Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/46—Hybrid immunoglobulins
  • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
  • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]

Definitions

• ANTIBODIES This invention relates to novel forms of antibody and their use as targeted cytotoxic agents, particularly in the treatment of neoplastic disease.
• bivalent antibody conjugate was constructed by chemical means but in a further application of the technique (Staerz and Bevan, Proceedings of the National Academy of Sciences of the USA, 1986, 83, 1453 and Immunology Today, 1986, 7, 241) hybrido a technology has been employed to produce a bi-specific antibody molecule.
• the bi-specific antibody exerts its effect by binding both to a tumour cell or other form of target cell, such as a virally infected cell, and to a T-cell thereby effecting destruction of the former by the action of the latter.
• the bi-specific antibody will in general utilise to any significant extent only one of the natural cell killing mechanisms of which the body is capable in order to kill the tumour cells. It is an object of the present invention to provide an improved method for the destruction of tumour cells utilising the natural killing mechanisms of the body including the use of effector cell retargeting. Accordingly the present invention comprises a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinity of the whole molecule.
• bi-specific antibody molecules of the present invention differ from those bi-specific antibodies of the 1985 Staerz et al, Perez et aj. and Liu et al papers in as far as, although being bi-specific, the present antibodies have the normal form of an antibody molecule in which two light chains and two heavy chains are present.
• the bi-specific antibodies described in 1985 were conjugates produced by the chemical cross linking of two normal antibody molecules and contained four light chains and four heavy chains.
• the present invention is further directed to the mitigation of this previously quite unrecognised problem.
• the 1985 Liu et al paper describes an antibody conjugate having one binding affinity for the human T3 complex and a second binding affinity for the idiotype of the surface immunoglobulin of a human B-lymphoma. Such an idiotype represents a true tumour specific transplantation antigen and is used to direct the antibody conjugate to tumour cells.
• the Liu et al antibody conjugate therefore essentially has anti-T-cell and anti-tumour cell affinities and, apart from its conjugate nature, may be distinguished from the bi-specific antibody molecules of the present invention which function against T-cells through binding with a further antibody having an anti-tumour cell, or other target cell affinity.
• the bi-specific antibody molecules described above are administered in conjunction with a separate antibody having a binding affinity for target cells. Such a procedure enables various of the body's cell killing mechanisms to be brought into play.
• the antibody having a binding affinity for target cells can bind to a target cell and effect killing thereof through an Fc receptor mediated killing mechanism, for example via antibody-dependent cell-mediated cytotoxicity (ADCC) involving K cells, neutrophils and acrophages, via phagocytosis by macrophages and cells of the reticuloendothelial system, or via complement activation.
• ADCC antibody-dependent cell-mediated cytotoxicity
• the resultant target cell/antibody system can bind further with the bi-specific antibody system through the immunoglobulin affinity of this system, the whole system then binding with a T-cell through the T-cell affinity of the bi-specific antibody system and utilising T-cell toxicity to destroy the target cell.
• the bi-specific antibody molecules of the present invention function by binding to T-cells in order to direct their toxicity and the T-cell binding affinity of the antibody molecule may be specific for any T-cell receptor which will cause killing.
• the receptor may be associated with the ability of the T-cells to kill either directly or indirectly through the assistance of other cell types or any other agent.
• the receptor may be one which is associated with the ability of cytotoxic T-cells to kill directly or with the ability of helper T-cells to assist killing by B-cells, or with any other indirect mode of killing (natural or artificial).
• receptors capable of activating direct killing are of particular interest, it will be appreciated that many receptors are associated with both direct and indirect modes of killing.
• the binding affinity may be directed against one or both of the ⁇ and ⁇ chains which comprise the T-cell antigen specific receptor termed Ti, and which are present on the vast majority of T-cells, or it may be directed against the receptor-associated CD3 unit (previously identified as T3) as a whole or against one of the individual chains thereof.
• T3 T-cell antigen specific receptor
• studies with human cells presently indicate that the T-cell receptor exists as a complex of two chains identified as ⁇ (M r about 50,000) and ⁇ (H r about 40.000) which are coded for by genes which are somatically rearranged in an analogous fashion to immunoglobulin genes. Each T-cell therefore possesses a unique rearrangement of genes coding for these two chains. These two chains are found in association with at least three other chains which comprise the CD3 complex and are identified as ⁇ (M r about 25,000), 5 (M r about 20,000) and ⁇ (M r about 20,000).
• Antibodies having a T-cell binding affinity may be identified by an assay procedure which we have developed. Host hybridomas as well as secreting antibody have small amounts of cell surface antibody. Thus, for example, a mouse hybridoma making antibody against rat IgG2b is capable of trapping a rat IgG2b antibody by virtue of the small amount of antibody against rat IgG2b on its surface.
• hybridoma cells are labelled with a radioactive label, for example 51 Cr, and then incubated with a mixture of T-cells and monoclonal rat IgG2b antibodies against T-cells, the hybridoma cells will bind to the antibody which will in turn bind to the T-cells, thereby leading to killing of the hybridoma and consequent release of the radioactivity.
• a radioactive label for example 51 Cr
• Such a procedure therefore provides a means of detecting rat IgG2b antibodies with an appropriate T-cell binding capacity (i.e. those capable of inducing killing).
• selection of an appropriate form of hybridoma such a screen can be conducted among antibodies of any species and class or subclass.
• the hybridomas producing antibodies detected in such an assay procedure may be used in preparing the bi-specific antibodies of the present invention by techniques described hereinafter.
• Anti-non human T-cell/anti-immunoglobulin bi-specific antibody molecules are of interest, particularly in a research context, for example as a model system in the investigation of the important requirements for cell killing with animal tumours such as those of the rat and mouse.
• the major area of interest of the present invention is in human medicine and bi-specific antibodies having a first binding affinity directed against human T-cells are therefore of particular interest, these most usually being used in conjunction with a separate antibody having a binding affinity for target cells present in the human body.
• assay procedures such as that described above it is possible to select from among hybridomas producing monoclonal antibodies having the ability to bind with human T-cells those which are capable of triggering T-cell cytotoxicity, one example of such an antibody being the rat
• IgG2b antibody produced by the hybridoma YTH 12.5.14.2 (and all related subclones) which is described in Example 2 hereinafter. This antibody binds to the T-cell antigen CD3 but other antibodies of different specificities may also be similarly selected.
• Other examples include the anti-human CD2 rat IgG2b antibodies produced by the hybridoma YTH 655(5)6 and YTH 616.7.10 (H.P. Tighe, Ph.D.
• hybridomas producing all of these antibodies may of course be used in derivative forms (as reclones or subclones) or that analogous hybridomas of similar specificity may be employed.
• the second binding affinity of the bi-specific antibody molecules according to the present invention may be directed against any of the variety of binding sites present in antibody immunoglobulins, being for example anti-allotype, anti-isotype (particularly -subclass), anti-species or to a lesser degree of preference, anti-idiotype, in its specificity.
• the word isotype is used in the art to designate a particular class and/or subclass so that in the rat, for example, IgM, IgGl ,
• IgG2a, IgG2b and IgG2c each constitute a separate isotype.
• an anti-isotype binding affinity is most usually anti-subclass, for example anti-IgGl etc. rather than anti-IgG, although it is possible for the affinity to be anti-class particularly when, as in the case of IgM, no subclasses exist.
• the immunoglobulin affinity of the bi-specific antibody molecule may also be directed against a site in the immunoglobulin exposed by the formation of a fragment thereof, for example the F(ab')2 fragment of the anti-target cell antibody as described hereinafter.
• the affinity may be for an artificially created site on the immunoglobulin produced by the attachment of a hapten thereto.
• Such an affinity for immunoglobulin based upon anti-hapten binding may be utilised, for example, with fluorescein isothiocyanate labelled or biotinylated immunoglobulins.
• the primary use of the bi-specific antibody molecules of the present invention is in indirect ECR where binding of the molecules to target cells is effected through a separate antibody molecule having a binding affinity for target cells.
• the immunoglobulin is not one which causes the bi-specific antibody molecule to be targeted to cells, in particular tumour cells, without the use of a separate anti-target cell antibody.
• the immunoglobulin therefore preferably is not one which functions as a cell surface antigen, and particularly is not an antigen present on the surface of tumour cells.
• immunoglobulin specifity are thus any specificity for an endogenously synthesised and expressed cell surface immunoglobulin, particularly one which appears on the surface of tumour cells.
• An anti-idiotype affinity is an especial case of a less preferred specificity, both as regards the functioning of many idiotypes as cell surface antigens and since an anti-idiotype can block antibody/antigen binding.
• a specific example of a less preferred specificity involving a cell surface immunoglobulin and idiotype is the idiotype of the surface immunoglobulin of a human B-lymphoma, such as that described by Liu et al, ibid, which functions as a tumour specific cell surface antigen.
• the immunoglobulin affinity of the latter is preferably as non-specific as possible.
• the bi-specific antibody molecule is intended for use in vivo and that any undesirable side reaction with the patient's body should be avoided in as far as possible.
• the simplest way of avoiding undesired reactions between the bi-specific antibody molecule and immunoglobulin molecules naturally present in the patient's body is for the immunoglobulin binding affinity to relate to a species other than that to which the T-cell receptor binding affinity relates.
• the latter species is man and the former may conveniently be another mammalian species, for example the rabbit and particularly the mouse or especially the rat.
• the bi-specific antibody will bind with any of the various classes of antibody as discussed hereinafter and particularly with both IgM antibodies and IgG antibodies, including the various subclasses of the latter.
• the binding affinity is not totally species specific so that, for example, binding to at least both of rat and mouse antibodies is possible.
• anti-immunoglobulin antibodies specific for the rat or mouse may be used a small group of anti-immunoglobulin antibodies specific for the rat or mouse and possibly even for different subclasses thereof.
• an important preference for the anti-immunoglobulin specificity is that it does not cause auto-reactivity.
• the anti-immunoglobulin specificity should exclude anti-rat IgG2b and anti-rat kappa.
• the bi-specific antibody molecule described in Example 2 hereinafter is a hybrid between the antibody YTH 12.5.14.2 (a rat IgG2b with a lambda light chain which is specific for the C03 antigen) and the antibody RG11/15.5 (a mouse IgG2a with a kappa light chain which is specific for the rat kappa lb allotype).
• This combination which avoids auto-reactivity, can be used in conjunction with any rat antibody containing a rat kappa lb allotype light chain.
• the anti-target cell antibodies used in conjunction with the bi-specific antibodies of the present invention may have a binding affinity for any antigen present on the surface of a target cell.
• the target cell may be any cell which may be beneficially removed from the body. Examples include virally infected cells (viruses themselves not normally being attacked by T-cells), the virus being of various types including the influenza and rabies viruses, and both parasitized cells and parasites themselves including those responsible for malaria, leprosy, trypanoso iasis and schisto iasis, as well as tapeworms and other parasitic worms such as helminths.
• the preferred target cells are however tumour cells, the anti-target cell antibodies having a binding affinity for any tumour-associated antigen.
• tumour-specific antigen i.e. an antigen found on tumour cells only and not on normal cells.
• B-cell Ig idiotype which has been employed in the treatment of B-cell malignancies such as BCLL is one of the very few examples of such antigens which exist and in practice the tumour-associated antigen will also exist on normal cells.
• the antigen is anomalously expressed at higher levels or in an appropriate way on tumour cells thereby allowing an enhanced level of antibody-antigen reaction with the tumour cells but even this is not completely necessary.
• anti-tumour antibodies of particular interest are antibodies against antigens which define clusters of differentiation (CD) of the haemopoietic system recognised by groups of monoclonal antibodies standardised and characterised by International Workshops on Human Leukocyte Differentiation Antigens (Paris 1980, Boston 1983, Oxford 1986), and antibodies to the common acute lymphoblastic leukaemia-associated antigen CALLA, the carcinoembryonic antigen as expressed on, human colon carcinoma, and the human melanoma-associated ganglioside GD3.
• a specific example is the human B-cell differentiation antigen CD19 which is expressed on normal B-cells and many malignant B-cells and B-cell lines.
• the anti-target cell antibodies may be produced by classical techniques as a polyclonal preparation but are more conveniently produced as monoclonal antibodies using hybridoma technology, for example as described in US Patent 4,172,124 and in a wide range of scientific papers present in the literature.
• the bi-specific antibody molecules described herein may be produced by the chemical linkage of the halves of two antibodies, which may be produced by classical techniques or by hybridoma technology, one of the first binding activity and another of the second binding activity.
• particularly preferred bi-specific antibody molecules according to the present invention are those produced directly by the techniques of hybridoma technology modified as necessary for the production of bi- rather than mono-specific antibodies.
• Such techniques for the preparation of bi-specific antibodies are described in European Patent Application 0068763 and PCT Application WO 83/03679 which relate broadly to bi-specific antibody molecules produced by hybridoma technology.
• Examples of myeloma starting materials for use both in the preparation of the bi-specific antibodies and also the antitarget cell antibodies are the Y3-Ag 1.2.3 myeloma of European Patent No. 001459 (C.N.C.M. No. 1-078), the YB2/3.0.Ag.20 myeloma of European Patent No. 0043718, the myeloma P3-X63-Ag8 (A.T.C.C. No.
• each hybridoma is either derived from a myeloma which does not express a light chain or is a myeloma light chain loss variant, i.e. being HL rather than HLK.
• the second hybridoma employed in our procedure is poisoned with a lethal dose of an irreversible biochemical inhibitor, for example diethylpyrocarbonate and particularly iodoacetamide.
• an irreversible biochemical inhibitor for example diethylpyrocarbonate and particularly iodoacetamide.
• Such an inhibitor poisons the cells but does not damage their DNA, which codes for immunoglobulin expression and also for the HPRT enzyme.
• Following the treatment of these cells they are washed to remove any excess of the inhibitor and are then ready for use, the overall structure of the cells remaining intact for several hours after the treatment, the fusion typically being carried out within 0.5 to 1 hour.
• Fusion of the two hybridomas will produce a fused cell system which possesses the DNA from both hybridomas and in which any short-term loss of vital enzyme function from the poisoned cells will be complemented by the enzymes derived from the other hybridoma.
• the fusion mixture is cultured in a medium free from inhibitors when cells of the unfused hybridoma which has been poisoned will gradually die whilst cells of the other unfused hybridoma and the fused cells will survive. Selection is then commenced with, for example, a HAT-containing medium when cells of the unfused hybridoma lacking TK or HPRT will die but the fused cells will survive since the enzyme deficiency is met by the DNA from the other hybridoma.
• Iodoacetamide has been found to cause cell death within 1 to 24 hours but it has been found that a greater level of hybridisation is generally obtained if selection is delayed for 2 to 3 days, possibly because HPRT expression derived from the poisoned cell requires a little time to occur fully. Moreover, best results have been obtained using an equal or higher proportion of untreated cells to iodoacetamide-treated cells, for example from 1:1 to 10:1.
• bi-specific antibody Although the use of the type of bi-specific antibody described herein of itself represents an improvement over the use of the bi-specific antibodies described in the art, in a preferred aspect of the present invention a further improvement may be obtained.
• a bi-specific antibody to induce killing of the T-cells to which it binds through the natural Fc receptor mediated killing mechanisms referred to hereinbefore.
• Staerz and Bevan papers described hereinbefore no reference is made thereto in the Staerz and Bevan papers described hereinbefore, this is a serious deficiency in the effector cell retargeting procedure of the prior art since that procedure kills tumour cells only through T-cell toxicity.
• the problem is less serious as other tumour killing mechanisms are also involved but it still reduces the efficiency of tumour cell killing which is likely to be achieved.
• the problem of T-cell destruction may be mitigated, and preferably substantially avoided, in one of two ways.
• the first of these ways is based on an appreciation that the problem can be overcome by the use of an appropriate combination of heavy chains in the bi-specific antibody molecules of the present invention.
• the overall structure of an immunoglobulin is determined by the interactions of the various globular domains of the individual chains with each other. These interactions consist of covalent bonds involving intra-chain and inter-chain disulphide bridges as well as non-covalent interactions involving both protein and carbohydrate groups.
• complement components and Fc receptors must bind to structures present in the different antibodies but different species, isotypes and allotypes of antibody have differences in portions of their protein sequences although they may have many similarities in other portions of these sequences.
• different immunoglobulins will interact differently with complement components and Fc receptors and, in addition, when hybrid antibody molecules are made the two heavy chains may differ in sequence at crucial points for their interaction with each other and this may influence the properties of different combinations.
• the immunoglobulins which constitute antibodies may be divided into several classes, the major ones of these being identified as IgG, IgA, IgM, IgD and IgE, of which some, in particular IgA and especially IgG, may be further divided into subclasses.
• the bivalent antibody molecules according to the present invention preferably contain two heavy chains of the same class but may be of different subclasses within that class and may be of the same or a different subclass but relate to different species, the human, rat and mouse being of most interest, although immunoglobulins of other species, for example the rabbit, may be employed. Combinations of rat and mouse and rat and rat immunoglobulins are preferred to the mouse and mouse combination in terms of the higher level of stability of the corresponding hybridomas.
• Fc receptor mediated killing mechanisms particularly ADCC
• the nature of the heavy chain providing the T-cell binding affinity is of prime importance
• the nature of the heavy chain providing the immunoglobulin binding affinity is also of importance.
• certain subclasses of immunoglobulin of a first species will not interact with the cell mediated effector mechanisms of a second species and with those subclasses which will interact when two heavy chains of the same species and subclass are present in the immunoglobulin it is possible to interfere with this interaction by replacing one of the two heavy chains by an appropriate selection from another species or subclass.
• the nature of the heavy chain providing the T-cell binding activity may be a more dominant factor in determining the level of complement activation which occurs than it is with ADCC, but the nature of the heavy chain providing the immunoglobulin binding activity can still exert an important effect as discussed hereinafter.
• the species to which the two heavy chains relate and the species to which the antibody is to be administered are all of importance.
• a particular heavy chain species subclass combination which does not produce killing of T-cells in the mouse or rat may well do so in the human, which is the species of choice as regards the T-cell binding affinity of the bi-specific antibodies.
• human heavy chains may conveniently be used for at least one of the heavy chains in the bi-specific antibody molecules of the present invention. Indeed, a preferred choice is the use of two human heavy chains of the same class but of a different subclass. However, the relative lack of availability of human myelomas as compared with mouse and rat myelomas can pose a problem and in practice these species may often therefore represent the mammalian species of choice for the heavy chains.
• the IgG class is divided into the subclasses IgGl , IgG2, IgG3 and IgG4 (the IgA class being divided into the subclasses IgA! and IgA2) whilst in the mouse and rat only the IgG class is divided into subclasses, these being IgGl, IgG2a, IgG2b and IgG3 in the mouse and IgGl, IgG2a, IgG2b and IgG2c in the rat (the similarly named subclasses not necessarily having similar properties in the mouse and the rat).
• each of the heavy chains is most likely to be of mouse or rat IgA or IgE, particularly IgM and especially IgG, the commonest situation being that they are each of an IgG subclass and especially mouse IgGl, IgG2a or IgG2b, and less commonly IgG3, or rat IgGl, IgG2a or IgG2b, and less commonly IgG2c.
• a heavy chain having T-cell binding activity the simplest course is to use a class or subclass, for example of the rat or mouse, which does not lead to the killing of T-cells in the species in question via an Fc receptor mediated mechanism.
• IgGl > IgG3 > IgG2a IgG2b and in the rat is IgGl > IgG2c > IgG2a > IgG2b.
• IgG2b preference in the mouse is IgG2b, IgG3 and IgGl > IgG2a and in the rat IgGl and especially IgG2a and IgG2c > IgG2b.
• the close interaction between the two heavy chains in bi-specific antibody molecules, but not in conjugates is such that even though the heavy chain of T-cell binding activity is one such as mouse IgG2a or rat IgG2b which will promote killing, it is possible to counteract this killing through the selection of a suitable form of heavy chain having immunoglobulin binding activity, in particular one of a different species or of a different isotype or allotype.
• the preferences for inactivating the rat IgG2b heavy chain are a rat IgG2a or especially a rat IgG2c heavy chain.
• rat IgG2b or mouse IgG2a combination with an alternative form of heavy chain either by species (for example rat IgG2b/mouse IgGl) or by subclass, is indicated since a rat IgG2b/rat IgG2b or mouse IgG2a/mouse IgG2a combination can generally be presumed to be effective in promoting killing through the ADCC mechanism and possibly also through the complement activation mechanism.
• the use of heavy chains of a different allotype or particularly a different species or isotype can also be of value when the anti-T-cell heavy chain may exhibit only an insubstantial level of effectiveness in causing killing (i.e.
• the achievement of the mitigation of the killing of T-cells may be assessed by a comparison with such bi-specific antibody molecules in which the heavy chain having the affinity for target cells is the same (species, isotype and conveniently allotype) as that having the affinity for T-cells.
• the resulting bi-specific antibody can then be tested to see if the T-cell killing ability of the first heavy chain/light chain combination is negated by combination with a heavy chain/light chain combination of the immunoglobulin type in question.
• a hybridoma producing a rat IgG2b antibody against T-cells with a hybridoma producing any irrelevant rat IgG2a or IgG2c antibody it can be tested whether the ability of the IgG2b heavy chain to induce killing by both the ADCC mechanism and through the complement activation mechanism is retained or not.
• every possible combination of the two different light chains and two different heavy chains can occur and in practice some antibodies of each type may be presumed to be obtained although not necessarily in equal proportions. It is only in the case where the bi-specific antibody molecule heavy chain having a T-cell binding affinity will not mediate the killing of T-cells by an Fc receptor mediated killing mechanism, such as ADCC or complement activation, that none of the types of antibody will be capable of destroying T-cells.
• types 2 and 4 will be toxic via this route irrespective of the nature of the immunoglobulin- binding heavy chain, and types 1 and 5 will be toxic in addition"to types 2 and 4 when the i rnunoglobulin-binding heavy chain is not such as to negate the activity of the other heavy chain. Accordingly even though the bi-specific type 1 monoclonal antibody molecules do not cause the killing of T-cells owing to the selection of an appropriate combination of heavy chains for the anti-T cell and anti-immunoglobulin halves of the molecule, types 2 and 4 will always possess the undesirable ability to kill T-cells.
• the order of value (for the anti T-cell/ anti-immunoglobulin combination) is IgG2b/IgG2b ⁇ IgG2b/IgG2a (or 2c) ⁇ IgG2a (or 2c)/IgG2a (or 2c) or IgG2a (or 2c)/IgG2b since the first is toxic to T-cells as the bi-specific molecule, the second produces toxic molecules in admixture with the bi-specific molecule but the third and fourth do not produce molecules toxic to T-cells and the other types of molecule present in admixture act only as a diluent to the bi-specific molecules.
• IgG2b/IgGl Other rat IgG combinations of particular interest for human therapy are IgG2b/IgGl , which should not be toxic to T-cells as the bi-specific antibody but will provide toxic type 2 and 4 molecules, and especially IgGl/IgG2b which should not be toxic to T-cells as types 1, 2, 4 and 5.
• IgGl/IgG2b which should not be toxic to T-cells as types 1, 2, 4 and 5.
• the four possible different combinations of IgGl with IgG2a or IgG2c may be used, which would all be expected to behave generally similarly to the IgG2a (or 2c)/IgG2a (or 2c) combinations described above.
• the combinations of similar isotypes IgGl/IgGl, IgG2a/IgG2a and IgG2b/IgG2b are generally somewhat less preferred (particularly IgG2a/IgG2a for the reasons given for rat IgG2b/rat IgG2b above) than all of the possible dissimilar isotype combinations of IgGl, IgG2a and IgG2b, which are of interest.
• Some of these dissimilar isotype combinations will of course be of greater interest than others on similar reasoning to that applied in the rat case so that, for example, combinations in which the anti-T cell binding is provided by mouse IgG2a will be expected disadvantageously to provide toxic type 2 and 4 molecules.
• the whole range of rat IgG/mouse IgG and mouse IgG/rat IgG combinations are also of some interest.
• the mixture of antibodies containing the bi-specific antibody of the invention which is produced by a hybridoma system derived from two fused hybridomas or two other fusion partners may be fractionated to enhance the proportion of the bi-specific antibody molecules therein and preferably substantially to separate the bi-specific antibody molecules from or at least to reduce the proportion of other species which are undesirable, i.e. any types of molecule which act as a diluent to the type 1 molecules and particularly any species which are toxic to T-cells by an Fc mediated receptor mechanism.
• the techniques used for such purification are broadly analogous to those described in UK Patent Application 2,144,147A where the product it is desired to purify is one of type 4, being a monovalent monoclonal antibody.
• Such procedures include the use of affinity chromatography, ion exchange chromatogrpahy and chromatofocussing, particularly using fast protein liquid chromatography (FPLC - Pharmacia Trade Mark) and high pressure liquid chromatography (HPLC) techniques.
• Affinity chromatography can be exploited using either an antigen-containing column (for example an immunoglobulin) which will select for those species of molecule having the correct combination of heavy and light chains for specificity or, alternatively, an anti-isotype or protein A column can be used to separate on the basis of isotype.
• Ion exchange chromatography relies on the fact that at different pHs the charge on a protein varies as different side chains ionize so that the binding of protein to a charged column can be affected by ionic strength.
• a powerful application of ion exchange cho atography involves the separation of fractions on a first column at a first pH followed by the use of a second column, at a second pH, the columns usually being of opposite charge so that cation exchange chromatography is followed by anion exchange chromatography, or vice versa.
• Chromatofocussing relies on the fact that at a particular selected pH, the protein has no net charge and will not bind to a charged column so that similar mixed proteins are separated on a basis of their pi.
• FPLC and HPLC offer different advantages and may be used in combination.
• the bi-specific antibody may generally be used in the form of a fragment retaining the binding affinities of the whole molecule. More importantly, however, a second way of avoiding the destruction of T-cell by the bispecific antibodies of the present invention is to use the antibody in the form of a fragment of the whole antibody which retains the binding affinities but not any T-cell killing capacity, particularly a F(ab')2 fragment.
• This approach depends on the fact that the F(ab')2 portion of the antibody molecule is still capable of binding to T-cells through one of its heavy chain/light chain combinations but the lack of the Fc portion of the molecule means that the killing of T-cells by an Fc mediated receptor mechanism cannot occur.
• a further advantage of using F(ab')2 portions is that, being smaller, they are more rapidly cleared by the kidneys.
• the preparation of the F(ab')2 portion of the antibody molecule requires the use of an appropriate enzyme system to effect cleavage at a suitable point in the heavy chains to thereby remove the Fc region of the heavy chains (or alternatively at least that of the anti-T cell heavy chain) whilst retaining the remaining portion of the two heavy chains, each linked to its light chain and also to the other heavy chain via disulphide bridges.
• an appropriate enzyme system to effect cleavage at a suitable point in the heavy chains to thereby remove the Fc region of the heavy chains (or alternatively at least that of the anti-T cell heavy chain) whilst retaining the remaining portion of the two heavy chains, each linked to its light chain and also to the other heavy chain via disulphide bridges.
• the techniques for the production of the F(ab')2 fragments of the bi-specific antibodies according to the present invention are broadly similar to those described in the literature for the preparation of the F(ab')2 fragments of mono-specific antibodies.
• the present invention thus includes (a) a F(ab')2 fragment of a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, and also (b) a process for the preparation of such a fragment which comprises treating the antibody molecule with a suitable enzyme system to effect cleavage thereof to yield this fragment.
• the present invention therefore further includes (a) a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, for use in surgery, therapy or diagnosis and also (b) the use of a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, for the manufacture of a medicament for use in the treatment of neoplastic or other disease.
• bi-specific antibodies and fragments thereof described herein may be formulated for use in various ways, which will however, usually involve the use of a physiologically acceptable diluent or carrier which will conveniently be sterile and preferably also pyrogen-free for certain uses.
• a physiologically acceptable diluent or carrier which will conveniently be sterile and preferably also pyrogen-free for certain uses.
• This may take various forms, for example phosphate buffered saline, saline, balanced salt solution and dextrose solution.
• phosphate buffered saline may be mentioned especially as often being suitable.
• composition may, if desired, be presented in unit dosage form, i.e. in the form of discrete portions containing a unit dose, or a multiple or sub-unit dose.
• anti-target cell antibodies described herein may also be utilised in fragment, for example F(ab')2, form although the advantages in doing this are less than those described above for the bi-specific antibodies.
• the anti-target cell antibodies, or fragments thereof may conveniently be formulated in a broadly similar manner to the bi-specific antibody molecule, or fragment thereof. Indeed, it may be appropriate either to incorporate the two types of antibody into the same composition or into a kit, although the former may result in conjugate formation as described hereinafter.
• the present invention thus includes (a) a composition comprising a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, and an antibody molecule having a binding affinity for target cells, or a fragment thereof having such an affinity, and also (b) a kit comprising in association a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, and an antibody molecule having a binding affinity for target cells, or a fragment thereof having such an affinity.
• a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule and an antibody molecule having a binding affinity for target cells, or a fragment thereof having such an affinity, for simultaneous, separate or sequential use in the treatment of neoplastic or other disease.
• Both types of antibody or fragment thereof may be administered in various ways, for example intravenously, intraperitoneally or possibly intracerebrally, the mode of administration being selected to be appropriate to the type and localisation of the tumour or other target cells and also for ease of administration by the clinician and for the safety of the patient.
• parenteral administration and particularly intravenous injection, will often be used.
• the two types of antibody may be employed simultaneously, separately or sequentially but it will usually be the case that the anti-target cell antibody will be administered either before or together with the bi-specific antibody and where the latter is the case, a suitable time delay will often be from 0.5 to 24 hours.
• dosages of the two types of antibody, or fragment thereof the exact dosages will depend upon the potency of the reagents, the tumour or other disease burden of the patient and the patient's body weight/surface area ratio.
• a dosage of between 1 to 25 mg of each antibody or fragment, for example approximately the same amount of each will often be suitable, conveniently used in a 7 to 10 day regimen involving 1 dose of each per day, i.e. a 10 day course of treatment involving the administration of a total dosage of 10 to 250 mg of each antibody or fragment to the patient. It will be appreciated however that dosages outside this range may be used where appropriate although a particular advantage of the present invention is the low dosages which may be used in many cases, i.e. towards the lower end of the range stated or even below it, thereby possibly even avoiding the setting up of an immune response to the bi-specific antibody molecules and thus allowing repeated usage.
• the present invention includes the use of a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, in conjunction with an antibody molecule having a binding affinity for target cells ⁇ or a fragment thereof having such affinity, in the treatment of neoplastic or other disease.
• neoplastic or other disease includes a method for aiding the regression and palliation of neoplastic or other disease which comprises administering to a patient in need thereof amounts which together are therapeutically effective in achieving such regression and palliation of a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin, or a fragment thereof retaining the binding affinities of the whole molecule, and an antibody molecule having an affinity for target cells, or a fragment thereof having such affinity.
• the bi-specific antibodies may be used in conjunction with the anti-tumour antibodies for the removal of neoplastic cells from bone marrow in. vitro, thereby allowing autologous bone marrow transplantation to be used in the treatment of malignancy.
• bi-specific antibody molecules of the present invention offers many benefits over direct ECR as envisaged by Staerz and Bevan.
• Possible variations upon the simple use of the bi-specific antibody molecules together with the anti-target cell antibodies in vivo or jn. vitro as described hereinbefore include the removal of some peripheral blood mononuclear cells from the patient and the.treatment of these cells briefly with anti-T-cell antibodies, such as the monoclonal antibodies YTH 12.5.14 or 12.5.14.2 described in Example 2, in order to activate the cells.
• a panel of anti-tumour monoclonal antibodies is then selected, for example against differentiation antigens, surface immunoglobulins, tumour associated antigens, etc., and appropriate antibodies or combinations of antibodies are used to target the tumour cells for lysis by activated effectors which are reintroduced into the body several hours later in the presence of the bi-specific antibody molecules, allowing excess anti-tumour cell antibodies to be eliminated.
• an important advantage of indirect ECR versus direct ECR is that both preactivated cytotoxic T-lymphocyte (CTL) and K-cell populations may be recruited to destroy a tumour target.
• CTL cytotoxic T-lymphocyte
• the efficient use of direct ECR requires that the Fc ⁇ Rlow on K-cells or Fc receptors on effector cells must be either blocked, for example with a suitable CD16 monoclonal antibody, or the heavy chains of the bi-specific antibody molecule must be selected so that the Fc portion does not bind to Fc ⁇ Rlow or other Fc receptors.
• the Fc portion of an anti-T-cell/anti-target cell bi-specific antibody molecule may be rendered inactive by producing F(ab')2 fragments.
• these processes eliminate the potential for K-cell mediated ADCC against a target cell coated with the bi-specific antibody.
• bi-specific antibody molecules selected for having a non-functional Fc portion mediate indirect ECR by focussing the CTL response against the same population of target cells.
• the bi-specific antibody molecules according to the present invention potentially allow the administration of different antibody treatments at various time intervals. This permits clearance of an excess of a first anti-target cell antibody so that a second anti-target cell antibody can home efficiently via its antiglobulin specificity to the tumour cells bound by the remaining first antibody. This will reduce the formation of aggregates which can be removed very efficiently by cells of the reticuloendithelial system, etc.
• indirect ECR the use firstly of an appropriate anti-target cell antibody will allow the activated K-cells to mediate ADCC against the target cell. This is followed by the administration of bi-specific antibody molecules when the CTL population is also recruited to kill the same target cell via indirect ECR.
• the bi-specific antibody molecules of the present invention may be used, either as the whole molecule or as a fragment thereof, in the preparation of bi-specific antibody conjugates having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for target cells.
• the bi-specific antibody molecules of the present invention provide an intermediate capable of use to prepare a range of bi-specific antibody conjugates having the same T-cell binding affinity but differing tumour cell or other target cell binding affinities.
• the present invention therefore includes a bi-specific antibody conjugate having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for target cells, characterised in that the conjugate - comprises a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin linked to an antibody molecule having a binding affinity for target cells, or a fragment thereof retaining the binding affinities of the whole conjugate.
• the bi-specific antibody molecules of the present invention are combined in vitro with an antibody having a binding affinity for target cells such as is described herein, the conjugate then being administered j_n vivo.
• the conjugates could be prepared by chemical linkage it is of course preferable to use the anti-immunoglobulin affinity of the bi-specific antibody molecule for the anti-target cell antibody to provide the linkage.
• the conjugates may therefore conveniently be prepared by the simple admixture of solutions of a bi-specific antibody molecule having a first binding affinity for a T-cell receptor capable of activating killing and a second binding affinity for immunoglobulin and an antibody having a binding affinity for target cells. It is preferred that the bi-specific antibody conjugates so prepared are not isolated but are administered directly to the patient. Conveniently, therefore, the preparation may be effected in a suitable medium for this purpose so that, for example, the two components may be contained in equal volumes of sterile physiological saline. The mixture may then be left for a suitable period for reaction to be complete but not to allow any substantial degree of aggregation, such as for up to 1 hour, for example 30 minutes, and may then be administered, particularly parenterally.
• the hybridoma is hypoxanthine-guanine phosphoribosyl transferase positive (HPRT " * * ) and expresses one spleen cell-derived light chain and a second myeloma-derived light chain of the kappa-la allotype.
• myeloma light chain loss variants by cell cloning on semi-solid agar (Clark and Waldmann, ibid) and then assaying for the loss of rat kappa-la allotype expression using a sensitive red cell haemagglutination assay (Clark, Methods in Enzymology, 1986, 121, 548-556).
• the variant selected is cloned on semi-solid agar and then maintained in culture in Iscoves modification of Dulbecco's medium (IMDM - Gibco Europe) supplemented with 1 to 5% v/v foetal calf serum (FCS) and buffered with bicarbonate using 5% CO2 in air.
• the bicarbonate is replaced by extra hepes buffer and NaCl to maintain the ionic strength.
• the BALB/C myeloma cell line NSI/Ag 4.1 is fused with spleen cells from a SJL mouse strain immunised with pooled rat IgG in complete Freund's adjuvant (Springer et aJ, Hybridoma, 1982, 1, 257-273).
• the selected hybridoma produces a monoclonal antibody having specificity for a particular rat allotype but this is not one corresponding to the immunoglobulin produced by the hybridoma (1), thereby avoiding auto-reactivity.
• the hybridoma (1) Prior to fusion the hybridoma (1) is treated in phosphate buffered saline (PBS) with 10 mM iodoacetamide for 30 minutes at 0°C.
• PBS phosphate buffered saline
• the HPRT 4 " iodoacetamide-poisoned cells are washed with HEPES buffered IMDM to remove excess inhibitor and then mixed with the HPRT" hybridoma (2) cells in three different proportions:- (a) 3.5 x 10 7 cells hybridoma (1) + • 3.5 x 10 6 cells hybridoma (2) (10:1); (b) 3.5 x 10? cells hybridoma (1) + 3.5 x 10? cells hybridoma (2) (1:1); and (c) 3.5 x 10?
• cells hybridoma (1) + 3.5 x 10 s cells hybridoma (2) are mixed in HEPES buffered IMDM and pelleted at 200 x g.
• Cell fusion is induced by treating the cell pellet for 2 minutes with 1 ml of a 50% w/v solution of polyethylene glycol 1500 in PBS whilst stirring (Clark and Waldmann, ibid) .
• the cells are then washed once with HEPES buffered IMDM, resuspended in bicarbonate buffered IMDM containing 5% v/v foetal calf serum, plated out into 24 x 2 ml culture well plates at four concentrations from 8 x 10 ⁇ to 2 x 10 ⁇ and cultured at 37°C under 5% C ⁇ 2 « On the following day a control containing iodoacetamide-treated but infused cells is typically totally
• the hybridoma (1) is 8-azaguanine resistant and the hybridoma (2) is poisoned with iodoacetamide in step (3) and in a second variant 6-thioguanine is used instead of 8-azaguanine in either the original or first variant procedures, and in a third variant the hybridoma (2) is a myeloma light chain loss variant as well as or instead of the hybridoma (1), or one or both hybridomas are derived from a myeloma which does not express a light chain. non-viable, whilst in the cultures of the fused cells the majority of cells are observed to be viable.
• HAT selective medium - Clark and Waldmann, ibid
• a human T-cell leukaemia cell line such binding indicating the presence of both the heavy and the light chain of the hybridoma (1).
• Functional anti-rat immunoglobulin activity is detected using a rat antibody with anti-mouse Thy-1 antigen or other irrelevant specificity (which is of the appropriate allotype in relation to hybridoma (2)) fixed to the microtitre wells.
• Antibody capable of binding to such an antibody is detected with biotinylated YA 9/36.39 (Clark, Ph.D.
• the plate binding assay is again employed to screen for antibodies containing mixed immunoglobulin chains derived from both parental cells, the biotinylated YA 9/36.69 being replaced by a biotinylated monoclonal antibody specific for the rat IgG sub-class of the hybridoma (1).
• Cells from wells which are strongly positive in all three assay procedures are cloned twice in semi-solid agar and a suitable clone is selected, this being maintained in culture as for the mono-specific hybridoma (1).
• the anti-rat Ig hybridoma (2) is replaced by a hybridoma specific for Ig of another species, for example the mouse or the human.
• one or both of the hybridomas (1) and (2) are replaced by a hybridoma derived from a human myeloma, for example as described in European Patent Applications 0062409 and 0148644, in UK Patent 2086937 and in US Patent 4529694.
• Example 1(3) The procedure of Example 1(3) was followed using as hybridoma (1) the hybridoma YTH 12.5.14 (this is identical to the sister clone YTH 12.5.22 described by Cobbold and Waldmann, Nature, 1984, 308, 460-462) and as hybridoma (2) the hybridoma RG 11/15.5 (Springer et al, ibid).
• YTH 12.5.14 produces a rat IgG2b monoclonal antibody and expresses a myeloma-derived kappa light chain of the la allotype.
• the myeloma chain loss variant YTH 12.5.14.2 secretes a single light chain unreactive with all tested anti-rat kappa reagents and which may therefore be presumed to express a light chain of the lambda class.
• RG 11/15.5 produces a mouse IgG2a monoclonal antibody specific for the rat kappa lb allotype light chain and expresses a kappa light chain.
• the fusion mixture was screened using biotinylated NORIG 7.16.2 (Hale et al. Journal of Immunology, 1985, 134. 3056), a reagent specific for rat IgG2b in the third assay procedure.
• the bispecific anti-human CD3/anti-rat kappa lb allotype light chain hybridoma LHC 49.18.2 was selected following the cloning procedure.
• Example 1 The procedure of Example 1 was followed again using as hybridoma (2) the hybridoma RG 11/15.5 referred to in Example 2 and in this case using as hybridoma (1) the hybridoma 0KT3.5.2 which is a redone of the mouse IgG2a anti-human CD3 hybridoma 0KT3 referred to in US Patent 4361549 and deposited with the ATCC, Rockville, USA, under the accession number CRL 8001.
• 0KT3.5.2 has identical properties to 0KT3, which is derived from the mouse myeloma P3-X63-Ag8Ul , that secretes a mouse kappa light chain, through a fusion with spleen cells from a CAF1 mouse immunised with E rosette positive-purified human T-cells.
• the fusion mixture was screened with an immunoglobulin isotype assay using a suitable rat IgG antibody with a kappa lb allotype light chain fixed to the microtitre wells and biotinylated YA9/36.69 to detect RG11/15.5 specificity and complement mediated lysis of human T-cells to detect 0KT3 specificity by indirect fluorescence.
• the bi-specific anti-human CD3/anti-rat kappa lb allotype light chain hybridoma LHD 6.23 was selected following the cloning procedure.
• Example 1 The procedure of Example 1 was followed using the hybridoma 0KT3.5.2, which is described in Example 3, as the hybridoma (1) and as hybridoma (2) the hybridoma NORIG 7.16.2 which is an HL secreting hybridoma derived by a fusion between spleen cells from a BALB/c mouse immunized with rat anti-pertussis/pertussis immune complexes and the non-producing mouse myeloma NSO/u (NORIG 7.16.2 is a subclone of identical properties to the NORIG 7.16 described by Hale et ⁇ , ibid).
• NORIG 7.16.2 produces an anti-rat IgG2b mouse IgG2a monoclonal antibody expressing a kappa light chain.
• the 8-azaguanine resistant HL clone used was NORIG 7.16.2 AG.
• the screening procedures involved complement mediated lysis of human T-cells to detect 0KT3 specificity by indirect fluorescence and an ELISA assay using plates coated with an irrelevant rat IgG2b antibody.
• NORIG 7.1.62 specificity was detected using a specific biotinylated anti-mouse Ig reagent followed by streptavidin-peroxidase (both from Amersham pic, England).
• streptavidin-peroxidase both from Amersham pic, England.
• the bi-specific anti-human CD3/anti-rat IgG2b hybridoma LHB 63.10 was selected. Exampl e 5
• the bi-specific hybridoma produced in Example 1, 2, 3 or 4 is maintained in low serum culture (IMDM containing 1% v/v FCS) for 48 hours at 37°C using 5% CO2 in air.
• the culture supernatant is then concentrated by precipitation with ammonium sulphate added to a level of 50% w/v.
• the precipitate is redissolved in the minimum volume of water and is desalted into 20 mM tris buffer at pH 8.0 on Sephadex G25.
• the antibody is fractionated by hpic ion exchange chromatography on a TSK-5PW column (LKB Ltd.). Pooled fractions are desalted into PBS for assay.
• Venous blood was collected from healthy donors and was defibrinated using glass beads.
• Mononuclear cells were isolated from the interface following density gradient centrifugation on Ficoll-Hypaque and were then washed into bicarbonate buffered IMDM containing 5% v/v FCS. These cells were divided into tissue culture bottles at 1 x 10 6 cells per ml in the same culture medium, the bottles having been pre-coated with anti-human CD3 antibodies, in particular those produced by the hybridoma YTH 12.5.14.2 of Example 2. After 3 days in culture at 37°C the effectors were subjected to a 7 day expansion in the same medium containing 10 U/ml rIL2 (Cetus).
• the blast cells produced in this way also contain 10-15% Fc receptor positive cells (as detected by Fc rosetting) which are able to mediate ADCC.
• Fc receptor positive cells as detected by Fc rosetting
• These ADCC effectors were inhibited by pre-incubating the once-washed effector cells in the same medium with a 1/600 dilution of the anti-Fc receptor antibody CLB-Fcr gran I (Tetteroo et al, Leucocyte Typing II, 1985, Volume 3, page 27 - Springer Verlanger, New York) for 15 minutes at room temperature.
• This anti-CD16 antibody blocks K-cell mediated ADCC.
• the mouse Thy-1 positive cell line BW 5147 (CRL 1588, PHLS European Collection of Animal Cell Cultures, Porton Down, England) was used as the target cell line.
• Cells were maintained in exponential phase in IMDM containing 2% v/v FCS until required for assay. Approximately 5 x 10 6 cells were then spun down at 200 x g and the pellet resuspended in 150 ⁇ l IMDM containing 300 ⁇ Ci 51 Cr-sodium chromate. The cells were incubated at 37°C for 1 hour and were washed into HEPES buffered IMDM. The target cells were then washed and divided into two tubes.
• the cells in the first tube were resuspended in 500 ⁇ l of HEPES buffered IMDM medium for analysis of direct ECR and the cells in the second tube were resuspended in a similar volume of the same medium containing approximately 100 ⁇ g/ml of anti-Thy-1 monoclonal antibodies derived from the hybridoma YBM 29.2.1 (see Cobbold et al, ibid) for analysis of indirect ECR. After a 1 hour incubation at room temperature the cells in the second tube were washed once to remove excess YBM 29.2.1 and were resuspended in the same- medium at a level of 2 x 10 ⁇ viable cells/ml.
• effector cells Two-fold dilutions of effector cells were set up in 50 ⁇ l volumes in U-bottom microtitrre wells and equal vo!8 hybridoma, (abelled Thy-1 coated or uncoated target cells were added in a 50 ⁇ l volume of HEPES buffered IMDM medium to provide an effector:target cell ratio ranging from 32:1 to 0.5:1.
• Suitable dilutions of the antibodies to be tested for effector cell retargeting were added in a 100 ⁇ l of HEPES buffered IMDM to the wells and incubation was effected at 37°C for 4 hours, after which 100 ⁇ l of medium per well was collected for a determination of the released radioactivity by measurement of gamma radiation using a Phillips gamma counter, model PW 4800.
• Each of the BW 5147 and EL-4 target cells were used in both unlabelled and labelled forms as descirbed in (A) using for labelling monoclonal antibodies from the anti-Thy-1 rat IgG2c, klb hybridoma YBM 29.2 as in (A) or alternatively, monoclonal antibodies from the anti-Thy-1 rat IgG2b, klb hybridoma YTH 154.7.7 (Cobbold et al, Molecular Biology and Medicine, 1983, 1, 285-304) or the anti-LC/7200 rat IgG2a, klb hybridoma YBM 42.2 (Stenning et aj_, Molecular Biology and Medicine, 1983, 1, 95-115).
• the labelled target cells were added to microtitre plates containing two-fold dilutions of effectors and a 1/100 dilution of supernatant was added (the cultures being obtained as in Example 5) of one of
• FIG. 3a and 3b show the results obtained with BW 5147 cells coated with YBM 29.2.1 and YBM 42.2 antibodies, respectively.
• Figures 3c and 3d show the results obtained with EL-4 cells coated with YBM 29.2.1 and YBM 42.2 antibodies, respectively.
• the results obtained with both types of target cells coated with YTH 154.7 antibodies were similar to those obtained with YBM 29.2.1-coated target cells and are therefore not illustrated. Unlabelled target cells of both types were, as expected, not killed in the presence of any of the three types of supernatant.

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