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WO1988003565A1 - Anticorps - Google Patents

Anticorps Download PDF

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Publication number
WO1988003565A1
WO1988003565A1 PCT/GB1987/000781 GB8700781W WO8803565A1 WO 1988003565 A1 WO1988003565 A1 WO 1988003565A1 GB 8700781 W GB8700781 W GB 8700781W WO 8803565 A1 WO8803565 A1 WO 8803565A1
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WIPO (PCT)
Prior art keywords
fragment
cell
antibody molecule
cells
molecule
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PCT/GB1987/000781
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English (en)
Inventor
Michael Ronald Clark
Herman Waldmann
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National Research Development Corporation
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Application filed by National Research Development Corporation filed Critical National Research Development Corporation
Publication of WO1988003565A1 publication Critical patent/WO1988003565A1/fr
Priority to DK348888A priority Critical patent/DK348888A/da

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    • 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]
    • 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
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]

Definitions

  • ANTIBODIES This invention relates to novel forms of antibody and their use as targeted cytotoxic agents, particularly in the treatment of neoplastic disease.
  • neoplastic disease still remains an intractable problem despite the fact that a very wide range of cytotoxic agents has now been developed for use in the treatment of the disease.
  • One recent approach utilises the phenomenon of effector cell retargeting (ECR) to destroy tumour cells.
  • ECR effector cell retargeting
  • a bi-specific antibody is constructed which has both an anti-T cell and an anti-tumour antigen activity.
  • Staerz et a_l Nature, 1985, 314. 628 and Perez et al_, Nature, 1985, 3Jj_. .
  • a bi-specific 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, 1, 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.
  • effector cell retargeting does however disregard one important disadvantage of such an approach.
  • a bi-specific antibody to induce killing of the T-cells to which it binds through one of the natural Fc receptor mediated cell killing mechanisms of which the body is capable, for example via antibody-dependent cell-mediated cytotoxicity (ADCC) involving K cells, neutrophils and macrophages, via phagocytosis by macrophages and cells of the reticuloendothelial system, or via complement activation.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the present invention comprises a bi-specific antibody molecule having a first binding affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, or a fragment thereof retaining the binding affinities of the whole molecule.
  • bi-specific antibody molecules of the present invention differ from those bi-specific antibodies of the 1985 Staerz et a. and Perez et a_ 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 bi-specific antibodies described in the 1986 Staerz and Bevan papers consist of antibody molecules rather than conjugates, those authors failed to appreciate the problems inherent in the use of these molecules.
  • the present invention is based on an appreciation that the two heavy chains must be selected to mitigate killing of human T-cells by the bi-specific antibody molecule. Moreover, as described hereinafter, we have discovered that the close interaction between the two heavy chains in these molecules, but not in the conjugates, is such that even when the heavy chain having an affinity for a human T-cell receptor is of a type which would normally produce killing of the T-cells by natural Fc receptor mediated cell killing mechanisms, it is possible to counteract this killing through an appropriate selection of the heavy chain having an affinity for target cells.
  • 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 killing by 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 a. 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 C03 unit (previously identified as T3) as a whole or against one of the individual chains thereof.
  • T3 receptor-associated C03 unit
  • 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 ⁇ (M 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 C03 complex and are identified as ⁇ (M r about 25,000), ⁇ (M r about 20,000) and c (Mr about 20,000).
  • Antibodies having a T-cell binding affinity may be identified by an assay procedure which we have developed. Most hybridomas as well as secreting antibody have small amounts of cell surface antibody.
  • a mouse hybrido a 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.
  • hybrido a cells are labelled with a radioactive label, for example 51Cr, 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 51Cr
  • 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-human T-cell/anti-non human target cell bi-specific antibodies 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 (and a second binding affinity directed against target cells present in the human body) are therefore of particular interest.
  • the present invention thus further comprises a bi-specific antibody molecule having a first binding affinity for human T-cells and a second binding affinity for target cells present in the human body characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule.
  • the second binding affinity of the bi-specific antibody molecules according to the present invention may be directed against 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, trypanosomiasis and schistomiasis, as well as tapeworms and other parasitic worms such as helminths.
  • the preferred target cells are however tumour cells, the second binding affinity being against any tumour-associated antigen. The ideal situation would be for the affinity to be for a tumour-specific antigen, i.e.
  • the 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 usually 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.
  • the toxic effects of the treatment on the normal cells can be countered by the use of marrow transplants or of removal of the patient's own bone marrow prior to the treatment and its return thereafter, conveniently following separate treatment of the bone marrow in. vitro for the removal of tumour cells.
  • anti-tumour antibodies of particular interest are antibodies against antigens which define clusters of differentiation (CD) of the haemopoetic 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.
  • 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 in general, 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.
  • myeloma starting materials are the Y3-Ag 1.2.3 myeloma of European Patent No. 001459 (C.N.C.M. No. 1-078), the
  • 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 techniques used in the preparation of the bi-specific antibodies of the present invention closely parallel those described in U.K. Patent Application 2,144,147A which may also be applied in the case of the present invention, although the binding affinities of the fusion partners are of course different. We have, however, developed a variant of these techniques which has proved to be of especial value.
  • bi-specific antibody molecules As in previous techniques described for the preparation of bi-specific antibody molecules our procedure involves the fusion of two hybridomas, but in the present instance one of these produces antibodies directed against T-cells and the other produces antibodies directed against target cells.
  • the two hybridomas may be fused by the conventional procedures, as illustrated in the Examples, to produce a hybridoma secreting bi-specific antibody molecules having each of the specificities of the hybridomas from which they derive. It will be appreciated, however, that one of the most difficult stages of any hybridoma-producing fusion is the selection from the fusion mixture of the desired type of bi-specific hybridoma and the procedure we have developed involves features directed particularly to this end.
  • the first hybridoma used has a drug selectable marker which may conveniently be an enzyme deficiency, for example of thymidine kinase (TK) or particularly of hypoxanthi ⁇ e-guanine phosphoribosyl transferase (HPRT).
  • TK thymidine kinase
  • HPRT hypoxanthi ⁇ e-guanine phosphoribosyl transferase
  • Such hybridomas are obtained by selecting cells from a growth medium containing 5-bromouracy deoxyribose or 2-aminopurine (for TK) or 8-azaguanine or particularly 6-thioguanine (for HPRT).
  • Cells selected for growth on a medium containing the appropriate drug lack the enzyme in question and, in the case of both TK and HPRT, will therefore be incapable of growth in a medium containing hypoxanthine, a inopterin and thymidine (HAT) since the aminopterin blocks the main pathway for purine and pyrimidine synthesis and the lack of HPRT or TK removes the ability possessed by normal cells of utilising the hypoxanthine to make purines and the thymidine to make pyridimines.
  • hypoxanthine a inopterin and thymidine
  • 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,
  • hybridomas secreting monoclonal antibodies having the desired bi-specificity.
  • Such hybridomas may be cultured either i_n vitro or in vivo by conventional techniques in order to produce supplies of the monoclonal antibodies.
  • the mitigation, and desirably the substantial avoidance, of the killing of T-cells by the bi-specific antibodies of the present invention is based on an appreciation that this 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 In order to activate the killing mechanisms, 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. Thus different immunoglobuli ⁇ s 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 bi-specific 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, we have found that although the nature of the heavy chain providing the T-cell binding affinity is of prime importance, the nature of the heavy chain providing the target cell binding affinity is also of importance.
  • certain subclasses of i munoglobulin 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 - n - selection from another species or subclass.
  • the complement activation mechanism 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 target cell binding activity can still exert an important effect as discussed hereinafter.
  • a particular heavy chain species/subclass combination which does not produce killing of T-cells in the mouse or rat may well do so 1n 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 1s the use of two human heavy chains of the same class but of a different subclass.
  • 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 1s 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 1n 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 via an Fc receptor mediated mechanism.
  • 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.
  • ADCC killing of human T-cells is a heavy chain of the IgM class but such heavy chains are often particularly effective at producing killing through complement activation.
  • the preference in the mouse is IgG2b, IgG3 and IgGl > IgG2a and in the rat IgGl and especially IgG2a and IgG2c > IgG2b.
  • the heavy chain of T-cell binding activity is one such as mouse IgG2a or rat IgG2b which will promote killing, through the selection of a suitable form of heavy chain having target cell binding activity, in particular one of a different species or of a different isotype or allotype.
  • rat IgG2a the preferences for inactivating the rat IgG2b heavy chain are a rat IgG2a or especially a rat IgG2c heavy chain.
  • active heavy chains as rat IgG2b or mouse IgG2a a 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.
  • 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. being substantially ineffective), possibly acting at a low level through only one of these two mechanisms as may be the case for example with some mouse IgG isotypes.
  • isotype similar combinations may be used in such instances, the use of a difference of species or isotype will mitigate even the insubstantial level of effectiveness and provides a clear indication of suitability for the heavy chain combination.
  • the achievement of the mitigation of the killing of human 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 human T-cells.
  • the present invention thus also includes a bi-specific antibody molecule having a first affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells, for example target cells present in the human body, characterised in that the two heavy chains in the molecule are selected from different species, isotypes or allotypes to mitigate the killing of human T-cells by the molecule.
  • 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.
  • An alternative approach is to transfect cloned immunoglobulin genes into a hybridoma so that the cloned gene is expressed in the hybrid cell and mixed immunoglobulin molecules produced which can be assayed for activity in T-cell killing by both the ADCC and complement route.
  • bi-specific antibody molecule heavy chain having a T-cell binding affinity will not induce 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.
  • this heavy chain will cause the killing of T-cells via an Fc receptor mediated killing mechanism
  • types 2 and 4 will be toxic via this route irrespective of the nature of the target cell-binding heavy chain, types 1 and 5 only being prevented from also being toxic by the presence of an appropriate target cell-binding heavy chain which is such as to negate the activity of the other heavy chain.
  • 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-target cell halves of the molecule, types 2 and 4 will always possess the undesirable ability to kill T-cells unless the heavy chain they contain is inherently incapable of inducing Fc receptor mediated killing.
  • the antibody molecules of types 3 and 7 (type 6 usually being inactivated by the anti-T cell heavy chain) will contribute an added mode of target cell - killing to the T-cell mediated toxicity of the type 1 bi-specific antibody molecules (the antibody molecules of types 8, 9 and 10 are inactive as regards both T-cell and target cell binding , in view of their mismatched light and heavy chains).
  • types 3 and 7 contribute added target cell toxicity they may therefore conveniently be retained.
  • the mixture of antibodies containing the bi-specific antibody molecules 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 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/or compete therewith for binding to T-cells and particularly any species which are toxic to T-cells by an Fc mediated receptor mechanism, but not necessarily other species which are toxic to target cells.
  • Affinity chromatography can be exploited using either an antigen-containing column (for example an immunoglobuin) 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 chromatography 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.
  • rat IgG2b heavy chains are effective at inducing the killing of human T-cells by an Fc receptor mediated killing mechanism whilst IgG2a and IgG2c heavy chains are not and, moreover, will act to negate the toxic effect of an IgG2b heavy chain when combined therewith in the same bi-specific antibody molecule.
  • a rat IgG2b anti-T-cell/rat IgG2b anti-target cell bi-specific antibody is thus itself expected to be toxic to human T-cells whilst a rat IgG2b anti-T-cell/rat IgG2a (or 2c) anti-target cell bi-specific antibody is not itself expected to show such toxicity.
  • this latter type of antibody will be obtained in admixture with type 2 and 4 antibodies which will show such toxicity and are therefore preferably removed.
  • a rat IgG2a (or 2c) anti-T-cell/rat IgG2a (or IgG2c) anti-target cell bi-specific antibody should neither be toxic to human T-cells itself nor be obtained in admixture with type 2 and 4 antibodies which show such toxicity so that the advantage of fractionation is only in the removal of other antibodies acting as a diluent to the bi-specific antibody.
  • the fourth type of bi-specific antibody which is rat IgG2a (or 2c) anti-T-cell/rat IgG2b anti-target cell, has a particular advantage over the other types in that the bi-specific antibody should not be toxic to human T-cells itself, nor should the type 2 and 4 antibodies, but the type 3 and 7 antibodies should contribute an added target cell toxicity through Fc receptor mediated killing mechanisms (type 6 will be inactivated against target cells by the IgG2a (or 2c) anti-T cell heavy chain) and no fractionation is required unless this is to remove inactive diluent antibodies.
  • IgG2b/IgG2b ⁇ IgG2b/IgG2a (or 2c) ⁇ IgG2a (or 2c)/IgG2a (or 2c) ⁇ IgG2a (or 2c)/IgG2b.
  • 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 but should provide molecules of types 3 and 7 which contribute target cell toxicity.
  • 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.
  • the bi-specific antibody may generally be used in the form of a fragment retaining the binding affinities of the whole molecule, particularly a F(ab')2 fragment.
  • F(ab')2 portions of the antibody molecule can have certain advantages it also has disadvantages so that, being smaller, their half life in serum is shorter and, in particular, the antibody molecules of types 3 and 7 will not contribute the Fc-mediated toxicity discussed above in the F(ab')2 form.
  • 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 an antibody molecule having a first binding affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, 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.
  • fractionation of the different antibody molecules of types 1 to 10 as discussed hereinbefore is still of value where the F(ab')2 fragment is used in order to remove other species acting as a diluent and/or as competitors for binding to T-cells.
  • the fractionation may be carried out either before, or preferably after, formation of the F(ab')2 fragment.
  • the present invention therefore further includes (a) an antibody molecule having a first binding affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, or a fragment thereof having the binding affinities of the whole molecule, for use in surgery, therapy or diagnosis and also (b) the use of an antibody molecule having a first binding affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, or a fragment thereof having the binding affinities of the whole molecule, for the manufacture of a medicament for use in the treatment of neoplastic or other disease.
  • the bi-specific antibody molecules 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.
  • the 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.
  • the bi-specific antibody molecules 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. In general, however, parenteral administration, and particularly intravenous injection, will often be used. As regards dosages of the bi-specific antibody molecule 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 the antibody molecule or fragment 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 the 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 an antibody molecule having a first binding affinity for a human T-cell receptor capable of activating killing and a second binding affinity for target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, or a fragment thereof having the binding affinities of the whole molecule, in the treatment of neoplastic or other disease.
  • the present invention includes a method for aiding the regression and palliation of neoplastic or other disease which comprises administering to a patient in need thereof an amount therapeutically effective in achieving such regression and palliation of an antibody molecule having a first binding affinity for human T-cells and a second binding affinity for tumour or other target cells characterised in that the two heavy chains in the molecule are selected to mitigate the killing of human T-cells by the molecule, or a fragment thereof having the binding affinities of the whole molecule.
  • the bi-specific antibodies may be used 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.
  • the present invention is illustrated by the following Examples. Exampl e 1
  • the Lou rat myeloma cell line Y3-Ag 1.2.3 (CNCM, 1-078) is fused with spleen cells from a DA rat immunised with human lymphocytes according to the procedure described by Clark and Waldmann, Methods in Hematology, 1986, ]_3, 1-20, and the fusion mixture worked up as described therein selecting for hybridomas producing monoclonal antibodies having specificity for the human CD3 antigen by reactivity with all human peripheral T-cells, cross inhibition with mouse monoclonal antibodies such as UCHT-1 (Burnset et al_, Journal of Immunology, 1982, 1_29_, 1451) and 0KT-3 (US Patent 4361549), and immunoprecipitatio ⁇ .
  • the selected hybridoma producing an antibody of such specificity 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. Selection is made for 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, 1_21, 548-556).
  • the variant selected is cloned on semi-solid agar and then maintained in culture in Iscoves modification of Oulbecco'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.
  • IMDM Oulbecco's medium
  • FCS foetal calf serum
  • bicarbonate is replaced by extra HEPES buffer and NaCl to maintain the ionic strength.
  • the Lou rat myeloma cell line Y3-Ag 1.2.3 (CNCM, 1-078) is fused with spleen cells from a DA rat immunised with the mouse Thy-1 antigen according to the procedure described by Cobbold et aj., Molecular Biology and Medicine, 1983, 1, 285-304, selecting for hybridomas producing monoclonal antibodies having specificity for the Thy-1 antigen as described therein using an assay based directly upon binding to this antigen.
  • the selected hybridoma producing a monoclonal antibody having specificity for the mouse Thy-1 antigen is then selected further for a variant of this hybridoma which is HPRT negative by effecting culture in increasing concentrations of medium containing the selective drug 6-thioguanine (Clark and Waldmann, ibid).
  • the selected HPRT" variant is cloned and cultured as for the hybridoma (1).
  • the cells of the HPRT+ * hybridoma (1) Prior to fusion the cells of the HPRT+ * hybridoma (1) (5 x l ⁇ 6) are washed into phosphate buffered saline (PBS) by centrifugation at 200 x g and are resuspended in 10 ml PBS containing 5 mM iodoacetamide. The cells are incubated on ice for 30 minutes and are then washed into HEPES buffered IMDM.
  • PBS phosphate buffered saline
  • iodoaceta ide-poisoned cells are mixed with cells of the hybridoma (2) (5 x 10 7 ), the cell mixture is washed once with HEPES buffered IM0M and the mixed cells are then 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 washed once with HEPES buffered IMDM and are resuspended in bicarbonate buffered IMDM containing 5% v/v foetal calf serum, then being plated out into 48 x 2 ml culture wells and cultured at 37°C under 5% CO . On the following day a control containing iodoacetamide-treated, but unfused, cells is
  • the hybridoma (1) is 6-thio-guanine resistant and the hybridoma (2) is poisoned with iodoacetamide in step (3) (see Example 5), in a second variant 8-azaguani ⁇ e is used instead of 6-thioguanine 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. typically totally non-viable, whilst in the cultures of the fused cells the majority are observed to be viable.
  • HAT selective medium Clark and Waldmann, ibid the HPRT " hybridoma (2) cells being non-viable in this medium.
  • Culture in the HAT selective medium is continued for 2 or more weeks and the following assays are then employed to screen for hybrid cell lines producing bi-specific antibody.
  • a series of mouse monoclonal antibodies specific for rat immunoglobulin chains is employed as isotyping reagents in a rapid red-cell linked haemagglutinin assay (Clark, ibid) .
  • MAR 18.5 (Lanier et al_, Hybridoma, 1982, 1, 125-131), which is specific for all rat kappa light chains, RG11/15.5 (Springer et a1_, Hybridoma, 1982, 1, 257-273). which is specific for rat kappa lb allotype light chains, N0RIG1.1.6 and NORIG7.16.2 (Hale et al_, Journal of Immunology, 1985, 134, 3056), which are specific for the rat IgG2b isotype, and N0RIG31.12.14 (Hale et al_, ibid), which is specific for the rat IgG2c isotype.
  • the same reagents are also used 1n sandwich enzyme-linked immunoassays as follows.
  • a relevant anti-isotype monoclonal antibody is used to coat plastic microtitration plates by incubating a serum free culture supernatant at 100 ⁇ l per well overnight at 4°C. Unbound antibody is then washed off with PBS and the wells are filled with blocking buffer (PBS plus 1% w/v bovine serum albumen and 0.1% w/v sodium azide) and the coated plates are stored at 4°C until needed. 100 yl of the supernatants to be tested are incubated in the wells for 1 hour at room temperature and unbound antibody is washed away with PBS containing 0.1% w/v bovine serum albumen.
  • Bound antibody is then detected using a second anti-isotype monoclonal labelled with biotinyl succini ide ester followed by subsequent layers of streptavidin-peroxidase (Amersham) and finally the enzyme substrate ortho-phenylenediamine, the colour change of the reaction being determined at 492 ⁇ m.
  • Cells from wells which are positive in both 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 bi-specific hybridoma produced in Example 1 is maintained in low serum culture (IMDM containing 1% v/v FCS) for 48 hours at 37°C using 5% CO2 in air and 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 desalted into 50 mM malonate buffer at pH 5.5 on Sephadex G25. This preparation is then filtered through a 0.2 micron filter and is subjected to FPLC (Pharmacia) ion-exchange chromatography on a Mono S column (Pharmacia).
  • Bound protein is eluted using a linear salt gradient of 0 to 1 M sodium chloride, antibody- containing peaks being determined using immunoglobulin isotype specific assays of the type described in Example 1 and the pooled fractions of breakthrough and bound/eluted material are each desalted into PBS by gel filtration through Sephadex G25 and are stored at 4°C until required.
  • the anti-Thy-1 hybridoma (2) is replaced by a hybridoma specific for a target cell present in the human body, particularly a tumour cell.
  • one or both of the hybridomas (1) and (2) are replaced by a hybridoma derived from a human myeloma, for example a myeloma 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 (l)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 YBM 29.2.1 (Cobbold et a]_, ibid).
  • YTH 12.5.14 produces a rat IgG2b monoclonal antibody and expresses a myeloma-derived kappa light chain of the la allotype.
  • YBM 29.2.1 is an HL hybridoma which is a myeloma chain loss variant of YBM 29.2. It produces a rat IgG2c monoclonal antibody and expresses a kappa-lb allotype light chain.
  • the 6-thioguanine resistant variant was YBM 29.2.1TG6.
  • the fusion mixture was screened using the appropriate isotyping reagents as listed in Example 1.
  • Example 1 A procedure similar to that of Example 1(3) was followed using as hybridoma (1) the hybridoma YTH 12.5.14.2TG101 and as hybridoma (2) the anti-human CD!9 hybridoma 8EB1BU12. In this case, however, it is the hybridoma (1) which is HPRT" and the hybridoma (2) which is poisoned with iodoacetamide.
  • YTH 12.5.14.2TG101 is a 6-thioguanine resistant variant of the myeloma chain loss variant YTH 12.5.14.2 which is described in Example 3.
  • SEB1BU12 produces a mouse IgGl antibody which, rather than being specific for the mouse Thy-1 antigen as is the hybridoma (2) used in the main procedure of Example 1, is instead specific for the human CD19 antigen, this being a human B cell differentiation antigen expressed on both normal B cells and many malignant B-cells (the source of the 8EB1BU12 hybridoma was Ling and McLennan of Birmingham University, England).
  • the wells showing growth were selected by the detection of CD3 binding to human T-cells and of CD19 binding to human B-cells using fluorescein isothiocyanate (FITC) labelled anti-rat mouse immunoglobulin (Hudson and Hay, "Practical Immunology", Blackwell Scientific Publications, 1976, page 11). On the basis of this selection, well number 1 was cloned and then re-cloned on semi-solid agar and the clone SHR 1.6.1 selected for use.
  • FITC fluorescein isothiocyanate
  • Example 5 Low serum culture super ⁇ atants from the hybridoma SHR 1.6.1 of Example 5 were produced and processed as described in Example 2 but, instead of using a Mono-S column for purification, the filtered preparation was subjected to ion exchange chromatography on a TSK-SPW(LKB) column under HPLC with a pH 5.8, 50 mM malonate buffer and a 0 to 1 M sodium chloride gradient. The various peaks were dialysed into phosphate buffered saline (PBS) to yield the following concentrations of material after dialysis:
  • PBS phosphate buffered saline
  • Tubes 10,11 and 12 pooled 2 1.43
  • cytotoxic effector cells 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 plated out at 5 x 10° cells per ml in the same culture medium containing 100 ⁇ g/ml of a mitogenic rat IgG2c monoclonal antibody such as YTH 361.1. After 3 to 5 days in culture the cell blasts were washed and used in assays for cytotoxicity.
  • the potency of these effectors in mediating cytotoxicity could be demonstrated by using the anti-CD3 secreting hybridoma YTH 12.5.14.2 described in Example 3 as a target cell line in 5 ⁇ Cr release assays.
  • 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.
  • the ADCC effectors were inhibited by preincubating the washed effector cells with 1 ⁇ g/ml of the anti-Fc receptor antibody CLB-Fcr gran I (Tetteroo et a]., Leucocyte Typing II, 1985, Volume 3, page 27 - Springer Verlanger, New York) for 15 minutes at room temperature. The antibody was not washed away prior to plating the cells out in the assay.
  • the mouse Thy-1 positive thymoma cell line EL-4 (A.T.C.C. reference number TIB40) 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° cells were then spun down at 200 x g and the pellet resuspended in 200 ⁇ l IMDM containing 150 ⁇ Ci 51 Cr-sodium chromate. The cells were incubated for 37°C for 45 minutes and were washed into HEPES buffered IMDM. Suitable dilutions of the antibodies to be tested for effector cell retargeting were prepared in 100 ⁇ l volumes in round bottomed micro-titre wells.
  • the radiolabelled EL-4 targets were then added at a level of 10 4 cells per well in a volume of 50 ⁇ l of HEPES buffered IMOM containing 5% v/v heat inactivated FCS and washed cytotoxic effector cell blasts prepared as under (1) were added at a suitable effector to target ratio in a volume of 50 ⁇ l of HEPES buffered IMDM containing 5% v/v heat inactivated FCS.
  • the cell mixture was incubated at 37°C for 4 hours and 100 ⁇ l of supernatant was harvested to determine the released radioactivity (all of the assays being carried out in replicates of 3 or 4) by measurement of gamma radiation using a Phillips gamma counter model PW 4800.
  • Example 4 Supernatants from cells in the stationary phase of the cultures prepared as described in Example 2 were utilised for the study of the YTH 12.5.14.2 and YBM 29.2.1 antibodies, and 1:1 mixtures thereof, whilst the bound/eluted preparation of Example 4 was utilised for the SHN 20.12 antibodies.
  • the starting concentration of antibody was estimated from the antibody isotyping assays to lie in the range of 0.01 to 0.1 mg/ml.
  • the antibodies were assayed for their ability to effect the killing of EL-4 targets through human blasts as described in (2) above (the antibody YTH 361.1 having been used in the procedure of (1) above).
  • the YBM 29.2.1 antibody does not mediate the killing of target cells in the presence of the effector cells but that the YTH 12.5.14.2 antibody does show a small but significant level of mediation which is probably due to a bystander effect of T-cell activation by this antibody (rat IgG2b isotype antibodies such as YTH 12.5.14.2 are very effective in ADCC and the human blast cells contain 10-15% Fc receptor positive cells).
  • the action of the YTH 12.5.14.2/YBM 29.2.1 mixture will be seen to be little different from that of YTH 12.5.14.2 alone.
  • the bi-specific antibody SHN 20.12 derived therefrom shows very efficient killing of the targets.
  • the hybrid supernatant therefore shows a much improved level of killing to the mixture of the parental antibodies despite the fact that the mixture of antibodies which is present together with the bi-specific antibodies includes combinations which would be expected to compete for binding to target or effector cells.
  • the effect of titrating effector to target ratio at a constant antibody concentration (1:100 v/v dilution of supernatant) is shown in Figure 3 (the designations being similar to Figure 1).
  • the potency of the SHN 20.12 antibodies at directing target cell killing can be seen to be very high, effective killing being achieved at relatively low effector to target ratios and at very low antibody concentrations.
  • rat IgG2b/IgG2b antibodies are used for comparative purposes only and, not showing mitigation of T-cell toxicity, do not fall within the scope of the invention.
  • YTH 12.5.14 provides a myeloma chain loss variant YTH 12.5.14.2 which is again described under Example 3.
  • YTS 154.7.7 is a myeloma light chain loss variant of the parent hybridoma YTS 154.7 which produces a rat IgG2b monoclonal antibody and, like the hybridoma YBM 29.2.1 of Example 3, expresses a kappa-lb allotype light chain.
  • the 6-thioguanine resistant variant of YTS 154.7.7 was YTS 154.7.7.TG9.40.
  • the fusion mixture was screened using the appropriate isotyping reagents as listed in Example 1.
  • Human effector cells were prepared as described in Example 7(1) and were labelled with 51 Cr as described for the mouse target cells in Example 7(2). Dilutions of the purified antibody fractions Into HEPES buffered IMDM were made in volumes of 100 ⁇ l in micro-titre wells. Labelled cells were added at 10 4 cells per well in a volume of 50 ⁇ l and finally 50 ⁇ l of autologous human serum (from the original donor of the mononuclear effector cells) was added as a complement source. The plates were incubated at 37°C for 1 hour and 100 ⁇ l of supernatant was harvested to determine released radioactivity.
  • Example 7 A variation of the effector cell retargeting assay of Example 7 was performed in which some (20%) of the human T-cell blast population was labeled with radioactive chromium as described for the mouse target cells of Example 7(2), and these cells were then added to dilutions of the antibodies to be assayed. Finally, unlabelled human blast effector cells were added so that the final cell numbers per well were as for the effector cell retargeting assays described above and the cultures were incubated for 4 hours at 37°C before harvesting the supernatant to determine released radioactivity (the effector cells were not treated with CLB-FcR gran I in this procedure) .
  • FIG 5a It will be seen that the antibodies caused lysis of the human effector cells at concentrations in the same range as those which caused mouse target cell lysis (compare with Figure 4). However, it should be noted that for the same antibody concentrations the YTH 12.5.14.2 parental antibody shows a titre (3 x 10 -7 mg/ml, estimated as the concentration giving 15% specific cytotoxicity) which is about 30 fold greater than the breakthrough and bound/eluted fractions of the bi-valent antibody SHM 15.3 (compare with 10"5 mg/ml value) and about 10 fold greater than the breakthrough fraction and 100 fold greater than the bound/eluted fraction of the bi-valent antibody SHN 20.12 (compare with 3 x lO -6 and 2 x 10"5 mg/ml values, respectively).
  • the antibody YTH 12.5 shows a marked prozone over the range tested. It will be seen that of the four SHN 20.12 and SHM 15.3 preparations the lowest level of lysis is observed for the SHN 20.12 bound/eluted fraction which is also the fraction giving the highest cytotoxic titre against the mouse target cells. (It 1s believed that the cytotoxicity observed in both (a) and (b) for the breakthrough and bound/eluted fractions of SHN 20.12 1s attributable to contaminants as illustrated in Figure 1, rather than to the b1-spec1f1c antibody molecules.)
  • Example 7(2) but replacing the mouse Thy-1 positive thymoma cell line EL-4 by a human CD19, HLA-class II positive cell line HWLCL produced by the transformation of human B-cells with Epstein-Barr virus.
  • the human effector cells were used in a ratio to the HWLCL target cells of 10:1 and the antibodies for test were used in a range of dilutions from 1:10 to 1:10° for culture supernatants and 1:10 2 to 1:10 7 for column fractions.
  • the following antibody preparations were assayed: supernatants from cells in the stationary phase of cultures prepared as described in Example 2 from (a) the parental hybridomas YTH 12.5.14.2 and 8EB1BU12 (a 1:1 v/v mixture of the supernatants), and (b) the hybridoma SHR 1.6.1 of Example 5; and column fractions produced by the purification of SHR 1.6.1 as described in Example 6 corresponding to (c) tubes 2-6 (breakthrough), (d) tube 9, (e) tubes 10-12, (f) tube 13, and (g) tube 14.
  • Example 8(4)(a) The assay procedure was based on that described in Example 8(4)(a) using the column fractions (d), (e) and (f) as described in (1) above, (h) supernatant from cells in the stationary phase prepared as described in Example 2 for the hybridoma SHL 45.6.1 referred to in Example 8(4)(a) and (i) a monovalent preparation from the hybridoma SHL 45.6.1 also referred to in Example 8(4)(a).
  • each of the preparations was diluted on a 1:3 v/v basis six times commencing with the following concentrations of antibody 1n the first wells which are diluted 1:3 v/v in the first dilution for the first determination: (d) 10.0 ⁇ g, (e) 23.8 ⁇ g, (f) 17.8 ⁇ g, (h) 16.7 ⁇ g and (i) 13.2 ⁇ g.
  • the results obtained are shown in Figure 7 from which it will be seen that the lowest level of complement mediated lysis results from preparation (d)(fraction 9). It is noteworthy that preparation (d) also gives the highest level of ECR among the fractions.
  • the results obtained indicate that the mouse IgGl heavy chain negates the toxicity of the rat IgG2b heavy chain.
  • Monospecific a ⁇ ti-CD3 rat IgG2b/rat IgG2b antibodies will however be markedly toxic and it is likely that preparation (d)(fraction 9) contains substantially pure bi-specific antibody but in preparation (e)(fractions 10-12) the bi-specific antibody 1s contaminated with monospecific anti-CD3 antibody thereby explaining the substantially increased T-cell toxicity of this preparation.

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Abstract

Des molécules d'anticorps bispécifiques, présentant une première affinité de liaison avec un récepteur de leucocytes T humains capables d'activer la destruction et une seconde affinité de liaison avec des cellules cibles, se caractérisent par le fait que les deux chaînes lourdes de la molécules sont sélectionnées de façon à atténuer la destruction de leucocytes T humains par la molécule. Lesdites molécules cytotoxiques et leurs fragments qui conservent les affinités de liaison de toute la molécule sont utiles dans le traitement de certaines maladies, telles que notamment les affections néoplastiques, virales et parasitiques.
PCT/GB1987/000781 1986-11-05 1987-11-04 Anticorps WO1988003565A1 (fr)

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JPH01501200A (ja) 1989-04-27
GB8626412D0 (en) 1986-12-03
GB8725811D0 (en) 1987-12-09
AU616871B2 (en) 1991-11-14
GB2197322A (en) 1988-05-18

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