WO2001033225A2

WO2001033225A2 - B-cell superantigen mediated antibody-ligand dissociation

Info

Publication number: WO2001033225A2
Application number: PCT/GB2000/004217
Authority: WO
Inventors: Ingrid Randen
Original assignee: Dynal Biotech Asa; Dzieglewska, Hanna
Priority date: 1999-11-05
Filing date: 2000-11-03
Publication date: 2001-05-10
Also published as: EP1240516A2; GB9926289D0; WO2001033225A3; AU1158501A

Abstract

The present invention provides a method of reversal of a linkage between an antibody and its target ligand comprising reacting said antibody-target ligand linkage with a B-cell superantigen, or a fragment thereof, which acts to reverse said linkage. The present invention further provides the use of this method in various isolation and purification methods, including cell detachment procedures, together with kits for carrying out such methods.

Description

Method

The present invention relates to a method of reversal of a linkage between an antibody and its ligand, and the use of this method in various procedures including cell detachment procedures. Kits for carrying out the method of the invention are also provided.

In biochemistry and related fields it is often desirable to form a linkage between an antibody molecule and its target ligand. Such linkage may be used for example in isolation or purification of substances or cells, bacteria or viruses etc. from a mixture of components, or in the immobilisation of substances on solid supports. Although the formation of such linkages between antibody and ligand allows for such isolation, purification or immobilisation, problems often arise if it is then desired to further manipulate the isolated or purified substance or cell population by breaking or reversing the antibody-ligand linkage.

While affinity binding systems are reversible, the linkages formed between antibodies and their ligands, e.g. antibody-antigen linkages are very strong and difficult to reverse without having a destructive effect on the entities involved.

Known methods to try and break such linkages involve alterations in pH, high salt concentration, etc. which reduce the interaction strength by modifying the conformation of the linked molecules. If however, this conformational change is not reversible and the linked molecules do not recover their functionality when the cleavage conditions are removed, then these methods are not particularly useful for techniques where further manipulation or analysis of a functional antibody or ligand or the viable cellular entity to which either of these components may be attached, is desired.

Unfortunately, in the case of antibody-ligand, e.g. antibody-antigen binding, which must have an affinity or association constant in the order of at least K_A = 10⁵ M^"1 to be effective, while the antibody molecule can usually recover its native activity following such treatments to break the linkage, the antigen frequently is irreversibly altered and its functional properties lost. This is clearly a problem in many methods, and is particularly a problem in the separation of cells, bacteria or viruses etc. when it is important to maintain viability or infectivity.

W091/15766 discloses a method of cleaving an antigen/anti-antigen linkage joining two particles which involves reacting the linkage with a secondary antibody which binds to the anti -antigen. A reagent (i.e. a "secondary antibody" preparation) for use in such a method is marketed by Dynal AS, Oslo, Norway under the trade mark DETACHaBEAD. Although this method has the advantage that the linkage is broken under mild conditions thus avoiding the destruction or disruption of the proteins involved and the functional properties associated therewith, it has the disadvantage that the molecular tool required for breaking the linkage, i.e. the secondary antibody, has some specificity for the particular linkage concerned, i.e. may not work for all anti -antigen antibodies which might be used, linked to their corresponding particular antigen. Also the development of such a tool requires immunisation and is therefore inconvenient, time consuming, labour intensive and costly.

Surprisingly, it has now been found that the reversal of a linkage between an antibody and its target ligand/antigen can be effected by reacting the linkage with a B cell superantigen. These B-cell superantigens, functioning as "displacement ligands" to disrupt or reverse the antibody-target antigen/ligand linkage, have the advantage that they are more general molecular tools than the secondary antibodies of the prior art method. A single species of B cell superantigen can be used to reverse many different antibody-target ligand linkages. The advantages of this more general method of reversing antibody-target ligand linkage are self evident and include the advantages of being more convenient, less time consuming and labour intensive to develop and therefore less costly. Significantly, as mentioned above, the B cell superantigen "displacement ligand" avoids the need for immunisation and is simpler and quicker to prepare than the antibody-based DETACHaBEAD reagent .

Moreover, in common with the prior art method, the method of reversal of linkage between an antibody and ligand of the present invention is carried out under mild conditions which do not lead to the destruction or loss of activity of either the antibody or target ligand components involved in the linkage.

Thus, the present invention provides a method of reversal of a linkage between an antibody and its target ligand comprising reacting said antibody-target ligand linkage with a B-cell superantigen, or a fragment thereof, which acts to reverse said linkage.

"Reversal" or "reverse" as used herein refers to the disruption or negation of a previously formed antibody- ligand linkage and can be effected by any suitable means. "Reversal" therefore includes the interruption, disruption, destabilisation, or physical breakage of the linkage. What is required, is that the linkage between the antibody and its ligand (the antigen) is disrupted or broken to allow separation of the respective entities (i.e the antibody and its antigen) . The "displacement ligand" (i.e. the superantigen) may physically break or cleave the linkage, or it may destabilise the linkage in a sufficient manner to allow it to be cleaved, or reversed, or for the two linked entities to be separated. Furthermore, in a population of linkages, it may not be necessary for each and every linkage to be disrupted, as long as a sufficient or significant proportion are "reversed" e.g. where substantially all of the linkages are "reversed" . "Substantially" in this context, may be taken to mean that at least 70% (or more preferably at least 75, 80, 85, 90 or 95%) of the linkages are reversed. It will be understood in this regard that the method of the invention relies on a biological system, and as such absolute precision and uniformity of behaviour can never be guaranteed, and 100% reversal may not always be achieved. In any such system, some tolerance must be allowed for, and this is a principle accepted in the art. In the linkage reversal system of the present invention, utility may be preserved even through reversal may not be 100% complete .

"Linkage" as used herein refers to any interaction by which an antibody and its target ligand may be associated. Such association may for example involve a physical association between parts of the antibody and target ligand and may also involve so-called "weak" interactions such as hydrogen bonds, Van der Waal's forces and oppositely charged ionic interactions. Such an interaction or "linkage" between an antibody and its target ligand may be of any strength providing that the strength of interaction is sufficient for the antibody to be associated with its target ligand. The strength of interaction may thus be weak, for example of the order of about K_A = 10⁴ to 10⁶ M^"1 or it may be strong, for example of the order of or greater than about K_A = 10¹² NT¹, or between these two extremes. In the case of an an ibody-ligand interaction, the strength of interaction is usually strong, i.e. of the order of or greater than K_A = 10⁸ M^"1.

The functional definition of the B cell superantigen herein as a "displacement ligand", means that said superantigen can react or interact with the linkage formed between an antibody and its target ligand and lead to the "reversal" of this linkage. Whilst not wishing to be bound by theory, the B cell superantigen may thus react with, for example by binding to, any part of either the antibody component or the target ligand component of the antibody-target ligand linkage and may act to reverse the linkage by any means, for example by competitive or any other displacement of the antibody or target ligand, by steric hindrance or by disruption of the configuration of either the antibody or target ligand component of the linkage, or by any other mechanism which may destabilise the linkage, or by any combination of such means. Fragments of B cell superantigens are also included, providing these fragments retain the ability to react or interact with the linkage as that described above.

A number of different B cell superantigens are known and described in the literature. Any of these, or their fragments, may be used according to the present invention as indeed may any molecule, or a fragment thereof, exhibiting the properties and characteristics of a B cell superantigen, for example as described below. Such superantigens may occur naturally in different native forms e.g. in different allelic variants, or due to species or geographic variation etc. Moreover, they may also be used as functionally equivalent variants or derivatives of the native molecules, which may differ in their amino acid sequence, for example by truncation or sequence extension (e.g. from the N- or C-terminus or both) or other amino acid deletions, additions or substitutions. It is known in the art to modify the sequences of proteins or peptides, whilst retaining their useful activity and this may be achieved using techniques which are standard in the art and widely described in the literature e.g. random or site-directed mutagenesis, cleavage and ligation of nucleic acids etc. All such analogues, variants or derivatives of B cell superantigens are included in the scope of this invention, and are subsumed under the term "a B cell superantigen" .

Advantageously, the B cell superantigen or fragment thereof interacts with or binds to the antibody component in the antibody-target ligand linkage, and more preferably interacts with the Fab region of said antibody, most preferably with the variable (v) domain included in the Fab region of said antibody.

Representative B cell superantigens include, for example, Staphylococcal protein A (SPA), HIV-1 gpl20 envelope protein (gpl20) , capsular polysaccharide of Haemophilus influenzae type b, sialoprotein pFv (pFv) , Peptostreptococcus agnus protein L (protein L) , red blood group antigen (I/i) or human CD4 or a fragment thereof. Most preferably, the B cell superantigen used as a displacement ligand is SPA or a fragment thereof.

As indicated above any fragment of SPA may be used providing such fragments retain the ability to react or interact with the antibody component of the linkage, but exemplary fragments are those comprising one or more of the homologous domains termed the E, D, A, B and C domains of the SPA molecule, which are known to bind to the Fab region of antibody molecules.

Superantigens are molecules which were initially described for T cells and in contrast to conventional antigens, do not require presentation in the context of MHC molecules to induce T-cell stimulation. Instead superantigens bind to the Vβ segment of the T cell receptor (TcR) outside the MHC-peptide-TcR groove. Because of this property, the number of T cells responding to a superantigen exceeds that of conventional antigens by several tenfold.

Analogous superantigens directly activating B cells have been described, examples of which are indicated above (see, for example, Silverman, G.J., Immunology Today, 1997, 18(8), 379-386 and Zouali, M. Immunology Today, 1995, 16(8), 399-405). The characteristics of these antigens are that they 1) stimulate a high frequency of B cells, 2) target B cells employing particular variable gene segment products, and 3) bind to immunoglobulins outside the sites which confer specificity of binding to conventional antigens.

The antigen binding part of an immunoglobulin comprises the variable regions of both the heavy and light chain. The huge diversity of antibodies in mouse and man (and indeed in other species) is produced by the combinatorial rearrangement of a relatively small number of gene segments. The variable region of the light chain consists of variable (V_L) and joining (J_L) gene segments. The variable region of the heavy chain consists of variable (V_H) , diversity (D) and joining (J_H) gene segments. During rearrangement the different segments join together forming the antigen binding part of the antibody. The complementarity-determining regions (CDRs) of the antigen binding part of the antibody are in direct contact with the antigen and show tremendous diversity while the framework regions (FRs) comprise more conserved structures.

Human V_H segments can be classified into seven families, V_H1-V_H7, with different members of the same family being at least 80% homologous at the nucleotide sequence level . Human V_L segments can be either k (kappa) or λ (lambda) . There are six human V_κ families and nine human V_λ families.

The mouse V_H repertoire can be divided into fourteen different families, each containing between one and fifteen germline genes. One exception is the V_H1 family (J558) which contain between sixty and one thousand genes. The mouse V_L repertoire has 18 V_κ gene families and only three V_λ genes.

The B cell superantigen Staphylococcal protein A (SPA) is a bacterial membrane protein of Staphylococcus aureus, one of the more commonly found bacteria in humans. SPA has two distinct binding sites on immunoglobulins . The binding to the Fc region of most IgG molecules is well established and much exploited. However, in addition an "alternative" binding site has been localised to the Fab region of at least human and mouse immunoglobulins.

As mentioned above Protein A comprises five homologous domains named E, D, A, B and C sharing more than 80% homology at the protein level. All five domains bind both Fab and the Fc region of IgG immunoglobulins. However, the Fab-binding sites seem to be structurally different from the Fey binding sites because chemical modification of SPA can eliminate Fc binding while the Fab interaction remains intact. The Fab binding of SPA is independent of immunoglobulin isotype and specificity. Because of the latter property SPA has been classified as a B cell superantigen.

The mechanism for B cell superantigen reversal of the antibody-ligand linkage is unknown. However, in order for the particular superantigen to reverse the antibody-target ligand linkage, the antibody linked to the target ligand must comprise an appropriate variable (V) segment in its Fab region. Thus, whilst not wishing to be bound by theory, it is possible that the mechanism of action of the B cell superantigens involves competitive displacement of the target ligand from the ligand binding region, a steric interruption of ligand binding due to the fact that the B cell superantigen binds to the Fab region of an immunoglobulin outside the sites which bind conventional antigens, or by the binding of the B cell superantigen inducing a conformational change in either the antibody or the linked target ligand to destabilise or reverse the interaction between the two components and thus break the linkage.

Different B cell superantigens are known to interact with different variable region gene segments of immunoglobulin molecules and this binding is independent of immunoglobulin isotype or specificity. For example SPA interaction with human immunoglobulins has been mapped to the heavy chain variable region gene segments primarily belonging to the V_H3 family. V_H3 is the largest of the human V_H families and also the predominantly expressed. SPA interaction with V_H3 gene segments is thought to involve conserved residues at the end of CDR2 and beginning of FR3. The predominant expression of the V_H3 gene segment family means that SPA can be used as a displacement ligand in the methods of the present invention to reverse a significant proportion of antibody-target ligand linkages.

SPA interaction with mouse V_H genes seems to be confined to the V_H6 (J606) - and V_H7 (J107) -families which share amino acid homology with the human V_H3 derived genes. SPA is also known to react with the Fab regions of immunoglobulins from other species such as rat, avian, porcine and other mammalian species. The structure and arrangement of the V gene families in these species are not well studied. However, it can be seen that the methods of the invention are not limited to reversal of the linkage of antibody-target ligand interactions from any particular species and that the main requirement for the functioning of the methods of the present invention is that the B cell superantigen in question can interact with and reverse the linkage formed between the antibody and the target ligand.

The HIV gpl20 envelope protein superantigen also interacts with the V_H3 family of variable segments. The capsular polysaccharide of Haemophilus influenza type b interacts with a subgroup of the V_H3 family, Fv interacts with members of the V_H3 and V_H6 family, protein L interacts with the V_κl , V_κ3 and V_κ4 families, the red blood group antigen interacts with the V_H4 family (and in particular the V_H4-21 germline gene within the V_H4 family) , and human CD4 interacts with the Fab region of human antibodies.

Thus, in preferred methods of the invention the particular B cell superantigen for use as displacement ligands in the method is chosen depending on the particular variable region gene segment included in the Fab region of the heavy or light chains of the antibody involved in the antibody-target ligand linkage concerned.

In a preferred embodiment of the invention, when the variable region gene segment of the antibody in the antibody-target ligand linkage is a member of the V_H3 family, the displacement ligand is SPA or a fragment thereof. Clearly however, when the variable region gene segment of the antibody in question is a domain other than a member of the V_H3 family, the superantigen to be used as a displacement ligand is chosen accordingly, to interact with the particular variable region gene segment present. In addition, B cell superantigens other than SPA (e.g. HIV gpl20) have been shown to interact with the V_H3 family of variable region gene segments, or sub-members thereof. Thus, these superantigens may be used as displacement ligands where the variable region gene segment is a member of the V_H3 family.

The determination of the appropriate B cell superantigen for use in the methods of the present invention can clearly be determined by trial and error. However, a more elegant way to determine which B cell superantigen to use is to identify the nature of the variable region gene segment in the Fab region of the antibody concerned. Sequence data for the various variable region gene segments comprising human and mouse antibodies as well as immunoglobulin sequence data from other species is available. Thus, the type of variable region gene segment of a particular antibody can be determined by sequencing all or part of the variable region gene segment present using methods which are well known and documented in the art .

Alternatively, antibodies to many of the different variable region gene segments found in human and mouse immunoglobulins are available and thus the particular variable region gene segment present in a particular antibody involved in an antibody-target ligand linkage could be determined by appropriate immunological methods well known and documented in the art.

Antibodies for use in methods of the present invention may be of any species, class or subtype providing that such antibodies are capable of forming a linkage with a particular target ligand and that this linkage can be reversed or broken by reacting it with a displacement ligand as described above.

Thus antibodies for use in the present invention include :

(a) any of the various classes or sub-classes of immunoglobulin, e.g. IgG, IgA, IgM, IgD or IgE derived from any animal e.g. any of the animals conventionally used, e.g. sheep, rabbits, goats, or mice,

(b) monoclonal antibodies

(c) intact antibodies or "fragments" of antibodies, monoclonal or polyclonal, the fragments being those which contain the binding region of the antibody, e.g. fragments devoid of the Fc portion (e.g. Fab, Fab', F(ab')₂, Fv) , the so called "half molecule" fragments obtained by reductive cleavage of the disulphide bonds connecting the heavy chain components in the intact antibody.

(d) antibodies produced or modified by recombinant DNA or other synthetic techniques, including monoclonal antibodies, fragments of antibodies, "humanised antibodies", chimeric antibodies, or synthetically made or altered antibody-like structures. Also included are functional derivatives or "equivalents" of antibodies e.g. single chain antibodies. The method of preparation of fragments of antibodies is well known in the art and widely described in the literature and hence modifications and derivatives will not be described herein.

Preferred antibodies for use in the present invention will comprise a Fab region which can react with a B cell superantigen as described above. Most preferably the antibody Fab region comprises a variable region gene segment which can interact with a B cell superantigen as described above, for example the Fab region comprises a V_H3 segment which can interact with for example SPA or the HIV gpl20 envelope protein.

Most preferred antibodies for use in the present invention are human antibodies or fragments thereof which comprise a V_H3 segment in their Fab regions, or mouse antibodies or fragments thereof which comprise a V_H6 (J606) or V_H7 (J107) segment in their Fab regions.

Ligands for use in the methods of the present invention may be any entity which forms a linkage or interacts with an antibody, and which linkage can subsequently be reversed by a displacement ligand. Preferred ligands for use in the methods of the present invention include antigens and smaller molecules such as haptens .

The appropriate ligands and antibodies will be chosen depending on the use to which the method of the invention is desired to be put. For example, if the method is to be used to isolate or purify a particular cell population (or type of virus or bacteria etc.) from a mixture of cells then the ligand chosen may be a particular structural molecule e.g. a peptide, protein, glycoprotein, lipid or carbohydrate etc. associated with the surface of the cell or a signalling molecule or any other functional cell surface molecule etc. Ideally, for use in the present invention any chosen ligand will have available a suitable specific antibody with which it interacts to form the required linkage. However, if no such antibody exists, or if the antibodies which are available are unsuitable for any reason then such antibodies specific for a particular ligand may be produced by methods well known and documented in the art .

The step of "reacting" the linkage with the B cell superantigen (or fragment thereof) may take place in any convenient or desired way. Thus, the linkage, conveniently in an aqueous medium, may simply be contacted with the superantigen, e.g. the superantigen may simply be added to a sample containing the linkage. The "reaction mixture" or sample may simply be allowed to stand under appropriate conditions (e.g. the sample/ reaction mixture may be incubated under appropriate conditions) for a time interval to allow the superantigen to bind, and one or both of the components of the linkage may then be separated.

Representative conditions may include for example incubation at 4°C to 37°C e.g. room temperature, for a time interval of 2 minutes to 2 hours e.g. 5-60 minutes.

In many situations it may be sufficient simply to add the superantigen and incubate the reaction mixture to achieve release. However, in some cases, it may be desirable to assist "reversal" of the linkage for example by gentle stirring or mixing e.g. pipetting in order to assist breakage of a linkage destabilised by the superantigen binding.

In a further aspect of the invention, either or both of the antibody and target ligand components of the linkage may be associated with or attached to other entities. Alternatively, they may be provided with means for attachment to a further entity. Such means may constitute or comprise for example an affinity binding partner e.g. biotin, binding to the corresponding binding partner of the affinity binding pair e.g. streptavidin, provided on the entity to be attached. For example, in methods of cell separation as discussed above and below, the target ligand component of the linkage may be associated in some way with the surface of the cell. Furthermore, either the antibody or the target ligand component of the linkage may be attached to a solid phase, for example a solid support such as glass, plastic, tissue culture plastic, a matrix such as sepharose, solid supports such as iron or other metals, a solid particle such as for example a magnetic or non-magnetic bead.

The solid support may thus be any of the well known supports or matrices which are currently widely used or proposed for immobilisation, separation etc. As mentioned above, these may take the form of particles, sheets, dip-sticks, gels, filters, membranes, fibres, capillaries, or microtitre strips, tubes, plates or wells etc.

Conveniently the support may comprise glass, silica, latex or a polymeric material such as for example nitrocellulose. Preferred are materials presenting a high surface area for binding e.g. of cells or other moieties for separation. Such supports will generally have an irregular surface and may be for example be porous or particulate eg. particles, fibres, webs, sinters or sieves. Particulate materials e.g. beads are generally preferred due to their greater binding capacity, particularly polymeric beads.

Conveniently, a particulate solid support used according to the invention will comprise spherical beads. The size of the beads is not critical, but they may for example be of the order of diameter of at least 1 and preferably at least 2 μm, and have a maximum diameter of preferably not more than 10 and more preferably not more than 6 μm. For example, beads of diameter 2.8 μm and 4.5 μm have been shown to work well.

Monodisperse particles, that is those which are substantially uniform in size (e.g. size having a diameter standard deviation of less than 5%) have the advantage that they provide very uniform reproducibility of reaction. Monodisperse polymer particles produced by the technique described in US-A-4336173 are especially suitable .

Non-magnetic polymer beads suitable for use in the method of the invention are available from Dyno Particles AS (Lillestrøm, Norway) as well as from Qiagen, Pharmacia and Serotec.

However, to aid manipulation and separation, magnetic beads are preferred. The term "magnetic" as used herein means that the support is capable of having a magnetic moment imparted to it when placed in a magnetic field, and thus is displaceable under the action of that field. In other words, a support comprising magnetic particles may readily be removed by magnetic aggregation, which provides a quick, simple and efficient way of separating the particles, and is a far less rigorous method than traditional techniques such as centrifugation which generate shear forces which may disrupt cells.

Thus, using the method of the invention, the magnetic particles may be removed onto a suitable surface by application of a magnetic field e.g. using a permanent magnet. It is usually sufficient to apply a magnet to the side of the vessel containing the sample mixture to aggregate the particles to the wall of the vessel and to pour away the remainder of the sample.

Especially preferred are superparamagnetic particles for example those described by Sintef in EP-A- 106873, as magnetic aggregation and clumping of the particles during reaction can be avoided, thus ensuring uniform cell, protein and nucleic acid extraction. The well-known magnetic particles sold by Dynal AS (Oslo, Norway) under the trade mark DYNABEADS, are particularly suited to use in the present invention.

Functionalised coated particles for use in the present invention may be prepared by modification of the beads according to US patents 4,336,173, 4,459,378 and 4,654,267. Thus, beads, or other supports, may be prepared having different types of functionalised surface, for example positively or negatively charged, hydrophilic or hydrophobic.

The attachment of either component of the linkage to a solid phase allows easy manipulation of the individual component originally attached to the solid phase and also the linked components. Thus, the attachment to some kind of solid phase can enable the separation of the linked components from the rest of the components in the mixture. This can be achieved for example by carrying out washing steps, or if the components are attached to magnetic beads, using a magnetic field to effect physical separation of the linked component from the rest of the components in the mixture .

Once separation or any other required manipulation of the linked antibody-target ligand complex has been carried out, the linkage can then be reversed and the individual components released according to the methods of the present invention.

In particular, where one of the linked components is a molecule on a cell surface, the cell can be bound to a solid support and subsequently liberated with its functional potential/viability unaffected. In a similar way, where the target ligand of the linkage is a substance (for example a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, or any signal substances such as hormones, toxins etc.) isolated from a complex mixture of substances (for example a cell lysate or a culture supernatant) , the ligand can be bound to an antibody on a solid support and subsequently liberated with its functional potential/viability unaffected.

The term "cell" is used herein to include all prokaryotic (including archaebacteria and mycoplasmas) and eukaryotic cells (including insect cells and fungal cells) and other entities such as viruses and sub- cellular components such as organelles. Representative "cells" thus include all types of mammalian and non- mammalian animal cells, plant cells, insect cells, fungal cells, protozoa, protoplasts, bacteria and viruses .

Thus it can be seen that the new method of the invention may be used in a large number of fields, for example in the fields of analysis, diagnosis, and in the purification and isolation of individual components of an antibody-target ligand linkage as defined above, or in the purification and isolation of larger biological entities, for example cells (e.g. mammalian cells, bacteria or viruses) which are associated with or have as an integral part of the cell surface a component of an antibody-target ligand linkage as defined above. The method of the invention may thus find application in any method or procedure which relies upon or utilises antibody binding to a target ligand. For example, the method may be used in the isolation of infectious agents such as bacteria or viruses in order to quantitate them or characterise their infectivity, toxicity or susceptibility to drug treatment. The method can also be used for isolation of malignant cells or cell populations specific for different diseases and to characterise these cells further without interference from other contaminating cells. Also, the method may be used to isolate protective cell populations from an individual or from a group of individuals; the isolated population can then be expanded and/or potentiated before being returned to the patient under treatment. Such protective cell populations can for example be monocytes/macrophages, lymphocytes or bone marrow stem cells .

As regards analytical applications, the method of the invention may be used in quantification and morphological analyses or in immunochemical staining studies employing both immunological and non- immunological markers .

As regards the purification or isolation of individual components of an antibody-target ligand linkage, the method of the invention can be used to pull out a target ligand or indeed an antibody from a mixture of components contained for example in a cell, bacterial or viral lysate, or a culture supernatant of any cell, bacteria or virus etc., or other biological samples such as for example biological fluids derived from a human or animal source including whole blood, serum, plasma, saliva, urine, milk and organ, tissue or cellular extracts or secretions etc. The sample may thus be any material containing cells, or cellular material or indeed any desired biological or clinical or analytical moiety including for example foods and allied products, clinical and environmental samples. Thus, the sample may be a biological sample, which may contain any viral or cellular material, including all prokaryotic cells including Cyanobacterium and mycoplasma, eukaryotic cells, viruses including bacteriophages, protoplasts and organelles. Such biological material may thus comprise all types of mammalian and non-mammalian animal cells, plant cells, insect cells, algae, fungi, bacteria, protozoa etc. Representative samples thus include whole blood and blood-derived products such as plasma or buffy coat, urine, faeces, cerebrospinal fluid or any other body fluids, tissues, cell cultures, cell suspensions etc., and also environmental samples such as soil, water or food samples.

The sample may also include relatively pure or partially purified starting materials, such as semi-pure preparations obtained by other cell or biomolecule separation processes.

Alternatively, biological or chemical libraries for example phage display libraries which display the library constituents on their cell surfaces can be used as sources of individual components of an antibody- target ligand linkage.

In such methods the appropriate component of the antibody-target ligand linkage (i.e. the known component for which a binding partner is desired to be isolated or purified) is usually immobilised on a solid phase as described above so that the relevant sample from which the appropriate entity is to be purified can be brought into contact with the solid phase under conditions which allow the antibody-ligand linkage to be formed. The linked entities can then be separated from the other components of the mixture by methods as described above and the entity in question (either the target ligand or antibody) purified by reversing the linkage using the method of the invention.

Thus, according to a further aspect of the invention there is provided a method of isolating or purifying a target ligand from a sample (e.g. mixture of components) , wherein an antibody which can form a linkage with the target ligand is attached to a solid phase before or after binding to said target ligand, whereby the solid phase and attached target ligand are separated from the sample (e.g. from the other components present) and the target ligand is released from said solid phase by the addition of a B cell superantigen or fragment thereof.

In an alternative embodiment of the above method an appropriate ligand may be attached to a solid phase and used to purify or isolate an antibody from a mixture of components. Similar methods may be applied to the isolation and subsequent liberation of sub-cellular components such as mitochondria and nuclei, and macromolecules such as proteins, nucleic acids, oligosaccharides, glycoproteins, lipids, carbohydrates, peptides, or any signal substances such as hormones, toxins etc .

Preferably the solid phase for use in such methods comprises magnetic particles.

As previously mentioned, the method of the invention is particularly suited to cell isolation, particularly in the possible selection of desirable cells using antibodies directed against the cells to be isolated (as opposed to negative selection procedures where unwanted cells are removed from a cell preparation using antibodies specific for the unwanted cells) .

It has been proposed to positively isolate desired cells using magnetic beads coated with an antibody directed against a cell surface ligand/antigen, thus binding the desired cells, with the magnetic particles and attached cells then being separated from other cells by magnetic aggregation and the cells being liberated from the magnetic particles to leave a positively selected population of cells. In particular in WO91/09938 of Dynal AS there is described such a method for the selection of haemopoietic stem cells from bone marrow and other mixed cell populations. In such methods, the magnetic particles are advantageously the superparamagnetic, monodisperse particles sold as Dynabeads (Dynal AS, Oslo) .

In many prior art methods to positively isolate cells using magnetic particles, to liberate cells from the particles, the cell/particle "rosettes" have been incubated overnight at 37°C to effect separation of the cells from the particles. In some cases the cells detach from the particles, but in many cases they do not, and such poor recovery makes difficult the isolation of poorly represented cell sub-populations. The new method of the invention is thus particularly suited for detaching the bound cells from the magnetic particles .

According to a yet further aspect of the invention therefore is provided a method of positively isolating a target cell type from a mixed population of cells wherein an antibody is bound to a target ligand on said target cell, said antibody being attached to a solid phase before or after binding to said target cells, whereby the solid phase and attached cells are separated from the other cells present and the target cell is released from said solid phase by the addition of a displacement ligand (e.g. a B cell superantigen), or fragment thereof . In a preferred embodiment of the invention the solid phase comprises magnetic particles and the magnetic particles and attached cells are isolated from the mixed population of cells by magnetic aggregation.

Although particularly suited to the isolation of haemopoietic stem cells, the method of the invention may be applied to the isolation of any prokaryotic or eukaryotic cells or viruses from biological or artificial media, including whole blood, buffy coat and cell suspensions obtained by density gradient centrif gation .

The amount of displacement ligand required for optimal cleavage will of course vary depending upon the entities bound, their ratio, and the number or quantity of entities e.g. cells requiring isolation, and can readily be determined according to need. For example, in the case of positive cell selection mentioned above, the ratio of magnetic particles to target cells may vary in different systems and with different applications, and different amounts of the displacement ligand will accordingly be required to detach the cell from the particles .

Conditions for detachment may also be varied as appropriate. Thus rosetted cells, suspended in a suitable medium, may simply be incubated with the displacement ligand at ambient temperature. Alternatively the temperature may be reduced e.g. to 4°C or raised e.g. to 37°C. Best results are obtained by incubating on an apparatus providing both gentle tilting and rotation or pipetting.

The various reactants in the method of the invention are conveniently supplied in kit form. Thus in a yet further aspect, the present invention provides a kit comprising:

(i) an antibody against a target ligand either attached to a solid support, preferably magnetic particles, or provided with means for such attachment (e.g. biotin or some other moiety, binding to a corresponding binding partner e.g. streptavidin provided on the solid support) ; and

(ii) a B cell superantigen, or fragment thereof, which is capable of interacting with said antibody.

Examples of solid support, antibodies and B cell superantigens etc. for use in such kits are discussed above .

In an alternative embodiment part (i) of the kit may comprise a solid support carrying a target ligand which can interact with an antibody to form an antibody- target ligand linkage, said target ligand either being provided attached to the solid support, or provided with means for such attachment as described above for the antibody components of the kits.

The invention will now be further described by way of the following non-limiting Example (s).

Example 1 :

The following abbreviations are used in the below described experiments :

BSA: Bovine Serum Albumin

ELISA: Enzyme Linked ImmunoSorbent Assay

FCS: Fetal Calf Serum

HLA: Human Leukocyte Antigen

PBS: Phosphate Buffered Saline

To evaluate how prevalent the preferred method of the invention utilizing SPA as the displacement ligand may be, a number available IgM antibodies from our laboratory were tested for the binding to SPA in a direct binding ELISA assay. The SPA used in all experiments was purchased from Calbiochem, product number 539203, recombinant, E. coli.

ELISA method:

1) Coat: 10 μg/ l SPA in PBS, 100 μl/ well, overnight 4°C

2) Block: PBS/1% BSA, 200 μl/well, 20 min room temperature

3) Incubate with various IgM Mabs of different concentrations in PBS/0.05% Tween 20, 1 hr/37°C or overnight, 100 μl/well

4) Add goat anti-mouse IgM alkaline phosphatase conjugate (Sigma #A-9688, lot 125H8840 or mouse anti-rat K and λ (Sigma, A-1062), cone: 1:10000, 100 μl/well, and incubate for 1 hr, 37°C.

5) Add 100 μl of phosphatase substrate (Sigma) /well , incub 30 min, 37°C. O.D. was measured in an ELISA reader (Dynatech Laboratories) .

3x washes with PBS/0.05% Tween 20 between each step Results

Antibody SPA binding 0. D.

Mouse antibodies

5C2 (anti-CD8) neg (0.040)

AB4 (anti-DR) neg (0.029)

AB1 (anti-CD19) pos (>D

66.1 (anti-CD4) pos (>D

BRA 4F1 (anti-CD15) pos (>D

HKB1 (anti-DR, DQ, DP) pos (>D

Bl 3C5 (anti-CD34) pos (0.443)

2MN 2B7 (anti-CD56) neg (0.017)

Rat antibodies

CD8H5 ( nti -mouse CD8) neg (0.000)

L3T4 (anti -mouse CD4) neg (0.071)

B220 (anti-mouse pan B) pos (0.931)

Thyl .2 (anti-mouse pan T) neg (0.000)

Rat -anti -Mouse IgM pos (0.523)

Of the 7 SPA binding IgM antibodies, 3 have been tested for antigen release by SPA. In addition, an IgG antibody which is known to bind SPA in the Fab region has been tested in the same way. All four antibodies tested showed that SPA can reverse antibody-antigen binding .

Example 2 :

10⁷ Dynabeads coated with IgM antibodies with specificities for either CD4 (66.1) or HLA class II (HKB1) were incubated with 10⁶ peripheral blood lymphocytes for 10 minutes at 4°C. 70% of the CD4 positive cells and 99% of the HLA class II positive cells were rosetted. Cells were released by SPA by incubating the rosetted cells for 45 minutes at room temperature. Stock solution of SPA was 20 mg/ml and different amounts were diluted into PBS to a total volume of 200 μl . PBS alone was used as a negative control. Released cells were measured by flow cytometry.

Results :

Example 3 ;

This assay was performed to detect the effect of SPA on CD19 rosetted cells and to compare release by SPA to our existing DETACHaBEAD procedure (W091/15766) for CD19. CD19 positive cells were the Reh cell line (ATCC CRL- 8286) . Rosetting and SPA release were performed as in Example 2 except that dilutions were done in culture medium RPMI (Bio-Whittaker) /l% FCS instead of PBS. 93% of the Reh cells were rosetted. DETACHaBEAD was used as in our standard protocol (W091/15766) (10 μl DETACHaBEAD + 190 μl RPMI/FCS) . RPMI/FCS was used alone as a negative control.

Results :

* The concentration of antibodies in the DETACHaBEAD antiserum is not known. Example 4 ι

The aim of this assay was to evaluate the effect of SPA on a different isotype (IgG) and to see whether the release effect is concentration dependent. 10⁷ Dynabeads coated with monoclonal antibody 561 (anti-CD34, IgG2a) were incubated for 15 minutes at 4°C with 10⁶ tHL60 cells bearing the CD34 antigen on its surface. Ninety-eight percent of the cells were rosetted. Different amounts of SPA from a stock solution of 23.5 mg/ml were diluted in RPMI/FCS to a final volume of 200 μl and incubated with the cells for 40 minutes at room temperature. RPMI/FCS was used alone as a negative control. Released cells were measured in a Coulter Counter.

Results :

Conclusion:

The above examples document that B cell superantigen reversal of antibody-antigen binding is independent of antibody specificity and isotype. The mechanism functions on a wide range of antibodies as more than 50% (8/14) of randomly collected antibodies bound SPA. All SPA interacting antibodies tested for reversal of antigen binding demonstrated the effect. The yield after SPA release is comparable to the DETACHaBEAD product . SPA as a release reagent as described in the above experiments is an anti-Fab reagent and will remove the attached antibody (and bead) from the antigen (and cell) .

Claims

1. A method of reversal of a linkage between an antibody and its target ligand comprising reacting said antibody-target ligand linkage with a B-cell superantigen, or a fragment thereof, which acts to reverse said linkage.

2. The method of claim 1, wherein the B-cell superantigen, or a fragment thereof, interacts with or binds to the antibody component in the antibody-target ligand linkage.

3. The method of claim 2 wherein the B-cell superantigen, or a fragment thereof, interacts with the Fab region of the antibody component.

4. The method of claim 3 wherein the B-cell superantigen, or a fragment thereof, interacts with the variable (v) domain included in the Fab region of said antibody.

5. The method of any one of claims 1 to 4 , wherein the B-cell superantigen is selected from the group comprising Staphylococcal protein A (SPA) , HIV-1 gpl20 envelope protein (gpl20) , capsular polysaccharide of Haemophilus influenzae type b, sialoprotein pFv (pFv) , Peptostreptococcus magnus protein L (protein L) , red blood group antigen (I/i) or human CD4.

6. The method of claim 5, wherein the B-cell superantigen is SPA or a fragment thereof.

7. The method of claim 6, wherein the fragment of SPA comprises one or more of the homologous domains termed the E, D, A, B and C domains of the SPA molecule.

8. The method of claim 5, wherein the particular B- cell superantigen is chosen depending on the particular variable region gene segment included in the Fab region of the heavy or light chains of the antibody involved in the antibody-target ligand linkage.

9. The method of any one of claims 1 to 8, wherein the antibody in the antibody-target ligand linkage is a human antibody or fragment thereof which comprises a V_H3 segment in the Fab region, or a mouse antibody or fragment thereof which comprises a V_H6 (J606) or V_H7 (J107) segment in the Fab region.

10. The method of claim 9, wherein the B-cell superantigen chosen is SPA and the variable region gene segment of the antibody in the antibody-target ligand linkage is a member of the V_H3 , V_H6 or V_H7 families.

11. The method of any one of the previous claims wherein either the antibody or the target ligand component of the linkage is attached to a solid phase.

12. The method of claim 11 wherein the solid phase comprises magnetic particles.

13. The method of any one of claims 1 to 12, wherein the target ligand is a protein, peptide, nucleic acid, oligosaccharide, glycoprotein, lipid, carbohydrate, hormone or toxin .

14. The method of claim 13, wherein the ligand is a cell surface molecule.

15. Use of the method as defined in any one of claims 1 to 14 in the isolation or purification of a cell population from a mixture of cells, wherein a component of the antibody-target ligand linkage is a molecule associated with the surface of the population of cells being isolated or purified.

16. Use of the method as defined in any one of claims 1 to 14 in the isolation or purification of a component of an antibody-target ligand linkage from a mixture of components .

17. Use of the method as defined in any one of claims 1 to 14 in the analysis or diagnosis of a sample.

18. A method of isolating or purifying a target ligand from a sample, wherein an antibody which can form a linkage with the target ligand is attached to a solid phase before or after binding to said target ligand, whereby the solid phase and attached target ligand are separated from the sample and the target ligand is released from said solid phase by the addition of a B cell superantigen or fragment thereof.

19. A method of isolating or purifying an antibody from a mixture of components, wherein a target ligand which can form a linkage with the antibody is attached to a solid phase before or after binding to said antibody, whereby the solid phase and attached antibody are separated from the sample and the antibody is released from said solid phase by the addition of a B cell superantigen or fragment thereof.

20. A method of positively isolating a target cell type from a mixed population of cells wherein an antibody is bound to a target ligand on said target cell, said antibody being attached to a solid phase before or after binding to said target cells, whereby the solid phase and attached cells are separated from the other cells present and the target cell is released from said solid phase by the addition of a B cell superantigen or fragment thereof.

21. The method as claimed in any one of claims 18 to 20 wherein the solid phase comprises magnetic particles.

22. A kit comprising:

(i) an antibody against a target ligand either attached to a solid support, or provided with means for such attachment ; and

23. A kit comprising:

(i) a target ligand which can interact with an antibody to form an antibody-target ligand linkage, said target ligand being either attached to a solid support, or provided with means for such attachment; and

24. The kit of claim 22 or claim 23 wherein the solid support comprises magnetic particles.

25. The kit of claim 22 or claim 23 wherein said means for attachment is a biotin moiety.