vivo B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture.

In a third aspect the present invention es a method for producing antibodies specific for an antigen of interest, comprising: - selecting from an ex vivo B cell culture at least one B cell capable of producing antibody with a higher thermal stability and/or ance to aggregation and/or chemical stability as ed to the e thermal ity or resistance to aggregation or chemical stability of antibodies produced by said ex vivo B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture, with a method according to the second aspect; - culturing said at least one B cell into a B cell culture; and - obtaining antibodies produced by said B cell culture.

In a fourth aspect the present invention provides a method for producing antibodies specific for an antigen of st, comprising: - selecting from an ex vivo B cell culture at least one B cell capable of producing antibody with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the e thermal stability or resistance to aggregation or chemical stability of antibodies produced by said ex vivo B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture, with a method according to the second aspect; - optionally allowing ion of said selected at least one B cell into a B cell culture; - determining the amino acid sequence of the heavy chain and/or light chain of said antibody with a higher stability; and - expressing a nucleic acid le encoding the heavy chain and/or light chain of said antibody in a second cell.

In a fifth aspect the present invention provides a method for fying at least one mutation in the amino acid sequence of the heavy chain and/or light chain of an antibody as compared to the amino acid sequence of the heavy chain and/or light chain of a reference antibody, which on promotes thermal stability and/or resistance to aggregation and/or chemical stability of said antibody, comprising: - ing from an ex vivo B cell culture at least one B cell capable of producing antibody with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to ation or chemical stability of antibodies produced by said ex vivo B cell e. and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture, with a method according to the second aspect; - optionally allowing expansion of said selected at least one B cell into a B cell culture; - determining at least part of the amino acid sequence of the heavy chain and/or light chain of an antibody produced by said ed at least one B cell; - comparing said amino acid sequence with at least part of the amino acid ce of the heavy chain and/or light chain of a nce antibody, y identifying at least one mutation in the amino acid sequence in the heavy chain and/or light chain of said antibody which on promotes thermal stability and/or ance to aggregation and/or chemical stability of said antibody.

Also described is a method for producing a B cell, preferably a B cell culture, capable of producing antibody for an antigen of st comprising: a) selecting at least one B cell e of producing antibody specific for said antigen of interest or at least one B cell capable of developing into a B cell capable of producing antibody specific for said antigen of interest; b) inducing, enhancing and/or maintaining expression of BCL6 and inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid in said at least one B cell; c) allowing expansion of said at least one B cell into a first B cell culture; d) selecting at least one B cell from said first B cell culture with a binding avidity for said antigen of interest that is higher than the average binding avidity for said antigen of interest of B cells in said first B cell culture; e) preferably allowing expansion of said at least one B cell selected in step d) into a second B cell culture; f) determining the stability of antibodies produced by said at least one B cell selected in step d) or by said second B cell culture; and g) selecting at least one B cell capable of producing antibodies with a higher stability as compared to the average ity of antibodies produced by B cells of said first B cell culture. Preferably, said at least one B cell selected in step g) is expanded into a further B cell culture.

Further bed is a method for selecting from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies ed by said ex vivo B cell culture, the method comprising: a) providing an ex vivo B cell culture capable of producing antibody specific for an antigen of interest; b) selecting at least one B cell from said ex vivo B cell culture with a binding avidity for said antigen of interest that is higher than the average binding avidity of B cells of said ex vivo B cell culture for said antigen of interest; c) determining the stability of dies produced by said at least one B cell selected in step b); and d) selecting at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies produced by B cells of said ex vivo B cell e.

As used herein fic for an antigen (of interest)” and "capable of ically binding an antigen (of interest)" refer to the interaction n an antibody and its antigen, meaning that said antibody preferentially binds to said antigen over other antigens. Thus, although the antibody may non-specifically bind to other antigens, the affinity of said dy for its antigen is significantly higher than the non-specific affinity of said antibody for any other antigens. A B cell capable of producing antibody specific for an n of st is for instance obtained by isolation of memory B cells from peripheral blood, followed by staining with labelled antigen and isolation of antigen-bound B cells as bed by Kwakkenbos et al. (Nature medicine, 16(1), 123–128. doi:10.1038/nm.2071; Methods 2013 doi:10.1016/j.ymeth.2013.07.002; patent application WO 2007/067046).

A method described herein involves determining the binding avidity of at least one B cell and determining the average binding avidity of B cells of the B cell culture from which the at least one B cell originates for an antigen of interest.

Subsequently, a B cell is selected with a higher binding avidity than the average binding avidity of B cells of the B cell culture. As used herein the term “binding avidity” refers to the accumulated strength of all interactions contributing to the binding of a B cell to an antigen of interest. A factor that contributes to the binding of a B cell to an antibody of interest is the number of B cell receptors expressed on the cell surface of the B cell. Indeed, it was confirmed in that when ed for binding capacity for a given n, B cells sorted for a relatively low binding capacity to an antigen did express less immunoglobulin on the surface of the B cells as compared to B cells sorted for higher binding.

Nevertheless, when the B cells with higher antigen binding were cultured, they appeared to produce antibodies that had a higher affinity for the antigen as compared to antibody produced by the B cells with lower antigen binding. In it was further surprisingly found that if such higher affinity B cell selected from the first population based on its antigen g is expanded into a second tion of B cells, the B cells in this second population retain the higher affinity of the selected B cell d of returning to the average affinity of B cells in the first population as would be expected. Hence, affinity of secreted antibody for the antigen is also correlated with binding capacity of a B cell for a given n. In it was therefore concluded that B cells that e antibodies with a high affinity for an antigen of interest can be selected and further cultured while maintaining high ty based on high antigen g of the selected B cell to the antigen.

The present disclosure provides the insight that another factor that correlates with the binding avidity of a B cell for a specific antigen is the stability of antibody produced by said B cell. The present inventors surprisingly found that, in addition to a subset of the B cells ed for having a high binding avidity for an n that indeed es dies with a high binding affinity for the antigen as described in , another subset of B cells selected for having a high g avidity for the antigen produces antibodies that have a high stability. It was further found that the highly stable antibodies produced by this subset of B cells with a high binding avidity do not necessarily have a high affinity for the antigen. For instance, a subset of B cells with a high binding avidity for an n produces antibodies that are highly stable and that have an average affinity for the n; a second subset of B cells with a high binding avidity for an antigen produces antibodies that are highly stable and that have a high ty for the antigen; a third subset of B cells with a high binding avidity for an antigen produces antibodies that have an average stability and a high affinity for the antigen; a fourth subset of B cells with a high binding avidity for an antigen produces dies that are instable but have a high affinity for the antigen; etc.

The present disclosure for the first time establishes a correlation between the binding avidity of B cells for an antigen of interest and the ity of antibodies produced by these B cells. Further, the present disclosure shows that spontaneous mutations in the genes ng antibodies occur while the antibodies maintain their specificity and/or affinity for a specific n, for which a pre-selection can be made by determining the binding avidity of B cells producing the antibodies. r, the present disclosure for the first time describes the selection of ity of antibodies by determining the characteristics of B cells producing the dies.

Without being bound to any theory, it is believed that differences in the affinity of antibodies for an antigen of interest or differences in stability of antibodies within a population of monoclonal B cells may result from processes mediated by Activation Induced Cytidine Deaminase (AID). n-activated naïve and memory B cells in the germinal centre undergo extensive proliferation, accompanied by somatic hypermutations (SHM) and switch recombination (CSR) of Ig genes mediated by AID. AID deaminates deoxycytidine residues in immunoglobulin genes, which triggers antibody ification. The expression of AID in (a B cell which will develop into) an antibody producing B cell allows the generation of novel immunoglobulins that harbor mutations that were not present in the original B cell before transduction with BCL6 and an anti-apoptotic c acid. Thus, culturing B cells in which somatic hyper mutation is induced by expression of AID allows the generation of immunoglobulin variants which, for example, have a higher or lower affinity for an antigen of interest, or that are more stable.

Although describes that culturing of B cells wherein somatic hypermutation due to AID activity is induced may result in antibody variants which have a higher stability, it is also clear from that the described selection methods wherein B cells are ed based on their binding capacity only do not specifically select for stable antibodies. d, ses that variants with a higher affinity for the antigen of interest are ed using such selection methods. Accordingly, WO 72814 repeatedly refers to “high affinity B cells ing to the invention”. Contrary, the present disclosure describes selection methods for ically identifying B cells which have a higher stability.

A method described herein, using B cells, thus provides the advantage that the stability of antibodies can easily be taken into account and improved already during the early stages of development of antibodies, for instance therapeutic antibodies. This reduces or obviates the need to improve stability of antibodies that have already been selected based on their binding and/or affinity characteristics by introducing mutations in the nucleic acid encoding the antibody and testing the resulting antibodies for their stability much later in the development s. Such recombinant methods to e stability of antibodies specific for an antigen of interest currently used generally first require determination of the amino acid ce of the dy. Subsequently one or more mutations are introduced into the sequence of the nucleic acid encoding the antibody, at multiple possible locations in the nucleic acid sequence so that a large number of mutated antibodies can be produced. Then, the genes containing one or more mutations need to be expressed in a cell followed by production of antibodies in producer cells. Finally, the mutated antibodies are tested for their ity in order to ine whether antibody with an improved stability is obtained. Such a process for improving the stability of an antibody is elaborate and time-consuming.

A method described herein allows the selection of stable antibody in a straight- forward and less elaborate process without the need of molecular engineering in the same stage of development as selecting for g avidity and/or affinity of the antibody. Using a method described herein antibodies are produced in ex vivo B cell cultures. Once B cells capable of ing antibodies specific for an antigen of interest have been obtained the B cells can be cultured during which differences in affinity and stability occur as a result of mutations introduced during such culturing. The B cell culture thus consists of a vast amount of B cells which are all specific for the antigen of interest but which vary in the affinity for the antigen and in the stability of the antigen. In this e of antigen specific B cells, a small subgroup of B cells will have a particularly high stability as compared to the average stability of B cells in the B cell culture. Before the present disclosure, considering the small amount of B cells producing antibodies with the desired high stability, selection for stability of antibodies would have required testing of antibodies produced by a large number of B cells in order to identify antibodies with the desired high ity. As detailed above, the t disclosure provides the insight that a correlation exists between a high binding avidity of B cells to an antigen of interest and a high stability of antibodies produced by these B cells.

Thus, the subset of B cells capable of producing highly stable antibodies within the subset of B cells with a high g y for a specific n is much larger than the subset of B cells capable of producing highly stable antibodies within the entire B cell culture. Hence, when selecting B cells which have a high binding avidity for an antigen, at the same time a pre-selection of B cells capable of producing highly stable antibodies is made. Antibodies obtained from only a limited number of B cells now need to be tested for stability. This is advantageous e the selection of high g avidity B cells is relatively fast, easy and less expensive as compared to the testing of antibodies for their stability.

The methods described herein wherein production of antibodies by ex vivo B cell cultures are used allow the fast, easy and cost effective selection of antibodies having a specificity for an antigen of interest which in addition have a high stability. I.e. the present invention allows for the inclusion of stability as a parameter in the selection of antibodies at an early stage of development. Preferred methods for ing stable ex vivo B cell cultures from which stable dies are selected in accordance with the present sure are the methods as for instance described in , which is incorporated herein by reference.

In a method as sed in , a tion of B cells obtained from a human individual is maintained in culture using BCL6 nucleic acid and an anti- apoptotic nucleic acid (or compounds increasing the expression of such nucleic acids) and subsequently ed. This results in human B cells, which are capable of both proliferating and ing antibody for a prolonged period of time (up to > 6 months). In a method described herein, these B cells are tested for their binding avidity for a specific antigen. One or more B cells selected for having a high binding avidity are preferably further expanded into a further B cell e. During culturing, the stabilized B cells produce dy, which is ed into the culture medium. In a method described herein, these antibodies are tested for stability, and optionally for affinity for the antigen. For these test procedures, a minimum antibody concentration of approximately 100 ng/ml culture medium is typically used. The time required for obtaining this minimal concentration of antibodies after a high binding avidity B cell has been selected and expanded depends on the mammal from which the B cells was originally isolated. For stabilized human B cells, such antibody concentration is typically obtained after 15-20 days of culturing starting from a single B cell. Therefore, using human B cell cultures, antibody is harvested at least 15-20 days after starting a single B cell 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 a single B cell 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 starting a single B cell culture. When using rabbit B cells, an antibody concentration of at least 100 ng/ml is already obtained after 11-12 days after starting a single B cell culture. As the skilled person will appreciate, the desired antibody concentration may be obtained at an earlier time point if culturing is started from more than one B cell.

Preferably, in a method described herein the stability of dies produced by the at least one B cell selected as having a binding avidity higher than the average g avidity of B cells in the B cell culture from which the at least one B cell is obtained the stability of antibodies produced by a B cell culture after ion of said at least one B cell ed as having a higher binding y is determined within four months from selecting said at least one B cell having a g avidity higher than the e binding avidity of B cells in the B cell culture from which the at least one B cell is ed. More preferably said stability is determined within three months from selecting said at least one B cell having a binding avidity higher than the average binding avidity of B cells in the B cell culture from which the at least one B cell is obtained, more preferably within two months. Most preferably, said stability is determined within one month from selecting said at least one B cell having a binding avidity higher than the average binding avidity of B cells in the B cell culture from which the at least one B cell is obtained, such as within between 12 and 30 days, more preferably within 12-25 days.

A B cell capable of ing antibody is defined as a B cell which is capable of producing and/or secreting antibody or a onal part thereof, and/or which cell is capable of developing into a cell which is capable of producing and/or secreting antibody or a functional part thereof. A functional part of an antibody is defined as a part which has at least one same property as said antibody in kind, not necessarily in amount. Said functional part is preferably capable of binding a same antigen as said antibody, albeit not necessarily to the same . A functional part of an antibody preferably comprises a single domain antibody, a single chain dy, a FAB fragment, a nanobody, an y, a single chain variable fragment (scFv), or a F(ab')2 nt.

The binding avidity of a B cell described herein to an antigen of interest can be measured using any method known to a person skilled in the art. For instance, an antigen of interest is labelled with, for example, a fluorescent label.

Detection of binding can subsequently be ined by various techniques, among which fluoresce copy and Fluorescence Activated Cell Sorting (FACS). FACS allows the separation of cells in a sion for instance on the basis of size and/or the scence of d antigen bound to the B cell receptor expressed on the cell e of B cells.

Selecting at least one B cell with a high binding avidity for an antigen of interest from a of B cell culture, preferably from a monoclonal B cell line, can be performed using any method known to a person skilled in the art. Selection of at least one high-affinity B cell described herein is for instance performed by cell sorting for instance using FACS (see above), for instance during the same method in which binding avidity is measured, or by limited dilution. Limited dilution comprises the serial dilution of a suspension of cells, for instance B cells, until a single cell is present in a given volume. Subsequently, the binding avidity of each B cell (after expansion of single cells to a tion) is tested to allow selection of a B cell producing antibodies with a high affinity for antigen.

Selecting at least one B cell with a binding avidity higher than the average binding avidity of the B cell culture from which the B cell is obtained, ably involves determining the binding y of the B cell and determining the average binding avidity of B cells from the B cell culture. Subsequently, the binding avidity of the at least one B cell is compared with the average binding avidity of B cells from the B cell culture and a B cell is selected that has a higher binding y than the average binding avidity of B cells from the B cell culture.

The term “stability” as used herein ably refers to the chemical and/or physical stability of an antibody, for ce stability during production and/or storage of antibodies. Thus, stability as used herein preferably is chemical stability and/or physical ity, more preferably stability during production and/or storage of antibodies, more preferably thermal stability and/or resistance to aggregation. During production and storage dies in liquid formulations, such as pharmaceutical compositions, are susceptible to a variety of processes that influence the physical and/or chemical ties of the antibodies. Such processes e degradation, aggregation, oxidation, and fragmentation of the antibodies.

Such processes are detrimental to the cy of antibodies because they result for ce in a decrease of the amount of functional antibodies in the formulation, and/or by reducing the antigen binding properties of the antibodies. Antibodies that are at least in part resistant to one or more of such processes are referred to as stable antibodies. Hence, determining the stability of antibodies produced by at least one B cell in a method described herein preferably comprises determining the resistance of said dies to ation, aggregation, oxidation and/or fragmentation. Further, antibodies produced by at least one B cell with a higher stability as compared to the average stability of antibodies produced by B cells of a B cell culture selected in accordance with a method described herein thus preferably are antibodies that have a higher resistance to degradation, ation, oxidation and/or fragmentation as compared to the e resistance to degradation, aggregation, oxidation and/or fragmentation of antibodies produced by said B cells of a B cell culture. As used herein the term “a higher resistance to degradation, aggregation, oxidation and/or fragmentation” of an antibody as compared to the average resistance of antibodies means that the antibody exhibits less of a reduction or increase in molecular weight and/or alteration in structure within a given period of time under comparable conditions. The result of a higher resistance to degradation, aggregation, oxidation and/or fragmentation is that the loss of ty of such antibodies within a given period of time is less as compared to antibodies having an e ance to degradation, aggregation, oxidation and/or fragmentation. Thus, poor stability of antibodies can result in subsets of non-functional antibodies, such as antibodies which have the propensity to form aggregates, antibody degradation products and chemically modified antibodies. In addition to losing their antigen-binding properties, such aggregates are potentially dangerous and/or immunogenic when administered to a patient. Poor stability may further for instance result in denaturation of antibodies which also results in a loss of function.

A "stable" antibody as used herein preferably refers to an antibody which essentially retains its al and/or chemical stability and/or biological ty upon storage. Various methods are available in the art for measuring ity of proteins, including antibodies. For instance, stability can be measured at pre-determined temperatures for pre-determined periods of time. In the Examples two es of such methods for determining the stability are ed.

The first method involves determining the thermal stability of dies, and is also referred to as dynamic scanning fluorescence or DSF. In this method, the unfolding of antibodies upon heating is determined. As the antibodies are heated, they unfold and a fluorescent dye is able to bind to the dies as they unfold.

The dye becomes fluorescent when it binds to the unfolded antibodies and fluorescence is measured over time. For instance, the unfolding of the antibodies is measured over a temperature range of 30-95°C. Another method detailed in the Examples measures the tendency of antibodies to ate. This method involves separation of antibody monomers and ates of antibodies using gel chromatography. For instance, the tendency of antibodies to aggregate over time can be measured, whereby the amount of aggregated antibodies is measured after pre-determined periods of time of storage at a pre-determined temperature. For instance aggregation of antibodies is ed after 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 weeks or months of storage or before and after 0.5, 1, 1.5 and 2 years of storage.

Storage is for instance at room temperature, at temperature of 4°C to 7°C or at temperature of -80°C to -20°C. Stability of antibodies as used herein thus preferably refers to al stability and/or physical stability, more ably stability during production and/or storage of antibodies. Stability of antibodies as used herein most ably refers to thermal stability and/or resistance to aggregation. Preferably antibodies are selected that are stable for at least 1 year, more preferably for at least 18 months, and even more preferably for at least two years, at temperatures of between -80 and 80°C. More preferably antibodies are selected or B cells capable of producing antibodies are selected, which antibodies are stable for at least 1 year, more preferably for at least 18 months, and even more preferably for at least two years, at temperatures of between -80 and 80°C, preferably between -80 and 65 °C, more preferably between 80 and 65 °C, such as at room temperature, at a temperature of 4°C to 7°C or at a temperature of -80°C to -20°C. Selected antibodies preferably have a shelf life of at least t least 1 year, more preferably for at least 18 months, and even more preferably for at least two years in a liquid formulations or in solid, e.g. freeze dried, formulations. Antibodies are considered stable for the indicated periods of time if they show no substantial ation, unfolding and/or denaturation during these periods of time. With “no substantial aggregation, unfolding and/or denaturation” is meant that at most 20% of the antibodies, more ably at most 10% of the antibodies, more preferably at most 5%, more preferably at most 2% of the antibodies aggregates, unfolds and/or denatures during the indicated period of time. Most preferably at most 1% of the antibodies aggregates, unfolds and/or denatures during the indicated period of time.

Selecting at least one B cell capable of ing antibody with a stability higher than the average stability of antibodies ed by the B cell culture from which the B cell is obtained in accordance with the methods described herein ably involves determining the stability of antibodies ed by the B cell and ining the average stability of dies produced by B cells from the B cell culture. Subsequently, the stability of antibody produced by the at least one B cell is compared with the average stability of antibodies produced by B cells from the B cell culture and a B cell is ed that is capable of producing antibody that has a higher stability than the e stability of antibodies produced by B cells from the B cell culture.

The average binding avidity of a B cell culture or of a population of B cells is herein defined as the average of the binding avidity of all individual B cells in said culture or population, respectively. A B cell selected from a B cell culture with a high binding avidity, preferably from a monoclonal B cell culture, is preferably selected from the upper 40% of the B cells of said B cell culture with respect to binding avidity, preferably from the upper 30% of the B cells of said culture more ably from the upper 25% of the B cells of said B cell culture, more preferably from the upper 20% of the B cells of said B cell culture, more preferably from the upper 15% of the B cells of said B cell culture, more preferably from the upper 10% of the B cells of said B cell culture, more ably from the upper 5% of the B cells of said B cell culture. Most preferably, one high binding avidity B cell is selected from the upper 1% of the B cells of a B cell culture with respect to binding y.

The average stability of dies produced by B cell of a B cell culture or of a population of B cells is herein defined as the average of the stability of antibodies produced by all individual B cells in said culture or population, respectively. A B cell selected from a B cell culture capable of producing dies with a high stability in accordance with the present disclosure is preferably selected from the upper 40% of the B cells of said B cell culture with respect to the stability of the antibodies produced by said B cells, preferably from the upper 30% of the B cells of said e more ably from the upper 25% of the B cells of said B cell culture, more preferably from the upper 20% of the B cells of said B cell culture, more preferably from the upper 15% of the B cells of said B cell culture, more preferably from the upper 10% of the B cells of said B cell culture, more preferably from the upper 5% of the B cells of said B cell culture with respect to the stability of the antibodies produced by said B cells. Most ably, a B cell capable of producing stable antibodies is selected from the upper 1% of the B cells of a B cell e with respect to the stability of the antibodies produced by said B cells.

Whether or not an antibody produced by at least one B cell selected in accordance with the present disclosure or produced in accordance with the present disclosure has a higher ity as compared to the average ity of dies produced by B cells in the first (ex vivo) B cell culture is for instance determined using one or more of the assays described herein for determining stability of antibodies, or with alternative methods for determining stability of antibodies known in the art. If a difference in stability is observed in one or more of these assay’s and the antibody selected in accordance with the present disclosure or produced by an antibody selected in accordance with the present disclosure shows a higher stability than antibodies produced by B cells in said first (ex vivo) B cell culture, it is concluded that the antibody selected in accordance with the present disclosure or ed by an antibody selected in accordance with the present disclosure has a higher stability as compared to the average stability of antibodies produced by B cells of said first (ex vivo) B cell culture.

The e ity of antibody produced by a B cell culture, preferably by a monoclonal B cell line, cultured from at least one B cell selected or produced in accordance with the present disclosure is preferably at least 1.1 times the average stability of antibodies produced by B cells in the first (ex vivo) B cell culture from which the B cell capable of ing highly stable antibodies was selected, more preferably at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3.0, 3.5, 4.0, 5.0, 10.0, 20, 50, 100 times, or more, than the average ity of antibodies produced by B cell in said first (ex vivo) B cell culture. Such factor of is for instance established using one or more of the assays described herein for ining stability of antibodies. For instance, in the assay described herein in which aggregation of antibody is measured, it is possible to calculate the percentage of antibodies in a sample that has aggregated. An antibody selected in accordance with the present disclosure or produced by an antibody selected in ance with the present disclosure is for instance said to have a 1.2 times higher stability if the percentage of aggregated dy in a sample is 1.2. times lower than the percentage of aggregated dy produced by B cell in said first (ex vivo) B cell culture.

A method described herein preferably further comprises determining the affinity for the antigen of interest of dies produced by a B cell selected for having a binding avidity that is higher than the average binding avidity of the (ex vivo) B cell culture from which the B cell is selected. Said affinity is preferably compared with the average affinity of dies produced by B cells from said B cell culture. The average affinity for an antigen of interest of an antibody produced by a B cell culture or by a population of B cells is herein d as the average of the affinities for said antigen of st of the antibodies produced by all individual B cells in said culture or population, respectively. A method described herein thus preferably further ses determining the affinity for the n of interest of antibodies produced by said at least one B cell ed for as having a high binding y or produced by the B cell culture from which said at least one B cell is selected; and selecting at least one B cell capable of producing antibodies with a higher affinity for the antigen as compared to the average affinity of antibodies produced by B cells of the B cell culture or by the ex vivo B cell culture from which said at least one B cell is selected.

In another preferred embodiment, however, a B cell is selected that is capable of producing antibodies which have an affinity for the antigen of interest that is similar to, or less than, the average affinity for said antigen of interest of antibodies ed by the B cell culture or by the ex vivo B cell culture from which said at least one B cell is selected. Such lower or average affinity is often sufficient in order to have sufficient functionality of the antibodies, such as their therapeutic activity. A high ity of the antibodies on the other hand is an absolute requirement for commercial production and (therapeutic) use of antibodies specific for an antigen of interest. I.e. it is more preferred to select an antibody with a method in ance with the present sure that has a particularly high stability but an average affinity or an affinity that is slightly less than average than to select a high affinity antibody that is unstable or has a relatively low stability. Now that the present disclosure establishes a correlation between the binding avidity of a B cell for an antigen of interest and the ity of antibodies produced by the B cell, it has become possible to select such highly stable antibody with average affinity for the n over an unstable dy with a high affinity already at an early stage in antibody screening and development ses.

Described is a method for producing a B cell capable of producing antibody for an antigen of interest comprising: a) selecting at least one B cell capable of producing antibody specific for said antigen of interest or at least one B cell capable of developing into a B cell capable of producing antibody specific for said antigen of interest; b) inducing, ing and/or maintaining sion of BCL6 and inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid in said at least one B cell; c) allowing expansion of said at least one B cell into a first B cell culture; d) selecting at least one B cell from said first B cell e with a binding avidity for said antigen of interest that is higher than the average binding y for said antigen of st of B cells in said first B cell culture; e) preferably allowing expansion of said at least one B cell selected in step d) into a second B cell culture; f) determining the stability,, and affinity for said antigen of interest, of antibodies produced by said at least one B cell selected in step d) or by said second B cell culture; and g) selecting at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies produced by B cells of said first B cell culture and with an affinity for said antigen of st that is similar to, or less than, the average affinity for said antigen of interest of or of antibodies produced by said first B cell culture.

Further described is a method for selecting from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies produced by said ex vivo B cell culture, the method comprising: a) ing an ex vivo B cell culture capable of producing antibody specific for an antigen of interest; b) selecting at least one B cell from said ex vivo B cell e with a binding avidity for said antigen of interest that is higher than the average binding avidity of B cells of said ex vivo B cell culture for said antigen of interest; c) determining the stability, and ty for said antigen, of interest of antibodies produced by said at least one B cell selected in step b); and d) selecting at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies produced by B cells of said ex vivo B cell culture and with an affinity for said antigen of interest that is similar to, or less than, the average affinity for said antigen of interest of or of antibodies produced by said ex vivo B cell culture.

The affinity of an antibody can be determined using any method known to a person skilled in the art. The affinity of an antibody is for instance determined using Enzyme-linked sorbent assay (ELISA), Surface n Resonance (such as Biacore) or Octet (ForteBio). Surface Plasmon Resonance (SPR) and Octet are techniques to e biomolecular interactions in real-time in a label free environment. For SPR, one of the interactants, for instance an antibody, is immobilized to the sensor surface, the other, for instance antigen, is free in solution and passed over the surface. Association and dissociation is measured in arbitrary units and preferably displayed in a gram. Any change in the number of molecules bound to the biosensor tip causes a shift in the interference pattern that can be measured in real-time. Using Octet the interference pattern of white light ted from two surfaces, a layer of immobilized n on the sor tip, and an al reference layer is analyzed. The binding between a ligand lized on the biosensor tip surface, for instance an antibody, and a n in solution, for instance an antigen of interest, produces an increase in optical thickness at the biosensor tip, which results in a wavelength shift which is a direct e of the change in thickness of the biological layer. ELISA comprises immobilizing a protein, for instance the antigen of interest, on the surface of the solid support, for example a 96-well plate, and applying a sample to be detected or quantified on the solid support. Alternatively, a capture antibody is d on the surface of a solid support after which a sample containing the protein to be detected or quantified is applied to the immobilized capture antibody allowing the protein of st to bind. Non-binding proteins are than washed away. Subsequently a specific antibody conjugated to a label or an enzyme (or a primary antibody followed by a secondary antibody conjugated to a label or an enzyme) is added to the solid support. Preferably the affinity constant (KD) of an antibody produced by a B cell according to the present disclosure is ined.

Selecting at least one B cell capable of producing dy with an affinity for a ic antigen higher than, similar to, or lower than the average affinity of antibodies produced by the B cell e from which the B cell is obtained, preferably involves determining the ty of antibody produced by the B cell for the antigen and determining the average affinity of antibodies produced by B cells from the B cell culture for the antigen. Subsequently, the affinity for the antigen of antibody produced by the at least one B cell is compared with the average affinity for the antigen of antibodies produced by B cells from the B cell culture and a B cell is ed that is e of producing antibodies that have a higher, similar, or lower affinity than the average affinity of antibodies produced by B cells from the B cell culture.

A B cell culture or an ex vivo B cell culture in ance with the present disclosure preferably is a monoclonal B cell culture. An example of a B cell culture or an ex vivo B cell culture in accordance with the present disclosure is a cell line of B cells, preferably monoclonal B cells. Hence, a B cell culture or an ex vivo B cell culture in accordance with the present disclosure is most preferably a monoclonal B cell line. Allowing expansion of a B cell selected on its binding or stability properties into a B cell culture is for ce lished by allowing expansion of said B cell until a population of B cells is obtained.

Non-limiting examples of a B cell used or selected in a method bed herein include B cells derived from a human individual, a rodent, a , a llama, a pig, a cow, a goat, a horse, an ape, a chimpanzee, a e and a gorilla.

Preferably, said B cell is a human cell, a murine cell, a rabbit cell, an ape cell, a chimpanzee cell, a macaque cell and/or a llama cell. Most preferably, said B cell is a human B cell or a rabbit B cell. Preferably, a B cell capable of producing antibody specific for an antigen of interest that is selected in accordance with a method bed herein in which expression of BCL6 and an anti-apoptotic nucleic acid is induced, enhanced and/or maintained is a memory B cell, for instance a human memory B cell or a rabbit memory B cell. Particularly preferred is a eral blood memory B cell. Peripheral blood memory B cells are easily obtained, without much discomfort for the individual from which they are obtained, and have been demonstrated to be very suitable for use in a method described .

Within a, preferably monoclonal, B cell culture of B cells capable of ing antibody specific for an antigen of interest, it is possible to select at least one, optionally more than one, such as for instance 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 50, 100, 103, 104, 105 or 106 B cells with a binding avidity for said antigen of interest that is higher than the average binding avidity of said B cell culture for said antigen of interest. As described in , a subset of B cells in a given B cells culture produce dies ic for the antigen of interest shows a higher binding to said antigen, which is correlated to the fact that antibodies produced by said subset of B cells have a higher affinity for the antigen than the average affinity of antibodies produced by said B cell culture. The present disclosure provides the insight that r subset of B cells that have a higher binding avidity for the antigen produce antibodies which have a higher stability than the average stability of antibodies ed by said B cell culture. As r described in the B cell cultures ed after culturing antibody producing B cells which produce high affinity antibodies contains B cells of which the antibodies maintained the ability to bind antigen with a higher affinity than the average B cell in the original B cell culture. The same applies to the subset of B cell producing antibodies with high stability: B cell cultures obtained after culturing such high stability antibody producing B cells contains B cells that produce antibodies with a higher stability than the e B cell in the original B cell culture. Single B cells can thus be isolated from a given B cell e on the basis of their higher binding avidity by methods known in the art and be expanded to a new B cell culture of which the average stability of ed dies is higher than the average stability of B cells in said given B cell culture.

In a method described herein preferably a single B cell is selected that is specific for an antigen of interest, for instance from a polyclonal B cell population obtained from an individual. The single B cell is subsequently preferably ed into a monoclonal B cell culture. This is for instance achieved using a method as described in , which is discussed herein before. Hence, a monoclonal B cell line specific for an antigen of interest is obtained. In principle, all B cells in the monoclonal B cell line produce essentially the same antibodies specific for said antigen, although small differences in the stability of antibodies may be present n B cells of said monoclonal B cell line, i.e. some B cells in the monoclonal culture produce antibodies with a stability which is slightly higher than the average stability and some B cells in the monoclonal culture produce dies with a ly lower stability. The B cell culture becomes slightly heterogeneous again. In accordance with the t sure at least one B cell with a higher g avidity than the average binding avidity is selected from the monoclonal B cell line. Of all the B cells with a higher binding avidity than the average binding avidity, a subset produces antibodies with a higher stability than the average stability of antibodies produced by B cells in the B cell culture. In accordance with the present disclosure subsequently at least one of such B cells producing antibodies with a higher stability than the average stability is selected.

A selected B cell is or ed B cells are uently preferably cultured into a second, preferably monoclonal, B cell culture. The t disclosure provides the insight that this second, preferably monoclonal, B cell culture produces antibodies with an average stability that is higher than the average stability of the original (monoclonal) B cell culture. As described above, it was found that the high stability of antibodies produced by a selected B cell is ined after ing, even if culturing takes place during a prolonged period of time. Thus, antibodies produced by the second (monoclonal) B cell culture ed in accordance with the present disclosure have a higher average stability than antibodies ed by the first (monoclonal) B cell culture.

As detailed herein before, ably one B cell is selected that is capable of producing antibodies with a stability that is higher than the average stability of antibodies produced by the B cell culture from which the B cell is selected. In another embodiment, more than one of such B cells capable of producing antibodies with such higher stability is selected, for instance 2, 3, 4, 5, , 15, 25, 50, 100, 103, 104, 105, 106 B cells. The B cells are for instance selected from a polyclonal B cell culture or from a biological . The B cells are subsequently expended into a B cell culture, for instance using a method as described in . The obtained B cell culture is in this case thus a (second) polyclonal B cell e. Thereafter, a monoclonal B cell culture is ably produced. This is for instance done by selecting a single B cell from said (second) polyclonal B cell culture using Fluorescence Activated Cell g (FACS) or limiting dilution, as described herein, and expanding said selected single B cell to a monoclonal B cell culture. Then, preferably at least one B cell with a higher binding avidity than the average binding avidity of the monoclonal B cell culture and which is capable of producing antibodies with a higher stability than the average stability of antibodies produced by the monoclonal B cell culture is ed. The selected B cell is preferably subsequently ed into a second monoclonal B cell culture, after which antibodies produced by said second onal B cell line can be obtained. ably, the amino acid ce of at least part of the heavy chain and light chain of the antibodies are determined and compared with the amino acid sequence of (the relevant part of) the heavy and light chain of the antibodies produced by the (ex vivo) B cell culture from which a B cell was originally selected. This way the mutation(s) in the amino acid sequence that promotes the increased stability of the antibody can be identified. A preferred method further comprises expressing a nucleic acid molecule encoding the heavy chain and/or light chain of the antibody with increased stability in a second cell.

Said second cell is preferably a led producer cell, such as for ce a cell of a Chinese hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line, which are preferably adapted to commercial antibody production. Proliferation of such producer cells results in a producer cell line capable of producing stable antibodies according to the present disclosure.

In one embodiment described herein, after the step of selecting at least one B cell producing high stability antibodies from said already monoclonal B cell e, said at least one B cell is allowed to expand into a B cell culture, preferably a monoclonal B cell line, again, after which another step of selecting at least one B cell producing high stability antibodies from said new B cell culture, preferably from said new monoclonal B cell line, is performed. By ing the steps of allowing expansion of a selected B cell into a B cell culture and ing at least one B cell on the basis of its binding y and/or on the basis of the stability of antibodies produced by it, it is possible to generate high affinity antibody producing B cells. Preferably, by repeating the steps of expansion and ion as described above, it is possible to increase with each selection cycle the stability of antibody produced by the resulting B cell culture.

In a red embodiment in a method for selecting from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher stability than the average stability of antibodies produced by said ex vivo B cell culture according to the present disclosure step a) comprises selecting at least one B cell capable of producing antibody specific for said antigen of interest or selecting at least one B cell capable of developing into a B cell e of producing antibody specific for said n of interest and allowing expansion of said at least one B cell into said ex vivo B cell culture. Preferably, allowing ion of said at least one B cell into said ex vivo B cell culture and selecting at least one B cell from said ex vivo B cell culture capable of ing antibody with a binding avidity for said antigen of interest that is higher than the average binding avidity of B cell receptors or antibodies produced by said ex vivo B cell culture for said antigen of interest in step b) are ed at least once. Said steps may be repeated twice, three times, four times, five times or even more times. r described is a method for selecting from an ex vivo B cell culture at least on B cell capable of producing antibodies with a higher stability as compared to the average ity of antibodies produced by said ex vivo B cell culture in accordance with the present disclosure is thus described wherein step a) comprises selecting at least one B cell capable of producing dy specific for said antigen of interest or selecting at least one B cell capable of developing into a B cell capable of producing antibody specific for said antigen of interest and allowing expansion of said at least one B cell into said ex vivo B cell culture, and wherein allowing expansion of said at least one B cell into said ex vivo B cell culture and selecting at least one B cell from said ex vivo B cell culture capable of producing antibody with a binding avidity for said n of interest that is higher than the average g avidity of B cell receptors or antibodies produced by said ex vivo B cell culture for said antigen of interest in step b) are repeated at least once. Said steps are for instance ed once, but preferably twice, three times, four times, five times or even more times.

A method described herein preferably comprises inducing, enhancing and/or maintaining expression of BCL6 in a B cell and/or a (ex vivo) B cell culture.

BCL6 s a transcriptional repressor which is required for normal B cell and T cell development and maturation and which is required for the formation of germinal centers. BCL6 is highly expressed in germinal center B cells whereas it is hardly expressed in plasma cells. BCL6 inhibits differentiation of activated B cells into plasma cells. In a method according to the t disclosure, BCL6 expression product remains present in the B cells of an ex vivo culture. The presence of BCL6 together with the presence of an anti-apoptotic nucleic acid, prolongs the replicative life span of the B cells. Expression of BCL6 is preferably induced, enhanced or maintained by administering a BCL6 expression-promoting compound to the B cell(s) used for culturing, or by culturing B cells in the presence of such compound. s compounds capable of directly or indirectly enhancing expression of BCL6 are known in the art. Such nd for instance comprises a Signal Transducer of Activation and ription 5 (STAT5) protein, or a functional part or a functional derivative thereof, and/or a nucleic acid sequence coding therefore. STAT5 is a signal ucer capable of enhancing BCL6 expression. There are two known forms of STAT5, STAT5a and STAT5b, which are encoded by two different, tandemly linked genes. Administration and/or tion of STAT5 results in enhanced levels of BCL6. Hence, STAT5, or a functional part or a onal derivative thereof is capable of ly increasing expression of BCL6. Described is therefore a method according to the t disclosure comprising providing the B cell(s) with STAT5, or with a functional part or a functional derivative thereof, or providing the B cell(s) with a nucleic acid molecule encoding STAT5, or a onal part or a functional derivative thereof, or culturing said B cell in the presence of STAT5, or a functional part or a functional derivative thereof.

The presence of STAT5 directly ses the amount of BCL6. It is also possible to indirectly se expression of BCL6. This is for instance done by regulating the amount of a certain compound, which in turn is capable of directly or indirectly activating STAT5 and/or increasing expression of STAT5. Hence, in one embodiment the expression and/or activity of endogenous and/or exogenous STAT5 is increased. It is for instance possible to indirectly enhance sion of BCL6 by culturing a B cell in the presence of interleukin (IL) 2 and/or IL4 which are capable of activating STAT5, which in turn increases expression of BCL6.

It is, r, preferred to provide a B cell with a c acid molecule encoding BCL6, or a functional part or a functional derivative thereof. This way, it is possible to directly regulate the concentration of BCL6 in said B cell. Also described is therefore a method according to the present disclosure comprising providing said B cell with a nucleic acid le encoding BCL6, or a functional part or a functional derivative thereof. In one embodiment, said nucleic acid molecule is constitutively active, meaning that BCL6, or a functional part or a onal derivative thereof, is continuously expressed, independent of the presence of a regulator. In another embodiment, said nucleic acid molecule is ble, meaning that the expression thereof is regulated by at least one inducer and/or repressor. This way, expression of said nucleic acid molecule is regulated at will. For instance, Tet-On and Tet-Off expression systems (for example Tet-On® and Tet-Off® Advanced Inducible Gene Expression Systems, Clontech) can be used for inducible expression of a c acid ce 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 ed of the Escherichia coli Tet repressor protein (TetR) and the Herpes simplex virus transactivating domain VP16. tTA tes transcription of a nucleic acid sequence of interest under the control of a tetracycline-responsive element (TRE) comprising the Tet operator (TetO) DNA ce and a promoter sequence, for instance the human cytomegalovirus (hCMV) er. A c acid sequence encoding, for instance, Bcl6, or a functional part or a functional derivative thereof, can be placed downstream of this promoter.

In the Tet-off system, tTA binds to TRE in the absence of TC or dox and transcription of a nucleic acid sequence of interest is activated, whereas in the presence of TC or dox tTA cannot bind TRE and sion of a nucleic acid sequence of interest is inhibited. In contrast, the Tet-on system uses a reverse tTA (rtTA) that can only bind the TRE in the ce of dox. Transcription of a nucleic acid sequence of interest is inhibited in the absence of dox and activated in the presence of dox. Alternatively, inducible expression is executed using a hormone inducible gene expression system such as for ce an ecdysone inducible gene expression system (for example RheoSwitch®, New England s) (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 ne receptor, a heterodimer consisting of the ecdysone or (Ecr) and id X receptor (RXR) is formed in the presence of an ecdyson t selected from ecdysone, one of its analogues such as muristerone A and erone A, and a non-steroid ecdysone agonist. In the presence of an agonist, Ecr and RXR interact and bind to an ne se element that is present on an expression te. Exaperssion of a c acid sequence of interest that is placed in an expression cassette downstream of the ecdysone response element is thus induced by exposing a B cell to an ecdyson agonist.

As another example described herein inducible expression is ed using an arabinose-inducible gene sion system (for example pBAD/gIII kit, Invitrogen) (Guzman, L. M. et al. Bacteriol 177, 4121–4130 (1995)). Arabinose is a monosaccharide containing five carbon atoms. In cells transfected with the arabinose-inducible promoter PBAD expression 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 molecule encoding) a BCL6 protein, or a functional part or functional derivative thereof, wherein the activity of said BCL6, or functional part or functional derivative is regulated by at least one inducer and/or repressor. A non-limiting example is a fusion protein wherein a regulatory element is fused to a sequence encoding at least part of BCL6. For ce, an en receptor (ER) is fused to BCL6, resulting in fusion protein ER-BCL6. This fusion protein is inactive because it forms a complex with heat shock proteins in the cytosol. Upon administration of the exogenous inducer 4 hydroxy-tamoxifen (4HT), the fusion protein ER-BCL6 dissociates from the heat shock proteins, so that the BCL6 part of the fusion protein becomes active.

A method described herein preferably comprises inducing, enhancing and/or maintaining expression of an anti-apoptotic nucleic acid in a B cell and/or a (ex vivo) B cell culture. As used herein, the term apoptotic nucleic acid molecule” refers to a nucleic acid molecule, which is capable of delaying and/or preventing apoptosis in a B cell. Preferably, said anti-apoptotic nucleic acid molecule is capable of delaying and/or preventing apoptosis in a plasmablast-like B cell, which is capable of both proliferating and producing antibody. Preferably, an anti-apoptotic nucleic acid molecule is used which ses an exogenous c acid molecule. This means that either a nucleic acid sequence is used which is not naturally expressed in B cells, or that an additional copy of a naturally occurring nucleic acid sequence is used, so that expression in the resulting B cells is enhanced as compared to natural B cells. Various anti-apoptotic 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 antiapoptotic member of the Bcl-2 family e anti-apoptotic Bcl-2 proteins are good apoptosis ters 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 functional part or functional derivative thereof, is preferred because Bcl-2, , Bcl-w, A1, Bcl2L10 and Mcl-1 are generally ated with the outer mitochondrial membrane. They directly bind and inhibit the pro-apoptotic proteins that belong to the Bcl-2 family to protect mitochondrial ne integrity.

Preferred is therefore a method according to the present disclosure, wherein said anti-apoptotic nucleic acid molecule comprises an anti-apoptotic gene of the Bcl2 , preferably Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a functional part or a functional derivative thereof. ably, expression of Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, is induced, enhanced or maintained by administering at least one compound, capable of promoting expression of any of these anti-apoptotic genes, to B ), or by culturing B cells in the presence of such compound(s). Further bed is ore a method ing to the present disclosure, comprising: - providing said B cell with 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; and/or - culturing said B cell in the presence of 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.

More preferably, however, a B cell is ed 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 functional parts and functional derivatives thereof. This way, it is possible to directly enhance the amount of expression product in said B cell. Also described is therefore a method according to the present disclosure, comprising providing said 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, 0, and functional parts and functional derivatives thereof. In one ment, said nucleic acid molecule is constitutively active, g that said nucleic acid molecule is continuously expressed. In another embodiment, said nucleic acid molecule is inducible, meaning that the expression thereof is regulated by at least one inducer and/or repressor. miting examples of inducible c acid expression systems known in the art are described herein before.

In a particularly preferred embodiment said anti-apoptotic nucleic acid molecule s Bcl-xL or Mcl-1, or a functional part or a functional derivative thereof, most preferably Bcl-xL or Mcl-1. Described herein, a combination of BCL6 and Bcl-xL is particularly well capable of increasing the ative life span of B cells, thereby forming long term cultures of the resulting plasmablast-like B cells.

The same holds true for a combination of BCL6 and Mcl-1. Most preferably, said anti-apoptotic nucleic acid s Bcl-xL or a functional part or a functional derivative thereof, and most preferably encodes Bcl-xL.

A functional part of BCL6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or 0 is a proteinaceous molecule that has the same capability - in kind, not necessarily in amount - of increasing the replicative life span of a B cell as compared to natural BCL6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, respectively. Such functional part is for instance devoid of amino acids that are not, or only very little, involved in said lity.

For instance, functional parts of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10 are d herein as fragments of Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, respectively, which have retained the same kind of anti-apoptotic characteristics as full length Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, respectively (in kind, but not necessarily in amount). Functional parts of , Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10 are typically shorter fragments of , Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10, respectively, which are capable of delaying and/or preventing apoptosis in a B cell. Such functional parts are for instance devoid of sequences which do not significantly contribute to the anti-apoptotic ty of BclxL , Mcl-1, Bcl-2, A1, Bcl-w and 0. A functional part of BCL6 is lly a shorter fragment of BCL6 which is capable of increasing the replicative life span of a B cell.

A functional derivative of BCL6, , Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10 is defined as a BCL6, Bcl-xL, Mcl-1, Bcl-2, A1, Bcl-w or Bcl2L10 protein, tively which has been altered but has maintained its lity (in kind, not necessarily in amount) of increasing the replicative life span of a B cell. A functional derivative is provided in many ways, for instance h 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 able label or with an inducible compound.

Furthermore, a method according to the present disclosure is described, further comprising providing said B cell with IL21 and CD40L. Preferably, said IL21 is murine or human IL21, most preferably murine IL21. Said CD40L is also preferably murine or human CD40L, most preferably human CD40L.

Besides increasing BCL6 expression and the expression of an anti- apoptotic nucleic acid molecule, it is also advantageous to induce, enhance and/or maintain expression of Blimp-1 in a B cell. This enhances antibody production of said B cell. Also described is a method according to the present disclosure, wherein the method further comprises ng, enhancing and/or maintaining expression of Blimp-1 in said B cell. Blimp-1 expression is ably induced or enhanced.

The extent of expression of Blimp-1 in a B cell can be regulated in a variety of ways. For instance a B cell is provided with a nd, which is capable of directly or indirectly increasing expression of Blimp-1 Additionally, or alternatively, a B cell is cultured in the presence of a compound capable of directly or indirectly increasing expression of Blimp-1. Further described is a method according to the present disclosure, further comprising: - providing said B cell with a compound capable of directly or indirectly increasing expression of 1; and/or - culturing said B cell in the presence of a compound capable of directly or indirectly increasing sion of Blimp-1.

Said compound capable of increasing expression of Blimp-1 preferably comprises IL21. Hence, described is a method wherein B cells are cultured in the presence of IL21, at least during part of the e time.

Another preferred compound capable of increasing Blimp-1 expression comprises 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 involved in B cell development and differentiation. STAT3 is e of upregulating Blimp-1 expression. In another preferred , a B cell is thus provided with a nucleic acid molecule ng STAT3 or a functional part or a onal derivative thereof, ably wherein the expression of said nucleic acid molecule is regulated by an exogenous inducer of repressor, so that the extent of STAT3 expression is regulated at will. For instance, one of the earlier mentioned inducible expression systems is used. For ce, a fusion product sing STAT3, or a functional part or a functional derivative, and ER is used and a B cell is provided with a nucleic acid molecule encoding an estrogen receptor (ER) and STAT3 as a fusion protein ER-STAT3. This fusion n is inactive 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 y-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 expression.

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 expression of Blimp-1 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. A functional derivative is provided in many ways, for instance through vative amino acid substitution wherein one amino acid is substituted by another amino acid with generally similar properties (size, hydrophobicity, etc), such that the l functioning is not sly affected. Alternatively, a functional derivative for instance comprises a fusion protein with a detectable label or with an inducible compound.

Since STAT3 is capable of increasing expression of Blimp-1 it is also possible to ctly increase sion of Blimp-1 by administering a compound capable of sing the activity and/or sion of STAT3. In one embodiment, a B cell is therefore ed with a compound that is capable of enhancing the activity of STAT3, so that sion of Blimp-1 is ctly enhanced.

STAT3 is activated in a variety of ways. Preferably, STAT3 is ted by providing a B cell with a cytokine. 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. Moreover, Toll-like receptors (TLRs), which are involved in innate immunity, are also capable of ting STAT3. In a preferred method described herein a B cell is therefore cultured in the presence of IL21, IL2, IL6, IL7, IL10, IL15 and/or IL27. Most preferably IL21 is used, since IL21 is particularly suitable for enhancing antibody production of B cell cultures described herein. IL21 is capable of upregulating Blimp-1 expression, even when Blimp-1 expression is counteracted by BCL6.

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

In yet another embodiment, expression of Blimp-1 is increased by providing a B cell with a ssor of cytokine signalling (SOCS) n and/or by activating a SOCS protein 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. E47 is a transcription factor that belongs to a family of 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 spliced differently. E proteins have been described as tumor suppressors. One of the specific targets of E47 are the Socs1 and Socs3 genes.

Also described are isolated or recombinant B cells obtainable with a method according to the presence disclosure. Such ed or recombinant B cells preferably comprise an exogenous anti-apoptotic nucleic acid sequence and an exogenous nucleic acid sequence encoding BCL6 or STAT5, or a functional part or a functional derivative thereof. Further described is therefore an isolated or recombinant B cell, comprising an ous nucleic acid sequence encoding BCL6 or STAT5, or a functional part or a functional derivative thereof, and an exogenous anti-apoptotic nucleic acid sequence. As explained before, said exogenous nucleic acid le either contains a nucleic acid ce that does not naturally occur in B cells, or an additional copy of a natural B cell nucleic acid sequence. , Mcl-1, Bcl-2, A1, Bcl-w and Bcl2L10, are preferred poptotic nucleic acid molecules. Preferred is therefore an isolated or recombinant B cell, which comprises an exogenous nucleic acid sequence encoding BCL6 or STAT5, or a functional part or a functional derivative thereof, and an exogenous nucleic acid sequence encoding Bcl-xL or Mcl-1 or Bcl-2 or A1 or Bcl-w or Bcl2L10, or a functional part or a functional derivative f. Said nucleic acid sequence ng BCL6 or STAT5, or a functional part or a functional derivative thereof, and said exogenous poptotic c acid sequence may be present on one nucleic acid molecule. atively, these sequences are present on at least two different nucleic acid molecules.

Described is a method for producing a B cell capable of producing antibody for an antigen of interest comprising: a) selecting at least one B cell capable of producing antibody specific for said antigen of interest or at least one B cell capable of developing into a B cell capable of producing antibody specific for said antigen of interest; b) inducing, ing and/or maintaining expression of BCL6 in said B cell by providing said B cell with a nucleic acid molecule ng BCL6 and/or STAT5, or a functional part or a functional derivative thereof, inducing, enhancing and/or ining expression of Blimp-1 in said B cell, preferably by ing said B cell with a nucleic acid molecule encoding STAT3, or a functional part or a functional derivative thereof or by culturing said B cell in the presence of IL-21, and inducing, enhancing and/or maintaining expression of a gene encoding an antiapoptotic molecule of the BCL2 family, preferably Bcl-xL or Mcl-1, in said at least one B cell; c) allowing expansion of said at least one B cell into a first B cell culture; d) selecting at least one B cell from said first B cell culture with a g avidity for said antigen of interest that is higher than the average binding avidity for said antigen of interest of B cells in said first B cell culture; e) ably allowing expansion of said at least one B cell selected in step d) into a second B cell e; f) determining the stability of antibodies produced by said at least one B cell selected in step d) or by said second B cell culture; and g) ing at least one B cell e of producing dies with a higher stability as compared to the average stability of dies produced by B cells of said first B cell culture.

Further described is a method for selecting from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies produced by said ex vivo B cell culture, the method comprising: a) providing an ex vivo B cell culture capable of producing antibody specific for an n of interest; b) selecting at least one B cell from said ex vivo B cell culture with a binding y for said antigen of interest that is higher than the average binding y of B cells of said ex vivo B cell culture for said antigen of interest; c) determining the stability of antibodies produced by said at least one B cell selected in step b); and d) selecting at least one B cell capable of producing antibodies with a higher stability as compared to the average ity of antibodies produced by B cells of said ex vivo B cell culture, further comprising inducing, enhancing and/or maintaining sion of BCL6 in said ex vivo B cell culture by providing said ex vivo B cell culture with a nucleic acid molecule ng BCL6 and/or STAT5, or a functional part or a functional derivative thereof, inducing, enhancing and/or ining expression of Blimp-1 in said ex vivo B cell culture, preferably by providing said ex vivo B cell culture with a c acid molecule encoding STAT3, or a functional part or a functional derivative thereof or by culturing said ex vivo B cell culture in the presence of IL-21, and inducing, enhancing and/or maintaining expression of a gene encoding an anti-apoptotic molecule of the BCL2 family, preferably Bcl-xL or Mcl-1, in said ex vivo B cell culture.

A method according to the present disclosure is preferably used for generating a cell line of B cells capable of producing stable antibodies that is stable for at least one week, preferably at least one month, more preferably at least three months, more ably at least six months so that commercial production of antibodies with high stability has become possible. Preferably a stable cell line capable of producing monoclonal stable antibodies is produced. This is preferably performed by using memory B cells that have for instance been isolated from a sample by selection for CD19 (B cell marker) and cell e IgG and/or CD27 (to mark memory . Furthermore, a memory B cell capable of specifically binding an antigen of interest is for instance selected in a binding assay using said antigen of interest. Subsequently, BCL6 and an anti-apoptotic nucleic acid, preferably BclxL or Mcl-1, are ably co-expressed in said B cell, resulting in a population of cells specific for said antigen of interest. Preferably only one memory B cell is used and expanded into an (ex vivo) B cell culture in a method as described herein, so that a B cell e producing monoclonal antibodies (a onal B cell line) is obtained.

In one ment, a B cell, preferably a memory B cell, that originates from an individual which had been previously exposed to an antigen of interest, is used in a method according to the t disclosure. However, this is not necessary. It is also possible to use a B cell from an individual that has not been exposed to said antigen of interest. For instance, a B cell is used that is specific for another antigen but shows cross-reactivity with the antigen of interest. As another example, a B cell is used that is selected from a naïve B cell population of an individual. The naïve B cell population of an individual may contain B cells that show reactivity with an antigen of interest even though the individual has not been exposed to said antigen of interest. Such B cell from a naïve B cell population is for instance selected using ed antigen of st.

Also described is a method for producing antibodies specific for an antigen of interest, comprising: - ing from an ex vivo B cell culture at least one B cell capable of ing antibody with a higher stability as compared to the average stability of antibodies produced by said ex vivo B cell culture with a method according to the present disclosure; - culturing said at least one B cell into a B cell culture; and - obtaining antibodies produced by said B cell culture.

Further described is a method for producing antibodies specific for an antigen of interest, comprising: - selecting from an ex vivo B cell culture at least one B cell capable of producing antibody with a higher ity as compared to the e stability of antibodies ed by said ex vivo B cell culture with a method according to the present disclosure; - optionally allowing expansion of said selected at least one B cell into a B cell culture; - determining the amino acid sequence of the heavy chain and/or light chain of said antibody with a higher stability; and - expressing a c acid molecule encoding the heavy chain and/or light chain of said antibody in a second cell. Said second cell is preferably a so-called producer cell, such as for instance a cell of a Chinese hamster ovary (CHO), NSO (a mouse myeloma) or 293(T) cell line, which are preferably adapted to commercial antibody production. Proliferation of such producer cells results in a producer cell line capable of producing stable antibodies according to the present disclosure. ably, said producer cell line is suitable for producing antibodies for use in . Hence, said producer cell line is preferably free of pathogenic agents such as pathogenic micro-organisms.

Further described is a method for identifying at least one mutation in the amino acid sequence of the heavy chain and/or light chain of an antibody as compared to the amino acid sequence of the heavy chain and/or light chain of a reference antibody, which mutation promotes stability of said antibody, comprising: - selecting from an ex vivo B cell culture at least one B cell e of producing dy with a higher stability as compared to the average ity of antibodies produced by said ex vivo B cell culture with a method according to the present disclosure; - optionally allowing expansion of said selected at least one B cell into a B cell culture - determining at least part of the amino acid sequence of the heavy chain and/or light chain of an antibody produced by said selected at least one B cell; - comparing said amino acid sequence with at least part of the amino acid sequence of the heavy chain and/or light chain of a nce antibody, thereby identifying at least one on in the amino acid sequence in the heavy chain and/or light chain of said dy which mutation promotes stability of said antibody.

The amino acid sequence of at least part of the heavy chain and/or the light chain of an antibody selected in accordance with the present disclosure or of an antibody produced by a B cell selected in accordance with the present disclosure can be determined using any method known in the art, such as by mass spectrometry or Edman degradation reaction. Preferably the amino acid sequence of at least the mentarity determining regions (CDRs) of the heavy chain and/or light chain of the antibody are determined, more ably the CDRs of the heavy chain and of the light chain.

A reference antibody is preferably an antibody produced by the first B cell culture or ex vivo B cell culture as ed to herein from which a B cell with a high binding avidity is ed in accordance with the present disclosure. Hence, said reference antibody is preferably an antibody produced by a first B cell culture obtained in step c) of a method for producing a B cell capable of producing antibody for an antigen of interest in accordance with the present disclosure. In another preferred embodiment, said reference antibody is an antibody produced by an ex vivo B cell culture capable of producing antibody specific for an antigen of interest provided in step a) of a method for selecting from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher stability as compared to the average stability of antibodies ed by said ex vivo B cell culture in accordance with the present disclosure.

Also described are isolated or recombinant B cells, B cell cultures and populations of B cells, preferably monoclonal B cell lines, obtained by a method according to the present disclosure. Such B cells e of producing stable antibodies are preferably stable for at least one week, preferably for at least one month, more preferably for at least three months, more preferably for at least six months, meaning that the B cell is capable of both replicating and producing antibody, or capable of replicating and developing into a cell that produces antibody, during said time periods. B cells selected or ed in accordance with the present disclosure preferably comprise cells producing IgM, IgG, IgA, or IgE, preferably IgG. A B cell selected or produced in accordance with the present sure is particularly suitable for use in producing an antibody producing B cell line. B cells capable of ing highly stable antibodies selected or produced in accordance with the present disclosure are preferably ed ex vivo and antibody is preferably collected for further use. Alternatively, the amino acid sequence of B cells capable of ing highly stable antibodies selected or produced in accordance with the present disclosure is ined and a producer cell line is provided with a c acid molecule encoding the heavy and/or light chain of the antibodies in order to produce and t stable dies. Described is therefore a method according to the present disclosure further sing determining at least part of the amino acid sequence of the heavy chain and/or light chain of said at least one B cell that is capable of producing antibodies with a higher stability.

Antibodies ed from a B cell or from a B cell culture or monoclonal B cell line ed and/or produced in accordance with the t disclosure are also described. Stable antibodies or functional parts f produced with a method according to the present disclosure are useful for a wide variety of applications, such as for instance therapeutic, prophylactic and diagnostic applications, as well as research es and ex vivo experiments.

Features may be bed herein as part of the same or separate aspects or embodiments of the present invention for the purpose of clarity and a concise description. It will be appreciated by the skilled person that the scope of the invention may include embodiments having combinations of all or some of the features described herein as part of the same or separate embodiments.

The term “comprising” as used in this specification and claims means “consisting at least in part of”. When interpreting statements 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 ent or claim may also be present. Related terms such as “comprise” and “comprised” are to be interpreted in similar manner.

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an ion 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 t application. That subject matter should be readily identifiable by a person skilled in the art and may assist in putting into practice the invention as d in the claims of this application.

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

Brief description of the drawings Figure 1. Selection and isolation of subclones (outliers) with increased binding to labeled H1 or H3 compared to the average binding of the influenza group 1 and influenza group 2 cross ve HA specific parental clone AT10_004. Cells were d with Alexa-647 labeled HA H3- or H1 n together with IgG-PE or Lamda-PE antibody. Following three subsequent rounds of enrichment for sed antigen binding, AT10_004 cells single cell cloning was performed with a cell sorter.

Figure 2. Selection of subclones with increased H1 or H3 antigen binding compared to the al AT10_004 clone. AT10_004 cells were labelled with a fluorescent CD19 antibody and mixed with non-labelled subclone cells. This mix of cells was stained with lambda-PE and 647 labelled H1 or H3. Shown is the intensity of antigen binding related to the BCR expression of the subclones compared to the al AT10_004 cells.

Figure 3. Dynamic scanning fluorescence (DSF) melt curves of the recombinant al and mutant AT10_004 antibodies. Whereas the parental AT10_004 shows a bimodal melt curve, the subclones with the light chain mutations (S30N (not shown), D50G and S92Y) show a single peak at high temperature only. The P100dF mutation does not influence the DSF curve.

Figure 4. A). Gel filtration chromatogram overlays of recombinant parental and mutant AT10_004. Recombinant AT10_004 antibody solutions were applied on to a gel filtration column to detect the presence of aggregated antibodies. B) Antibody monomers of AT10_004 and LC mutant D50G were purified using gel filtration (panel 1 and 3) and stored at -20ºC. After 2 months antibody ons were thawed and analyzed for the presence of antibody aggregates (panel 2 and 4).

Figure 5. In vitro nza A virus neutralization of H1N1 (A/Hawaii/31/2007) and H3N2 (A/Netherlands/177/2008) virus on MDCK-SIAT cells by recombinant antibodies. Top panels show H3N2 neutralization using low (left panel) or high (right panel) viral titers. Bottom panel shows the lization of H1N1 virus by the different (mutant) antibodies.

Figure 6. In vitro influenza A virus neutralization of H1N1 (A/Hawaii/31/2007) virus on MDCK-SIAT cells with stabilized recombinant AT10_004.8 (HC: P100dF) mutant antibodies.

Examples Generation and characterization of Influenza Hemagglutinin B cell clone AT10_004 Human memory B cells were immortalized using the BCL6 / Bcl-xL technology described by Kwakkenbos et al. e medicine, 16(1), 123–128. doi:10.1038/nm.2071; Methods 2013 doi:10.1016/j.ymeth.2013.07.002; patent application ). The fication and characterization of the crossreactive anti-influenza Hemagglutinin specific B cell clone AT10_004 is described in patent applications and . The binding of the AT10_004 antibody to the different Hemagglutinin proteins was tested using a solid phase ELISA or by using a FACS assay with virus infected cells (described in patent application ). 04 shows strong binding to most Group 2 Hemagglutinin proteins but only modest binding to Group 1 lutinin proteins is detected (Table 1).

Table 1. Binding of AT10_004 in ELISA or FACS-based assay to different nza group 1 and group 2 Hemagglutinin (HA) subtypes.

Influenza strain Subtype Group Binding capacity A/California/07/2009 H1 1 + A/Netherlands/602/2009 H1 1 - A/New Caledonia/20/1999 H1 1 + A/Hawaii/31/2007 H1 1 + A/Vietnam/1203/2004 H5 1 - ey/Turkey/2004 H5 1 + A/Hong 073/1999 H9 1 + A/Aichi/2/1968 H3 2 ++ A/Wyoming/03/2003 H3 2 ++ A/Netherlands/177/2008 H3 2 ++ A/Swine/St.Oedenrode/1996 H3 2 ++ A/Swine/Ontario/01911-1/1999 H4 2 + A/Chicken/Italy/1067/1999 H7 2 ++ A/Netherlands/219/2003 H7 2 ++ A/Chicken/Netherlands/621557/2003 H7 2 ++ A/Duck/Hong Kong/786/1979 H10 2 - A/Duck/AUS/341/1983 H15 2 + Selection of subclones with increased n binding In patent application it is shown that, within the heterogeneous subpopulation of a monoclonal B cell clone, cells with increased antigen binding capacity can be selected using a combination of antigen staining (H3-Alexa-647) and BCR staining. BCR staining was med with antibodies that bind to the heavy- or the light chain of the BCR. High H3 staining and equal or low BCR staining indicate higher antigen binding capacity of the BCR of that particular subclone, whereas low H3 staining and equal or high BCR staining indicates low n binding per BCR.

In the present study, HA-specific B cell clone (AT10_004) was cultured for 2-3 weeks to produce millions of cells, ng an unbiased heterogeneous B cell population, before an antigen-BCR staining was performed. Cells that showed increased antigen binding to the soluble labeled HA protein of H1 or H3 (H1 HA: A/New Caledonia/20/1999 or H3 HA: A/Wyoming/03/2003) were selected and sorted on a cell . After 3 rounds of sorting and growing, FACS analysis was performed on these cells to determine differences in antigen binding. Cells that were sorted three times for increased antigen binding show a clear shift in antigen staining ed to non-selected cells (Figure 1). These sorted cells were then subjected to single cell cloning using a FACSAria III (BD Biosciences). The clones were cultured for 2-3 weeks to allow expansion and then tested for sed antigen g compared to the parental clone.

Antigen competition assay usly differences in antigen binding were detected by staining the exact same number of cells with BCR antibody and labeled antigen to have similar cell/staining on-ratio. In the t experiments subclones that show increased antigen binding are detected using an antigen competition experiment.

AT10_004 cells were harvested and stained with Pe-Cy7 labelled CD19 antibody on ice. After 15 minutes the cells were washed, seeded at 40.000 cells per well and 100 µl of subclone cells is added. Subsequently the cell mix is washed and stained with labelled antigen (H1 HA: A/New Caledonia/20/1999 or H3 HA: A/Wyoming/03/2003) and labelled BCR antibody. Cells are incubated for 1-3 hours on ice, washed and measured on a FACSCanto (BD Biosciences). The amount of labelled antigen binding to CD19 positive cells (the parental 04 cells) is compared with the amount of labelled antigen bound to the ne (CD19 negative). Plotted in Figure 2 are 6 nes that show increased antigen binding to either H1- or H3 antigen.

Cloning and sequence analysis of selected subclone antibodies of AT10_004 A panel of subclones was selected based on enhanced antigen g compared to the parental AT10_004 clone. Of these subclones we isolated total RNA with the RNeasy® mini kit (Qiagen), generated cDNA, performed PCR, and performed sequence analysis. To produce recombinant human IgG we cloned the heavy and light variable regions in frame with human IgG1 and Kappa constant regions into a pcDNA3.1 (Invitrogen) based vector and ently transfected 293T cells. We ed recombinant IgG from the culture supernatant using MAbSelect Sure columns (GE Healthcare).

Sequence analysis of the ed AT10_004 subclones revealed that several of the subclones have identical mutations (Table 2), either as a single mutation or in combination with additional mutations. Interestingly, all of the subclones that show enhanced H3 binding have a d light chain. From the 17 H3 selected subclones, 11 have the light chain D50G mutation while the other 6 show the light chain S92Y on. All of the identified subclones (6) that show enhanced H1 binding contain the heavy chain P100dF mutation. Three of these subclones harbor a light chain mutation in addition. In the experiments described below we analyzed the effect of light chain S30N mutation detected in clone AT10_004.10.

Table 2. Description of the molecular teristics of the subclones that were selected for sed antigen-binding capacity after 3 rounds of antigen specific sorting. From these subclones, recombinant antibodies were produced. Mut HC and Mut LC indicate mutations in the heavy or light chain respectively. Amino acid numering according to Kabat.

AT10_004 stability g We tested the structural ity of the recombinant al and mutant AT10_004 antibodies with dynamic scanning fluorescence (DSF) (Phillips and Hernandez de la Pena, 2001, The Combined Use of the Thermofluor Assay and ThermoQ Analytical Software for the Determination of Protein Stability and Buffer zation as an Aid in Protein Crystallization. Hoboken, NJ, USA: John Wiley & Sons, Inc. doi:10.1002/0471142727.mb1028s94), using an iCycler RealTime PCR instrument (BIORAD) (Figure 3). In the DSF assay, antibodies are subjected to increasing temperature, g their structure to unfold. Protein unfolding is measured with a fluorescent reporter dye (SyPro, Invitrogen). The DSF meltcurve of the original AT10_004 antibody shows a multimodal pattern, representing the unfolding of the antibody subdomains (Fab, CH2 and CH3) at different temperatures (Garber and Demarest, 2007, Biochemical and Biophysical Research Communications, 355, 751-757). Mutants containing the light chain mutations S30N (not shown), D50G and S92Y lack the first “unfolding peak” at ± 60 °C observed in the parental antibody (Figure 3), indicating that these selected mutants have an increased thermostability.

Next, we analyzed the presence of aggregates in the recombinantly produced, purified antibody solutions by gel tion, on a Superdex200 gel filtration column (GE Healthcare). The gel filtration analysis clearly shows that ates are present in ed AT10_004 (Figure 4a). AT10_004 mutants with a light chain mutation (S30N, D50G or S92Y) do not form these aggregates (Figure 4a). We further analyzed the effect of light chain mutations on the stability of AT10_004 by looking at formation of aggregates during long-term storage. Antibody monomers (AT10_004 and mutant LC: D50G) were ed with gel filtration (Figure 4b, panel 1 and 3) and stored for two months at -20 °C. After storage, the amount of aggregates in each sample was determined with gel filtration. AT10_004 contains more ates than mutant LC: D50G (Figure 4b, panel 2 and 4). Similar results are obtained with light chain s S30N and S92Y (data not shown).

These s t that the light chain mutations (S30N, D50G and S92Y) we have obtained enhance the thermostability of the AT10_004 antibody and that the mutations prevent formation of aggregates.

Virus neutralization To determine whether the obtained mutant antibodies had different neutralizing capacities compared to the parental 04 antibody, an in vitro neutralization assay was performed. The assay was performed on MDCK-SIAT cells (Matrosovich M. et al., 2010, Journal of Virology, 77(15), 425). MDCK-SIAT cells were grown in DMEM/8%FCS/PS/G418 in a 96 well plate (CellCarrier Plate, PerkinElmer) to 80-100% confluency. Neutralization assays are performed in m/PS/G418/Trypsin medium without FCS or BSA. Fifty µl of recombinant mAb was mixed with 50 µl of virus suspension ID50/50 µl or 1000TCID50/50 µl) of H3N2 (A/Ned/177/2008) or H1N1 (A/Hawaii/31/2007) nza and incubated for 1 h at 37°C. The suspension was then transferred in multiply into 96-well plates containing MDCK-SIAT cells in 100 µl m/PS/G418/Trypsin. Prior to use the MDCK-SIAT cells were washed twice with 150 µl PBS. The plates were then centrifuged for 15 minutes at RT at 2500 rpm and placed at 37°C / 5% CO2. After 24 h cells were washed twice with PBS, fixed with Formalin (37% formaldehyde in water) for 10 minutes at RT, washed twice with 150 µl PBS and stained with DAPI and an antibody against the r protein of the Influenza virus TC, Abcam) at RT. After 30 minutes cells were washed twice with 150 µl PBS and 100 µl of PBS + 50 % glycerol was added to the wells. Viral infection of the MDCK-SIAT cells was ed and analyzed on the Operetta (PerkinElmer) using a 10x objective. To quantify neutralizing capacity of the mAbs the number of infected cells was counted ive for DAPI and NPFITC ). IC50 values were calculated with Prism (GraphPad software). Antibodies containing the LC: D50G or LC: S92Y mutation maintained H3N2 neutralizing capacity at low TCID50 and even have increased neutralizing capacity as evidenced by a lower IC50 when the neutralization assay was performed with a higher viral dose (> 3000TCID50) (Figure 5 and Table 3). No increase in H1 neutralizing ty for these light chain mutants was detected (tested up to 150 µg/ml). The AT10_004 subclones that were identified by selection with labeled H1 antigen (AT10_004.8, AT10_004.9 and AT10_004.10) showed reduced H3N2 neutralizing capacity (Figure 5 and Table 3) however, AT10_004.8 and AT10_004.9 showed increased H1N1 neutralizing potency. Whereas the original AT10_004 antibody or the light chain s show no inhibition of H1N1 infection antibody, AT10_004.8 shows a marked effect on H1N1 infection.

Table 3. IC50 values for H3 neutralization of recombinant AT10_004 antibody and the different recombinant antibody subclones of AT10_004 at two virus concentrations.

Clone Selection Mut Mut IC50 H3(1) IC50 H3(2) HC LC (ng/ml) (ng/ml) 004 - 436 2163 004.2 H3 D50G 433 1230 004.5 H3 S92Y 424 1028 004.8 H1 P100dF 3127 5174 004.9 H1 P100dF, 2118 5732 T107S 004.10 H1 A24T, S30N 922 2328 A52aV, P100dF, T110I (1) 300-500 TCID50 (2) 3000-5000 TCID50 Surface plasmon nce (SPR) analysis SPR analysis was performed on an IBIS MX96 SPR imaging system (IBIS Technologies BV, Enschede, The Netherlands) as bed before (Lokate et al., 2007, J Am Chem Soc. 129(45):14013-8). In this experiment, dies (1.0 μg/ml in coupling buffer: 10 mM MES-NaOH, pH 4.5 + 0.05 % Tween20) were directly immobilized on an Easy2Spot ilm gel-type SPR-sensor (Ssens, Enschede, The Netherlands) using a continuous flow microspotter device (Wasatch Microfluidics, Salt Lake City, UT, USA). After ng, the sensor was de-activated with 50 mM ethanolamine, pH 8.5. After de-activation, several blank injections cycles were done, each consisting of 45 minutes injection with empty assay buffer (PBS + 0.05 % sodium azide, 0.05 % Tween20 and 0.01 % BSA) followed by 2 minutes incubation with regeneration buffer (50 mM glycine-HCl, pH 2.0).

The SPR analysis consisted of multiple cycles of concatenated injections with H3 or H1 proteins. Each cycle consisted of an association step (10 min), in which recombinant influenza HA3- or H1-proteins (H1 HA: A/California/07/09; H3 HA: A/Wyoming/03/2003), diluted in assay buffer, are injected on the coated sensor; a dissociation step (15 min), in which the sensor is d with assay buffer, and, lastly, a wash with regeneration buffer (2 min), to remove any remaining bound e from the sensor. SPR data was ed using Sprint re (version 1.6.8.0, IBIS Technologies BV, Enschede, The Netherlands). Binding constants were fitted using Scrubber2 software (Biologic Software, Campbell, Australia).

Subclones with a stabilizing mutation in the light chain (clones AT10_004.2 (D50G), AT10_004.5 (S92Y) & AT10_004.12 ) all have an H3 affinity similar as the original AT10_004 clone (Table 4). The H1-affinity of these subclones is also r to the original 04 clone (no more then 50% deviation) (Table 4). The H1-induced HC: P100dF mutation increases binding affinity to H1 and reduces H3- binding (Table 4); this corresponds to the neutralizing capacity of this subclone (AT10_004.8) for these s. When subclone AT10_004.8 (P100dF) is stabilized by incorporating one of the stabilizing LC mutations we have identified, its affinity for H1 increases r (Table 4); this is most evident for mutant AT10_004.13 (LC: D50G). This mutant (HC: P100dF + LC: D50G) also shows an improved neutralizing capacity for H1, compared to the HC: P100dF mutation alone e Table 4. SPR analysis of the g of recombinant AT10_004 and AT10_004 subclones to recombinant H1 and H3 HA.

Clone Selection Mut Mut Affinity (KD) Affinity (KD) HC LC H1 (pM) H3 (pM) 004 - 660 (± 100) 39 (± 4) 004.2 H3 D50G 970 (± 130) 35 (± 1) 004.5 H3 S92Y 490 (± 10) 34 (± 3) 004.8 H1 P100dF 240 (± 90) 150 (± 60) 004.12 S30N 540 (± 70) 32 (± 1) 004.13 P100dF D50G 28 (± 2) 100 (± 40) 004.14 P100dF S92Y 110 (± 40) 34 (± 2) 004.15 P100dF S30N 41 (± 20) 29 (± 1)

Claims

Claims 1. An ex vivo method for producing a B cell e of producing antibody for an antigen of interest comprising: a) selecting at least one B cell capable of producing dy specific for said antigen of interest or at least one B cell capable of developing into a B cell capable 5 of producing antibody specific for said antigen of interest; b) providing said at least one B cell with a nucleic acid encoding BCL6 and with an anti-apoptotic nucleic acid of the BCL2 family; c) allowing ion of said at least one B cell into a first B cell culture; d) selecting at least one B cell from said first B cell culture with a binding avidity 10 for said n of interest that is higher than the average binding y for said antigen of interest of B cells in said first B cell culture; e) optionally allowing expansion of said at least one B cell selected in step d) into a second B cell culture; f) determining the thermal ity and/or the resistance to aggregation and/or 15 the chemical stability, and the ty for said antigen of interest, of antibodies produced by said at least one B cell ed in step d) or by said second B cell culture; and g) selecting at least one B cell capable of producing antibodies with a higher thermal stability and/or resistance to aggregation and/or chemical ity as 20 compared to the average l stability or resistance to ation or chemical stability of antibodies produced by B cells of said first B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said first B cell culture.
2. A method for isolating from an ex vivo B cell culture at least one B cell capable of producing antibodies with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by said ex 30 vivo B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture the method sing: a) providing an ex vivo B cell culture capable of producing dy specific for an antigen of st; 5 b) selecting at least one B cell from said ex vivo B cell culture with a binding avidity for said antigen of interest that is higher than the average binding avidity of B cells of said ex vivo B cell culture for said antigen of interest; c) determining the thermal stability and/or the resistance to aggregation and/or the chemical stability, and the affinity for said antigen of interest, of antibodies 10 produced by said at least one B cell selected in step b); and d) isolating at least one B cell capable of producing antibodies with a higher thermal stability and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by B cells of said ex vivo B cell culture, and with an 15 affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of st of antibodies produced by said ex vivo B cell culture.
3. A method according to claim 2 wherein B cells of said ex vivo B cell 20 e comprise a nucleic acid encoding BCL6 and an anti-apoptotic nucleic acid of the BCL2 family.
4. Method according to claim 2 or 3 wherein step a) comprises selecting at least one B cell capable of ing antibody ic for said antigen of interest or 25 selecting at least one B cell capable of developing into a B cell e of producing antibody specific for said antigen of interest and allowing expansion of said at least one B cell into said ex vivo B cell culture.
5. Method ing to claim 4, wherein allowing expansion of said at least 30 one B cell into said ex vivo B cell culture and ing at least one B cell from said ex vivo B cell culture capable of producing antibody with a binding avidity for said antigen of interest that is higher than the average binding avidity of B cell receptors or antibodies produced by said ex vivo B cell culture for said antigen of interest in step b) are repeated at least once.
6. Method according to any one of claims 1-5 wherein the thermal stability 5 and/or the resistance to aggregation and/or the chemical stability of antibodies produced by said at least one B cell selected as having a binding avidity higher than the average g y of B cells in said first B cell culture or in said ex vivo B cell culture or the thermal stability and/or the resistance to aggregation and/or the chemical stability of antibodies produced by said second B cell culture is 10 determined within four months from selecting said at least one B cell having a binding avidity higher than the average binding avidity of B cells in said first B cell culture or in said ex vivo B cell culture.
7. Method according to any one of claims 1-6 wherein the thermal stability 15 and/or the ance to aggregation and/or the chemical stability of antibodies produced by said at least one B cell selected as having a binding avidity higher than the average binding avidity of B cells in said first B cell culture or in said ex vivo B cell culture or the l stability and/or the resistance to ation and/or the chemical stability of antibodies produced by said second B cell culture is 20 determined within one month from selecting said at least one B cell having a binding avidity higher than the average binding avidity of B cells in said first B cell e or in said ex vivo B cell culture.
8. A method according to claim 1 or 3, wherein said anti-apoptotic nucleic 25 acid comprises a gene ng Bcl-xL or Mcl 1, or a onal part thereof.
9. Method according to any one of claims 1-8 further comprising ly or indirectly inducing, enhancing and/or maintaining the amount of Blimp 1 expression product in said at least one B cell or in said ex vivo B cell culture.
10. A method according to any one of claims 1-9, wherein said at least one B cell or said ex vivo B cell culture originates from an individual which had been previously exposed to said antigen of interest.
11. Method according to any one of claims 1-10, further comprising determining at least part of the amino acid sequence of the heavy chain and/or light chain of said at least one B cell that is capable of producing antibodies with a 5 higher thermal stability or resistance to aggregation or chemical stability.
12. A method for producing antibodies specific for an antigen of interest, sing: - selecting from an ex vivo B cell culture at least one B cell capable of producing 10 antibody with a higher thermal ity and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or ance to aggregation or chemical stability of antibodies produced by said ex vivo B cell culture, and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said 15 ex vivo B cell culture, with a method according to any one of claims 2-11; - culturing said at least one B cell into a B cell culture; and - obtaining dies produced by said B cell culture.
13. A method for producing antibodies specific for an antigen of interest, 20 comprising: - selecting from an ex vivo B cell culture at least one B cell e of producing antibody with a higher l ity and/or resistance to aggregation and/or chemical stability as compared to the average thermal stability or resistance to aggregation or chemical stability of antibodies produced by said ex vivo B cell 25 culture, and with an ty for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell e, with a method according to any one of claims 2-10; - optionally allowing expansion of said ed at least one B cell into a B cell culture; 30 - determining the amino acid sequence of the heavy chain and/or light chain of said antibody with a higher stability; and - expressing a nucleic acid molecule ng the heavy chain and/or light chain of said antibody in a second cell.
14. A method for identifying at least one mutation in the amino acid ce of the heavy chain and/or light chain of an antibody as compared to the amino acid sequence of the heavy chain and/or light chain of a nce antibody, 5 which mutation promotes thermal stability and/or resistance to aggregation and/or chemical ity of said antibody, comprising: - isolating from an ex vivo B cell culture at least one B cell capable of producing antibody with a higher thermal stability and/or resistance to aggregation and/or chemical ity as compared to the average thermal stability or resistance to 10 aggregation or chemical stability of dies produced by said ex vivo B cell culture. and with an affinity for said antigen of interest that is the same as, or less than, the average affinity for said antigen of interest of antibodies produced by said ex vivo B cell culture, with a method according to any one of claims 2-10; - optionally allowing expansion of said selected at least one B cell into a B cell 15 culture; - determining at least part of the amino acid sequence of the heavy chain and/or light chain of an antibody produced by said selected at least one B cell; - comparing said amino acid sequence with at least part of the amino acid sequence of the heavy chain and/or light chain of a reference dy, thereby identifying at 20 least one mutation in the amino acid sequence in the heavy chain and/or light chain of said antibody which on promotes thermal stability and/or resistance to aggregation and/or chemical stability of said antibody.
15. An ex vivo method according to claim 1, substantially as herein described 25 with nce to any example thereof, and with nce to the accompanying drawings.
16. A method according to any one of claims 2-14, substantially as herein described with reference to any example thereof, and with reference to the 30 accompanying drawings. AT10_OO4 3 rounds of n Parental affinity sorting § Outliers Lambda Single cell sorting Outliers H1 antigen binding— W0 2015/1 15892 ‘BCR Parental control H3 antigen g I Parental AT10_004 N H1 or H3 selected AT10_004 WO 15892 20000 A 15000 g AT10_004 8 10000 5000 40 50 60 70 80 90 Temperature (°C) 20000 A15000 E, AT10_004 8 10000 :3: LC: $92Y 5000 40 5O 60 70 80 90 Temperature (°C) 20000 315000 mo...e%4 § 10000 x 93 HO: P100dF o i:1:;:»:-: ) 5000 ...... ........... ................ ........ --------llllllllllllllllllll ........... nnnnnn 40 50 60 7O 80 90 Temperature (°C) Cont’d PCT/NL20 15/050054 Om. E A 28.9 999 8635mm Solo + Zomw ck H0.. Om. HoEocoE ‘ 0.8:: 99.9 8835mm <v + >Nmm .QE voolo E.< H0.. 320:50:05 0.8.8 989 voolo + Conn. Fr< ”04 PCT/NL20 054 116.12, mEGEOCOE g , 2 voono 35:88.: 88am: / £< ON 3998 N {1 u_ wLmEocoS— umccsq 8.9m emEocoE on 38869 / voono 605.8. F: €85 \WJL 20 15/050054 emEocoE q 2“ 080 35:88.: N0.. 3998 PEoO 29:95.)— umccsq 8.9m .OE QwEocoE q f. 605.8. 88969 ”3 £85 8J9) AT10_004 Parental AT10_004 LC mutants AT10_004.8 H1 selected AT10_004.9 H1 selected AT10_004.10 H1 selected Negative control mAb 000 TC|D50 cells 100 infected \I01 Percentage N01 101 102 1o3 104 105 1o1 102 1o3 10:1 1o5 Concentration mAb (ng/ml)— cells 1 00 s‘\\\\\\“““ \\\\\\\-\\\\x\\\\§\\\\\\.\\\\\\\\\ infected g AT10_004 LC mutants ‘ \\\“ 04.8 H1 selected 50 ‘. .& AT10_004.9 H1 selected Percentage . AT10_004.10 H1 ed 10 50 100 Concentration mAb (pg/ml) F1C3.£5 I] VH2 P100dF LC: S3ON 9 HC: P100dF LC: S92Y I HC: P100dF LC: DSOG (004.13) & AT10_004 LC mutants \\§\V AT10_004.8 H1 selected (HC: ) 100 §\\ ,. W‘W“\\\W\ kw,“\\ \§\.\\\\\ &“C“ cells \\ \\\§\\\\ infected 50 Percentage N01 10 50 100 Concentration mAb (ng/ml) FIG. 6