JPH01501201A - antibody - Google Patents

Abstract

(57) [Abstract] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] birth-birth This invention provides a novel type of antibody and targeted cytotoxic agents in the treatment of ic disease The present invention relates to the use of the antibody as a toxic agent.

Although numerous cytotoxic agents have been developed for the treatment of neoplastic diseases, However, difficult problems remain in the treatment of this type of disease. One recent app As a roach, 27 ector cell retargeting phenomenon [pheno menon or effector cellreLargeting (E Attempts have been made to utilize the CR). In this approach, anti-T cells and bispecific antibodies with antitumor antigen activity are formed. First application of this technology Examples include [Staerz et al., Nature ), 1985, vol. 314, p. 628: Perez et al. 1985, vol. 316, p. 354: Liu et al., Proceedings. National Academy of Sciences of the National Academy of Sciences Ninis-1--(Proceedings or the National Academy of 5 sciences of the U.S.A), 19 85, p. 8648], the bivalent antibody Funshugate was formed by chemical methods. However, in subsequent applications of the technology [Steltz and Bevan ( B. Evan), Proceedings of the National Academy of Sciences The Sciences of the Ninisny, 1986, Volume 83, No. 1 453 pages and Immunology Today ), 1986, Vol. 7, p. 241], double singularity using the hyperdoma technique. sexual antibody molecules are produced. Bispecific antibodies target tumor cells or another by binding to target cells (e.g., virus-infected cells) and T cells. effect, whereby the action of the latter brings about the destruction of the former.

However, effector cell retargeting as described in the above-mentioned literature etc. Exploitation of phenomena comes with various inherent disadvantages. In particular, bispecific antibodies are generally The natural killing mechanism of cells in the body chanism), which utilizes only the mechanism that kills tumor cells to some extent. However, it is an object of this invention to We present an improved method to destroy tumor cells by utilizing the cells' natural killing mechanism. It is to provide.

The invention therefore includes a first binding affinity for T cell receptors that can activate lethal mechanisms. dual binding affinity and a second binding affinity for immunoglobulins. A specific antibody molecule and its seven lag molecules that possess the binding affinity of the antibody molecule. Contains

Bispecific antibodies according to the invention are bispecific, with two light chains and two heavy chains. have a normal form of the antibody molecule present. Therefore, the bispecific antibody according to the present invention The body molecule was published in 1985; and the bispecific antibody molecules described in the paper by Liu et al. 1985 The bispecific antibodies described in these papers, published in Funschgate formed by chemical cross-linking of four types of light chains and four types of Has the stalwarts of species. Published in 1986; described in the paper by Stelz and Bevan. Bispecific antibodies are composed of unconjugated antibody molecules; The binding affinity of the child is different from the binding affinity of the antibody molecule according to the invention. Of course, the authors also discuss the use of these molecules in their killing of T cells. are not aware of the inherent problems in using The present invention further provides that It will be aimed at solving this kind of problem that did not exist before. Published in 1985, Liu et al. The paper includes binding affinity for human T3 complex and human B-lymphoma. has a second binding affinity for the idiotype of surface immunoglobulins. Antibody conjugates have been described. This type of idiotype is a true tumor. To target antibody conjugates to tumor cells in response to tumor-specific transplantation antigens. used. Therefore, the antibody conjugates described in the paper by Liu et al. has anti-T cell and anti-tumor cell affinity, and its conjugate Apart from the properties, the bispecific antibodies according to the invention, i.e. anti-tumor cells or other Acts on T cells by binding to another antibody that has target cell affinity It is distinguished from bispecific antibodies that

In the primary use of these antibodies, the bispecific antibody molecules described above target cells. is administered with another antibody that has binding affinity for. People like this The method can activate the body's various cell killing mechanisms. First, the target Antibodies with binding 1 affinity for cells bind to target cells and bind to Fc receptors. mediated lethality mechanisms, e.g. by antibody-dependent cells including neutrophils and macrophages. Antibody-clependen tcell-mediated cyLotoxicity)], macrophages and kill target cells by phagocytosis or complement activation by cells of the reticuloendothelial system. let Second, the resulting target cell/antibody system is Further binding by affinity to the bispecific antibody system, the entire system It binds to T cells through the T cell affinity of the isomeric antibody system and takes advantage of T cell toxicity. to destroy target cells.

The bispecific antibody molecules according to the invention act by binding to T cells and thereby However, the T cell binding affinity of the antibody molecule may also cause lethal effects. ; may be specific for any T cell receptor. Therefore, like this receptors, either directly or with the aid of Ell type cells or other mediators. They may also have T cell functions that indirectly bring about lethal effects. Also, acceptance The body uses the function of cytotoxic Tm cells or Bm cells to directly cause lethal effects. the function of helper T cells, either natural or artificial, that assist in the lethal effect; may have the function of producing a lethal effect by another indirect mode of stomach. Of particular interest are receptors that can activate direct lethality; It is associated with both direct and indirect lethal modes.

In particular, the binding affinity involves the T cell antigen-specific receptor (Ti) and is highly It may be directed to one or both of the α chain and β chain present on many T cells. , or the entire CD3 unit bound to the receptor (73 above) or the unit may be directed to one of the individual chains contained in the set. using human cells I; Studies have shown that T cell receptors are a (Mr: approximately 50,000) and β (M 40,000) exists as a complex of two chains identified as and these chains are somatically expressed in a manner similar to that of immunoglobulin genes. The genetic code is specified by the genetically rearranged genes.

Therefore, each T cell contains genes that specify the genetic code for these two chains. It involves a unique rearrangement. These two chains are linked to at least three chains, including the CD3 complex. chain of ff1f, that is, γ (Mr: about 25,000), δ (Mr: about 20 ．． 000) and c (Mr: approximately 20,000). was found.

Antibodies with T cell binding affinity for known or novel receptors are , may be identified by an assay method developed by the present inventors. most hybrids The cells contain small amounts of cell surface antibodies as well as secreted antibodies. For example, rat IgG2b Mouse hybridomas that produce antibodies against are present on the surface of the rat Trapping rat IgG2b antibodies with small amounts of antibodies against IgG2b be able to. Label hybridoma cells with a radioactive label, e.g. Poppy, then a mixture of T cells and monoclonal rat IgG2b for T cells. When cultured with the compound, hybridoma cells bind to the antibody, which in turn binds to T cells. binding, thereby potentially killing the hybridoma; as a result, radioactivity is released. Therefore, such methods require adequate T cell binding capacity (i.e., killing). provides a means for detecting rat IgG2b antibodies having the ability to induce IgG2b antibodies. suitable Therefore, selecting hybridomas with a specific morphology s) and class or subclass of antibodies ( screen). detected by such assays. Hybridomas that produce antibodies can be produced using the method described below! From this, the present invention may be used when preparing bispecific antibodies.

Anti-non-human cell/anti-immunoglobulin bispecific antibody molecules are of particular research relevance. Cell killing using animal tumors, such as rat and mouse tumors, is important in It is important as a model system when investigating important requirements for But long An important primary field of application of the present invention is the field of human medicine. Therefore, human T-cells Bispecific antibodies with a first binding affinity directed toward cells are of particular interest; Most commonly, antibodies such as used in combination with another antibody that has the same Use assay methods as described above By this, monoclonal antibodies having the ability to bind to human T cells are produced. It is possible to select hybridomas that can induce T cell cytotoxicity. be. Such antibodies include hybridoma YTH1 described in practical example 2. Rat I produced by 2, 5, 14, 2 and all related subclones An example is gG2b antibody. This antibody binds to T cell anti-lCD3, but in a different way. Other antibodies with specificity may be selected in a similar manner. Other antibodies include The following are exemplified = hybridomas YTH655(5)6 and YTH61 Anti-human CD2 rat IgG2b antibody produced by 6,7,10 [Thailand] E(H,P.

Tighe's doctoral thesis [Models for cell surface antigens that affect leukocyte function] "Noclonal Antibodies", University of Cambridge (1987)], Example 3. Anti-human CD3 mouse IgG2b anti produced by hybridoma 0KT3 A group of anti-CD3 mice listed by Karl et al. gG2b antigen [Leukocyte Typing ■, Volume 1, Human T lymphocytes, Reinhertz ( Reinherz et al., eds., Springer Verlag. , 1985, p. 137] and [Human Leukocyte Differentiation Antigen, Leukocyte Typing ■ Proceedings of the 3rd International Research Meeting and Conference on Leukocyte Differentiation Antigens and Leukocyte Differentiation Antigens [MacMy Edited by McM. Chael, Oxford University Press, 1987] The various antibodies described in and certain anti-CD2 antibodies. All these antibody-producing hybridomas are also Of course, in the form of derivatives, reclone or subclone c1one) or a similar high one with similar specificity. Bridema may also be used.

The second binding affinity of the bispecific antibody according to the invention is that the antibody immunoglobulin may be directed to any of the various binding sites present in the protein, e.g. Chiarotypes, antiisotypes (especially subclasses), antispecies and Antiidiotypes may also be used, although their specificity and specificity are somewhat inferior. (“I The term "sotype" is used in the field to refer to a particular class and/or subtype. It is used to specify the class, so in the case of rats, for example IgM, IgG 1. IgG2a, IgG2b and IgG2c each constitute a separate isotype. Ru. In this case, anti-isotype binding affinity is most commonly For example, anti-1gG1 is more effective than anti-IgG, but in the case of IgM, As such, when there are no subclasses, the affinity is particularly It is possible to ) This type of binding affinity for immunoglobulins In addition to affinity, the immunoglobulin affinity of bispecific antibody molecules is , 7 fragments of the antibody molecule, e.g. 7 fragments of an anti-target cell antibody as described below. Substances in immunoglobulin brought about by the production of fragment F(ab')2, etc. It may also be directed towards the target. Alternatively, the affinity binds to immunoglobulins. to the site artificially formed on immunoglobulin by binding ten. It may be for the other person. Anti-hapten binding-based immunoglobulin The affinity of species for example with fluorescein intiocyanate Labeled immunoglobulin or biotinylated (biotinylat) ed) May be used with immunoglobulin.

Although the specificity of immunoglobulins is wide-ranging, there are certain advantages and disadvantages. main departure The main application of bispecific antibody molecules according to Akira is indirect ECR, in which case Binding of the antibody molecule to a target cell has binding affinity for the target cell. This is done through another antibody molecule. Therefore, the immune system to which the bispecific antibody molecule binds It is also possible to make epidemic globulin such that it provides direct ECR. However, preferably the immunoglobulin is double-specific, without the use of another anti-target cell antibody. By not allowing the isomeric antibody molecules to target cells, especially tumor cells. be. Preferably, the immunoglobulin does not act as a cell surface antigen. , especially those that are not antigens present on the surface of tumor cells. immunoglobulin special The less isomeric type is endogenously synthesized and expressed cell surface immunoglobulin, Particularly specificity for those occurring on the surface of tumor cells. anti-idioty affinity is due to the function of many idiotypes as cell surface antigens and Less specificity in blocking antiidiotypic antibody/antigen binding This is a special case of of specificity, including cell surface immunoglobulins and idiotypes. A particularly inferior example is the human B-ri as described in the above-mentioned paper by Liu et al. The idiotype of surface immunoglobulins in lymphomas that is associated with tumor-specific cell surface antibodies. An example is one that functions as a source.

However, when using anti-target cell antibodies together with bispecific antibodies, the former The latter immunoglobulin affinity when selected within the maximum tolerance range is preferably made as non-specific as possible. However, such a preferable choice The bispecific antibody molecules are used in vivo and the desired provided that harmful side reactions are avoided as much as possible. In practice, bisingularity The desired relationship between antibody molecules and immunoglobulin molecules naturally present in the patient's body The easiest way to avoid a non-responsive response has to do with T cell receptor binding affinity. relating immunoglobulin binding affinity to species other than the It is. Generally, the former species is humans and the latter species is other mammalian species, e.g. Bits and especially mice or especially rats. Thus, e.g. bispecific anti- The body is exposed to various classes of antibodies, particularly IgM antibodies and IgM antibodies, as described below. It is convenient to react with G antibodies (including various subclasses of the latter).

Furthermore, binding affinities are not specific for whole species, e.g. It would also be advantageous if binding to both rat and mouse antibodies was possible.

However, under real conditions this requires too many requirements, and rats also or small groups specific to mice and these different subclasses. It is appropriate to use anti-immunoglobulin antibodies.

The important thing about anti-immunoglobulin specificity is that it does not lead to autoreactivity. That's what it means. For example, the bispecific antibody molecule itself interacts with rat IgG2b heavy chain. If the protein is composed of rat kappa light chain, the anti-immunoglobulin specificity indicates that the anti-rat I gG2b and anti-rat kappa should be excluded. The two described in Example 2 below. The heavy specific antibody molecule is the antibody YTHI 2.5.14.2 (specific for CD3 antigen). rat IgG2b with typical lambda light chain) and antibody RGl/15.5 ( Mouse Ig with kappa light chain specific for rat kappa Ib allotype It is a hybrid with G2a). This combination does not cause self-reactivity. tion with any of the rat antibodies with the rat kappa Ib allotype light chain. Can be used together.

Regarding anti-target cell antibodies used in combination with bispecific antibodies according to the invention, these has binding affinity for any antigen present on the surface of the target cell. You can leave it there. The target cell may be any cell advantageously harvested from the body. good. Examples of such cells include virally infected cells (the virus itself are not normally attacked by T cells). Various types of infectious viruses Viruses, such as influenza and rabies viruses, parasitic cells and parasites The parasite itself is the cause of malaria, leprosy, trypanosomiasis, and schistosomiasis, etc. Examples include parasites such as tapeworms, tapeworms, and other parasites such as tapeworms. But long , the preferred target cells are tumor cells, and the anti-target cell antibodies are directed against any tumor-related It also has binding affinity for antigens. The ideal situation is that tumor-specific anti- antigens, i.e. antigens that are present only on tumor cells and not on normal cells. It is to have affinity. Used in the treatment of cellular malignancies such as BCLL The B-cell 1g idiotype currently available is one of the few existing antigens of this type. In fact, tumor-related antigens also exist on normal cells. There will be. Preferably, the antigen is present at a higher concentration or It is aberrantly expressed in appropriate conditions, thereby increasing the level of antibody-antigen response using tumor cells. Although the bell is raised, this is not entirely necessary. for example, Even in extreme cases where there is no differentiation between tumor MMi cells and normal IrnMjJ cells, , the toxic effects on normal cells associated with the treatment, the use of bone marrow transplants, or the 31! ,R The patient's own bone marrow is harvested prior to in vitro processing to remove tumor cells. After attaching, it is possible to antagonize the return to the original state. As a particularly important anti-tumor antibody Examples include: International Conference on Human Leukocyte Differentiation Antigens (Paris, (1980; Boston, 1983; Oxford, 1986) of the hematopoietic system recognized by a group of standardized and characterized monoclonal antibodies. Antibodies to antigens that define clusters of differentiation, and systemic acute lymphoblastic leukemia disease-associated antigen (GALL), carcinoembryonic antigen expressed in human colon carcinoma and human An antibody against ganglioside GD3, which is associated with human melanoma. What is special is human B-cell differentiation antigen CD19, which is present in both normal and many malignant B-cells. It is expressed on cells and B cell lines.

Antigenic cellular antibodies may be prepared as polyclonal preparations by traditional techniques. However, hybridoma technology, such as U.S. Pat. No. 4,172,124 and and using hybridoma techniques described in the extensive literature in the field. Therefore, it is convenient to produce it as a monoclonal antibody.

The bispecific antibody molecules described herein can be produced using traditional or hybridoma techniques. Two antibodies may be prepared by the method, namely, an antibody having a first binding activity and May be prepared by chemical conjugation of each half of the antibody with a second binding activity . However, particularly preferred bispecific antibody molecules according to the invention are monospecific through hybridoma technology modified for the production of bispecific antibodies rather than antibodies. It is directly produced. Such techniques for preparing bispecific antibodies are Bispecific antibodies, generally produced primarily by hybridoma technology European Patent Application No. 0068763 and PCT Application No. WO3 relating to molecules No. 3103679. It is described in each description. Bispecific antibodies and anti-target cells Myeloma starting materials used for the preparation of both antigens include European Patent No. 0O1 Y3-Ag1.2.3 myeloma (C,N) disclosed in No. 459.

C, M, No, I 078), disclosed in European Patent No. 0043718 YB2/3.0. Ag, 20 myeloma, myeloma P3-X63-Ag8 (A, T, C, C, No, CRL 1 597), myeloma NS 1/1-Ag4 1 (A, T, C, C, No, T I B l 8) and myeloma P3 [Stelz et al. , Journal of Immunology ogy), 1985, Vol. 134, pp. 3994-4000], and -Ropper Patent Application No. 0062409, No. 0148644, British Patent No. 20 86937 and U.S. Patent No. 4,529,694. Examples include various human myeloma. Each hybridoma has bone marrow that does not express light chains. Myeloma or myeloma light chain loss mutant, i.e. derived from HL rather than HLK. preferable. The technology used to prepare bispecific antibodies according to the invention is described in a UK patent application It is very similar to the technology described in No. 2144147A, and the latter Although the binding affinities of the fusion partners are different, it is applied in the case of the present invention. Good too. However, the inventors have developed a particularly useful variation of these techniques. , in the method described above for the preparation of bispecific antibody molecules, two hives are In the case of the present invention, +j, the process of fusion of two hybridomas is included. One of them produces antibodies directed against T cells, the other against immunoglobulins. Produce antibodies. As explained in the Examples, two types of hybridomas are commonly used. Prepare hybridomas that secrete fused and bispecific antibody molecules by the method of The antibody molecules may have specificity for each hybridoma from which they are derived. have One of the most difficult steps in the fusion method to produce hybridomas by selecting the desired type of bispecific hybridoma from the fusion mixture. The method developed by the present inventors includes features particularly in this regard.

The first hybridoma used in this method contains enzymes such as thymidine kinase ( TK) or especially hypoxanthine-guanine phospholiposyl transferer The drug may have a drug selection label that may be deficient in enzyme (HP RT) or the like. child Hybridomas such as 5-bromouracil deoxylipose or 2- Aminopurine (for TK) or 8-azaguanine or especially 6-thioguanine (for HPRT) by selecting cells from a growth medium containing (for HPRT).

Cells selected for growth in media containing the appropriate drug are deficient in the enzyme in question. In both TK and HPRT, hypoxanthine, aminopterin and It cannot grow on a medium containing thymidine (HAT). Because Amino Pterin inhibits the major pathway to purine and pyrimidine synthesis and also inhibits HPRT. Or, due to TK deficiency, the function of normal cells, which is to produce purines, is impaired. The ability to utilize thymidine to produce hyboxanthin and pyridine is lost. This is because

The second hybridoma used in this method is an irreversible biochemical inhibitor, e.g. Subjected to treatment with lethal doses of ethylpyrocarbonate and especially iodoacetamide. and thereby suppress that function.

This class of inhibitors suppresses cell function, but inhibits immunoglobulin expression and HPRT enzymes. It does not damage the cell's DNA, which specifies the genetic code for the target. For such processing The cells are washed to remove excess inhibitor before being used. cell The overall structure of the remains intact for several hours after treatment, with fusion typically occurring at 0. It is done within 5 to 1 hour.

Fusion of two hybridomas preserves DNA from both hybridomas. A fused cell line with A brief suppression of active enzyme function from the cell is induced from the other hybridoma. complemented by the enzyme After the fusion of two hybridomas, the fusion mixture is Cultured in medium containing no inhibitors. In this case, unfused cells that have been subjected to poisoning treatment Hybridoma cells gradually die, but other unfused hybridoma cells and and the fused cells survive. Selection is initiated using, for example, a HAT-containing medium.

In this case, unfused hybridoma cells lacking TK or HPRT die and lyse. Synthetic cells survive, but this is because the enzyme deletion is carried by DNA from other hybridomas. This is because it is brought about by Iodoacetamide causes cell death within 1-24 hours. However, higher levels of hybridization generally delay selection by 2-3 days. , which is sufficient for HPRT expression from poisoned cells to occur. This is probably because it takes some time. Furthermore, the best results are The ratio of treated cells to iodoacetamide treated cells is 1 or more, e.g. 1:1. -10:l.

After removing both types of unfused hybridoma cells, the cell mixture contains A hybrid that secretes a monoclonal antibody with the desired bispecificity by a conventional method. isolate the polymer. Such hybridomas can be harvested in vitro or by conventional methods. Monoclonal antibodies may be produced by culturing in vivo.

The use of bispecific antibodies of the type described herein is per se well known in the art. Although this is an improvement over the use of bispecific antibodies known in the art, the present invention In a preferred embodiment, this can be further improved. bispecific anti The body binds to T cells by the natural Fc receptor-mediated killing mechanism described above, and the T cells It is possible to induce cell death. Stelz and Bevan's paper mentioned above There is no mention of this in the previous 27th edition. This is a major flaw in the vector cell retargeting method. This is because in this method, This is because tumor cells are killed only by T cell toxicity. In the case of the present invention, Although this issue is less serious since other tumor killing mechanisms are involved, The achievable lethal efficiency of Therefore, in a preferred embodiment of the present invention, Can the problem of T cell destruction be alleviated by one of the following two methods? , preferably substantially avoided.

The first method involves the use of a suitable combination of key players in a bispecific antibody molecule according to the invention. It is based on the recognition that this problem can be overcome by using the Immunology The overall structure of Robulin is determined by the interactions of the various globular domains of the individual chains be done. These interactions are due to covalent bonds and disulfide bridges within and across the steel. and non-covalent interactions involving both protein and carbohydrate residues. lethal To activate the mechanism, complement components and Fc receptors must be present in different antibodies. It must be connected to the structural part. However, different species of antibodies, Sotypes and allotypes have differences in their protein sequences. However, there are many similarities in other parts of these sequences. follow Immunoglobulins interact differently with complement components and Fc receptors; Ru. Furthermore, when hybrid antibody molecules are formed, these interactions The two heavyweights differ in the sequence of very important parts, and for this reason Characteristics of different combinations are affected.

The invention therefore includes a first binding affinity for T cell receptors that can activate lethal mechanisms. an antibody having a second binding affinity for an immunoglobulin in A molecule in which two key players act on the body's natural mechanisms that lead to the destruction of Tm cells. Contains antibody molecules selected such that the ability of the antibody molecule to utilize lethal mechanisms is reduced. be caught.

The immunoglobulins that make up antibodies can be classified into several classes, with the main are identified by IgG, IgA, IgM, IgD and IgE. However, among these, IgA in particular and IgG in particular may be further classified into subclasses. . The bivalent antibody molecule according to the invention preferably has two heavyweights of the same class, may be of different subclasses within the same class, and may be the same or may be of different subclasses but relate to different species . Humans, rats and mice are the most important, but other species such as rabbits Other immunoglobulins may also be used. rat and mouse and rat and rat The combination of immunoglobulins is derived from mouse and mouse combinations. Also preferred from the viewpoint of stability of the corresponding hybridoma.

Regarding the Fc receptor-mediated lethal mechanism, particularly ADCC, the present inventors have obtained the following findings.

That is, the ability of the heavy chain to provide T cell binding affinity is fundamentally important; The ability of the heavyweight to provide immunoglobulin binding affinity is also important. First kind of immunity Certain subclasses of epiglobulin have a second class of cell-mediated effector mechanisms. It does not interact with the immune system, and two key species of the same species and subclass are immunoglobulins. When using subclasses that interact when present in the Replace one of the heavyweights with a random selection from another species or subclass It is possible to prevent this interaction by Regarding the complement activation mechanism Therefore, the ability of key players to confer T cell binding activity determines the level of complement activation that occurs. may be a more important factor than in the case of ADCC, but The ability of the heavyweights to provide immunoglobulin binding activity plays an important role, as explained below. I bring you.

Selecting an appropriate combination of key players to utilize in a bispecific antibody molecule according to the invention If selected, the species in which the two heavyweights are related and the species to which the antibody is administered are all heavyweights. We must recognize that this is an important factor. For example, a mouse or Key species and subclass combinations that do not kill T cells in rats are selected as species in relation to the T cell binding affinity of the bispecific antibody. The same applies to humans as well. As previously mentioned, human stalwarts are may be used as at least one of the key players in a bispecific antibody molecule, but more Preferably, two human heavyweights of the same class but different subclasses are used. do. However, human myeloma is difficult to obtain compared to mouse and rat myeloma. In practice, it is difficult to use these animal species as heavy-duty mammal species. Good too.

In humans, the IgG class includes IgG1.1gG2.1gG3 and 1gG4. (The IgA class is divided into rgAl and 1gA2 subclasses.) In the case of mice and rats, only the IgG class is classified into subclasses. In the case of mice, it is classified into IgG1, IgG2a, IgG2b and 1gG3. In the case of rats, IgG1. IgG2a5 IgG2b and IgG2a (subclasses with similar names are not necessarily classified in mice and rats). (It does not have similar characteristics). In practice, each heavyweight is usually a mouse. or rat IgA or IgE, especially IgM, especially 1gG, most IgG subclasses in general, specifically mouse IgG1. IgG2a or Ig G2b (1gG3 is less common) or rat IgG1. IgG2a The most common is IgG2b. (IgG2a is less common).

The simplest way to select heavyweights with T cell binding activity is through Fc receptor-mediated mechanisms. does not lead to T cell death in problematic species, such as rats or mice. By using classes or subclasses. In particular, to avoid ADCC lethality. A simple method is to use 1gM class! I chain is used, but this kind of M chain can be extremely effective in causing lethality through complement activation. active and one From a general availability standpoint, stalwarts of the IgG subclass are usually selected. Complement activity In mice, the priority to avoid lethality due to sexualization is IgG1>1gG3. >IgG2a=IgG2b, and in the case of rats, IgG1>IgG2a)Ig G2a>IgG2b. Other Fc receptor-mediated lethal mechanisms, especially A The priority for avoiding lethality due to DCC is IgG2b% 1g in the case of mice. G3 and IgG1 > IgG2a, and in the case of rats, IgG1, especially IgG 2a and rgG2C>IgG2b. However, the conjugate ( There is a close relationship between the two key players in the bispecific antibody molecule, rather than a conjugate. A heavyweight with T-cell avidity that interacts closely with mouse I to promote lethality. gG2a or rat IgG2b, but with immunoglobulin binding suitable forms of the active compound, especially different species or different isotypes. Or, by selecting allotypes, this lethality can be prevented. This knowledge was obtained. Among other rat IgG subclasses, rat IgG2b Preferred heavy chain inactivators are rat Ig (y2a or especially rat IgG 2a heavy chain. Active heavyweights like rat IgG2b or mouse IgG2a When using a combination of species (e.g. rat IgG2b/mouse Ig G) or combinations with other forms of heavyweights based on subclasses are exemplified. However, this is a rat IgG2b/rat IgG2b or mouse IgG2a/ma The mouse IgG2a combination is generally activated by the ADCC and complement activation mechanisms. This is because it is thought to be effective in promoting lethality. different allotypes or especially the use of different species or isotypes, may be harmful to anti-T cell heavyweights. If it shows only a weak level of effectiveness in causing death, i.e. it is not effectively effective. It is also effective when there is no fruit, for example in the case of mouse IgG isotype. , are effective at low levels by only one of two mechanisms. like this Similar combinations of isotypes may be used in some cases, but different Using different species or isotypes may reduce efficacy even at low levels. As a result, a suitable heavy chain combination can be obtained. In general, T cell lethality is low. The extent to which a heavy agent with affinity for target cells has an effect on T cells The same stalwart (species, isotype and preferably allotype) as the stalwart with affinity may be evaluated by comparison with bispecific antibody molecules that are

In addition to utilizing the aforementioned knowledge, the present invention can be achieved by utilizing a simple test method. Appropriate key combinations for use in bispecific antibodies may be determined.

The bispecific antibody molecule has a specific antibody against T cells that can itself induce T cell killing. When using a specific monoclonal antibody heavy chain/light chain combination, this A hybridoma producing a monoclonal antibody of to produce a specific type of immunoglobulin, a monoclonal antibody with It can be fused with hybridomas. The double singularity obtained in this way The T-cell killing ability of the first heavy chain/light chain combination is the key to the problem. In combination with the heavy chain/light chain combination of immunoglobulins of will be subjected to a test to see if it can be canceled out. For example, Hybridomas producing IgG2b antibodies were isolated from either unrelated rats. By fusion with a hybridoma that produces gG2a or IgG2a antibody Thus, IgG2 induces lethality by both ADCC and complement activation mechanisms. It can be investigated whether the b-heavy chain mechanism is preserved. Another method is to clone Transfection of modified immunoglobulin genes into hybridomas.

In this case, the cloned gene is expressed within the hybrid cell and the mixed Regarding immunoglobulin molecules, T-cell lethality can be achieved by both ADCC and the complement pathway. evaluate.

Such methods are advantageous for identifying particularly useful stalwart combinations. but generally species differences or isotypes, i.e. classes or subclasses. This difference sufficiently reduces T cell killing by bispecific molecules and also Many differences in types also lead to this result. Selecting a key combination Other related criteria will be discussed later.

Appropriate combinations exist within bispecific antibodies that do not result in T-cell lethality However, the methods available for preparing such antibodies, especially hybridoma A complex mixture of antibodies can be obtained by the method using the technique, which Some of these exhibit T cell toxicity that bispecific antibodies do not exhibit. bone marrow Bispecific antibody molecules were generated from two hybridomas that do not express tumor-derived light chains. Various types of different antiseptics that may be present in the mixture obtained during preparation The body is shown in Figure 1. Theoretically, two different light chains and two different heavy chains Although all possible combinations can occur (in Figure 1, T cell binding Chains with activity are indicated by black symbols, chains with immunoglobulin activity are indicated by white symbols. ), but in reality some of each type are obtained in unequal proportions. It is thought that In cases where neither type of antibody destroys T cells, A bispecific antibody molecule with cell-binding affinity has been shown to induce Fc receptor-mediated lethality. mechanisms, such as ADCC or complement activation, in the absence of mediating T cell killing. It is only. This heavyweight causes T cell death through an Fc receptor-mediated lethal mechanism. In some cases, types 2 and 4 are The immunoglobulin-binding heavyweights are toxic by the route of activating other heavyweights. In addition to types 2 and 4, types l and and 5 are also toxic. Therefore, type 1 bispecific monoclonal antibody molecules are A heavyweight against Tm cells and the anti-immunoglobulin half of the molecule. Type 2, which does not cause death of T cells by selecting an appropriate combination and 4 always have the undesirable function of killing T cells.

Bispecific antibodies containing different rat IgG combinations can be used in human therapy. If used, order of utility for anti-T cell/anti-immunoglobulin combinations. The position is IgG2b/TgG2b < IgG2b/IgG2a (or 2c) < IgG 2a (or 2c) / IgG 2a (or 2c) or IgG2aC or 2c)/IgG2b. Because the first con As a bispecific molecule, the combination is toxic to T cells and Nation generates toxic molecules in a mixture with bispecific molecules and or the fourth combination does not produce molecules that are toxic to T cells. and the other type of molecule present in the mixture is a diluent for the bispecific molecule. It only fulfills its role as a. Other rat gG complexes of particular importance in human therapy Bination is a bispecific antibody that is not toxic to T cells, but IgG2b/IgG1 and especially type I, which donates toxic molecules of 2 and 4; 2.4 and 5 are IgG1/IgG2b non-toxic to T cells.

Alternatively, four possible combinations of IgG1 and IgG2a or IgG2a These generally include the IgG2a (or 2 c)/I gG 2 Shows similar behavior to the a (or 2c) combination. It is expected that For mice, similar isotype combinations IgG1/IgG1. IgG2a/IgG2a and IgG2b/IgG2b is an important IgG1. Possible dissimilar isotypes of IgG2a and IgG2b They are generally somewhat less useful than combinations (particularly the rat For the same reason as in the case of IgG2b/rat IgG2b, IgG2a/IgG2a ). Some of these dissimilar isotype combinations are more important than others for similar reasons as in rats, so e.g. The combination in which anti-T cell binding is provided by mouse IgG2a is of type This is disadvantageous as it is expected to result in toxic molecules of 2 and 4. rat Ig G/mouse IgG and mouse IgG/rat IgG combinations is also important.

In yet another aspect of the invention, two fused hybridomas or two according to the invention produced by hybridoma lines derived from other fusion partners. The antibody mixture containing the bispecific antibodies is subjected to a fractionation treatment to separate the bispecific antibody molecules. The content of bispecific antibody molecules may be increased, and more preferably, the bispecific antibody molecules may be substantially separation or dilution to other undesired species, i.e. type I molecules. molecules that act as agents and are toxic to T cells, particularly through Fc-mediated receptor mechanisms. At least reduce the content of species exhibiting sexual characteristics.

The technology used for such purification treatment is described in British Patent Application No. 2144147A. (The product to be purified is a type 4 monoclonal antibody. ) is almost the same. Affinity chromatography is used for such methods. the use of ion-exchange chromatography and isoelectric focusing chromatography; In particular, high-performance protein liquid chromatography [FPLC: Pharmacia Co., Ltd. rmacia trademark] and high pressure liquid chromatography.

Affinity chromatography provides the correct combination of heavy and light chains for specificity. an antigen-containing column (e.g., immunoglobulin) that selects molecular species with May be exploited by using or anti-isotype or Protein A columns may also be used to perform separations based on isotype. Io Exchange chromatography is based on the following findings. i.e. different pH In , the charge on a protein changes as different side chains are ionized, so the charge Protein binding to the column is affected by ionic strength. ion exchange chroma Effective use of chromatography involves the collection of fractions on the first column (first pH). This is a method in which treatment is carried out on a second column (second pH) following separation of the sample. child In the case of cation exchange chromatography, the columns usually have opposite charges. Anion exchange chromatography treatment is performed after the feed treatment, or Perform this process in the reverse order. Based on the following findings of isoelectric focusing chromatography It's a spider. That is, at a certain selected pH, proteins have a net charge. mixtures of similar proteins based on their pr. separated. FPLC and HPLC have different advantages, so they can be used together. You may.

As mentioned above, appropriate selection of anti-T cell heavyweights that do not exhibit T cell toxicity or different Combination of toxic anti-T cell heavyweights of anti-immunoglobulin heavyweights of species or isotypes tion provides bispecific antibody molecules that substantially avoid T-cell killing. It is generally possible to achieve the initial objective of the invention to. The above purification technology By utilizing other components that are toxic to T cells, It is possible to remove components resulting in a bispecific antibody molecule-containing composition that exhibits can.

Bispecific antibodies are generally fragments that possess the binding affinity of the entire molecule. It may also be used in the form of an agent. However, the bispecific antibodies according to the invention A second method to avoid destruction of T cells that retains binding affinity but 7 fragments of total antibodies that do not have cell-killing ability, especially F(ab'), This method uses antibodies in the form of a drug. This method uses F(ab ’) = moiety binds to T cells through one of its heavy/light chain combinations However, the absence of the Fc portion of the antibody molecule allows for This is based on the knowledge that T cells do not die. F(ab’)z part Another advantage of using N is that the 7-ragment is smaller and therefore more quickly removed from the kidneys. It means being excreted.

An appropriate enzyme system is used to prepare the F(ab')z fragment of the antibody molecule. It is necessary. Using such an enzyme system, the heavyweights are cut at appropriate points and the heavyweights are removed. The Fc region (or at least the region of the anti-T cell heavy chain) is removed, while the two The remaining parts of the heavy weight are retained, and each remaining part is linked to its light weight through disulfide bridges. Connect to chains and other heavyweights.

Different species and isotypes have different amino acid sequences in the cleavage region of immunoglobulins. Since the amino acid sequence in question has a Enzymes must be used. In addition, the two heavy chains are species or isotypes. If some, but not all, cysteines in one chain group must be linked to the cysteine group in the other chain via a bridge, and the F Consider the possibility of a cleavage mode that generates a Fab rather than an (ab')2 fragment. Must. Therefore, it maintains two types of specificity that are not the T cell killing ability of whole antibodies. F(ab'), fragments or other 7 lacking only one Fc region To generate the fragment, use an enzyme appropriate for the bispecific antibody system in question ( (e.g. pepsin, papain, v8 protease, etc.) and conditions for using the enzyme. (pH1 temperature and time). of products with the above-mentioned properties. If the appropriate enzyme and conditions for its use are selected as determined by the preparation, the present invention The technique for preparing bispecific antibody F(ab') fragments is based on monospecific As described in the literature on the preparation of F(ab'')2 fragments of sexual antibodies. The technology is almost the same as the one currently used.

Accordingly, the present invention includes: (a) a first binding antibody to a T cell receptor capable of activating lethality; affinity and a second binding affinity for immunoglobulins. F (ab') of a heavy specific antibody molecule, a fragment, and (b) the antibody molecule is cleaved to produce the fragment by treatment with an appropriate enzyme system. A method of manufacturing the 7-ragment is included.

The classification of different antibody molecules of types 1-10 as described above is F(ab').

Also useful when using fragments. Because it is toxic to T cells Removal of other types indicating ~10 dilutes the content of a single active type 1 in the preparation, while type 2.4 and 5 compete for binding to T cells, types 3.6 and 7 are immunoglobulin This is because they compete for phosphorus. Other types, especially types 2 to 7, are It is advantageous to reduce or substantially eliminate it compared to type 1. This kind of separation The processing may be performed before generating F(ab'') fragments, but It is preferable to do this after.

The bispecific antibodies and fragments thereof that are the subject of the present invention can be used in medical fields. Until now, there had been no proposal to use it.

The invention therefore includes (a) bispecific antibodies for use in surgery, therapy or diagnosis; body molecules or fragments thereof that are directed against T cell receptors that can activate lethality. a first binding affinity for the immunoglobulin and a second binding affinity for the immunoglobulin. a bispecific antibody molecule that has the binding affinity of the entire molecule or and (b) the treatment of neoplastic or other diseases. bispecific antibody molecules or their seven fragments for use in the manufacture of pharmaceuticals for use in The first binding affinity for T cell receptors that can activate lethality. bispecific with a second binding affinity for T and immunoglobulins of a specific antibody molecule or a fragment thereof that retains the binding affinity of the entire molecule. Includes usage.

When using the bispecific antibodies and fragments thereof described herein may be formulated in a variety of ways, but usually at physiologically acceptable dilutions. agent or carrier. Such diluents and carriers must be sterilized. However, for certain applications it is preferable that the material does not contain a heat source. stomach. It is available in various forms such as phosphate buffered saline, saline, balanced salt solutions and grape It may also be a sugar solution or the like. However, phosphate buffered saline is often suitable.

Such compositions may optionally be prepared in unit dosage form (i.e., separate containers containing unit doses). ) or as multiple or subunit dosage forms. good.

The anti-target cell antibodies described herein also include fragments, such as F(ab'), etc. can be used in the form of . Additionally, anti-target cell antibodies or 7 fragments thereof can be used as bispecific antibody molecules or may be prepared in much the same way as its fragments. Two types of anti- body may be combined in the same composition or kit, but in the former case, as described below. As will be seen, this results in the formation of a Funschgate. Therefore, the present invention includes (a ) primary binding affinity for T cell receptors that can activate lethality and immunity a bispecific antibody molecule with a second binding affinity for globulin or a fragment thereof that possesses the binding affinity of the entire molecule, as well as to the target cell. an antibody molecule that has a binding affinity for or an antibody molecule that has a binding affinity for and (b) a T cell receptor capable of activating lethality. A first activation affinity for the body and a second binding affinity for immunoglobulins. Bispecific antibodies that have binding affinity or that maintain the binding affinity of the entire molecule. 7 fragments, as well as binding affinity for target cells. Antibody molecules or kits containing fragments with said affinity. be caught. Additionally, the present invention includes methods for simultaneously treating neoplastic diseases or other diseases. , products used separately or sequentially to activate T cell receptors capable of activating lethality. a first binding affinity for the host and a second binding affinity for the immunoglobulin. Binding affinity of a bispecific antibody molecule or the entire molecule with affinity a fragment thereof that possesses as well as binding affinity for the target cell. a product comprising an antibody molecule or a fragment thereof having said affinity for included.

Both types of antibodies or their 7 fragments can be administered in a variety of ways, e.g. intravenously. The drug may be administered by internal administration, intraperitoneal administration, intracerebral administration, or the like. administration The formula depends on the type and location of the tumor or other target cells and the ease of administration by the clinician. It may be selected as appropriate depending on the patient's safety, etc. However, in general, Oral administration, especially intravenous injection, is often used. Two types of antibodies at the same time may be used separately or sequentially, but the anti-target cell antibody is usually administered first. or simultaneously with the bispecific antibody; in the latter case, the lag time is between 0.5 and It is 24 hours. Regarding the dosage of the two types of antibodies or their seven fragments. The exact dosage depends on the efficacy of the drug and the patient's suffering from tumor or other diseases. The amount to be used as a guideline depends on the degree of 1 to 25 mg of each antibody or fragment, e.g. It is appropriate to administer the drug in equal amounts, and administer it once a day for 7 to 10 days. (If administered for 10 days, the total administration of each antibody or fragment to the patient The dosage is 10-250111 g).

Dosages may be outside this range if appropriate, but may take advantage of the features of the invention. The point is that lower doses can often be used. That is, within the above dosage range. bispecific antibody molecules even at low or lower doses. The development of an immune reaction against the drug is avoided, and administration can be repeated.

The present invention includes lethal activity in the treatment of neoplastic or other diseases. primary binding affinity for Tm cell receptors and immunoglobulins that can be a bispecific antibody with a second binding affinity for or a binding affinity of the whole molecule. 7 fragments with binding affinity to target cells. in combination with an antibody molecule that has the affinity or its 7 fragments that have the affinity For example. In particular, the present invention includes neoplasia or other diseases. A method to assist in alleviating and temporarily suppressing the symptoms of T cells that can activate lethality. Binding affinity for receptors and secondary binding for immunoglobulins Binding affinity of the bispecific antibody molecule or the whole molecule with affinity J, which has an affinity for target cells. antibody molecule or its 7 fragments having this affinity to alleviate the symptoms and and administering a therapeutically effective amount to achieve temporary suppression. . However, bispecific antibodies can be used in combination with anti-tumor antibodies to treat bone marrow in vitro. removal of neoplastic cells from the body and transplantation of autologous bone marrow for the treatment of malignant tumors You may.

When the bispecific antibody molecules according to the invention are used for indirect ECR, Stelz and Peptide It offers many advantages over direct ECR as recognized by Van . The aforementioned bispecific antibody molecules can be used as anti-target cell antibodies in vivo or in vitro. A variation of the combination method involves collecting peripheral blood mononuclear cells from the patient and injecting the cells with anti-T. Cellular antibodies, e.g. monoclonal antibody YTHI as described in Example 2 2.5 ．． Activate the cells by short treatment with 14 or 2.5.14.2. This includes: Anti-tumor monoclonal antibodies can be used, for example, against differentiation antigens, surface immunoglobulins, etc. Antibodies or combinations of antibodies selected against antigens, tumor-associated antigens, etc. used to target tumor cells and, after a few hours, detect the presence of bispecific antibody molecules. The activated effector reintroduced into the body lyses the cells and eliminates excess Anti-tumor cell antibodies are removed. In addition to having a wide range of applications, indirect ECR is Compared to standard ECR, pre-activated cytotoxic 1973 cells (CTL) and It has the advantage of being able to recruit cell populations to destroy tumor targets. anti The Fc portion of the bispecific antibody molecule, which has anti-target cell affinity for T cells, is activated. CD16 or other effector cell populations (e.g. mononuclear leukocytes) on cells during sexualization. If it binds to CD3 on Fc receptors and CTLs contained in neutrophils and neutrophils, etc. For example, the presence of bivalent CD3 Mab makes the antibody unsuitable for therapeutic use. As seen between CTLs and K1m cells or mononuclear leukocytes in the presence of This is because it will lead to mutual lethality between the two. Therefore, direct ECR can be used effectively. In order to use FcγR1°W on K cells or Fc receptors on effector cells, The cells can be blocked, for example, with a suitable CD16 monoclonal molecule, or or a bispecific antibody molecule in which the Fc portion is Fc7R1ow or other Fc receptors. It must be selected so that it does not bind to the body. Alternatively, anti-T cell/anti-target The Fc portion of a cellular bispecific antibody molecule is modified by generation of F(ab')2 fragments. You can make it inactive. However, these methods are limited to bispecific antibodies. The function of cell-mediated ADCC to coated target cells is abolished. ECR and It is effective to use ADCC in combination to set a standard for target cells and kill them. Additionally, killing of K cells can be achieved using indirect ECR; This is because the antigenic cellular antibody can be administered separately from the bispecific antibody portion. child It has been shown that ADCC effectively mediates cell killing in cases such as Anti-tumor cells or other antigenic cell antibodies of the rat IgG2b isotype It is preferable to select other isotypes of rat antibodies as well as other isotypes of IgG2b antibodies. Techniques for class conversion to Yi Tutaib are known. processed effector details When the cells are injected into the patient's body, they become activated and reach the tumor site, producing antibodies. Coated target cells are killed by ADCC. Therefore, it has a non-functional Fc portion. Bispecific antibody molecules selected to target C to the same target cell population Mediates indirect ECR by adjusting standards to TL responses.

Bispecific antibody molecules according to the invention allow administration of different antibodies at different time intervals. treatment, thereby allowing excess primary antigenic cell antibodies to be excreted. Second, antigenic cellular antibodies, due to their antiglobulin specificity, can effectively reach tumor cells that bind to the first antibody. This allows the reticuloendothelium to The production of aggregates, which can be removed very efficiently by cells such as Ru. Furthermore, when using indirect ECR, appropriate antigenic cell antibodies should be used. Upon activation, cells mediate ADCC against target cells. then two A bispecific antibody molecule is administered, which also recruits the CTL population and indirectly promotes EC. The same target cells are killed by R.

In addition to the above-mentioned uses, the bispecific antibody molecules according to the invention may also be used to activate lethality. a first binding affinity for the T cell receptor and a second binding affinity for the target cell. When preparing bispecific antibody conjugates with binding affinity, It may be used as a molecule or as a fragment thereof.

In this case, the bispecific antibody molecule according to the invention has the same binding affinity for T cells. have different binding affinity for tumor cells or other target cells. Intermediates that can be used to prepare bispecific antibody conjugates with affinity I will provide a.

Therefore, the present invention includes a first binding affinity for a T cell receptor that can activate lethality. a bispecific antibody compound with a second binding affinity for a target and a second binding affinity for the target. conjugate, the first binding affinity for a T cell receptor capable of activating lethality. The immune system is bound to an antibody that has binding affinity and binding affinity for the target cell. Bispecific antibody molecules, or or its 7 fragments that retain the binding affinity of the entire conjugate. Includes patent routes characterized by having.

In this alternative use, the bispecific antibody molecules according to the invention may be used as described herein. in vitro with antibodies that have binding affinity for target cells such as The conjugate is then administered into the body. This kind of conjugate is Bispecific for target cell antibodies, which can also be prepared by chemical conjugation The bond is formed by utilizing the anti-immunoglobulin affinity of the anti-immunoglobulin antibody molecule. preferable. This type of conjugate is therefore useful for T cell receptors that can activate lethality. a first binding affinity for an immunoglobulin and a second binding affinity for an immunoglobulin. A solution containing bispecific antibody molecules with specificity and binding to target cells. Prepared by simply mixing solutions containing antibodies with binding affinity It is easy to do. Bispecific antibody conjugates thus prepared It is preferred that the drug be administered directly to the patient without being isolated. Therefore, such a preparation It is convenient to carry out the reaction in a medium suitable for this purpose, for example, when the two components are It may be stored in an equal volume of sterilized physiological saline. The mixture is completely reacted and A suitable period of time during which no aggregation occurs, for example up to 1 hour (for example 30 minutes) and then administered, especially parenterally.

Bispecific antibody molecules and anti-target cell antibodies and the aforementioned preferences for their use. Another embodiment is when these antibodies are used as components of bispecific antibody conjugates. It can also be applied when For example, the F(ab')27 lag of a bispecific antibody molecule or other 7 fragments are anti-target cell antibodies or their F(ab') = 7 fragments. or in combination with other 7-ragments. The therapeutic formulation is similar. Just do it. The same applies to dosage, but in this case, Since a conjugate is a combination of two separate components, a conjugate The appropriate dose for each component is the sum of the doses for each component. should be kept in mind.

The invention is illustrated by the following examples.

Example 1 Monoclonal antibodies specific for human CD3 antigen and rat immunoglobulin /- Preparation of hybridoma (1) Anti-CD3 component Lou La La Myeloma Cell Line Y3-Ag1.2.3 (CNCM, I-078 ) and human lymphocyte-immunized lung cells from Clark (C1a). According to the method described in the miscellaneous works of R.K.R.K. and Waldmann. [Methods in Hemato] 1986, Vol. 13, pp. 1-20], the fusion mixture is The reactivity with all human peripheral cells, UCHT-1 Burnset et al., Journal of Immunology, 198 2, Vol. 129, p. 1451] and 0KT-3 (U.S. Pat. No. 4,361,549). Cross inhibition using mouse monoclonal antibodies such as ion) and specificity for the human CD3 antigen defined by immunoprecipitation. Hybridomas producing monoclonal antibodies were selected. This/1 Ibrid Hyboxanthin-guanine phosphoribosyltransferase positive (HPRT”), the first light chain derived from lung cells and the kappa 1a allotype. expresses a second light chain derived from myeloma.

Cell cloning on semi-solid agar for a modified form of myeloma that has lost its light chain. (see Clark and Waldman's above), and then A hemagglutination assay was used to assess the loss of expression of the rat buckwheat 1a allotype. (Clark, Methods in Hematology, 1986, Vol. 121, No. 54) (See pages 8 to 556). Cloning of selected variants on semi-solid agar This was added to Dulbecco's Iscobus modified medium [IMDM (I5co ves modification of Dulbecco’s medium m) - Gibco Europe], i.e. fetal calf In an air atmosphere supplemented with 5% v/v serum (FCS) and containing 5% CO3, Cultures were grown in media modified by buffer treatment with bicarbonate.

After a short treatment of cells, bicarbonate was added to excess HEPES buffer and Na CQ was substituted to maintain ionic strength.

(2) Anti-depressant immunoglobulin component BALB/C myeloma cell line NSI/Ag4. ], Complete 70 Indoor Adjuva SJL mouse strain immunized with rat IgG pooled in fused with lung cells [Springer et al., hybrid 1982, Vol. 1, pp. 257-273].

The selected hybridoma is a model with specificity for a particular rat allotype. Produces noclonal antibodies, which are produced by hybridomas (1) Since it does not correspond to immunoglobulins, autoreactivity is avoided. HPR Selection for the T-negative variant of this hybridoma was performed using the selective drug 8-A. This was done by incubating increasing concentrations of zaguanine-containing medium (clara (See above miscellaneous notes by Ku and Waldman). Cloning selected HPRT-variants The hybridoma (1) was cultured [the first stage of this treatment]. In the variant, hybridoma (1) is resistant to 8-azaguanine and Ibridoma (2) is poisoned using iodoacetamide in step (3). In the second variant, 6-thioguanine is substituted for 8-azaguanine. processing or in the first variant, and in the third variant, knotburid Similar to hybridoma (1), or instead of hybridoma (1), myeloma light chain deletion mutation It is atypical, and one or both hybridomas are derived from myeloma that does not express light chains. It is something that will come].

(3) Hybridoma-bibridoma cell fusion Before fusion, hybridoma ( 1) in phosphate buffered saline (PBS) with 10mM iodoacetamide. The mixture was treated at 0° C. for 30 minutes. HPRT” iodoacetamide poisoned cells Wash with HEPES-buffered IM buffer 1 to remove excess inhibitor, then HPR T-hybridoma (2) cells were mixed in three different proportions: (a)3. 5 x l O’ cell piperid? (1) +3.5X10' cells/% (Brido (2X10:1), (b) 3.5xlO' cell hybridoma (1) +3. 5x10y hybridoma (2Xl:1), and (c) 3.5x10' cell battery. Ibrid? (1) +3.5XlO” cell hybridoma (2) (1:10) . In each case, cells were mixed in 1 D in HEPES buffered IM buffer and pelleted. It was molded (200×g). Cell fusion was performed using 50% W/V polyethylene glycol 1 The cell pellet was treated with 1 mQ of 500 PBS solution for 2 minutes under stirring. Therefore, this was done (see the above miscellaneous article by Clark and Waldman). HEPES cells After washing once in buffered IM buffer 1 D, the resuspended in carbonate-buffered IMDM and mixed with four types of Transfer to a culture well plate (24X 2mff) at a concentration of The cells were cultured at 37°C under ambient air. The next day, the iodoacetamide-treated fusion Controls containing cells showed no overall viability, but fusion In cell cultures, the majority of cells were found to be viable.

After 24 hours of incubation, hypoxanthine, aminopterin and thymidine Selection was initiated using HAT selection medium; Clark and Waldman's HPRT-hybridoma (2) cells are not viable in this medium. did not show. Culture in HAT selection medium for 2 weeks or longer After a long period of time, bispecific antibodies can be produced using the assay described below. 1/1 hybrid cell line was selected. Immunofluorescence analysis of functional human CD3 antibody was detected using the human T-cell leukemia cell line HPB-AL. The bond to L was shown [Adrian and Hutton , Journal of Clinical Investigation of C11nical Investigation), 1983, No. 71 Vol. ), indicating the presence of both heavy and light chains.

Functional anti-rat immunoglobulin activity is derived from anti-mouse Thy-1 antigen or other A rat antibody of unrelated specificity immobilized in the wells of a microtiter (this This is an appropriate allotype for hybridoma (2)). Ta. Antibodies that can bind to such antibodies include reagents specific for mouse IgG2. was detected using biotinylated YA9/36.39 [Dr. paper (University of Cambridge, 1982)]. Using a plate binding assay We screened antibodies with a mixture of immunoglobulins derived from both parental cells ( In this case, biotinylated YA9/36.69 is used in hybridoma (1) by a biotinylated monoclonal antibody specific for the IgG subclass. Replacement I ;). showed a strong positive response in the three assay treatments; Cells were subjected to cloning twice on semi-solid agar, and appropriate clones were selected. This was then cultured as in the case of monospecific hybridomas (1). In a variation of the anti-porous Ig hybridoma (2), the were replaced by hybridomas specific for human or human Ig. No. In the second variant method, which may be used in combination with the first variant method, hybridoma (1) and (2) from human myeloma, for example in a European patent. Application No. 0062409, Application No. 0148644, British Patent No. 2086937 and hives induced as described in U.S. Pat. No. 4,529,694. Replaced with ridoma. ].

T-IgG2b anti-human CD3/mouse TgG2a anti-rat immunoglobulin 2 Preparation of heavy specificity hybridoma LHC49,18,2 This preparation was carried out as described in Example 1 (3). ), however, hybridoma (1) is hybridoma YTHl. 2.5. l　4　[This is a miscellaneous essay by Cobbold and Waldman ( Nature, 1984, Vol. 308, pp. 460-462) It is the same as Sister Chron YTH12, 5, and 22. ] and hive Hybridoma RGI 1/15.5 (Sbringer et al. (see the miscellaneous text above) was used. YTHl 2.5.14 is rat IgG2b model produces noclonal antibodies and expresses allotypic kappa light chains derived from myeloma . Mutant YTH12,5,14,2, which loses the myeloma chain, was found to be The lambda class secretes a single light chain that is inactive to rat kappa reagent. It is thought to express light chains.

RGI I/15.5 is specific for the rat kappa 1b allotype light chain. A mouse IgG2a monoclonal antibody is produced and expresses a kappa light chain.

The fusion mixture is used as a specific assay for rat IgG2b in the third assay. Biotinylated NORI G7.16.2 was used to select [Hμ(H ale) et al., Journal of Immunology, 1985, Vol. 134, No. 3 See page 056]. By subjecting it to a cloning process, the bispecific anti-human C D3/anti-Rattokatsupa lb allotype light chain hybridoma LHC49,18,2 selected.

Example 3 Mouse IgG2a anti-human CD3/mouse gG2a anti-rat immunoglobulin double Preparation of specific hybridoma LHD6.23 This preparation was carried out according to Example 1. became. However, the hybridoma (2) is the hybridoma described in Example 2. RG 11/15°5 was used, and hybridoma (1) was Dormer 0KT3.5.2 [described in U.S. Pat. No. 4,361,549, AT Mouse deposited at CC (Rockville, USA) under accession number CRL8001. Hybrid that is a lephron of IgG2a anti-human CD3 hybridoma 0KT3 I used the 0KT3-5.2 is mouse myeloma P3-X63-Ag8U It has the same properties as 0KT3 derived from IIC, and is E rosette positive purified. By fusion with mouse lung cells of CAF immunized with human T cells, mouse secretes kappa light chain.

The fusion mixture is immobilized in microtiter wells with the kappa 1b allotype light chain. Immunoglobulin isotype assay using appropriate rat IgG antibodies RGII/15.5 by biotinylated YA9/36.69 Detect specificity and indirect fluorescence of KT3 specificity by complement-mediated lysis of human T cells Detection was performed using By cloning process, bispecific anti-human CD3 /Anti-Rattokatsupa lb allotype light chain hybridoma LHD6.23 was selected. .

Example 4 Mouse IgG2a anti-human CD3/mouse IgG2a anti-rat immunoglobulin double Specific hybridoma LHB63. Preparation of IO This preparation was according to Example 1. It happened. However, the hybridoma (1) is the hybridoma described in Example 3. The hybridoma 0KT3.5.2 was used, and the hybridoma (2) was a rat anti- Immunized by pertussis/pertussis immune complex: B A L B / C mice induced by fusion with mouse lung cells and non-productive mouse myeloma NSO/u. Hybridoma NORI G7゜16, an HL-secreting hybridoma derived from ．． 2 was used (NORIG 7.16.2 was described in the aforementioned miscellaneous paper by Heμ et al. A subclone with similar characteristics to NORI G7.16). NORI G7.16.2 is an anti-mouse 1gG2b mouse IgG2a expressing kappa light chain. Produce monoclonal antibodies. HL-clone with 8-azaguanine resistance and NORIG 7.16°2Ag was used. After fusion, by indirect fluorescence Sorting procedures including complement-mediated lysis of human T cells to detect 0KT3 specificity and EL I using plates coated with and irrelevant rat IgG2b antibody SA7　It was okay. NORIG7.1.62 specificity is Tinylated anti-mouse Ig reagent and streptavidin-peroxidase [am- obtained from Amersham plc, England]. Ta. By cloning process, bispecific anti-etoCD3/anti-rat gG2b Hybridoma LHB63. I chose IO.

Example 5 Production of monoclonal antibodies from bispecific hybridomas Examples 1, 2.3 and The bispecific hybridoma prepared in step 4 was cultured in low serum blood (containing 1% v/v FC5). IMDM) in an air atmosphere containing 5% CO2 at 37°C for 48 hours. did. The culture supernatant was precipitated by adding ammonium sulfate at a concentration of 50% w/v. concentrated by treatment. After redissolving the precipitate in a minimum amount of water, Sephadex Desalting was performed on G25 using 20 mM buffer (pH 8,0).

The antibody was subjected to HPLC ion exchange chromatography on a TSK-5PW column (manufactured by LKB). It was subjected to a fractionation process using graphics. Pooled fractions were assayed. Desalted using PBS.

Example 6 Effector of rat IgG2b/mouse IgG2a bispecific monoclonal antibody -Activity test in cell retargeting (A) Venous blood collected from healthy donors was subjected to defibrillation treatment using glass beads. Ta. Density gradient on F1coll-Hypaque Mononuclear cells were isolated from the interface by centrifugation and these were incubated with 5% v/v FC5. Washed with bicarbonate buffered IMDM containing . These cells were treated with anti-human C D3 antibody, especially produced by the hybridoma YTH12,5,14,2 of Example 2. Add the same medium 1III1 + warm water into tissue culture bolts pre-coated with the resulting antibodies. It was distributed in 1X10' cells. After 3 days of culture at 37°C, the effector was 7 days in the same medium containing IOU/mQ of IL2 [Cetus] It was subjected to an expansion treatment.

Blasts prepared in this way possess Fc receptor positivity that can mediate ADCC. containing 10-15% of Fc cells [Fc rosetting ]. These ADCC effectors were isolated using the same medium. The effector cells were washed twice and the anti-Fc receptor antibody CLB-Fcr Grande [ Tetteroo et al., Leucocyte typing (Leuc) ocyte T yping) II, 1985, Volume 3, Page 27, Spry springer, springer, new York) by pre-incubating for 15 minutes at room temperature with a 1/600 dilution of caused harm. This anti-CD16 antibody blocks cell-mediated ADCC.

(2) Mouse Thy as target cell line for effector cell retargeting assay -1 positive cell line BW5147 [CRL1588, PHLS Europe Collection Fist of ψAnimal Cell Cultures (European) Co11ecLionof Animal Ce1l Cu1tures), Po -Tondown, England] was used. Cells contained 2% v/v Fe2. The cells were kept in exponential growth phase in IMDM until used for assay. Approximately 5 X10' cells were spun down at 200Xg and the pellet was - resuspended in rMDM150p (1) containing sodium chromate (300 μci) After incubating the cells at 37°C for 1 hour, they were washed with HEPES-buffered IMDM. Ta. Target cells were then washed and distributed into two tubes. Inside the first tube Cells were diluted with 00 μg of HEPES-buffered IM buffer for direct ECR analysis. Resuspend cells in a second tube in a hive for indirect ECR analysis. Anti-Thy-1 monoclonal antibody derived from ridoma YBM29.2.1 500μ of the same medium containing about 100μg/mi2 Resuspend in g. After incubating for 1 hour at room temperature, cells in the second tube were incubated once. Excess YBM29.2.1 was removed by washing and resuspended in the same medium (2X lO'viable cells/m4).

Place a 2-fold dilution of effector cells into a microtiter well with a U-shaped bottom. Add 15 μa to a cell, add an equal volume of hybridoma, and target cells coated with labeled Thy-1. cells or uncoated target cells in 50 μQ of HEPES buffered IMDM medium; The effector to target cell ratio was 32:1 to 0.5:1. effector cell re Appropriate dilutions of antibodies used for targeting tests were added to HEPES-buffered IMDM1 wells. 00 μΩ and incubated at 37°C for 4 hours, then 1 00 μa of culture medium was collected, and the released radioactivity was measured using Feritubu Gamma Counter. (PW4800 model) was determined by measuring gamma radiation.

(3) Activity assay method for LHC49,18 antibody and 5HN20.12 antibody (a) The supernatant of the culture of ~(e) was used (the culture was obtained as in Example 5). : (a) Y3-Agl, 2.3 myeloma cell culture (control), (b) Parent hybridoma YTH12,5,14,2 and LHC49,18 hybrid (c) LHC49, Culture of 18 hybridoma cells, (cl) SHN20. ]2 hybridoma fine SHN 20.12 is a British patent application by Clark and Waldman. No. 8626412 is the basis for claiming priority, and the concentration patent application dated the same date as the present application Rat IgG2b anti-human CD3/rat prepared as described in Example 3. IgG2c anti-mouse Thy-] bispecific hybridoma), and ( e) Parent hybridoma YBM29-2.1 and 5HN20.12 hybrido 1:1 v/v mixture of cultures of YTHl 2.5.14.2.

In the first series of experiments, the assay method described in (2) above was used. However, In this case, the effector:target cell ratio was 15:1, and the supernatant (10 The dilution ratio of 0 μg) was 1/100 to 1/12800. Control Y3-A g1.2-3 cells and parent mixture contain unlabeled and labeled target cells. When we measured the concentration of ``Cr released using ``Cr'', it did not show any significant activity ( (See discussion below regarding parent mixtures). As expected, the 5HN20.12 antibody showed a significant “Cr released concentration” for both types of target cells, whereas 5HN Unlike the 20.12 antibody, the LHC49,18 antibody produces only indirect ECR. Cr release concentration was significant when labeled cells were used, but when unlabeled cells were used, For cells, the control and parent mixtures showed similar 5ICr released concentrations. Ta. In the second series of experiments, the effector:target cell ratio was adjusted to (2) above. The dilution ratio of the supernatant (100μQ) was 32:1 to 0.5:l as described, and the dilution ratio was 1/1. It was set as 100. The results obtained are shown in Figures 2a and 2b. Figure 2a and Figure 2b relates to unlabeled target cells and labeled target cells, respectively. Direct E The 5HN20.12 antibody that causes CR targets both uncoated and coated target cells. (For clarity, this plot is shown only in Figure 2a). and other plots are shown in Figure 2b), LHC49 leading to indirect ECR. , 18 antibodies are, as expected, effective only in the case of coated target cells. It was found that (for clarity, this plot is only shown in Figure 2b, Other plots are shown in Figure 2a). However, as shown in Figure 2b, LH Lethality produced by indirect ECR using the C49,18 antibody is shown in Figure 2a. produced by direct ECR using the 5HN20.12 antibody as shown in It is at least as effective as lethal force. (a small amount observed in the case of the parent mixture) The activity described in (A) above is thought to be due to a small number of naturally occurring microaggregates. The experiments were carried out using murine lymphoma BW5147 as the target cell, and BW5147 Mouse Thy-1 cells prepared in the same manner as described in (A) above. Diti lymphoma cell line E L-4 (A, T, C, C, deposit number: TI 6BW5147 and EL-4 target cells were unlabeled. In the case of (A), as described in (A) above, in the form of conjugation and in the form of labeling. Anti-Thy-1 rat IgG2c, Klb hybridoma YBM29゜2 such as monoclonal antibody or anti-Thy-1 rat IgG2b, Klb hybrid Doma YTH154,7,7 [Kottvold et al., Molecular Biology A Molecular B iology and Medi cine), 1983, Vol. 1, pp. 285-304] or anti-L C/7200 rat ■gG2a, 1b hybrid? YBM42.2 [Stenni S. Tenning et al., Molecular Biology and Medicine Monoclonal from 1983, Vol. 1, pp. 285-304] Antibodies were used for labeling.

Labeled target cells are placed into a microtiter containing a 2-fold dilution of effector. Add a 1/100 dilution of the supernatant to any of the following cultures (a) to (c). (Cultures were prepared as described in Example 5): (a) Y3~Agl , 2.3 Myeloma cell culture (control), (b) Parent hybridoma YT Cultures of H12,5,14,2 and LHC49,18 hybridomas: l v/v mixture and (c) culture of hybridoma LHC49,18.

Figures 3a and 3b are BW5147 coated with YBM29.2.1, respectively. Results obtained using cells and YBM42.2 antibody are shown. Figures 3c and 3c. 3d figure shows EL-4 cells coated with YBM29.2.1 and YBM42, respectively. ．． The results obtained using 2 antibodies are shown. coated with YTH154,7 antibody The results obtained using both types of target cells are similar to those obtained using YBM29.2.1. Not shown as the results are similar to those obtained using coated target cells. both ties As expected, the unlabeled target cells in the supernatants of the three supernatants It didn't die even if I left it there.

As is clear from Figures 3a to 3d, the control or parent pile body mixture The LHC49,1a antibody has two antigens, Thy-1 and BW5147 and EL-4 mediated by anti-target cell antibodies that bind to blood cell common antigen. Kill both at a good level.

Bamboo P=Except for 1 example (%) Bamboo different R, 4Ill sales suspension (%) Special blind system (%) ＃＋” (%) international search report 1'1+-ma-＾帥-一...”+IPC:/GB 871007112lsl #Mdarrogant+11^111m+nkinh+*, PCτ/Gelεfu/C0782 International Investigation report Gil　8700782

Claims (1)

[Claims] 1. Primary binding affinity for T cell receptors and immunity that can activate lethality. Bispecific antibody molecules, or or a fragment thereof that retains these binding affinities of the entire molecule. 2. The antibody molecule or its fragment according to claim 1, wherein the receptor is a human T cell receptor. gement. 3. The second binding affinity involves T cell receptor binding affinity. Claim 1 or 2, wherein the affinity is for an immunoglobulin of a species different from that of the species. The antibody molecule or fragment according to 2. 4. The second binding affinity is for rat and/or mouse immunoglobulin. 4. The antibody molecule or fragment thereof according to claim 3, which has an affinity for 5. The second binding affinity is the light chain or heavy chain immunoglobulin in the antibody molecule. Claims 1 to 4 have affinity for immunoglobulins that do not correspond to The antibody molecule or fragment thereof described in (1). 6. The two heavy chains are each derived separately from humans, rats or mice and are The antibody component according to any one of claims 1 to 5, which is of the IgG class of globulins. child or its fragment. 7. The two heavy chains in the molecule reduce the killing of human T cells by the molecule. The antibody molecule or flag thereof according to any one of claims 1 to 6, which is selected. ment. 8. Substantially, T cell killing promotes killing by Fc receptor-mediated lethal mechanisms. By using a first heavy chain with ineffective T cell binding affinity 8. The antibody molecule or fragment thereof according to claim 7, wherein the antibody molecule or fragment thereof is reduced. 9. The heavy chain is derived from rat and contains IgG2a, IgG2c or IgG1 protein. 9. The antibody molecule or fragment thereof according to claim 8, which is of Ras. 10. Two heavy chains each have isotypes related to anti-T cell/anti-target cell specificity IgG2a/IgG2a, IgG2a/IgG2c, IgG2c/IgG2a, IgG2o/IgG2c, IgG2a/IgG2b or IgG2c/IgG2 The antibody molecule or fragment thereof according to claim 9, which is b. ll. The heavy chain is a mouse-derived IgG2b, IgG1 or IgG3 supplement. 9. An antibody molecule or fragment thereof according to claim 8, which is of the class. 12. The first heavyweight with T cell binding affinity is the Fc receptor-mediated lethal mechanism. The first heavy chain is effective in promoting T-cell killing due to due to the presence of a second heavy chain with target cell binding affinity that further degrades the The antibody molecule or fragment thereof according to claim 7, wherein the antibody molecule or fragment thereof is reduced by: 13. Species, isotypes or allotypes in which the first and second heavy chains differ The antibody molecule or fragment thereof according to claim 12. 14. Claim 12 wherein the first heavyweight is rat IgG2b or mouse IgG2a. An antibody molecule or a fragment thereof as described in . 15. Claim 13 or 14, wherein the first and second heavy chains are of the same class. The antibody molecule is mounted on it. 16. The heavy chain is of the rat-derived isotype, and the first chain/second chain are IgG2b/IgG2a or IgG2b/IgG2c, respectively, Or, it is of the mouse-derived isotype and the first/second strand is of that type. Claim 15, each of which is IgG2a/IgG1 or IgG2a/IgG2b. An antibody molecule or a fragment thereof as described in . 17. Primary binding affinity for human T cell receptors that can activate lethality bispecific antibodies having a second binding affinity for immunoglobulins and immunoglobulins. a child in which the two heavy chains in the molecule are of different species or isotypes These binding affinities of the bispecific antibody molecule or the entire molecule are characterized in that The fragment that holds the property. 18. Two stalwarts were derived from different species of humans, mice and rats. The antibody molecule according to claim 17, or an fragment thereof. 19. Two heavyweights were derived, one from rats and the other from mice. or of a different rat or different mouse isotype. The antibody molecule or fragment thereof according to claim 17. 20. Any of claims 17 to 19, wherein the first and second heavy chains are of the same class. Antibody molecule described in Crab. 21. Both heavy chains are respectively elicited from rafts for anti-T cell/anti-target cell specificity. Isotypes derived from IgG2a/IgG2c, IgG2c/IgG2a, Ig G2a/IgG2b or IgG2c/IgG2b, or each Isotype IgG derived from mice for anti-T cell/anti-target cell specificity 1/IgG2b, IgG2b/IgG1, IgGI/IgG2a or IgG 21. The antibody molecule or fragment thereof according to claim 20, which is 2b/IgG2a. 22. Has two first heavy chains, at least one of which is associated with its corresponding light chain. The combined antibody molecule and its fraction that retains the binding affinity of the whole molecule. The antibody molecule or the antibody molecule according to any one of claims 12 to 16, which is substantially free of fragments. fragment. 23. It has two heavyweights with primary binding affinity, at least one of which is one of which is an antibody molecule in combination with its corresponding light chain and the binding affinity of the entire molecule. Any of claims 17 to 21 substantially free of fragments thereof possessing the same property. The antibody molecule or fragment thereof described in (1). 24. F(ab')2 fragments of whole molecules or T cell binding affinity Claims 1 to 23 are fragments of the entire molecule lacking only the Fc region of the heavy chain that has A fragment of an antibody molecule according to any of the above. 25. Primary binding affinity for human T cell receptors that can activate lethality bispecific antibodies having a second binding affinity for immunoglobulins and immunoglobulins. F(ab')2 fragment or T fragment of the entire molecule. A fragment of the entire molecule lacking only the key Fc region with cell-binding affinity . 26. A fragment according to claim 25 of an antibody molecule according to any of claims 3 to 6. . 27. a first binding affinity for a T cell receptor capable of activating lethality and Bispecific antibody conjugates with secondary binding affinity for target cells a first binding affinity for a T cell receptor capable of activating lethality; , and immunization bound to antibody molecules that have binding affinity for target cells. Comprising a bispecific antibody molecule with a second binding affinity for globulin A binding agent for a bispecific conjugate or a total conjugate characterized by The fragment that holds the identity. 28. The bispecific antibody molecule or fragment thereof according to any one of claims 1 to 26. antibody molecules or antibodies that have binding affinity for target cells. an antibody conjugate or an antibody conjugate having in combination with a fragment thereof having the is its fragment. 29. The antibody conjugate according to claim 27 or 28, wherein the target cell is a tumor cell. To. 30. An antibody molecule or conjugate according to any one of claims 1 to 29, or These fragments and a physiologically acceptable diluent or carrier composition containing. 31. Antibody molecules that have binding affinity for target cells or 31. The composition of claim 30, further comprising an allament with a tea. 32. An antibody molecule or an fragment thereof according to any one of claims 1 to 26, and An antibody molecule having a binding affinity for a target cell or said affinity A kit containing a fragment thereof having. 33. The composition according to claim 31 or claim 32, wherein the target cells are tumor cells. kit. 34. Claims 1 to 29 for use in surgery, treatment or diagnosis. Antibody molecules or conjugates or fragments thereof. 35. drugs for the treatment of neoplastic, viral or parasitic diseases; The antibody molecule or conjugate according to any one of claims 1 to 29 for producing or use of these fragments. 36. degeneration or degeneration of neoplastic, viral or parasitic diseases; 30. A method of aiding temporary suppression, comprising an antibody molecule according to any one of claims 1 to 29. or conjugates or fragments thereof as well as tumor cells, viruses Antibody molecules or binding affinity for cells or parasite cells Treat the degeneration or temporary suppression of the fragment that has a binding affinity. regression or temporary suppression of the disease, including administering the amount necessary to effectively bring about the How to assist. 37. 37. The method of claim 36, wherein the disease is a neoplasia disease. 38. The bispecific antibody molecule or fragment thereof according to any one of claims 1 to 26. 1. A method for producing an antibody, the method comprising culturing a hybridoma expressing the bispecific antibody molecule. and, if appropriate, then producing the antibody molecule by culturing the antibody molecule. bispecific antibody molecules or is the method for producing the fragment. 39. The bispecific antibody conjugate according to claim 27, 28 or 29 or A method for producing a fragment thereof, the method comprising: a first binding affinity for immunoglobulins and a second binding affinity for immunoglobulins. binding of a bispecific antibody molecule having a antibody molecules with affinity or one or both of these antibody molecules. fragment and, if appropriate, then the resulting conjugate. Bispecific antibody conjugates comprising processing to generate fragments or the method of manufacturing the fragment. 40. Bispecific antibody component produced by the method according to claim 38 or 39. children or conjugates or obvious equivalents thereof.