CH672796A5 - - Google Patents

Description

DESCRIPTION This invention relates to the production of a polydome and hybrid monoclonal antibodies which have dual specificities. It also relates to the hybridomas obtained and the hybrid monoclonal antibodies.

The antigen-antibody reaction has been routinely evaluated in a variety of practical applications and is being extensively researched to introduce its value in other, as yet untested, uses. E.g. For example, serum antibodies obtained by the immune response of a rage to an immunogen can be used in affinity purification procedures to isolate the immunogen from solutions in which it is present in minimal amounts.

In other circumstances, if the immunogen is an antigen associated with a disease, its presence in the patient's serum or other body fluid can be detected using immunoassay or immunometric techniques. E.g. the detection of HBsAg using radioimmunometric technology is one of the most common of the selected methods. In another context, the antibodies to ferridine obtained from white New Zealand rabbits and labeled with 131I are said to be promising for the treatment of liver tumors. (See Order et al, International Journal of Radiation Oncology, Biology and Physics, 6, 703 [1980].)

Serum antibodies, e.g. those of rabbits, murine species or other mammals are "poly-clonal" in nature because the host's immune system is stimulated to be a mixture of specific antibodies directed to various antigenic determinants or epitopes on the immunogen to which the Host responds, are directed to manufacture. The individual antibodies that make up the mixture are all the product of a B cell clone; moreover, each B cell secretes only one antibody species. The antibody produced by one clone differs from an antibody against the same antigen produced by another clone in that it has at least a slight difference between its peptide sequence and that of the other antibody. Thus, indeed, each antibody species is a different molecule and the differences in the peptide sequence between different species cause their general specificities as well as the individual epitopes they recognize and their affinities for the antigen.

A single B cell cannot be indefinitely grown using the cell culture techniques previously available to obtain antibody species that are excreted as a pure compound. Recently, however, Köhler and Milstein discovered and described a method by which a monoclonal antibody can be obtained favorably as a secretion product of a hybrid cell, which was referred to as a “hybridoma”. (G. Kohler and C. Milstein, Nature, 256,495 [1975].) Basically, the method involves the fusion of spleen cells taken from an immunized mouse with mouse myeloma cells to form the hybridoma.

Myeloma cells which do not produce or secrete their own immunoglobulin or parts thereof are preferred. Cell cultures obtained by cloning a single hybridoma secrete identical antibody molecules, which can then be easily obtained as pure chemical compounds. This technique is in contrast to the usual antibody production, for example from serum, where each antibody is only a single one from the components of an essentially inseparable antibody mixture of related but chemically distinguishable compounds.

Since it is a pure compound, the monoclonal antibody has a constant specificity for a specific center on the antigen molecule and a well-defined affinity. Thus, clones of different hybridomas can be "screened" to select the one that produces the monoclonal antibody with the most desirable properties for a given application. The immortality of the hybridomas guarantees an almost unlimited supply of the secreted antibodies, and reduces problems in connection with the change in antibody type and the overall affinity from animal to animal, which are used for the production of serum antibodies. Monoclonal antibodies obtained from hybridomas are for practical use in diagnostic kits. A selection of such kits is available from Hybritech, Inc.

Usually an antibody molecule has a single specificity, which is against the immunogen, against which the

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The host's immune system has responded by forming the antibody. The antibody is composed of two identical halves, each comprising a heavy and light chain pair, and each of which recognizes the other's same antigenic determinant. The arrangement of the heavy (H) and light chain (L) in an antibody molecule is shown below.

CS.

SV

An -S-S-disulfide bridge, which connects the two heavy chains together at the site of the cysteine residues, can usually be selectively cleaved in vitro by a mild reduction, and half the molecules are dissociated by subsequent acidification. The half molecules can then be recombined (renated), again in vitro, at neutral pH, the reunification takes place through a non-covalent interaction.

If antibodies of different specificities are subjected to selective cleavage of the disulfide bridges between the heavy chains and conditions are created for the subsequent renaturation, the reunification between the half molecules can take place randomly in such a way that a population of antibodies is generated, at least some of which are hybrids, in which half of the antibody molecule of one specificity is combined with half of an antibody molecule of different specificity. E.g. describes in Nisonoff et al, "The Antibody Molecule", Academic Press, New York (1975), on pp. 260-261 an in vitro production of a polyclonal anti-antibody hybrid of a rabbit anti-ovalbumin and anti-BGG antibody. Hybrid monoclonal antibodies have also been obtained using an analogous procedure. See D.M. Kranz et al, Proc. Nati. Acad. Be. USA, 78, 5807 (1981). At least theoretically, the hybrid antibody will have a dual specificity in one half of the antibody that will recognize and bind one antigenic determinant or epitope, while the other half will recognize a different epitope on the same or different antigen.

Although the hybrid antibodies can be obtained in the manner described above, the yields are often very low, the reactions used make them difficult to reproduce, and the hybrid antibodies usually suffer significant irreversible denaturation. Such denaturation can reduce immunoreactivity and it would be expected that various metabolic characteristics would be obtained in vivo. As a result, the hybrid antibody remains for the most part a laboratory curiosity that is difficult to obtain.

Dual specificity antibodies can also be made by conjugating pairs of intact antibodies, monoclonal or others, using various coupling or crosslinking agents such as Protein A (from Staphylo-coccus aureus), carbodiimide and bifunctional compounds such as N-succinimidyl-3 - (2-pyridyldithio) propionate to obtain dimers or higher antibody multimers, of which each member of the antibody pair contributes its specificity. E.g. Mandoche et al. have described the formation of multivalent antibodies which have dual specificities by a sequential reaction of antibodies with Protein A, which has been shown to be able to detect cell surface antigens in vitro. See J. Immunological Methods, 42, 355, (1981). According to their method, anti-5 bodies can be bound to the surface antigen with one specificity and the others to a rest, which allows the detection.

The synthesis of dual specificity antibodies by the foregoing techniques is complicated and therefore no commercial use has been made of them. Objects of the present invention are as follows:

a) Process for producing a polydome according to claims 1, 10 and 27

b) Process for the production of hybrid antibodies with 15 other specificity according to claim 28.

The present invention provides, among other things, a new fully biological process for the reliable production of hybrid monoclonal antibodies in good yields without denaturation. In this specification, the 20 "hybrid antibody" is used to refer to a single antibody molecule that has two different specificities. The individual specificities can be directed to the antigenic determinants on two different antigens or to different antigenic determinants (epitopes) on the same antigen. In addition, the term "antigen" also includes haptens unless otherwise stated.

According to the invention, hybrid antibodies with a dual specificity are obtained by fusing a hybridoma, preferably a selectively destructible hybridoma, which secretes an anti-301 body against a preselected antigenic determinate with a fusible B lymphocyte or a second hybridoma, the B lymphocyte or the second Hybridoma secretes a second antibody against a different antigenic determinant, whereby a second generation of Hybri-35 thorn (hereinafter "polydome") is formed. As used herein, the term "selectively destructible hybridoma" means a hybridoma which lacks or at least predominantly lacks the ability to survive in the medium in which the polydome is cultivated. It was unexpectedly found that the 4o polydome, in contrast to the mother hybridoma or the B-lymphocyte from which it was derived, secretes a population of identical antibodies with a single specificity, the polydome additionally containing a high percentage of a monoclonal hybrid antibody a dual specificity 45. Dual means the ability that the antibody can recognize the antigenic determinants that are produced by the individual antibodies generated by the mother cells and is able to combine with both determinants at the same time. The hybrid monoclonal antibodies obtained in this way are not subject to the undesirable denaturation which characterizes the hybrids obtained by the methods of chemical recombination of antibody half-molecules. Furthermore, the method according to the invention allows the hybrid reliable and in large quantities.

The antibodies obtained according to the invention are useful for immunodiagnostic and immunotherapy methods. In general, these methods use a monoclonal antibody or polyclonal antibody with a first specificity against a 60 target antigen and a second specificity against a substance, e.g. another antigen or hapten or which allows diagnosis based on the target antigen or which permits its release, or is itself an agent which is lethal for the target antigen or the tissue which is associated with it. 65 According to the present invention, a suitable selection of the mother cells can thus be used to obtain a polydome which secretes an antibody which has a specificity for a target antigen and a second specificity for one in diagnostics

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or therapy has useful rest. Alternatively, antibody half-molecules can be recombined using in vitro chemical means, or single intact monospecific antibodies can be coupled or cross-linked by chemical means to obtain antibody multimers (which can be dimer, trimer, or higher multimer) with dual specificity and which have the same or similar utility as hybrid monoclonal antibodies with the same dual specificity made in accordance with the present invention.

In the present description, the antibodies also include antibody fragments that have immunochemical properties, such as Fab or F (ab) 2 fragments.

The manner in which these and other items are accessible will become apparent from consideration in the following description of the preferred embodiments.

As indicated above, the method for obtaining a hybrid monoclonal antibody according to the present invention requires, as an origin, a hybridoma and preferably a selectively destructible hybridoma which secretes a monoclonal antibody against a preselected antigenic determinant or epitope. The use of a selectively destroyable hybridoma as an origin has the advantage that it prevents the cells obtained by fusing the selectively destroyable hybridomas with B lymphocytes or a second hybridoma, i.e. the polydome is overgrown by a population of the mother hybridoma when the cells obtained by the fusion process are cultured and a means is available by which the polydome cells can be isolated from the mother hybridoma cells.

Selectively destructible hybridomas useful in the invention are available from hybridomas that secrete an antibody that has one of the desired specificities and are by the classic Kohler-Milstein method, i.e. Hybridomas obtained by fusion of a myeloma cell and a B lymphocyte, such as those of mouse spleen cells.

According to one embodiment of the invention, a hybridoma is subjected to a re-selection process in order to obtain the hybridomas which are selectively destructible.

In general, selective destructibility is obtainable by reselecting a hybridoma which lacks a genetic component which is necessary for its survival in a chosen medium and in which medium the polydome is due to the genetic contribution of fusion partners of the selectively destroyable hybridoma, i.e. H. the B lymphocyte or second hybridoma, can be grown by fusion.

The currently preferred re-selection process involves culturing a hybridoma that secretes an antibody that has one of the desired specificities to be incorporated into the hybrid antibody in a growth medium containing 8-azaguanine. In such a medium, every cell which absorbs 8-azaguanine and can therefore grow lacks the enzyme hypoxanthine guanine phosphoribosyl-trans-ferase (HPRT). Clones of cells lacking this enzyme cannot grow in a medium containing hypoxanthine aminopterin thymidine (HAT). As a result, these can now be selectively destroyed in this medium.

A very similar re-selection procedure involves growing the hybridomas which secrete the desired antibody in a medium containing 6-thioguanine, another analogue of guanine, which is toxic to the cell if it is incorporated into the DNA. Again, certain cells that will grow in this medium lack the HPRT enzyme, and clones of these cells will necessarily be sensitive to the HAT medium.

Another re-selection process that can be used involves culturing cells of the selected hybrid dom cell strain in a medium containing one of the thymine analogues 5-bromouracyl-deoxyribose (BUdR) or 2-aminopurine. Only those cells that lack the enzyme thymidine kinase (TK) can grow in a medium that contains one of these two inhibitors. As in the case of cells lacking the HPRT enzyme, the cells lacking TK become

do not grow in a HAT medium.

Another method for obtaining a selectively destructible mother hybridoma involves irreversible enzyme inhibition using metabolic inhibitors. Among these, the so-called K ^ inhibitor is preferred. These inhibitors are analogs of the enzyme substrates which are converted by the target enzyme into a highly reactive molecule which reacts with the enzyme in its active center, resulting in the irreversible inhibition of the enzyme. E.g. Treatment of the selective hybridoma with an analog of glutamine, such as azaserine or 5-diazo-5-oxa-L-norleucine (DON), irreversibly inhibits the enzyme formylglycine amide ribonucleotide amidotransferase by forming a covalent bond with a cysteine residue in the active center of the enzyme. This inhibition will ultimately result in the death of the cell. However, the hybridoma can be saved by fusion with the second mother cell of the polydome, which provides the necessary enzyme.

In a preferred embodiment, the selectively destructible hybridoma is fused to a complementary B-lymphocytus, typically from spleen cells from a host that has been previously immunized with an antigen, which may be a hapten bound to a carrier protein, to produce an immune response in the host which produces antibodies that have the second specificity that is desired in the hybrid antibody. The host is usually a mouse, but rabbits or other animals and humans can be used, although interspecies fusion can result in a lower order of stability. The method of immunizing such a host is of course well known and details need not be given here.

The fusion of the selectively destructible hybridoma with the B lymphocyte to obtain the polydome can be carried out by combining two groups of cells in a medium known as a means for promoting cell fusion, such as polyethylene glycol or Sendi virus, according to known methods.

After the fusion, the cells are transferred to a medium such as the HAT medium for cultivation. The B lymphocytes only survive for a short period of time and the mother hybridoma cells cannot grow in the medium. However, the population of the polydome formed as a result of the fusion may be due to the complementation of the mother hybridoma by the B lymphocytus, e.g. by the genetic contribution of the ability to make missing enzymes, such as HPRT or TK, or the direct contribution of an inhibited enzyme in the mother hybridoma in the medium. Single polydome clones are cultured and screened to select those that secrete antibodies that have the desired dual specificity. Clones of polydomes whose antibodies have the desired dual specificity are subjected to further screening to select those whose specificity, i.e. the one obtained from the B lymphocytes and the affinity of which is most desired.

In a further embodiment, the polydome is obtained by fusing selectively destroyable hybridomas using a suitable fusion agent with a second hybridoma, which is also selectively destroyable. The second mother hybridoma is obtained in the same way as the first, i.e. by the process of reselection, irreversible enzyme inhibition or other suitable techniques. In such a case, the second hybridoma must be able to do the first

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complement. For example, if the first selectively destroyable hybridoma lacks the HPRT enzyme, the second must be able to contribute to the polydome a gene that enables the polydome to recognize HPRT. Similarly, if the second selectively destroyable hybridoma lacks the TK enzyme, a TK gene must be added to the polydome. A similar complementation must exist between the two hybridomas when the irreversible inhibition of an enzyme is carried out to give it selective destructibility. It is also possible to use as a mother hybridoma a hybridoma which has been subjected to a back selection process and as another to use a hybridoma which has been subjected to the process of enzyme inhibition.

The use of complementary selectively destructible hybridomas as primary cells for the polydomes has the advantage that both primary cells can be selected based on the specificities and affinities of the monoclonal antibody they produce, and in the case of a single hybridoma fusing with B-lymphocytes, none can Pre-fusion selection can be made among B lymphocytes to obtain those that produce an antibody of the desired specificity and affinity.

The fusion of the two selectively destructible hybridomas can be carried out using polyethylene glycol or using other fusion agents, again according to known methods. After the fusion, the cells are transferred into the growth medium, in which the two primary cells cannot grow, but however in which the polydomes resulting from the fusion have the ability to grow because of the complementary contributions of the primary cells.

Up to here we have discussed the selection procedures for obtaining selectively destroyable hybridomas for use as both of the hybridoma partners in a hybridoma-hybridoma fusion to form a polydome. However, the need for selective destructibility for the hybridoma cell can be avoided by giving the first hybridoma cell a dominant and a recessive label. The currently preferred method is the HAT ouabain selection. The drug ouabain is a specific inhibitor, the Na + -K + -activated ATPase of the plasma membrane. This enzyme is responsible for the introduction of K + into a cell and for the execution of Na + from the cell. Hybridoma cells which have been preselected to confer selective destructibility, e.g. HAT sensitivity, are grown in ouabain medium to select ouabain resistant cells. Clones of these cells are sensitive to HAT, but ouabain-resistant. In contrast, the hybridomas selected for fusion with it are ouabain sensitive, but can survive and grow in HAT. Alternatively, selection for ouabain resistance to destruction can be performed on the mother myeloma strain or on the hybridomas derived therefrom, followed by reselection or other techniques to confer selective destructibility.

Cells obtained by fusion of two hybridomas in polyethylene glycol or other fusion agents are transferred into the HAT medium, which contains ouabain in a lethal concentration for the second mother hybridoma. The selectively destroyable hybridoma cells can in the HAT medium, which e.g. HPRT or another enzyme is missing

do not survive, although they are ouabain resistant. However, the poly-dom cells can grow in the medium because they have the enzymes and the ouabain resistance that are necessary for survival. The previous method has the advantage that it is possible to obtain a polydome by directly fusing a selectively destructible mother hybridoma, which secretes an antibody with one of the specificities desired in the hybrid, with a second “off the shelf” hybrid dom which has an antibody which has the other specificity desired in the hybrid and does not require the use of techniques for conferring selective destructibility on the second mother hybridoma.

Yet another technique for obtaining a polydome that uses a universal original cell, i.e. one that has a positive and a negative label that can be fused to any off the shelf hybridoma involves the use of recombining DNA vectors that have different drug resistant labels. E.g. SV40 carrying a gene for neomycin can be used.

A currently preferred universal primary cell is one that is HAT sensitive neomycin resistant. The selected primary cell is preselected for HAT sensitivity and then transfected with an SV40 rector, which carries a gene for neomycin resistance. This procedure can also be reversed, with the transfection being carried out first. The hybridoma obtained can grow in the presence of neomycin, which is normally toxic to mammalian cells, but dies in the presence of HAT. However, off-the-shelf hybridomas grow in HAT, but die in the presence of neomycin. The products of the fusion of the primary cells therefore survive in the presence of HAT and neomycin. While the use of vectors to transmit neomycin resistance is currently preferred, vectors bearing genes that confer resistance to other drugs to mammalian cells can also be used.

Even if it is currently preferred, it is not essential for the method according to the invention for the production of polydomes from pairs of hybridomas that at least one of the original cell strains is selectively destructible. It is within the scope of the invention to fuse a pair of hybridomas, none of which are selectively destructible, but which secrete antibodies that have one of the desired specificities of the hybrid in the presence of a suitable fusion agent, followed by subcloning all cells before the Population of the unmelted mother hybridomas has increased to such an extent that the screening of the subclones to identify the polydomes is not practical. The subclones are then subjected to screening in order to obtain those which secrete antibodies with a dual specificity.

This method is best suited to the polydomes obtained when the fusion frequency of the original cell strains is high. In any event, and especially when the cell fusion frequency is low, the cells obtained from the fusion of hybridomas whose monoclonal antibodies are directed against various antigens can be screened using a cytofluorograph to identify the polydomes. To do this, samples of the two antigens are provided with different fluorescent residues whose fluorescence occurs at different wavelengths. E.g. one can be labeled with fluorescein and the other with rhodamine. The population of the cells of the fusion, which have preferably been cultivated overnight or during another suitable period to increase their number, are incubated with two labeled antigens. The cells are then screened using the cytofluorograph. These cells, which only fluoresce at one of the two wavelengths, are those of the cell strains from the mother hybridomas. However, when cells fluoresce at both wavelengths, they are polydomes that can be isolated and cloned.

In yet another embodiment, a polydome can be made directly by removing the nucleus of a first hybridoma that secretes a monoclonal antibody that has the specificity desired in the hybrid and inserting it into the citoplasm of a second hybridoma that has a monoclonal antibody with the second

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desired specificity. Of course, none of the mother hybridomas need to be selectively destructible to be used in this process. After the nuclear material is inserted, the cell is cloned to obtain a population of the polydome.

We have found that unlike the mother hybridomas or B lymphocytes which secrete a single antibody, the polydome cells obtained according to our invention secrete a mixture of antibodies, at least one of which is a hybrid antibody with dual specificity. Also produced by the polydome are relatively smaller proportions of antibodies of the same specificity as those produced by the primary cells used to obtain the polydome. The ratio of the hybrid to the monospecific antibody is about 2: 1: 1, which is the expected if the polydome produces equal proportions of all possible (H) chains as they do through the primary cells, which are combined in a random arrangement in the polydome itself.

The polydomes can be cultured in vitro or cultured in vivo genetically compatible animals or in nude mice to obtain large amounts of the hybrid antibody which is obtained from the culture medium or the ascitic fluid of the animal by known methods. See e.g. the protocols in Monoclonal Antibodies, edited by Kennett et al, Plenum Press, New York (1980), pp. 363-418.

The mixture of antibodies produced by the polydome can be separated to obtain the hybrid. For example, sequential affinity chromatography against the first and then against the other antigen for which the hybrid is specific allows it to be separated from the monospecific antibody contaminants. We have also found that simple ion exchange chromatography and electrophoresis techniques can also be used, at least in certain circumstances. If necessary, the charge difference for the ion exchange could be one of the characteristics of the antibody, which is taken into account when selecting the original cell strains.

example 1

A hybrid monoclonal antibody with a dual specificity for the hepatitis B surface antigen (HBsAg) and for the prostanoic acid phosphatase (PAP) was produced according to the present invention in the following manner:

A hybridoma that secretes a monoclonal antibody against PAP was grown in HAT medium for one week and then transferred and grown in a non-selective medium. After various periods of growth under non-selective conditions, 2 ml aliquots of cells were placed in a medium containing 10 ~ 4 M 8-azaguanine, which prevented the cells from growing by incorporating 8-azaguanine in their DNA instead of guanine . The cells lacking the HPRT enzyme survived and grew in this medium and these cells did not necessarily survive in HAT.

Clones that grew in the 8-azaguanine-containing medium were tested for HAT sensitivity and for anti-PAP production. A clone that still produced anti-PAP and had HAT sensitivity with a reverse sequence of less than 4 x 10 ~ 8 was subcloned. All subclones behaved like the mother clones.

Cells from one of the HAT-sensitive subclones were fused in polyethylene glycol with spleen cells fused from Balb / c mice hyperimmunized with HBsAg to obtain polydomes using Gerhard's fusion technique. See "Monoclonal Antibodies", mentioned above, on p. 370. The fusion generated 220 polydomes, which were screened to determine those secreted antibodies that showed specificity for PAP and also for HBsAg. Clones of two such polydomes were determined to produce the antibody and then ascites, which showed both specificities.

Subclones of the two polydomes continued to produce ascites, which showed both specificities, and gave triple bands on an Orstein-Davies PAGE, like those of the mother clones. The ascites of both clones were able to bind 125I-HBaAg and also 125I-PAP in radioimmunoassays and gave Ka values of about 109 for each, indicating the formation of antibodies with two specificities.

The data in Table 1 below show the results obtained from immunoassays using ascites from a clone of one of the polydomes compared to ascites from hybridomas secreting monoclonal antibodies or against IgE (used as a control), PAP and HBsAg, respectively Use of immobilized HBsAg as the solid phase and a number of radiolabeled antigens as the soluble phase.

A 200 μl sample of the ascites of the polydome and each of the three hybridomas were all incubated overnight with 12 polystyrene balls to which HBsAg was bound. The HBsAg spheres were obtained from Abbott Laboratories, North Chicago, Illinois. After washing, the triple sample was incubated for 4 hours with 100,000 cpm of the indicated I25I-labeled antigen. After a second wash, the balls were counted to determine the level of labeled antigen bound to the balls. In one set of experiments using radiolabelled PAP as the solution phase antigen, anti-PAP was added to the antigen before it was incubated with the beads. The antibody to PAP used for this purpose is the monoclonal antibody produced by the parental hybridoma used to make the polydome and is thus against the same PAP epitope as expected from the hybrid antibody has been.

Table 1

Results of the radioassays demonstrating the presence of hybrid antibodies with dual specificity in polydome ascites

Specificity cpm cpm cpm * PAP +

cpm of ascites

* HBsAg

* PAP

anti-PAP

* IgE

anti-IgE

15,340

2,145

2,930

3,290

anti-PAP

16,280

2,956

3,128

3,180

anti-HBsAg

73.02

2,973

2,870

3,330

guessed

Dual specificity

78,900

82,533

2,936

3,143

* with 125I-labeled antigen

The data in Table 1 indicate that only the radiation expected from non-specific binding was measured in the anti-PAP ascites when compared to that in the IgE control. The ascites containing the HBsAg antibody which, on the other hand, were bound to the labeled HBsAg antigen as expected, but showed non-specific binding when the other labeled antigens were tested. However, the ascites from the polydome clone bound labeled HBsAg and also labeled PAP, the former is attributed to the presence of some non-hybrid monospecific antibody for HBsAg in the ascites, and the latter becomes a hybrid that can bind and bridge the HBsAg to the ball and attributed to traces of labeled PAP in the solution. The experiment in which a mixture of labeled PAP and anti-PAP from the mother hybridoma was used confirms that the anti-PAP specificity of the hybrid is for the same epitope as the antibody secreted by the primary cell because of

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inhibition of the binding of the labeled PAP to the hybrid antibody by the mother antibody, only background radiation is observed.

To analyze the ascites from the polydomclone used in the comparative radioassays, polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis (IEP) were performed. Both indicated the presence of at least three species of antibodies. Preparative-scale DEAE ion exchange chromatography revealed three well-separated peaks, the middle one of which had a shoulder. Each of the peaks was homogeneous and was analyzed by PAGE and IEP and each corresponded to one of the bands of the original ascites.

The material corresponding to each of the DEAE peaks was checked for antigen binding using radiolabeled HBsAg and PAP. The first peak bound HBsAg, but not PAP. The middle peak and its shoulder bound HBsAg and also PAP and the last peak bound only PAP. The mean peak is thus the hybrid antibody, which has a dual specificity for HBsAg and PAP and contains at least two subspecies.

The hybrid antibody obtained as the middle peak of DEAE chromatography was radiolabeled with I25I. After labeling, 85 percent of the labeled antibody should be bound to PAP and 88 percent to HBsAg. The affinity of the hybrid for PAP was determined a little lower than that of the monoclonal antibody for PAP generated by the original cell strain. The difference in affinity was about the same as that observed by us between the monoclonal antibody and its Fab fragment.

DEAE chromatography shows that the hybrid antibodies contain more than 50 percent antibodies that are generated by polydome and roughly approach the 2: 1: 1 ratio, as predicted for statistical reasons, when the polydome synthesizes all possible heavy chains of the antibody , d. H. those that have a PAP or HBsAg specificity that are randomly combined in the cell to produce the hybrid antibody to which the two monospecific antibodies that have the same specificity as those that are mixed in smaller proportions through which primordial cells were produced. The existence of subspecies of the hybrid antibody suggests that they can differ in the composition of their light chain.

Example 2

The hybrid monoclonal antibodies, which have dual specificity for human IgD and prolactin, were produced according to the present invention by the fusion of two hybridomas, one of which was designed to contain two selectable genetic labels: sensitivity to HAT medium and resistance on Ouabain. This double-labeled hybridoma or so-called “universal primary cell” could then be fused with any other hybridoma. The polydomes obtained grew in the presence of HAT and ouabain, while each unfused primary cell died. The advantages of using a "universal original cell" have been described elsewhere here. To construct such a universal primordial cell, both selectable markings were introduced during the original construction of the hybridoma. The widely available HAT sensitive mouse myeloma P3.653 was selected to obtain this primary cell strain, for a second genetic marker, the ouabain resistance by introducing 1 mM ouabain into the growth medium. While most cells died, approximately 1 / 100,000 cells, by chance mutation, acquired resistance to the drug and survived and increased to form a new myeloma population that was HAT sensitive and ouabain resistant.

This HAT-sensitive, ouabain-resistant myeloma was then fused to spleen cells obtained from Balb / c mice hyperimmunized with IgD using the Gerhard technique previously mentioned. Hybrids were selected in HAT medium (without ouabain) and the clones were screened to produce the anti-IgD monoclonal antibody. Among the positive clones, one was selected for further studies that produced IgG against IgD. These clones were tested for the behavior of the ouabain resistance trait by adding 1 mM ouabain to the growth medium. About a third of the cells have retained this genetic label. As the culture grew exponentially in ouabain, the cells were subcloned. Ouabain-resistant subclones were tested for the continuous production of the anti-IgD monoclonal antibody. One of the subclones was further reselected following the procedure of Example 1 to obtain a population of cells sensitive to HAT. This subclone was grown under non-selective conditions for two weeks and then placed in a medium containing 6-thioguanine. As previously noted, the mechanism of the reaction of 6-thioguanine is similar to that of 8-azaguanine. Cells that incorporate 6-thioguanine in their DNA instead of guanine did not grow. Cells that lack the HPRT enzyme do not use the 6-thioguanine from the medium and can therefore grow, but are consequently sensitive to HAT. This population of the reselected subclone was then subcloned itself in a medium with 6-thioguanine and ouabain. The subclones were tested for continued production of the anti-IgD monoclonal antibody. A clone, which showed all the desired characteristics, grew in ouabain and 6-thioguanine as well as the production of the monoclonal anti-IgD for the so-called "universal primary cell" was selected. This universal primordial cell could then be fused with any other HAT-resistant, ouabain-sensitive hybridoma to produce a polydome, which is a hybrid antibody, one specificity of which was anti-IgD. For this purpose, we originally chose a mouse hybridoma that secreted a monoclonal antibody that was directed against prolactin. The antiprolactin monoclonal antibody is of the same subclass (IgGi) as the anti-IgD, which is expressed by the urine strain and which can be easily separated from this antibody by Ornstein-Davis gels. Such a separation indicates large charge differences on the antibodies and should allow the easy isolation of a hybrid antibody by DEAE-Sephadex chromatography.

107 cells of this HAT-sensitive, ouabain-sensitive cell strain were fused in polyethylene glycol with 107-antiprolactin-producing cells. The fused cells were grown for 3 days in the HAT medium, then again supplied with HAT + ouabain medium for 3 days and finally added back to the HAT medium. More than 600 clones emerged from this fusion: 66 clones were randomly selected for analysis. Of these clones, 66 showed anti-IgD and also anti-prolactin activity.

These clone supernatants were tested for the presence of hybrid antibodies by the following experiment. A polystyrene bead coated with another antiprolactin monoclonal antibody was incubated with 200 µl of a 100 mg / ml prolactin solution for 5 hours. The antibody used binds the prolactin at a pronounced center of the antibody produced by the fused hybridoma cell strain. The bead was washed, then incubated with the clone supernatant overnight. The following day, various washes followed, IgD labeled with 125I was added. The hybrid antibody bound to the bead by a functional arm

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was able to bind the radiolabeled IgD with the free anti-IgD functionality, whereas no antibody of the original type IgD-IgD or prolactin-prolactin could build this bridge between the prolactin bead and the i25I-IgD tracer. Results of a typical test for clones that produce hybrid bifunctional antibodies are shown in Table 2 below:

Table 2

Results of the immunometric tests show the presence of hybrid antibodies, which have a dual specificity for IgD and prolactin, in selected polydome supernatants and ascites.

Clonate No. cpm125I-IgD tracer bound

1 15339

2 16337

3 22886

4 23356

5 24434 anti-IgD 9357 anti-prolactin 8721

21 of the 36 clones showed pronounced bifunctional activity in this test. The reaction of ascites formed by two previously available clones was shown in the bifunctional test. These ascites contain antibodies that can be separated into three distinct bands on the Ornstein-Davis gels: Two bands match exactly with the antibodies generated by the mother hybridomas (anti-IgD and anti-prolactin). The third band migrated between the bands of the parent monoclonal antibodies as expected from the hybrid antibody.

That a hybrid monoclonal antibody against IgD and frolactim shows a dosed reaction in the test that provided the data in Table 2! by the data in the ® Table 3 using the antibody the »clonate No. 2 and as a control AntSköiper the Urzelli strains (EAnti-IgD * and anti-prolactin).

Table 3

Results? von-Tests ^ using hybrid antibodies and varying proportions of prolactin;

Prolactiir

Hybrid-

anti-

anti-

ng / ml

antibody

IgDl

Prolactin1

0

8010

6847

8020

10th

9169

7982

7405

50

13558

7783

8314

100

17599

7654

7844

i: emp from I2SF-FgE. bound

These requests) show that; the portion of prolactin bound by the antibody is dose sensitive, as is the portion of the labeled IgD that is bound increases with the dose of the prol'actm. In contrast, dose sensitivity was found when the mother antibodies to IgD and prolactin were used, as they cannot bridge the prolactin bound to the bead and the labeled IgD. Thus, the hybrid antibody can be used as a component of an assay for prolactin. Tailoring other hybrid antibodies provides similar opportunities for other tests.

Example 3

Using a technique similar to that in Example 2, a universal mother hybridoma is generated, which is a monoclonal

Antibody directed against the hapten-arsenate-arsenate dimer, which is both ouabain-resistant and sensitive to HAT. This hybridoma was fused with a hybridoma which secretes a monoclonal antibody with a specificity for the carcinoembrionic antigen (CEA), which is characterized both by embryonic tissues and by different types of carcinomas. Again, more than 600 clones were obtained from the fusion. Of 72 clones tested for their ability to bind CEA and Arsenal, 69 io both had binding activities. The clones with the greatest binding activity were selected for the enzyme-linked immunoadsorbent test (ELISA) of the hybrid antibody. For each test, a CEA solution (600 ng or 250 ng) was adsorbed in each well of a 15/96 well plastic microtiter plate. The next day, the unadsorbed material was washed out of the wells with PBS-Tween 20. The clone supernatants were added and incubated for 2-8 h at 35 0 C and then washed before the plate. CEA-CEÄ and CEA arsenate antibodies remain bound on plate 20 via the adsorbed antigen. The second antigen, arsenic acid-coupled to the enzyme alkaline phosphatase, was added to the wells at 35 ° C. for 3 hours. After a further wash with PSB-Tween, a chromagen substrate for alkaline phosphase, para-nitrophenyl phosphate was added to the wells and color developed for 48 hours. Absorbance in each well was measured at 410 nm. If present in the clone supernatant, the hybrid antibody, which is bound to the adsorbed CEA on the plate via a functionality, releases the other in order to bind the arsenic acid to the alkaline 3o phosphatase. The color of the chromagen substrate is only developed if the alkaline phosphatase coupled to the arsenic acid was bound. In this test, 3 out of 12 supernatants showed antibody activity as shown in Table 4.

35 Table 4

Demonstration of the hybrid antibody with dual specificity by the enzyme-bound Ikimunoadsorptions- (ELISÄ) test

Clone no.

Absorption at 490 'nm 600 ng CEA / joining

Absorption at 490 nm 250 ng CEA grouting

1

0.087, 0.105

0.022, 0.060

2nd

0.082, 0.054

0.023, 0.033

3rd

0.011, 0.017

0.041, 0.036

Anti

arsenate

0.010, 0.006

0.006.0.005

The present invention also provides methods for immunodiagnosis and immunotherapy using antibodies with a dual specificity, e.g. B. of hybrid antibodies obtained as described above, or from antibody half-molecules by the known technique of Niso-55 noff et al, as stated above or antibody multimers obtained by coupling or crosslinking individually monospecific antibodies. Preferably, the dual specificity antibody used in these methods is a hybrid antibody made in accordance with the present invention, as well as an antibody that can be reliably obtained as a substantially pure substance and does not undergo denaturation and which has uniform specificity and affinity for the antigen.

In the case of immunodiagnostic applications, one of the specificities of the hybrid or other antibody of dual specificity is directed against the target antigen whose detection is desired and the other against another antigen, which may be a hapten or other molecular genus

672 796

10th

the diagnosis allows. E.g. an antibody useful for immunohistology should have a first specificity for a suspect antigen, for example an antigen associated with a tumor, such as CEA, PAP or ferritin, and a second specificity for a hapten or antigen that participates in a color reaction, such as Enzvm. which triggers a color reaction in the presence of a suitable substrate. Among the suitable enzymes to which the second specificity of the antibody can be directed is prostanic acid phosphatase (PAP) horse radish peroxidase, glucose oxidase and alkaline phosphatase.

To perform the histological test, a tissue section is first treated with the antibody of dual specificity. Before this, the hybrid can already be linked to an enzyme that catalyses the color reaction. If this is not the case, the section is then treated with a second solution which contains the enzyme and rinsed after due incubation and then treated with a substrate which is subjected to a color change in the presence of the enzyme. The formation of the color produced by the enzyme and the substrate in the tissue sample is a positive indicator of the presence of the target antigen in the tissue. It has been found that the hybrid antibody against HBsAg and PAP, the production of which is described here, is bound to HBsAg on a test substrate (polyslylene beads) and to PAP in a simultaneous staining test using p-nitrophenylphosphate as the enzyme substrate. After incubation of the hybrid antibody with PAP and HBsAg, the addition of p-nitro-phenyl phosphate causes the spheres to be subjected to the characteristic color change from yellow to brown. As indicated in the example, the dual specificity antibody can also be used in immunoassays and immunometric assays. If the hybrid antibody to HBsAg and PAP, the preparation of which is described above, is used, an immunometric test for HBsAg can be carried out on an immobilized monoclonal antibody for HBsAg as a solid phase to remove HBsAg from a serum or other liquid sample from which it is suspected that it contains the antigen. The sample is incubated with a ball, bead, test tube or other substrate which has the Anti-HBsAg bound to its surface or coated with it. The incubation of the serum sample can be followed by an incubation with a solution of the hybrid, carried out simultaneously or before. In any event, if HBsAg is present in the sample, the result will be a sandwich of the immobilized HBsAg antibody, if present in the sample, and the hybrid antibody. As part of the test, PAP is allowed to bind to the hybrid antibody. This can be done during or after the sandwich is formed, or alternatively, performed in the antibody-PAP complex. After the sandwich is formed, the solid phase is washed to remove the sample residue and the unbound hybrid antibody, and then contacted with a solution containing a substrate such as p-nitrophenyl phosphate or a-naphthol phosphate, and which is subject to a color change in the presence of PAP. The appearance of the color change confirms the presence of the target antigen in the sample.

In such a test, the polyclonal anti-HBsAg bead of a commercial kit for the diagnosis of HBsAg, manufactured by Abbott Laboratories of North Chicago, Illinois (commercially available under the name "Aushia"), samples with different proportions of HBsAg incubated with the beads. The samples are the positive and negative controls of the commercial kit and two samples are diluted by diluting the positive controls with negative controls in the proportions one part negative control: two parts positive control or two parts negative control: one part positive control.

After incubating the samples with the beads, order

To bind HBsAg, the bead was washed and incubated with the hybrid antibody that is reactive to HBsAg and PAP. This allows the formation of a sandwich of immobilized antibody: antigen: and hybrid antibody. The bead was washed again and incubated with a solution of PAP. This incubation was followed by another wash and the bead was incubated with the substrate a-naphthol phosphate. PAP removes phosphate enzymatically. After an appropriate incubation, the substrate was removed from the bead and an indicator Fast Garnet GBC salt was added to it, which changes from clear to a reddish purple in the presence of the enzymatic reaction product. A color change was observed, which confirmed the presence of HBsAg in the samples. In the samples, the absorbance was measured at 570 nm and this data is shown in Table 5 below.

Table 5

HBsAg concentration absorption

(% of positive control) 570 nm

0 .031

34 .166

67,243

100,379

These data show dose sensitivity with the change in HBsAg concentration expected when the hybrid antibody bridges the HBsAg bound to the sphere and the PAP. This further demonstrates the utility of a hybrid antibody in an immunoassay.

Detection agents that are different from enzymatically catalyzed reactions are also possible. E.g. The second specificity of the hybrid or other antibody with a dual specificity can be directed against a hapten or antigen which is radiolabeled or which is fluorescent or which can be determined as a sandwich by other suitable means. A preferred method which uses a hybrid antibody or other antibody with a double specificity in an immunoassay is based on the phenomenon of fluorescence quenching. In such a test, one specificity of the antibody is directed against a target antigen and the other against, for example, a hapten which carries a fluorescent chromophore. The chromophore is either bound to the hapten or, in suitable cases, is the ~ hapten itself.

The test is carried out by incubating the antibody with serum or another sample in which the target antigen is suspected, to which a predetermined amount of the target antigen in which a predetermined amount of the target antigen labeled with a quenching chromophore has been added. The labeled antigen now competes with the target antigen of the sample, if present, because the antibody binding center is specific for the target antigen. Before, during or after this incubation, an amount of the hapten which carries the fluorescent chromophore is incubated with the antibody and binds to the other binding center.

The two chromophores are selected so that the first of them fluoresce at a wavelength that can be absorbed (quenched) by the other if they are close enough so that the photon emitted by the fluorescent is transmitted by the quentcher can be caught. To do this, the two chlorophores should be within 100 angstroms, preferably within about 50 angstroms. This positioning is achieved when the fluorescent chromophore is at an antibody binding center

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

11

672 796

is bound and the quentching chromophore is bound to the other via the added antigen. A suitable pair of chromophores include fluorescein as fluorescent chromophores and rhodamine as quentching chromophores.

The measured fluorescence changes inversely with the proportion of the native antigen in the sample, since in the absence of the native antigen, all of the antigen that is bound to the antibody is labeled by the quentchin chromophore and is brought into the position in which the fluorescence is absorbed by the chromophore carried by the hapten. The comparison of the measured fluorescence with that of the control samples, which contain a known proportion of the antigen, allows a qualitative and quantitative determination of the presence of antigen in the sample.

For example, this type of immunoassay can be used to determine the level of serum drugs, such as Dilantin, which must be closely monitored. In such a test, the target antigen would of course be Dilantin. It is obvious to the person skilled in the art that this method can also be used to determine other antigens, which in particular also include tumor-associated antigens.

Another preferred method that uses hybrid antibodies or other dual specificity antibodies is an immunoassay based on an enzymatic reaction. In a currently preferred method, one of the antibody specificities is of course directed to the target antigen and the other to an enzyme or hapten to which an enzyme is linked.

The test is carried out by incubating the antibody with a sample with the suspected target antigen, into which a predetermined amount of the target antigen has been put, which is modified by binding to a substance which interacts with the enzyme to give a detectable substance generate or generate the detection or allow the detection of the formation of the antigen-antibody complex in another way. The detection method can be, for example, fluorimetry, luminescence, spectrophotometry or the like.

In an alternative method, the added target antigen may have the enzyme bound to it, the antibody having one of its specificities directed against the substance which interacts with the enzyme or against a hapten to which the substance is bound.

This substance, which interacts with the En2ym, can itself be another enzyme. In such a case, one of the enzymes catalyzes the production of a product that is required by the other. Consequently, when the antibody targets the target antigen to which one of the enzymes is bound and also binds the other enzyme, the product of the first enzymatic reaction is formed in the vicinity of the second enzyme and can undergo a reaction caused by the last enzyme is catalyzed before a pronounced diffusion of the product into the surrounding medium can take place.

An example of such a method uses the two enzymes hexokinase (HK) and glucose-6-phosphate-dehydrogenase (G-6-PDH) according to the following reaction scheme.

HK

(1) Adenosine triphosphate + glucose

(ATP)

Adenosine diphosphate + glucose-6-phosphate (ADP)

(2) Glucose-6-phosphate + Nicotinaraid adenine dinucleotide

(NAD +)

6-6-PDH

^ Gluconolactone-6-phosphate + dihydronicotinamide-

adenine dinucleotide (NADH)

Using this reaction scheme, the added target antigen bound either HK or G-6-PDH and the hybrid antibody directed one of its specificities against the other (or at a hapten that carries it). The sample is added in addition to the hybrid antibody and the predetermined portion of the enzyme labeled antigen, glucose, ATP and the coenzyme NAD +. The hybrid antibody has preferably already bound the other enzyme to itself. On the other hand, this enzyme can be added to the sample with other reagents.

During incubation, the enzyme-labeled antigen competes for the native antigen, if any, in the sample for one of the hybrid antibody's active binding centers. The other enzyme is or is bound to the second binding center. This allows the binding of glucose-6-phosphate catalyzed by HK, with G-6-PDH present in close proximity. The latter converts the glucose-6-phosphate to gluco-nol-lactone-6-phosphate, a result that is accompanied by the reduction of NAD + to NADH. The NADH absorbs strongly at 340 nm and can therefore be detected spectrophotometrically. The proportion of shaped NADH varies inversely with the proportion of native antigen in the sample, i.e.

its highest production occurs when no target antigen can be found in the sample. The comparison of the proportion of NADH formed by the control sample allows a qualitative and quantitative determination of the presence of the antigen in the sample.

This type of test can be used to control the level of Dilantin or other drugs in the serum. In such cases, the drug is the target antigen. However, such a test can also be used to detect other serum antigens, 60 such as those associated with tumors or other diseases. In vivo immunodiagnosis can also be performed using a hybrid or other antibody with dual specificity. The antibody that has directed the first specificity against a target antigen, such as a tumor-associated anti-65 gene, and directed the second against a hapten to which a suitable radionuclide is bound, preferably one that emits y-rays, is the first Host organism administered. After enough time has passed,

672796

12

After the antibody has located the target center and the unbound antibody has been allowed to remove itself from the healthy tissue of the host organism, the hapten carrying the radionuclide is administered and bound by the localized antibody.

After a suitable period of time to allow the unbound hapten to excrete from the host organism, the host organism is scanned with a suitable camera to determine whether there are areas in which the radiation has been concentrated. If there are any, the presence of the target antigen in the host organism is confirmed and its location can be determined.

This method has several advantages over that using the monospecific antibody directed against the target antigen to which the radionuclide is directly bound. In such cases, the radionuclide must have a half-life that is long enough that a sufficient quantity is retained after the time required for the main localization of the antibody at the target center has expired. In addition, during this procedure, the antibody may remain in the liver or other non-target tissue for a period of time, which are then exposed to the radiation brought by the antibody.

The method, on the other hand, allows the use of radionuclides with a shorter half-life than those used with monospecific antibodies. Since the hapten-bearing radionuclide, which is a relatively small particle, has high in vivo mobility and moves rapidly through the host organism, and is able to bind the localized antibody to the target center or to the body without significant time in the non-target tissue spend ^, free. For these reasons, low half-life isotopes can be administered in amounts that pose minimal risks to healthy tissue, even if they are essential. Excess be administered.

The hapten is preferably an agent to which the radionuclide can be directly bound or which can form a complex with the radionuclide and its agent for the radionuclide which is bound to a hapten can be used for the latter purpose. Those skilled in the art will perceive that a wide variety of chelating agents and radionuclides are suitable for this purpose. Phenylarsenate, to which ethylenediaminetetraacetic acid (EDTA) is bound as a chelating agent, is a suitable hapten. A radionuclide for use with this hapten is inIn.

The dual specificity antibody can also be used in immunotherapy by designing to have specificity against a disease-bound antigen and the other against a hapten, which is or is a lethal agent, the agent lethal for that

Antigen or diseased tissue with which the antigen is associated and which it is desired to destroy. E.g. the antibody may have specificity against a tumor associated antigen such as PAP, carcinoembrionic antigen (CEA), ferritin or other such antigens and a second specificity directed to a hapten to which a radionuclide is bound, preferably one which has a or y radiation emits or one that contains a ricin A chain or other toxin or drug. Such drugs can include gelonin, a-amanitin, io diphtheria toxin A, methotrexate, dichloromethate rexate, daunomycin and chlorombucil. If the toxin or drug itself can function as a hapten, it doesn't need to be bound to another residue.

If a radionuclide is to be used as a lethal agent, 15 just as in the situation where they are to be used for in vivo immunodiagnosis, the hapten may have the radionuclide bound directly to it or the hapten may be a chelating agent which can form a complex with the radionuclide, or be bound to such. In such a case, the hybrid or other antibody of dual specificity is administered to the diseased host organism and the center of the affected tissue is located, and the host organism is allowed to get rid of the excess,

followed by administration of the hapten which binds to the antibody wherever it is located. This allows the use of a radionuclide with a short half-life which minimizes the risk of damage to healthy tissue even if the radionuclide-bearing hapten is substantially Excess is administered, since this excess is quickly removed from the body and cannot be located in substantial amounts in healthy tissue, because of the relatively small size of the hapten. It also eliminates or reduces the possibility that circulating target antigen will bind antibodies carrying a substance lethal to the tissue and convert it to healthy tissue, as can happen if the lethal agent is directly bound to a monospecific antibody. which is directed against the target antigen.

An example of a hapten to which a radionuclide is directly 40-linked is 6-211At-astatö-2-methyl-1,4-naphthoquinol-bis (disodium phosphate), which is described in the "International Journal of Applied1 Radiation and Isotopes" , 33, 75 (1982). The 2r'r-A6isfiefn Emittervoiï cc-Sfeahluiig .. The expert will recognize that there are a number of suitable radionuclides, 45 of them directly! can be bound to haptens or can be combined with a hapten by any means of the wide variety of chelating agents. Medium. ^ The previous description contains currently preferred embodiments. Variants are possible (slight deviation from the scope of the invention, which is only limited by the appended claims.

Claims (52)

672 796 2nd PATENT CLAIMS 1. A method for producing a polydome which produces a hybrid monoclonal antibody which has a dual specificity, characterized in that a hybridoma which produces a monoclonal antibody against a first antigenic determinant has a B-lymphocyte which has a monoclonal antibody excretes against a second antigenic determinant, is fused in the presence of a fusion-promoting agent. 2. The method of claim 1, wherein the hybridoma is selectively destructible. 3. The method of claim 2, wherein the hybridoma has been re-selected to obtain cells sensitive to a medium in which the polydome can be cultured. 4. The method of claim 3, wherein the deselection was performed by culturing cells of a hybridoma in a medium containing a member of the group consisting of 8-azaguanine, 6-thioguanine, 5-bromouracyl-deoxyribose or 2-aminopurine, wherein Hybridoma cells are obtained which are sensitive to a medium containing hypoxanthine aminopterin thymidine. 5. The method according to claim 2, wherein said selectively destroyable hybridoma was obtained by irreversible enzyme inhibition. 6. The method according to claim 5, wherein the inhibition has been obtained by using a metabolic inhibitor. 7. The method according to claim 6, wherein the inhibitor is a K ^ inhibitor. 8. The method according to claim 7, wherein the K ^ inhibitor is selected from azaserine or 5-diazo-5-oxa-L-norleucine. 9. The method according to any one of claims 1 to 8, wherein the B-lymphocyte is a mammalian spleen cell. 10. A method for producing a polydome which secretes a hybrid monoclonal antibody which has a dual specificity, characterized in that a first hybridoma which secretes a monoclonal antibody against a first antigenic determinant, with a second hybridoma which is a monoclonal antibody against a second antigenic determinant, is fused in the presence of a fusion promoter. 11. The method of claim 10, wherein the first and second hybridomas are selectively destructible. 12. The method of claim 11, wherein at least one of the hybridomas has been re-selected to obtain cells that are sensitive to a medium in which the polydome can be cultured. 13. The method according to claim 12, wherein both hybridomas have been re-selected. 14. The method according to claim 11, wherein the deselection was carried out by culturing cells of the hybridome in a medium containing 8-azaguanine, 6-thioguanine, 5-bromo-uracyl or 2-aminopurine, whereby hybridoma cells are obtained which are sensitive to a medium containing hypoxanthine aminopterin thymidine. 15. The method according to claim 10, wherein said selectively destroyable hybridomas were obtained by irreversible enzyme inhibition. 16. The method according to claim 15, wherein the inhibition was obtained by a metabolic inhibitor. 17. The method according to claim 16, wherein the inhibitor is a calcium inhibitor. 18. The method according to claim 17, wherein the inhibitor is selected from azaserine or 5-diazo-5-oxa-L-norleucine. 19. The method according to claim 10, wherein only the first hybridoma is selectively destructible. 20. The method of claim 19, wherein the selectively destructible hybridoma is selected to confer the ability to survive in a medium that is lethal to the other hybridomas. 21. The method of claim 20, wherein the selectively destroyable hybridoma is resistant to a medium containing ouabain. 22. The method of claim 21, wherein the hybridoma has been re-selected to obtain cells sensitive to a medium in which the polydome can be cultured. 23. The method according to claim 22, wherein the deselection was carried out by culturing cells of the hybri-lo dom in a medium which is a member selected from the group consisting of 8-azaguanine, 6-ihioguanine, 5-bromouracyl-deoxyribose or 2 Contains aminopurine, whereby hybridoma cells were obtained which are sensitive to a medium which contains hypoxanthine aminopterin thymidine. 15. The method according to claim 21, wherein the selectively destructible hybridoma was obtained by irreversible enzyme inhibition. 25. The method according to claim 24, wherein the inhibition was obtained by using a metabolic inhibitor. 20 tors. 26. The method according to claim 25, wherein the inhibitor is a Kcat inhibitor. 27. A method of making a polydome which is a hybrid monoclonal antibody with dual specificity 25, characterized in that the nucleus is removed from a first hybridoma cell, which generates a monoclonal antibody against a first antigenic determinant, and this nucleus into the citoplasm of a second hybridoma, which generates a monoclonal antibody against a second anti-30 genetic determinant , is used. 28. A method for producing a hybrid monoclonal antibody which has a dual specificity, characterized in that the antibody is isolated from cells of a polydome, which polydome according to the method according to one of the claims. 35 che 1-27 was obtained. 29. A polydome which produces a hybrid monoclonal antibody with a dual specificity, obtained by the method according to claim 1. 30. Polydome according to claim 29, characterized in that it is a hybrid antibody as a component of a Mixture of antibodies generated. 31. Polydome according to claim 30, characterized in that the mixture of antibodies generated contains the hybrid antibody and two species of monospecific antibodies. 45 holds. 32. Polydome according to claim 30, characterized in that the excreted hybrid antibody is comprised of two subspecies. 33. Hybrid, monoclonal antibody with a dual sensitivity, obtained by the method according to claim 28. 34. Antibody according to claim 33, characterized in that one of the dual specificities is directed against a target antigen and the other against a substance which allows the diagnosis of the target antigen. 55 35. Antibody according to claim 34, characterized in that the substance which allows the diagnosis is radiolabeled or is labeled with a radionuclide, a fluorescent part or with an enzyme. 36. Antibody according to claim 35, characterized in that 60 the substance is a hapten. 37. Antibody according to claim 36, characterized in that the substance is radiolabeled, the radionuclide being bound directly to the hapten. 38. Antibody according to claim 33, characterized in that 65 one of the specificities is directed against a target antigen and the other against a hapten, wherein the hapten is a lethal agent for the antigen or for the associated tissue or is bound to such. 3rd 672 796 39. Hybrid, monoclonal antibody according to claim 38, characterized in that the lethal agent is a radionuclide or a tissue toxin. 40. Hybrid antibody according to claim 36, characterized in that the radionuclide is bound to the hapten by means of a chelating agent. 41. Hybrid, monoclonal antibody according to one of claims 35, 36, 37 or 40, characterized in that the radio label is a gamma radiation emitter. 42. Hybrid, monoclonal antibody according to claim 35 or 36, characterized in that the substance is fluorescent. 43. Hybrid, monoclonal antibody according to claim 36, characterized in that the hapten is fluorescent. 44. Hybrid, monoclonal antibody according to claim 35, characterized in that the fluorescent residue is bound to the hapten. 45. Hybrid, monoclonal antibody according to claim 35, characterized in that the substance which allows a diagnosis is an enzyme. 46. Hybrid, monoclonal antibody according to claim 36, characterized in that the enzyme is bound to the hapten. 47. Hybrid, monoclonal antibody according to one of claims 34 to 37 and 40 to 46, characterized in that the target antigen is an antigen associated with a condition. 48. Hybrid, monoclonal antibody according to claim 47, characterized in that the antigen is a tumor-associated antigen. 49. Hybrid, monoclonal antibody according to claim 39, characterized in that the radionuclide is bound directly to the hapten. 50. Hybrid, monoclonal antibody according to claim 39, characterized in that the radionuclide is bound to the hapten by means of a chelating agent. 51. Hybrid, monoclonal antibody according to one of claims 39, 49 or 50, characterized in that the radionuclide is an α or β radiation emitter. 52. Hybrid, monoclonal antibody according to claim 38, characterized in that the lethal agent is a tissue toxin. 53. Hybrid, monoclonal antibody according to claim 52, characterized in that the tissue toxin is in a ricin A chain. 54. Hybrid, monoclonal antibody according to one of claims 38, 39, 49, 50, 51, 52 or 53, characterized in that the target antigen is an antigen associated with a condition. 55. Hybrid, monoclonal antibody according to claim 54, characterized in that the antigen is a tumor-associated antigen.