WO2014096389A1

WO2014096389A1 - Method for identifying antibody producing cells

Info

Publication number: WO2014096389A1
Application number: PCT/EP2013/077757
Authority: WO
Inventors: Daniel John Lightwood; Stephen Edward Rapecki; Dale Owen STARKIE
Original assignee: UCB SA
Current assignee: UCB SA
Priority date: 2012-12-21
Filing date: 2013-12-20
Publication date: 2014-06-26
Anticipated expiration: 2015-06-21
Also published as: GB201223316D0

Abstract

The present disclosure relates to a method for identifying a cell producing an antibody specific to a target antigen comprising the steps of: a) providing a population of antibody producing B cells, wherein said population has been selected on the basis of at least one B cell marker and/or at least one marker selected from the group consisting of an IgG, IgE and IgA marker, and two or more "de-selection" markers (not present on the target B cell), b) incubating the population of B cells from step a) with said target antigen and at least two distinct labels for said antigen such that the antigen is in a form labelled with a first label and/or a second label, c) screening the cell population for those cells capable of producing antibodies specific to the target antigen wherein the cells of interest are identified by at least one of each of the two or more distinct labels, and d) selecting the multi-antigen-labelled population of cells identified from step c), and e) optionally separating the population of cells selected in step d) into single cells.

Description

Method for Identifying Antibody Producing Cells

The present disclosure provides a method for identifying a B cell producing antibodies specific to a target antigen based on positive and negative selection criteria, wherein said method gives improved levels of confidence in the identification and selection of B cells specific to said antigen.

Methods for identifying antibody producing cells employing anti-antibody antibodies are disclosed in WO2004/051268. B cells markers such as CD19, CD138, CD20, B220, CD22, CD38, CD27 etc have been known for a number of years. Additionally techniques such as FACS (fluorescence-activated cell sorting) to sort B cells have been disclosed in applications such as WO2005/019824.

Antibodies are generally considered to be exquisitely specific to a given target. However, a certain amount of non-specific binding occurs and during antibody screening this requires extra steps and assays to isolate the B cells of interest. The process is further hindered by the fact the B cell population targeted is 1% or less of the hosts cells.

Good selection criteria for identifying antibody producing B cells is essential for generating good antibody leads for development of therapeutic antibodies. The present method is advantageous in that it eliminates cells which are not of interest and allows the resource and screening to be focused on the high quality cells of interest.

The present disclosure provides a streamlined method for identifying a cell producing an antibody specific to a target antigen comprising the steps of:

a. providing a population of antibody producing B cells, for example selected from plasma B cells, activated B cells and memory B cells, in particular B cells which express class switched affinity matured antibodies, wherein said population has been selected on the basis of at least one B cell marker and/or (in particular or) an IgG marker, and two or more de-selection markers,

b. incubating the population of B cells from step a) with said target antigen and at least two distinct labels for said antigen such that the antigen is in a form labelled with a first label and/or a second label,

c. screening the cell population for those cells capable of producing antibodies specific to the target antigen wherein the cells of interested are identified by at least one of each of the distinct antigen labels, and

d. selecting the multi-antigen- labelled population of cells identified from step c), and e. optionally separating the population of cells selected in step d) into single cells. Brief Description of the Figures

Figure 1 shows gating techniques for mouse B-cell sorting (antigen x)

Figure 2 shows gating techniques for mouse B-cell sorting (antigen z)

Figure 3 shows gating techniques for rabbit B-cell sorting (antigen y)

Detailed Description of the Disclosure

The specific reagents employed in the method of the present disclosure may vary depending on which species is employed as the host animal. Many reagents are available for where the host animal is murine. In one embodiment the host animal is a mouse, a rat or rabbit, in particular a mouse or rabbit.

In one embodiment the host animal is a mouse. In one embodiment the host animal is a rabbit. In one embodiment the host animal is a rat. Thus in one embodiment the method is suitable for identifying interesting antibodies generated in vivo, including antibodies that may have been generated in a human subject, for example isolatable from the serum, blood or tissue of said human.

The method described herein may require adaptation to reflect the specific species of host animal employed in the generation of antibodies. This adaptation is routine, for example the marker may be named differently for the particular species. However, the skilled person is well able to identify the corresponding marker in the given species or an alternative marker which performs the same function in that particular species. B cell markers include CD19, CD20, CD22, CD23, CD38, B220, CD40, CD43, CD138,

CXCR4, BCMA, IL-6R, B220, CD21 , CD35, CD24, CD23, CD40 and intracellular endoplasmic reticulum stains.

In one embodiment B cell markers include CD19, CD20, CD21 , CD22, CD23, CD24, CD 35, CD38, CD40, CD43, CD138, CXCR4, BCMA, IL-6R, B220 and intracellular endoplasmic reticulum stains, for example CD19, CD20, CD22, CD38, CD43, CD138, CXCR4, BCMA, IL- 6R, B220 and intracellular endoplasmic reticulum stains in particular in one embodiment the marker is selected from the group comprising CD19, CD20, CD22, CD38 and B220. Endoplasmic reticulum stains are dyes that are highly selective for the endoplasmic reticulum, for example the dyes available under the tradename ER-Tracker™ available from Life

Technologies (such as catalogue numbers E12353, E34250 and E341251). See also fluorescent staining of subcellular organelles: ER, golgi complex and mitochondria- Curr. Protoc. Cell Biol 2001 , May Chapter 4: Unit 4.4.In one embodiment B cells as employed herein includes cells known as plasmablasts.

In one embodiment the method comprises a step wherein a starting population of cells is pre- enriched for B cells, for example based on a marker B220 such as anti-B220 micro beads

(Miltenyi 130-049-501). B220 is also known as CD45, CD45R, T200, LY5, LCA and PTPRC. The murine version of the protein has the UniProt number P06800.

In one embodiment the method comprises a first enrichment step based on marker B220.

Suitable protocols for performing such an enrichment step are known in the art and may optionally include the use of a blocking agent, such as an Fc Block to prevent non-specific binding of anti-mouse B220 antibodies to Fc receptors on non-B-cells. In one embodiment the method comprises a first enrichment step based on marker B220 in combination with an Fc block, such as a mouse Fc Block.

In one embodiment the present methods comprise a pre-selection step employing marker CD 19.

In one embodiment the method comprises a first enrichment step based on marker B220 followed by a selection step based on the marker CD 19.

In one embodiment the B cell marker, for use in step a), is selected from the group comprising the following combination of markers: CD 19 & CD20, CD19 & CD22, CD19 & CD38, CD19 & B220, CD20 & CD22, CD20 & CD38, CD22 & B220, CD38 & B220, and CD138 & B220.

In one embodiment only a B cell marker or B cell markers are employed as positive markers to select the desired population.

Positive marker as employed herein refers to a marker present on cells which it is desirous to select and which can be used as a basis of the selection.

In one embodiment the marker is based on IgG as a marker for class-switched, affinity-matured IgG- producing cells that encompasses the B cell subset of interest. The technique exploits the observation that in an immunised animal, the IgG response becomes biased towards the immunogen (Reddy et al. (2010) Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells. Nature Biotechnology 28(9): 965-9) therefore, a certain percentage of IgG-secreting cells will possess reactivity towards that immunogen.

In some instances the IgG will be expressed on the surface of the cells. IgG as employed herein includes all sub-types thereof, such as IgGl , IgG2, IgG3 and IgG4. In one embodiment the agent specific to the IgG does not different between the sub-types thereof i.e. is specific to IgG generally.

In one embodiment the IgG marker, for use in step a), is a mixture of two or more IgG markers for different isotypes. In one example the IgG marker is a mixture of anti-IgGl , anti-IgG2A and anti-IgG2B.

The approach may also be readily applied to other markers of class-switched B cell, such as IgE or IgA. The technique exploits the observation that in an immunised animal, the IgG response becomes biased towards the immunogen (Reddy et al. (2010) supra).

Accordingly the present invention provides a method for identifying a cell producing an antibody specific to a target antigen comprising the steps of:

a. providing a population of antibody producing B cells, wherein said population has been selected on the basis of at least one B cell marker and/or at least one marker selected from the group consisting of an IgG, IgE and IgA marker, and two or more "de-selection" markers (not present on the target B cell),

c. screening the cell population for those cells capable of producing antibodies

specific to the target antigen wherein the cells of interest are identified by at least one of each of the two or more distinct labels, and

d. selecting the multi-antigen-labelled population of cells identified from step c), and e. optionally separating the population of cells selected in step d) into single cells.

In one aspect a method according to the present disclosure is provided wherein the IgG specific reagents (or markers) are antibodies or binding fragments thereof. In one embodiment at least two different antibodies or binding fragments specific to IgG are employed with distinct labels.

In one aspect a method according to the present disclosure is provided wherein the label or labels of the IgG specific reagents are fluorescent. Where two or more labels are fluorescent they may be chosen to have non-overlapping emission spectra. Alternatives to fluorescent labels may also be employed, these include enzymes (such as alkaline phosphatase, HRP and beta-galactosidase), phosphorescent, chemiluminescent dyes, gold, latex and magnetic particles, etc. In one embodiment an IgG specific reagent is the only positive marker employed in step a) to identify the B cell population. Thus in one embodiment only one or more IgG marker(s) are employed, for example because B cell markers for the host in which the antibodies are generated are limited. In one embodiment the B cell population can be identified employing a combination of at least one B cell marker and at least one IgG marker.

The combination of these markers can be employed which is suitable for identifying an antibody producing population of memory B cells, in particular B220 and an IgG marker.

In one embodiment the method according to the present disclosure employs in step a) a combination of markers B220, CD 19 and one or more IgG markers.

In one embodiment the method according to the present disclosure further comprises the step of identifying a B cell in the population employing a functional assay. Identification of antibody secreting cells may be identified using a functional screening assay, for example a binding assay or measuring a functional effect such as neutralisation of a given activity.

De-selection marker as employed herein refers to a marker on cells which are a sub-population that it is beneficial to exclude, i.e. a marker on cells that can be removed from the population that is retained based on the said marker.

Embodiments described herein referring to IgG markers may also be effected with an IgE or IgA marker or reagent.

In one embodiment a combination of IgG, IgE and IgA markers are employed, for example IgG and IgE, IgG and IgA, and IgE and IgA.

The negative selection criteria in the method of the present disclosure is important because it increases the concentration of the desired population of cells in the sample and allows more focussed analysis. In one embodiment the deselection markers are selected from the group comprising IgM, IgD, CD4+, CD8+, F4/80+, Grl+ and cells stained with a non-viability dye such as 7AAD. In one embodiment the method comprises de-selecting IgM+ and/or IgD+ populations of cells, because advantageously this focuses the population towards class switch affinity matured antibodies.

In one embodiment the method comprises de-selecting IgM+ populations of cells, because advantageously this focuses the population towards class switch affinity matured antibodies.

In one embodiment the method comprises de-selecting CD4+ and/or CD8+ populations of cells. This step eliminates T cells and thereby significantly contributes to enriching the population of cells for the desired population.

In one embodiment the method comprises de-selecting F4/80+ populations of cells. This is a marker for macrophages. Eliminating macrophages significantly contributes to enriching the population of cells for the desired population.

F4/80 (ηιφ)^" is a murine marker. In other species corresponding markers may be employed.

In one embodiment the method comprises deselecting GR-1⁺ populations of cells. This is a marker for granulocyte cells and eliminating these cells significantly contributes to enriching the population of cells for the desired population.

In one embodiment the method further comprises deselecting the population of cells which stain positive with 7-AAD or an equivalent dye. This dye penetrates cells which are non-viable, i.e. dead or dying cells. Removing non-viable cells from the population significantly contributes to enriching the population of cells for the desired population.

A combination of these markers for de-selection purposes is particularly advantageous because it greatly enriches the target memory B cell population thereby facilitating the isolation and/or recovery of the sequence information from the latter.

In one embodiment the B cell population selection is based on: a. cells positive for one or more markers selected from the group comprising CD 19, and IgG (such as IgGl and/or IgG2), and

b. de-selection of one or more markers selected from the group comprising IgM, IgD, CD4+, CD8+, F4/80+ and Grl+ , and wherein the cells are viable, for example unstained by dye such as 7AAD, particular wherein the cells are sorted sequentially based on the markers.

In one embodiment the B cell population selection is based on: a. cells positive for one or more markers selected from the group comprising B220, CD19, IgGl , IgG2A and IgG2B, and

b. de-selection of one or more markers selected from the group comprising IgM, IgD, CD4+, CD8+, F4/80+ and Grl+ , and

c. wherein the cells are viable, for example unstained by dye such as 7AAD, in

particular wherein the cells are sorted sequentially based on the markers.

The present method provides the selection of high quality antibody producing cells wherein the antibody repertoire of cells is not severely restricted or reduced by the selection process. This is advantageous because more and more it is important to identify so-called rare antibodies, such as function modifying antibodies. Many prior art selection processes narrowed the population of resulting cells in a way which eliminates the rare antibodies.

In one embodiment the de-selection markers consist of those described above.

The markers, described herein, may be employed sequentially, concomitantly or as a

combination.

"And/or" as employed herein refers to use of the elements in combination (and) and also in the alternative (or).

In one embodiment step a) further comprises an Fc block to prevent non-specific binding.

In one embodiment the markers are employed substantially sequentially, for example sorting based on the markers can be performed sequentially, that is the sorting is performed employing the first marker to provide a first subpopulation of cells and then further sorting is performed on this subpopulation of cells based on a second marker to provide a second small subpopulation of cells. This second subpopulation may be subject to sorting based on a further third marker to provide a third subpopulation and so on to provide a fourth and fifth subpopulation.

Sequentially as employed herein refers to use one after another. Concomitant as employed herein refers to use at the same time.

Combinations as employed herein refers to selection of certain markers, for example 3, 4 or 5, in particular combination for use at the same time

In one embodiment one or more markers are employed concomitantly, for example one or more de-selection markers are employed concomitantly. In one embodiment a combination of labelling agents for a given marker are employed in combination, for example two or more agents which selectively target IgG and which are independently detectable, for example because the labels associated with the agents (such as antibodies) emit light a different frequencies. This double labelling improves the selectivity of the method and eliminates non-specific binding of given labelling agent.

This multiple labelling may be employed with any one or more of the markers described herein, for example one or more positive markers described herein, such as all the positive markers described herein, or one or more negative markers as described herein. Cell sorting may be performed by any suitable method known in the art, for example FACS (fluorescence activated cell sorting) or MACS (magnetic cell sorting).

FACS is a form of fiow-cytometry, namely fluorescence activated cell sorting, which facilitates the separation of cells based on predefined parameters. FACS sorting of B cells in described in WO05/01924 incorporated herein by reference.

MACS is magnetic -activated cell sorting, which again facilitates the separation of sub-population of cells based on predefined parameters. MACS methods of sorting B cells is described in WO05/01923 incorporated herein by reference.

These technologies can be employed to sort cell populations into single cells.

The present method employs target antigen to identify cells which produce antibodies specific to the antigen.

Specific to an antigen as employed herein is intended to refer, for example to an antibody that only recognises the antigen to which it is specific or an antibody that has significantly higher binding affinity to the antigen to which it is specific compared to binding to antigens to which it is non-specific, for example at least 5, 6, 7, 8, 9, 10 times higher binding affinity.

Antibody affinity may be measured by routine techniques, for example surface plasmon resonance, such as BIAcore.

In one embodiment there is provided a method wherein the target antigen employed as a reagent is provided as two discrete populations one labelled with a first label and the second population labelled with a second distinct label, for example wherein the B cells in step b) are incubated with the two distinctly labelled populations of target antigen concomitantly. Alternatively, the B cells in step b) are incubated with a first labelled population of target antigen and sequentially incubated with the second distinctly labelled population of target antigen.

In one embodiment the antigen is provided unlabelled and at least two labels specific to the target antigen are introduced into the population or B cell population before, after or concomitant with the addition of unbound antigen, for example wherein the two labels specific for the target antigen comprise an antibody or binding fragment thereof specific to the target antigen. In this embodiment the two labels may be introduced concomitantly or sequentially with each other. In one embodiment the antigen is directly conjugated to a label or at least two distinct labels, such as fluorescent or magnetic labels. .

Directly conjugated as employed herein, for example refers to where the antigen is connected to the label via a bond, such as an amide or imide bond.

In one embodiment one or two, for example two, labels are fluorescent and in particular there are two distinct labels, which emit light at different frequencies i.e. the fluorophores have non- overlapping emission spectra and can therefore be can readily detected and distinguished from each other.

In one embodiment the unbound antigen is contacted with two distinct labels concomitantly. Thus in one embodiment in the method of the disclosure "labelled antigen", refers to where each antigen, for example all antigen molecules or the majority of antigen molecules bears at least two distinct labels.

In one embodiment antigen molecules bearing at least two distinct labels and cells identified in step a) of the method herein are mixed in a one-pot "reaction". In one embodiment antigen molecule, at least two labels specific thereto and cells identified in step a) are mixed in a one-pot reaction, without pre-incubation of the antigen with the label.

As employed herein antigen bearing a first label is one antigen form and antigen bearing a distinct label (a second label) is a different antigen form and so on. That is to say the antigen is the same and the labels on the two antigen populations are different. In one example each antigen form is only labelled with one type of label.

The same antigen as employed herein refers generically to the same protein or peptide or antigenic molecule employed, including different isotypes of the antigen and or antigen from different species. Advantageously, using for example primate antigen (or mouse, rat, rabbit etc) with one label and human protein with another label allows those cells/antibodies to be identified that bind both antigens i.e. cells/antibodies which cross-react. In one embodiment the antigen employed in the two populations are identical.

Distinct as employed herein refers to different, distinguishable, discrete and/or clearly defined populations or characteristic.

Thus in one embodiment the antigen is directly conjugated to the label. In one embodiment the antigen is conjugated to the label, for example conjugating using an amide bond, such as a maleimide.

In one embodiment the antigen is labelled with at least one antibody-conjugated to a label.

In one embodiment the method according to the disclosure comprises a step of identifying a high antibody producing cell.

Typically the cells of interest identified in step c) of the method are those that have bound labelled antigen and as such the cells of interest are a multi-antigen labelled population, in particular where both antigen forms with two distinct labels are bound and identified.

In one embodiment only those cells with double labels (i.e. two distinct labels) are identified in step c) and selected in step d).

In one example step d) of the method comprises selecting the cells of interest identified in step c) of the method. In one embodiment the method according to the disclosure comprises a further step wherein a B cell of interest is isolated, for example is manually or robotically picked, in particular after identification in a functional assay. Suitable techniques for isolating single B cells are known in the art and include the fluorescent foci method described in US7,993,864 and EP1570267B1.

In one embodiment the method comprises the further step of cloning at least the variable region of an antibody from a cell identified employing the method, for example employing PCR.

In one embodiment the method according to the present disclosure comprises a further step wherein an antibody or a variable region thereof from a B cell in the population is sequenced and/or amplified, for example employing PCR technology.

In one embodiment a method according to the present disclosure comprises cloning an antibody, variable region therefrom or at least one CDR therefrom.

In one embodiment the method comprises the further step of creating a library of CDRs, V- regions, single chains, antibodies or other antibody fragments.

The term 'antibody' as used herein generally relates to intact (whole) antibodies i.e. comprising the elements of two heavy chains and two light chains. The antibody may comprise further additional binding domains for example as per the molecule DVD-Ig as disclosed in WO 2007/024715, or the so-called (FabFv)₂Fc described in WO201 1/030107. Thus antibody as employed herein includes bi, tri or tetra-valent full length antibodies. Binding fragments of antibodies include single chain antibodies (i.e. a full length heavy chain and light chain); Fab, modified Fab, Fab', modified Fab', F(ab')₂, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (e.g. VH or VL or VHH), scFv, bi, tri or tetra-valent antibodies, Bis-scFv, diabodies, tribodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1 126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). The methods for creating and manufacturing these antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first disclosed in WO2009/040562 and the disulphide stabilised versions thereof, the Fab-dsFv was first disclosed in WO2010/035012. Other antibody fragments include the Fab and Fab' fragments described in International patent applications WO2005/003169, WO2005/003170 and

WO2005/003171. Multi-valent antibodies may comprise multiple specificities e.g. bispecific or may be monospecific (see for example WO 92/22583 and WO05/1 13605). One such example of the latter is a Tri-Fab (or TFM) as described in W092/22583. In one embodiment the method further comprises the step of expressing in a host cell an antibody or a binding fragment comprising at least one CDR identified in a B cell selected employing the method herein.

Suitable host cells include bacterial cells such as E. coli, yeast cells such as pichia, insect cells and mammalian cells such as CHO, HEK and the like.

Comprising in the context of the present specification is intended to meaning including. Where technically appropriate embodiments of the invention may be combined.

Embodiments are described herein as comprising certain features/elements. The disclosure also extends to separate embodiments consisting or consisting essentially of said features/elements.

Technical references such as patents and applications are incorporated herein by reference.

The present invention is further described by way of illustration only in the following examples, which refer to the accompanying Figures:

EXAMPLES

Example 1 Antigen-Specific Mouse Memory B-Cell Sorting (antigen X)

B cells from mice immunised with a human antigen (antigen X) protein were isolated from splenocytes using anti-mouse B220 MACS micro beads (Miltenyi 130-049-501) for B cell enrichment. This included the use of mouse Fc Block (Miltenyi 130-092-575) to prevent nonspecific binding of antibodies to Fc receptors on non-B cells. The following reagents were then used to identify the antigen-specific memory B cell subset. The subset of interest demonstrated positive staining with:

1. Anti-CD 19-Horizon V450 (BD 560375) (B cell stain)

2. A mixture of anti-IgGl -PE (BD 550083), anti-IgG2A-PE (Southern Biotech 115-09L) and anti-IgG2B-PE (Southern Biotech 1185-09L) (IgG+ B cell stains)

And negative staining with:

1. Anti-IgM-PE Cy 7 (BD 552867) (non-class switched B cell stain)

2. Anti-IgD-APC Cy7 (Bio-legend 405716) (immature B cell stain)

3. Anti-CD4 PercP (T-Cell stain) (biolegend 100432)

4. Anti-CD8 PercP (T-Cell stain) (Biolegend, 100732)

5. Anti-F4/80 PercP (Macrophage stain) (biolegend 123126)

6. Anti-Gr-1 PercP (Neutrophil stain) (biolegend 108426)

7. 7AAD (BD 51-68981E) for staining non-viable cells To identify antigen-specific memory B cells, the immunising antigen, in this case a human antigen protein (antigen X), was labelled with the two different fluorophores Alexafluor 488 (Invitrogen A30006) and Alexafluor 647 (Invitrogen A30009). These reagents were added to the multi-parameter B cell staining mix described above.

To confirm specific antigen binding, we also performed staining with Can-1 -human Fc, an irrelevant protein antigen, labelled with Alexafluor 488 and 647. As well as these reagents, a series of isotype control Abs for IgG staining, IgM staining and IgD staining were also included to confirm specificity: Rat IgGl PE isotype control (BD 550083), Rat IgG2a PE-Cy7 isotype control (BD 552784) and Rat IgG2a APC-Cy7 isotype control (BD 552770).

Compensation for the flow cytometry experiment was set with the following reagents:

Rat-anti-mouse-B220-PE (BD 553090)

Rat-anti-mouse-B220-FITC (BD 557669)

Rat-anti-mouse-B220-APC (BD 553092)

Rat-anti-mouse-B220-PercP (BD 553093)

Rat-anti-mouse-B220-PE-Cy7 (BD 552772)

Rat-anti-mouse-B220-APC-Cy7 (BD 552094)

Rat-anti-mouse-B220-Horizon V450 (BD 556042)

Following staining, B cells were assessed using a BD FACS Aria III multi parameter flow cytometer. The staining strategy and identification of the IgG+ antigen-specific B cell subset is illustrated in Figure 1. Gates run left to right and sort based on the thick gate in the final double antigen X plot.

The IgG+ antigen-specific (double positive for antigen on both Alexafluor 488 and 647(thick box in final double antigen X plot in Figure 1)) population, were sorted into a 96-well plate at one cell per well. 88 single cells were sorted per 96-well plate to enable a number of positive and negative controls to be included. Wells contained 20 ul of superscript III (Invitrogen) reverse transcription reaction mix. A reverse transcription reaction to generate cDNA from the single isolated B cell was performed immediately following sorting of the plate. Two rounds of PCR were then performed using antibody-specific oligonucleotides to amplify and recover antibody heavy and light chain variable region genes.

A 3° PCR was then performed enabling the combination of amplified variable regions, human CMV promoter fragment and mouse gamma 1 heavy constant or mouse kappa constant fragment to generate separate heavy and light transcriptionally-active linear PCR fragments. These DNA fragments were used directly for recombinant expression of mouse full-length IgG antibodies in a transient HEK-293 system. The resulting recombinant antibodies were then screened for antigen binding using a homogeneous fluorescence-based binding assay where human antigen X protein was immobilised on Superavidin^Tm beads (Bangs laboratories) coated with biotinylated goat anti-human Fc gamma antibody (Jackson). Table 1 summarises the heavy and light chain PCR recoveries from 2 sorted plates and the number that went onto demonstrate measurable antigen X-binding as a recombinant antibody in the antigen binding assay.

Table 1 - Recovery of heavy and light chain variable region genes and the number of recombinant IgG that went onto demonstrate binding to antigen in a fluorescence-based binding assay

As can be seen from Table 1 , thirty one antigen-specific recombinant mouse IgG molecules were generated from two 96-well plates. This suggests that the identified B cell subset contains antigen-specific B cells and the protocol described here enables the rapid recovery of recombinant antibodies that retain the natural heavy and light chain cognate pairing.

Example 2 Antigen-Specific Mouse Memory B-Cell Sorting (antigen Z)

B cells from mice immunised with a human antigen (antigen Z) protein were isolated from splenocytes using anti-mouse B220 MACS micro beads (Miltenyi 130-049-501) for B cell enrichment. This included the use of mouse Fc Block (Miltenyi 130-092-575) to prevent nonspecific binding of antibodies to Fc receptors on non-B cells. The following reagents were then used to identify the antigen-specific memory B cell subset. The subset of interest demonstrated positive staining with:

1. Anti-CD 19-Horizon V450 (BD 560375) (B cell stain)

And negative staining with:

8. Anti-IgM-PE Cy 7 (BD 552867) (non-class switched B cell stain)

9. Anti-IgD-APC Cy7 (Bio-legend 405716) (immature B cell stain)

10. Anti-CD4 PercP (T-Cell stain) (biolegend 100432) 11. Anti-CD8 PercP (T-Cell stain) (Biolegend, 100732)

12. Anti-F4/80 PercP (Macrophage stain) (biolegend 123126)

13. Anti-Gr-1 PercP (Neutrophil stain) (biolegend 108426)

14. 7AAD (BD 51-68981E) for staining non-viable cells

To identify antigen-specific memory B cells, the immunising antigen, in this case a human antigen protein (antigen Z), was labelled with the two different fluorophores Alexafluor 488 (Invitrogen A30006) and Alexafluor 647 (Invitrogen A30009). These reagents were added to the multi-parameter B cell staining mix described above.

To confirm specific antigen binding, we also performed staining with TEV, an irrelevant protein antigen, labelled with Alexafluor 488 and 647. As well as these reagents, a series of isotype control Abs for IgG staining, IgM staining and IgD staining were also included to confirm specificity: Rat IgGl PE isotype control (BD 550083), Rat IgG2a PE-Cy7 isotype control (BD 552784) and Rat IgG2a APC-Cy7 isotype control (BD 552770).

Rat-anti-mouse-B220-PE (BD 553090)

Rat-anti-mouse-B220-FITC (BD 557669)

· Rat-anti-mouse-B220-APC (BD 553092)

Rat-anti-mouse-B220-PercP (BD 553093)

Rat-anti-mouse-B220-PE-Cy7 (BD 552772)

Rat-anti-mouse-B220-APC-Cy7 (BD 552094)

Rat-anti-mouse-B220-Horizon V450 (BD 556042)

Following staining, B cells were assessed using a BD FACS Aria III multi parameter flow cyto meter. The staining strategy and identification of the IgG+ antigen-specific B cell subset is illustrated in Figure 2. Sorting was performed from the red gated populations labelled PI and P2. PI represents cells prior to the double antigen staining ie no antigen-specific sorting step. P2 represents the dual antigen-stained population. P2 represents only 1.06% of P 1 ; highlighting the importance of this enrichment step in isolating the antigen-specific B cells.

PI and P2 were sorted into separate 96 well plates one cell per well. Wells contained 20 ul of superscript III (Invitrogen) reverse transcription reaction mix. A reverse transcription reaction to generate cDNA from the single isolated B cell was performed immediately following sorting of the plate. Two rounds of PCR were then performed using antibody-specific oligonucleotides to amplify and recover antibody heavy and light chain variable region genes. A 3° PCR was then performed enabling the combination of amplified variable regions, human CMV promoter fragment and mouse gamma 1 heavy constant or mouse kappa constant fragment to generate separate heavy and light transcriptionally-active linear PCR fragments. These DNA fragments were used directly for recombinant expression of mouse full-length IgG antibodies in a transient HEK-293 system.

The resulting expression runs were tested for antibody expression as read out of successfully sorted wells and recoveries in an IgG ELISA where F(ab')2 fragment goat anti-mouse IgG Fc (Jackson immuoresearch code 115.006.008) at 2ug/ml before being blocked with 1%PEG in PBS. The transient supes were allowed to bind followed by a reveal of 1 :5000 Peroxidase labelled goat anti-mouse kappa (Southern Biotechnology Associates code 1050-05) and development with TMB. To assess levels of antigen specificity in the sorted cells the transients were tested for binding to antigen Z via ELISA where the antigen was directly coated onto the plate at 2ug/ml, before being blocked with 1%PEG in PBS. The transient supes were allowed to bind followed by a reveal of 1 :5000 goat anti mouse IgG Fc HRP and developed with TMB. The results from PI and P2 have been summarised in Table 2.

Table 2: Recoveries of recombinant antibodies from PI and P2 and numbers of

recombinant antibodies that show antigen specific binding as determined by ELISA binding to target antigen

As can be seen from Table 2, P2 generated 15 antigen-specific antibodies from one 96 well plate sort. This represented a 50% recovery of all wells that expressed antibody. This again demonstrated, with another antigen, that this double antigen stained population of cells contains the antigen-specific antibody producing B cells and that protocol described enables rapid recovery of those antigen-specific recombinant antibodies derived from those B cells. This claim is further substantiated by the recoveries seen in P 1 where cells were sorted based on all parameters of P2 except the dual antigen stain. This population shows expression of IgG to a comparable level to P2, however none of the antibodies derived from this population

demonstrated binding to the target antigen Z. This further demonstrates how essential the dual antigen staining is defining the antigen-specific B cell subset.

Example 3 Antigen-specific rabbit memory B-cell Sorting Antibodies specific for rabbit B cell markers are less readily available and for this reason the antibody panel was more restricted. We were also unable to apply a pre-enrichment step on beads (e.g. MACS) prior to flow cytometry. In this case, rabbit PBMCs from animals immunised with a mouse antigen (antigen Y) were used as a source of B cells. The following reagents were then used to identify the antigen-specific memory B cell subset. The subset of interest demonstrated positive staining with:

1. Anti-IgG-PE (southern Biotech 4090-09)

And negative staining with:

1. Anti-IgM Biotin (BD 550938) (pre conjugated with a streptavidin PE-Cy7 (BD 557598)) 2. Anti-CD4 (abd serotec MCA799G) labelled with PercP (Lightning Link PercP kit,

Innova Biosciences 718-0010)

3. Anti-CD8 (abd serotec MCA1576G) labelled with PercP (Lightning Link PercP kit, Innova Biosciences 718-0010)

4. Anti-T-Cell Ab (abd serotec MAB1680) labelled with PercP (Lightning Link PercP kit, Innova Biosciences 718-0010)

5. 7AAD (BD 51-6898 IE) non-viable cell stain

To identify antigen-specific memory B cells, the immunising antigen (antigen Y), was labelled with the two different fluorophores Alexafiuor 488 (Invitrogen A30006) and Alexafiuor 647 (Invitrogen A30009). To confirm specific antigen binding, we also performed staining with TEV protease, an irrelevant protein antigen expected not to bind, labelled with APC-Cy7 (Lightning Link APC-Cy7 Kit , Innova Biosciences 765-0010). These reagents were added to the multiparameter B cell staining mix described above. As well as these reagents, a series of isotype control Abs for IgG staining, IgM staining and IgD staining were also included to confirm specificity:

• Mouse IgGl PE isotype control (BD 345816)

• Mouse IgGl Biotin isotype control (BD 555747) Compensation in this experiment was done using BD compensation beads (BD 552844).

Following staining, B cells were assessed using a BD FACS Aria III multi parameter flow cytometer. The staining strategy and identification of the IgG+ antigen-specific B cell subset is illustrated in figure 3.

Three populations were sorted based on the expression of IgG and IgM (see figure 3):

1. IgG++/IgM-

2. IgG+/IgM- 3. IgG+/IgM+

Cells from the above populations were sorted into a 96-well plate at one or three cells per well. Wells contained 20 ul of superscript III (Invitrogen) reverse transcription reaction mix. A reverse trasnscription reaction to generate cDNA from the single isolated B cell was performed immediately following sorting of the plate. Two rounds of PCR were then performed using antibody-specific oligonucleotides to amplify and recover antibody heavy and light chain variable region genes.

A 3° PCR was then performed enabling the combination of amplified variable regions, human CMV promoter fragment and rabbit heavy constant or rabbit kappa constant fragment to generate separate heavy and light transcriptionally-active linear PCR fragments. These DNA fragments were used directly for recombinant expression of mouse full-length rabbit IgG antibodies in a transient HEK-293 system. The resulting recombinant antibodies were then screened for antigen binding using a homogeneous fluorescence-based binding assay where biotinylated mouse antigen protein was immobilised on Superavidin^Tm beads (Bangs laboratories). Table 3 summarises the heavy and light chain PCR recoveries from the different populations and the number that went onto demonstrate measurable antigen binding as a recombinant antibody in the antigen binding assay.

Table 3 - Results for Rabbit Memory B-Cell Sorting

As can be seen from Table 3 , thirteen antigen-specific recombinant rabbit IgG molecules were generated from four 96-well plates. This suggests that the identified B cell subsets contain antigen-specific B cells and the protocol described here enables the rapid recovery of recombinant antibodies from immunised rabbits that retain the natural heavy and light chain cognate pairing.

Claims

Claims:

1. A method for identifying a cell producing an antibody specific to a target antigen

comprising the steps of:

a) providing a population of antibody producing B cells, wherein said population has been selected on the basis of at least one B cell marker and/or at least one marker selected from the group consisting of an IgG, IgE and IgA marker, and two or more "de-selection" markers (not present on the target B cell),

b) incubating the population of B cells from step a) with said target antigen and at least two distinct labels for said antigen such that the antigen is in a form labelled with a first label and/or a second label,

c) screening the cell population for those cells capable of producing antibodies

d) selecting the multi-antigen-labelled population of cells identified from step c), and e) optionally separating the population of cells selected in step d) into single cells.

2. A method for identifying a cell producing an antibody specific to a target antigen

comprising the steps of:

a) providing a population of antibody producing B cells, wherein said population has been selected on the basis of at least one B cell marker or an IgG marker, and two or more "deselection" markers (not present on target B cell),

c) screening the cell population for those cells capable of producing antibodies specific to the target antigen wherein the cells of interest are identified by at least one of each of the two or more distinct labels, and

d) selection of the multi-antigen-labelled population of cells identified from step c), and e) optionally separating the population of cells selected in step d) into single cells.

3. A method according to claim 1 or 2, wherein the target antigen has been conjugated to at least two distinct fluorogenic labels to provide a form of the antigen labelled with a first label and a form of the antigen labelled with a second label.

4. A method according to any one of claims 1 to 3 wherein the cell population is incubated with one labelled antigen form followed by incubation with the second labelled antigen form.

5. A method according to claim 1 , wherein each antigen in step b) bears two distinct labels.

6. A method according to any one of claims 2 to 4, wherein the B cells in step b) are incubated with the two or more distinctly labelled antigen forms concomitantly.

7. A method according to claim 1 or claim 2, wherein the antigen is provided unlabelled and two or more labels are specific to the target antigen and are introduced into the population of B cells.

8. A method according to claim 7, wherein the two or more labels specific for the target

antigen comprise an antibody or fragment thereof specific to the target antigen.

9. A method according to claim 7 or 8, wherein the two or more labels are introduced

concomitantly.

10. A method according to claim 7 or 8, wherein the two or more labels are introduced

sequentially.

11. A method according to any one of claims 1 to 10 wherein one, two or more labels are

fluorescent.

12. A method according to claim 11 , wherein there are two fluorescent labels and the two

distinct labels emit light at different substantially non-overlapping frequencies.

13. A method according to any one of claims 1 to 12, wherein two or more labels (such as all the labels) are detected in step c).

14. A method according to any one of claims 1 to 13, wherein the method comprises the further step of cloning at least the variable region of an antibody from a cell identified employing the method, for example employing PCR.

15. A method according to any one of claims 1 to 14, wherein the method comprises a step wherein a starting population of cells is pre-enriched for B cells, for example based on a marker B220 such as anti-B220 micro beads (Miltenyi 130-049-501).

16. A method according to any one of claims 1 to 15, wherein the B cell population for use in step (a) has been selected based on: a. cells positive for one or more markers selected from the group comprising CD 19, and IgG (such as IgGl and/or IgG2), and

b. negative for one or more markers selected from the group comprising IgM, IgD, CD4+, CD8+, F4/80+, Grl+ and an anti-T cell antibody, and

c. wherein the cells are viable, for example unstained by dye such as 7AAD.

17. A method according to claim 16, wherein the cells are sorted sequentially based on the

markers.

18. A method according to any one of claims 1 to 17, which further comprises the step of

identifying a B cell of interest in the population employing a functional assay.

19. A method according to any one of claims 1 to 18, which further comprising the step of creating a library of CDRs, variable regions, antibodies or antibody binding fragments.