JP2562002B2 - Hybrid monoclonal antibody with bispecificity - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to an antibody having bispecificity. In another aspect, the invention relates to immunodiagnostic and immunotherapeutic methods. In yet another aspect, the present invention provides a hybridoma.
(hybridomas) and related monoclonal antibody technology.

[0002]

BACKGROUND OF THE INVENTION Antigen-antibody reactions are routinely used in a variety of practical applications and, although not yet understood, have been extensively studied to establish value in other uses.
For example, serum antibodies produced by the host animal's immune response to the immunogen can be used in affinity purification methods to separate the immunogen from solutions in which the immunogen is present in only small amounts. In other cases, where the immunogen is a diseased antigen, its presence in the patient's serum or other body fluids is assessed for its presence in an immunoassay or immunometric technique.
tech-niques). For example, detection of HBsAg using radioimmunoassay technology is the current method of choice. Also, I131-labeled serum antibodies against ferritin obtained from New Zealand white rabbits have been reported as showing promise for liver tumor treatment. (Order and others, International Journal of Radiation Oncology, B
Biology and Physics, 6 , 703 (1980)).

Serum antibodies are "polyclonal" in nature, eg, serum antibodies obtained from rabbits, mice, or other mammals. This is because when the host's immune system is stimulated, it produces a mixture of specific antibodies against different antigenic determinants or epitopes on the immunogen to which the host is responding. The individual antibodies that make up the mixture are each the product of a B cell clone, and each B cell secretes only one antibody species. Antibodies produced by one clone differ from antibodies to the same antigen produced by another clone, at least because there are subtle differences between the peptide order and the peptide order of the other antibody. . In fact, therefore, each antibody species is a distinct molecule, and differences in the order of peptides between different antibody species not only affect the specific epitopes they recognize and their affinity for their antigens, but also their overall specificity. Affect.

The growth of individual B cells in order to obtain antibody species which one B cell secretes as a pure compound is not possible by vague use of the tissue culture techniques currently available. More recently, Kohler
And Milstein have discovered and reported a convenient method for obtaining monoclonal antibodies as fused products of secreted cells called "hybridomas".
(C. Kohler and C. Millstein, Nature ,
256, 495 (1975)). Basically, this method consists of fusing splenocytes from immunized mice with mouse myeloma cells to form hybridomas. It is preferred that the myeloma cells do not produce or at least do not secrete their own immunoglobulins or parts thereof. Culturing the cells obtained by cloning a single hybridoma secretes the same antibody molecule, which is then readily available as a pure compound. This is very different from conventional antibody production, for example from serum. In this serum, any antibody is only one of the components of a related but almost inseparable mixture of distinct compounds.

Being pure compounds, monoclonal antibodies have a certain specificity for a unique site on an antigen molecule and a well-defined affinity. Thus, different hybridoma clones can be screened to select those that produce the monoclonal antibody with the most desirable properties for a given application. The persistence of a hybridoma ensures an almost unlimited supply of the antibody it secretes and alleviates the problems associated with variations in antibody titer and overall affinity for each animal used to produce serum antibodies. The monoclonal antibody obtained from the hybridoma is practically applied to, for example, a diagnostic kit. A selection of such kits is available from the assignee of this application, Hybritech. Inc.
Antibody molecules are generally considered to exhibit only one specificity,
Its specificity is exhibited against the immunogen that the host's immune system responds to by producing antibodies. An antibody consists of two identical halves, each having a heavy and light chain pair, each recognizing the same antigenic determinant as the other.

[0006]

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The -SS-disulphide bridge connecting the two (H) chains at the cysteine moiety can be selectively cleaved in vitro by mild reduction, half of which the molecule Separated by acidification. This half molecule can then re-associate (regenerate) in vitro at neutral pH, which re-association occurs by non-covalent interactions. If antibodies of different specificities are subjected to conditions that lead to selective cleavage of disulfide bridges between heavy chains and subsequent reprogramming, half-molecule recombination will occur randomly, resulting in a population of antibodies. Sometimes, at least some of them are hybrid in that half of the antibody molecules of one specificity are bound to half of the antibody molecules of a different specificity. For example, Nisono
ff) Outside, "Antibody Molecule",
Academic Press, New York (1975), 260-26
Page 1 describes in vitro production of polyclonal antibody hybrids of rabbit anti-ovalbumin antibody and anti-BGG antibody. Hybrid monoclonal antibodies have also been obtained using similar methods. D. M. Kranz (Kr
anz) Outside, Proc. Natl. Acad. Sci., USA , 78, 580
7 (1981). At least theoretically, one half of the antibody recognizes one antigenic determinant, or epitope, and binds to it, while the other half recognizes different epitopes of the same or another antigen, thus giving rise to the dual property. Will be shown.

Hybrid antibodies can be obtained as described above, but the yields are often very low and the reactions used for their production are difficult to reproduce, and the hybrid antibodies are usually prominent and irreversible. Suffering from degeneration. Such denaturation may reduce immunoreactivity, and is expected to show another new metabolism in vivo. As a result, hybrid antibodies remain an experimental subject of great difficulty today. Antibodies with dual specificity are found in various coupling compounds such as protein A (from Staphylococcus aureus), carbodiimides and bifunctional compounds such as N-succinimidyl-3- (2-pyridyldithio) propionate. Agents or crosslinkers are used to conjugate pairs of intact antibodies, monoclonal or otherwise, to the dimeric or higher multimers of which each member of the antibody pair imparts its specificity. It can also be produced by obtaining an antibody. For example, Mondoche et al. Reported the production of bispecific multivalent antibodies by a series of reactions of antibodies with protein A, which was shown to be able to detect cell surface antigens in vitro. ing. J. I
See mmunological Methods, 42, 355, (1981). According to these methods, one specific antibody binds to the surface antigen and the other binds to a moiety that allows detection. Synthesis of bispecific antibodies by the techniques described above has been complicated and far from commercial application.

DETAILED DESCRIPTION OF THE INVENTION First, the present invention relates to a hybrid monoclonal antibody having bispecificity, wherein the hybrid monoclonal antibody and two kinds of monospecific antibodies are about 2:
Obtained from a polydoma produced as a component of an antibody mixture containing in a ratio of 1: 1 and with sufficient charge difference to separate these antibodies by ion exchange method, A hybrid monoclonal antibody having bispecificity is provided. The present invention further provides a novel, fully biological method that ensures high yields of monoclonal hybrid antibodies without denaturation. Throughout this specification the term "hybrid antibody" is used to refer to a single antibody molecule having two different specificities. The individual specificity may be for antigenic determinants on two different antigens or for different antigenic determinants (epitopes) on the same antigen. Furthermore, unless otherwise stated, the term "antigen" also includes haptens. According to the method of the invention, a bispecific hybrid antibody can be fused with a hybridoma secreting an antibody against a preselected antigenic determinant, preferably a selectively destructible hybridoma, B-lymph. Second by fusion with a sphere or a second hybridoma
Generation hybridomas (hereinafter referred to as “polydoms”)
a) ")). The B-lymphocyte or second hybridoma secretes another antigen against a different antigenic determinant. As used herein," selectively. The term "destructible hybridoma" means a hybridoma that lacks, or at least lacks, the ability to survive in the medium in which the polydoma is cultivated. In contrast to the parental hybridoma or B-lymphocytes from which it was derived, which both secrete the same population of antigens with a single specificity, this polydoma is further dichotomous. Monochromes with heavy specificity, ie, the ability to bind to either or both of the antigenic determinants recognized by the individual antibodies produced by the parental cell, and both It secretes a high percentage of null hybrid antibodies, The monoclonal hybrid antibodies thus obtained have not undergone the undesired denaturation characteristic of hybrid antibodies obtained by the method of chemical recombination of antibody half-molecules. In addition, the method of the invention makes it possible to obtain hybrids reliably and in large quantities.

According to the present invention, there are also provided immunodiagnostic methods and immunotherapeutic methods using an antibody having bispecificity. Generally, these methods use a monoclonal or polyclonal antigen having a first specificity for a target antigen and a second specificity for a substance such as another antigen or hapten. The substance allows the diagnosis of the target antigen to be carried out, or the substance allows the delivery of the target antigen or an agent which is lethal to the tissue to which it binds, or itself acts as such. It is a substance. Therefore, by appropriate selection of parent cells, the specificity for the target antigen and the diagnostically or therapeutically useful moiety (mo
A polydoma secreting an antibody having a second specificity for iety) can be obtained according to the present invention. Alternatively, antibody half-molecules can be recombined using in vitro chemical methods, or individual fully monospecific antibodies can be chemically combined or cross-linked to produce the dual molecules produced by the present invention. It is possible to obtain multimers of antibodies that have the same bispecificity and the same or similar utility as the monoclonal hybrid antibody with specificity (which are dimers, trimers or higher multimers). it can.

As used in the present invention, the term "antibody" includes antibody fragments which have immunochemical properties such as Fab or F (ab) ₂ fragments. Therefore, an object of the present invention is to reliably and in high yield obtain a monoclonal hybrid antibody that has not been denatured during the production process. Another object of the invention is to provide an improved method of obtaining a monoclonal hybrid antibody. Yet another object of the present invention is to provide immunodiagnostic and immunotherapeutic methods using antibodies with bispecificity. The manner in which these and other objectives can be achieved will be apparent from the following description of preferred embodiments.

Description of the Preferred Embodiment As mentioned above, the method of obtaining a monoclonal hybrid antibody according to the invention requires a hybridoma as one parent, preferably a selectively destructible hybridoma, which has been preselected. It secretes a monoclonal antibody against an antigenic determinant or epitope. When a selectively destructible hybridoma is used as a parent, cells obtained by fusing the selectively destructible hybridoma with B-lymphocytes, i.e. a second hybridoma, the polydoma cultivate the cells obtained during the fusion There is an advantage that it can prevent the infestation due to the population of parental hybridomas and that the polydoma cells can be separated from the parental hybridomas. We have shown that the selectively destructible hybridomas useful in the present invention are hybridomas secreting antibodies with one of the desired specificities, myeloma, produced by the classical Kohler-Milstein method. It has been found that it can be obtained from hybridomas obtained by the fusion of B. typhi cells with B-lymphocytes such as those found in mouse splenocytes. According to one embodiment of the invention, such hybridomas are subjected to a back selection process to obtain hybridomas that are selectively destructible.

In general, selective disruption potential can be obtained by counter-selection against hybridomas lacking in particular the genetic elements necessary to survive in the selected medium, which medium is then , The polydoma produced by the fusion can be cultured therein by the genetic contribution from the fusion partner of the hybridoma which is selectively destructible, namely the B-lymphocytes or second hybridoma. The presently preferred counterselection method consists of culturing a hybridoma secreting an antibody with one of the desired specificities to be included in the hybrid antibody in a growth medium containing 8-azaguanine. In a medium like this,
All cells which take up 8-azaguanine and therefore are able to grow in the medium are devoid of the enzyme hypoxanthine-guanine phosphoribosyl transferase (HPRT). A clone of cells lacking this enzyme is hypoxanthine aminopuritin thymidine (HAT).
Cannot grow in a medium containing. Therefore, they are selectively destroyed in this medium.

A very similar method of counterselection is that hybridomas secreting the desired antibody are labeled with 6-thioguanine (DN
Growth in a medium containing another analog of guanine, which is toxic to cells if taken up by A). Similarly, some cells growing in this medium will have HP
Lacking the RT enzyme, clones of these cells inevitably had H
It is sensitive to AT medium. Yet another counter-selection method that can be used in the present invention is to grow cells of the selected hybridoma line in a medium containing either the thymine analog 5-bromouracil deoxyribose (BUdR) or 2-aminopurine. Consists of letting. Only those cells lacking the enzyme thymidine kinase (TK) can grow in medium containing either of these two inhibitors. As in the case of cells lacking the enzyme HPRT, cells lacking TK do not grow in HAT medium.

Another method for obtaining selectively destructible parent hybridomas involves irreversible enzyme inhibition using metabolic inhibitors. Of these inhibitors, the so-called K _cat inhibitors are preferred. These inhibitors are analogs of the enzyme's substrates that are converted by the target enzyme into highly reactive molecules that react with the enzyme at its active site, resulting in irreversible inhibition of the enzyme. For example,
Treatment of selected hybridomas with azaserine or 5-diazo-5-oxa-L-norleucine (DON) forms a covalent bond with a cysteine residue at the active site of the enzyme, resulting in the enzyme formyl glycinamide ribonucleotide. -Inhibits amide transferraza. This inhibition eventually leads to cell death. But,
This hybridoma can be rescued by fusing with a second parent of the polydoma that supplies the required enzyme. In a preferred embodiment, the selectively destructible hybridoma is fused with complementing B-lymphocytes, typically obtained as splenocytes taken from a host preimmunized with the antigen. To be done. At this time, the antigen may be a hapten bound to a carrier protein and is selected so that the host will generate an immune response which will produce an antibody having the second specificity required in the hybrid antibody. The host is usually a mouse, but rabbit, human and other animal species can also be used although interspecies fusions may exhibit poor stability. Methods of immunizing such hosts are, of course, well known and need not be discussed at length here.

The fusion of a selectively destructible hybridoma with a B-lymphocyte to obtain a polydoma promotes the fusion of two groups of cells according to known methods, such as polyethylene glycol or Sendai virus. Can be carried out by combining in a medium containing the drug known as. After performing the fusion, the cells are transferred to a medium such as HAT medium for culturing.
B-lymphocytes only survive for a short period of time and parental hybridomas cannot grow in this medium. However, the number of polydomas formed as a result of the fusion was inhibited by B-lymphocytes due to the genetic contribution of the ability to make a lost enzyme, eg HPRT or TK, or in the parental hybridoma. It can be grown in this medium for complementation of the parent hybridoma by the direct contribution of the enzyme. Individual polydoma clones are cultured and screened for those that secrete antibodies with the desired bispecificity. Polydoma clones whose antibodies exhibit the desired bispecificity are further screened to select those whose second specificity, ie specificity and affinity obtained from B-lymphocytes, is most desirable. .

In another embodiment, the polydoma is
The selectively destructible hybridoma is obtained by fusing a second hybridoma, which is also selectively destructible, using an appropriate fusion agent. The second parental hybridoma is obtained in the same manner as the first, ie by the method of counter-selection, irreversible enzyme inhibition or any other suitable method. In such cases, the second hybridoma must be able to complement the first. For example, if the first selectively destructible hybridoma lacks the enzyme HPRT, then it must be able to provide the polydoma with a gene capable of causing the polydoma to elicit HPRT. Similarly, if the second selectively destructible hybridoma lacks the enzyme TK, the first must provide the gene for TK to the polydoma. A similar complement must be present between the two hybridomas if irreversible inhibition of the enzyme is provided to confer selective destruction. It is also possible to use the hybridoma that has been subjected to the counter-selection method as the parent of one hybridoma and the hybridoma that has been subjected to the enzyme inhibition method as the other hybridoma. The use of complementary selectively destructible hybridomas as the parent for the polydoma has the advantage that both parents can be selected based on the specificity and affinity of the monoclonal antibodies they produce, while In the case of fusion of hybridoma with B-lymphocytes,
Pre-fusion selection between B-lymphocytes cannot be performed to obtain those that produce antibodies of the desired specificity and affinity.

The fusion of two selectively destructible hybridomas can be carried out using polyethylene glycol or other fusing agents, also according to known methods. After fusion, the cells are transferred to a growth medium in which the two parents are unable to grow, where the polydoma produced by the post-fusion fusion can grow due to the complementary contributions of the two parents. So far, we have discussed selection methods to obtain selectively destructible hybridomas for use as both counterpart hybridomas in hybridoma-hybridoma fusions to form polydomas. However, the second that should be destroyed selectively
Of the hybridoma parent can be eliminated by providing both the dominant and recessive genetic markers to the parent of the first hybridoma. The presently preferred method is the HAT-ouabain selection method. The drug ouabain is a specific inhibitor of plasma membrane Na ⁺ -K ⁺ activating adenosine triphosphate degrading enzyme (Arpase). This enzyme controls the import of K ⁺ into the cell and the export of Na ^{+ from} the cell. Selective destruction potential, eg HAT
Hybridoma cells that have been preselected and counterselected to sensitize are grown in ouabain medium to select for cells resistant to ouabain. Clones of these cells are HAT sensitive but ouabain resistant. In contrast, the hybridoma selected for fusion therewith is ouabain sensitive but can survive and grow in HAT. Alternatively, selection for ouabain resistance can be performed either first in the parental myeloma line or in hybridomas derived therefrom, followed by counterselection or other methods conferring selective destructive potential. it can.

The cells obtained by fusing the two hybridomas in polyethylene glycol or the other fusing agent are transferred to a second, HAT medium containing ouabain at a lethal concentration for the hybridoma parent. The selectively destructible hybridoma parent, even if resistant to ouabain, is also unable to survive in HAT medium, for example, because it lacks HPRT or other enzymes, however, the cells of the polydoma are enzyme and ouabain required for survival. Since it has resistance, it can grow in this medium. In the above method, the parent of the selectively destructible hybridoma secreting an antibody having one of the desired specificities for the hybrid antibody and the second secreting antibody having the other desired specificity for the hybrid antibody. The advantage is that a polydoma can be obtained by direct fusion with an off-the-shelf hybridoma of, and that there is no need to use any technique that gives the parent of the second hybridoma the possibility of selective disruption. Another technique for obtaining a polydoma, which uses a universal parent, that is, a parent that has both positive and negative genetic markers and can be fused to any off-the-shelf hybridoma, includes various techniques. Recombinant D carrying the drug resistance genetic marker of D
Includes the use of NA vectors. For example, SV ₄₀ having a neomycin resistance gene can be used.

The presently preferred all-purpose parent is HAT-sensitive-
It is neomycin resistant. The chosen parent is HA
After being inverse selected T susceptibility, it is at SV ₄₀ vector carrying the neomycin resistance gene infected (transfect). This procedure can be reversed to transfect first. The resulting hybridoma 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. Thus, the products of these parental fusions survive in the presence of HAT and neomycin.
While it is currently preferred to use a vector to transfer resistance to neomycin, it is also possible to use a vector that has a gene that confers resistance to other agents on mammalian cells. While presently preferred, it is not essential for the method of the invention to obtain a polydoma from a hybridoma pair that the parent is capable of selectively destroying at least one of the cell lines. After fusion of a pair of hybridomas secreting an antibody, none of which is selectively destructible, but having one of the required selectivities in hybrid antibodies, in the presence of a suitable fusing agent, they are not fused It is within the scope of the invention to perform subcloning of all cells before the parental hybridoma has increased to the extent that it is impractical to screen subclones to identify polydomas. Subclones are then screened to identify which one secretes the antibody with bispecificity.

This method is most suitable for obtaining a polydoma when the fusion frequency of parental cells is high. Anyway, especially when cell fusion is infrequent, cells whose monoclonal antibodies are derived from fusions of hybridomas to various antigens can be screened using cytofluorograh to identify polydomas. it can. To do this, samples of the two antigens fluoresce at different wavelengths, with different fluorescent moieties (moie).
ty). For example, one can be labeled with fluorescein and the other with rhodamine. The post-fusion treatment cells are preferably incubated overnight or another suitable period of time to increase their number, but are incubated in their entirety with the two labeled antigens. The cells are then screened using a cytofluorograph. Cells that fluoresce at only one of the two wavelengths will be from the parental hybridoma cell line. On the other hand, it is a cellular polydoma that exhibits fluorescence at both wavelengths and can be separated and subcloned.

In yet another embodiment, the nucleus is removed from a first cell that secretes a monoclonal antibody having one of the required specificities for a hybrid antibody and a second antibody that secretes a monoclonal antibody having a second required specificity. The polydoma can be obtained directly by inserting it into the cytoplasm of the two hybridomas. Of course, neither of the parental hybridomas need be selectively destructible for use in this method. After insertion of nuclear material, the cells are cloned to obtain a population of polydomas.
Unlike parental hybridomas or B-lymphocytes that secrete a single antibody, the cells of the polydoma obtained according to the invention secrete a mixture of antibodies, at least one of which has dual specificity. We have found that it is a hybrid antibody. With this polydoma,
Antibodies with the same specificity as the antibodies produced by the parental cells used to obtain this polydoma are also produced in relatively small amounts. The ratio of hybridization to monospecific antibody is about 2:
1: 1, which produces equal amounts of all possible (H) chains that the parental polydoma is synthesized by the parental cell that is randomly bound within the polydoma itself. This is the ratio that would be expected if

Polydomas can be cultured in vitro to obtain large amounts of hybrid antibodies or can be grown in vivo in either genetically matched animals or nude mice, which hybrid antibodies are known in the art. Recovered from the culture medium or animal ascites using the method. For example,
"Monoclonal Autibodiese" (edited by Knett and others, Plenum Press, New York (1980)) 363-4
See page 18, protocols. The mixture of antibodies produced by the polydoma can be separated to obtain hybrid antibodies. For example, its separation from monospecific antibody impurities can be performed by sequential affinity chromatography on the first and subsequent other antigens for which the hybrid antibody is specific. We have also found that simple ion exchange chromatography and electrophoresis techniques can be used at least in certain cases. If necessary, the difference in charge for ion exchange can be one of the characteristics of the antibody considered in selecting the parental strain.

[0024]

Example 1 Monoclonal hybrid antibodies with bispecificity for hepatitis B surface antigen (HBsAg) and prostatic acid phosphatase (PAP) were prepared according to the present invention as follows. Hybridomas secreting a monoclonal antibody against PAP were grown in HAT medium for one week and then transferred to a non-selective medium for growth. After various times of growth under non-selective conditions, 2 ml aliquots of cells were aliquoted to 10 ^-4.
The medium was placed in a medium containing M 8-azaguanine. This 8
-Azaguanines are their DNA instead of guanine
By being contained therein, it prevents the growth of cells. Cells lacking the HPRT enzyme survived and grew in this medium, but these cells did not necessarily survive in HAT. Clones grown in medium containing 8-azaguanine were tested for HAT sensitivity and anti-P
Tested for AP productivity. Still producing anti-PAP 4
One clone was subcloned that showed HAT sensitivity with a reversible frequency of less than × 10 ^-8 . All subclones behaved like parental clones. Using cells from one of the HAT-sensitive subclones in polyethylene glycol using the Gerhard fusion technique,
Polydomas were obtained by fusion with splenic cells obtained from Balb / c mice hyperimmunized with HBsAg. Above “Monoclonal
Antibodies ”, page 370. 220 by this fusion
, Were screened to determine which secreted antibody showed specificity for both PAP and HBsAg. It has been found that two such polydoma clones produce antibodies followed by ascites fluid that exhibits both specificities.

Both polydoma subclones produced ascites fluid exhibiting both specificities and, like those of the parental clone, gave a triple band on Orstein-Davis PAGE. Ascites fluid from both clones was found to bind ¹²⁵ I-HBsAg and ¹²⁵ I-PAP in a radioimmunoassay, giving a K value of about 10 ⁹ for each. Thus, the formation of bispecific antibodies was suggested. The data in Table I compares ascites fluid from one clone of this polydoma with ascites fluid from hybridomas secreting monoclonal antibodies against IgE (used as a control), PAP and HBsAg, respectively. Figure 3 shows the results obtained in an immunoassay using immobilized HBsAg as the solid phase and various radiolabeled antigens as the solution phase. A 200 μl sample of ascitic fluid from a polydoma and each of the three hybridomas were incubated overnight with 12 polystyrene spheres each loaded with HBsAg. The HBsAg sphere is from Abbott Laboratories (Abb) in North Chicago, Illinois.
ott Laboratories). After washing, triplicate samples were incubated for 4 hours with ¹²⁵ I-labeled antigen 100,000 cpm. After the second wash, the balls were counted to measure the labeled antigen bound to the balls. In one set of tests using radiolabeled PAP as a solution phase antigen, anti-PAP was added to the antigen before it was incubated with the balls. P used for this purpose
Since the antibody to AP is the monoclonal antibody produced by the parent hybridoma used to make the polydoma, it is directed against the same PAP epitope, as would be expected to be exhibited by the hybrid antibody.

[0026]

[Table 1] Table 1 Results of radioassay demonstrating the presence of a bispecific hybrid antibody in ascites of polydoma. Cpm Ascites specificity cpm cpm * PAP + cpm * 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 73,020 2,973 2,870 3,330 78,900 82,533 2,936 3,143 * ¹²⁵ I-labeled antigen

The data in Table I show that when compared to the data for the IgE control, only the radiation expected from non-specific binding is measured for anti-PAP. On the other hand, ascites containing HBsAg antibody bound to the labeled HBsAg antigen as expected, but showed non-specific binding when tested with other labeled antigens. However, ascites from polydoma clones was labeled with labeled HBsAg and labeled PA.
Bound to both P's. The former is due to the presence of a small amount of non-hybrid monospecific antibody to HBsAg in the ascites, and the latter is a hybrid that can bind and bridge HBsAg on spheres with trace labeled PAP in solution. Attribute. Anti-P from labeled PAP and parental hybridoma
Experiments using a mixture of APs confirm that the anti-PAP specificity of the hybrid is to the same epitope as the antibody secreted by the parent. I mean,
This is because only the baseline radiation is observed because the binding of the labeled PAP to the hybrid antibody was inhibited by the parent antibody.

Analysis of ascites from polydoma clones used in comparative radioimmunoassays was performed by polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis (I).
EP). Both showed the presence of at least 3 antibody species. Preparative scale DEAE ion exchange chromatography yielded three well-separated peaks, one in the middle of which had a shoulder. Each of the peaks was homogeneous as analyzed by PAGE and IEP, each corresponding to one of the bands in the initial ascites. The antigen binding properties of the substances representing each of the DEAE peaks were tested using radiolabeled HBsAg and PAP. The first peak bound HBsAg but not PAP. The peak in the middle and its shoulders are HB
It binds to both sAg and PAP, and the last peak is PAP.
Just joined. Therefore, the middle peak is HBsAg
Is a hybrid antibody having bispecificity for PAP and at least two subspecies.

The hybrid antibody, obtained as the middle peak of DEAE chromatography, was radiolabeled with ¹²⁵ I. After labeling, 85% of the labeled antibody binds PAP and 88% binds HBsAg. Hybrid PAP
Was found to be slightly lower than that of the monoclonal antibody to PAP produced by the parental strain. This 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 antibody contains more than 50% of the antibody produced by the polydoma, approximating the expected ratio 2: 1: 1 for statistical reasons. This 2: 1: 1 ratio indicates that the polydoma exhibits all possible antibody heavy chains, ie either PAP or HBsAg specificity, and is randomly combined intracellularly with the hybrid antibody. A small amount of two monospecific antibodies concomitant with this, which is expected on the basis of statistical reasons when formed with an antibody having the same specificity as the antibody produced by the parental cells Is. The presence of hybrid antibody subspieces suggests that they differ in their light chain composition.

Example 2 A monoclonal hybrid antibody having bispecificity for human IgD and prolactin was prepared according to the invention by fusion of two hybridomas. One of the hybridomas is constructed to contain two selectable genetic markers: sensitivity to HAT medium and resistance to ouabain. The advantages of utilizing such a "universal parent" were discussed elsewhere in this specification. This doubly labeled hybridoma or so-called "universal parent" can then be fused with any other hybridoma.
The resulting polydoma grows in the presence of HAT and ouabain, but kills all unfused parental cells. To form such a universal parent, both selectable labels were introduced during the initial formation of hybridomas. To obtain this parental cell line, the widely available HAT-sensitive mouse myeloma P3.653 was selected for a second genetic marker, ouabain resistance, by introducing 1 mM ouabain into the growth medium. . Most cells died, but about 1
/ 100,000 cells acquired resistance to the drug by unregulated mutation, survived, proliferated, and
A new myeloma population was formed that was sensitive and resistant to ouabain.

This HAT-sensitive, ouabain-resistant myeloma was then fused with splenocytes obtained from Balb / c mice hyperimmunized with IgD using the method of Garhard previously cited. Hybrids were selected in HAT medium (no ouabain) and clones were screened for production of monoclonal antibodies against IgD. Of the positive clones, those that produced IgG versus IgD were selected for further study. This clone was tested by adding 1 mM ouabain to the growth medium to maintain the ouabain resistance property. About 1/3 of the cells maintained this genetic marker. As this culture grew exponentially in ouabain, the cells further branched and gave rise to subclones. Ouabain resistant subclones were tested for continuous production of monoclonal anti-IgD antibodies. One of the subclones was further counterselected by the procedure of Example 1 to produce HAT
To obtain a cell population sensitive to. This subclone was grown for 2 weeks under non-selective conditions and then placed in a medium containing 6-thioguanine. As noted above, the mechanism of action of 6-thioguanine is similar to that of 8-azaguanine.
Cells that incorporate 6-thioguanine into their DNA instead of guanine do not grow. Cells lacking the HPRT enzyme do not utilize 6-thioguanine from the medium and can therefore grow but are consequently susceptible to HAT.

This back-selected subclone population itself was then subcloned in medium containing 6-thioguanine and ouabain. Subclones were tested for continuous production of monoclonal anti-IgD antibodies. One clone was selected which showed all the desired properties-growth in ouabain and 6-thioguanine and the productivity of monoclonal anti-IgD and was designated the so-called "universal parent". This universal parent then generated a polydoma that could be fused with any other HAT-resistant, ouabain-sensitive hybridoma to generate a hybrid antibody, whose monospecificity is anti-IgD. For this purpose, we first chose a mouse hybridoma that secretes a monoclonal antibody against prolactin. Anti-prolactin monoclonal antibodies are of the same subclass (IgG ₁ ) as anti-IgD generated by the parental strain and are easily separated from them by Ornstein-Davis gels. It is suggested that such a separation imposes a very different burden on the antibody. Therefore, separation of hybrid antibodies should be facilitated by DEAE-Sephadex chromatography.

10 ⁷ cells of the HAT-sensitive and ouabain-resistant cell line were
It was fused with 10 ⁷ cells producing anti-prolactin.
The fused cells were first grown in HAT medium for 3 days, then modified in HAT + ouabain medium for 3 days and finally re-introduced into HAT medium. Over 600 clones developed from this fusion. 66 clones were randomly selected for analysis. Of these clones, 36 showed anti-IgD and anti-prolactin activity. The supernatants of these clones were analyzed for the presence of hybrid antibodies by the following test method. Polystyrene beads coated with another anti-prolactin monoclonal antibody were incubated with 200 μl of 100 ng / ml prolactin solution for 5 hours. The antibody used binds prolactin at a site distant from that of the antibody produced by the fused hybridoma cell line. After washing the beads, they were incubated overnight with the supernatant of the clones. After several washes the next day, ¹²⁵ I-labeled IgD was added. The hybrid antibody bound to the beads by a single functional arm was able to bind IgD radiolabeled with the free anti-IgD functionality, although both parent-type antibodies, IgD-IgD and prolactin-prolactin. This bond could not be formed between the prolactin beads and the ¹²⁵ I-IgD tracer. Typical analytical results for clones producing hybrid bifunctional antibodies are shown in Table 2 below.

[0034]

TABLE 2 TABLE 2 IgD and hybrid antibodies with dual specificity for prolactin, a result of the demonstration to have immunoassay that they are present in the supernatant and ascites of Polydomas selected Clonate # cpm bound ¹²⁵ I-IgD Tracer 1 15339 2 16337 3 22886 4 23356 5 24434 Anti -IgD 9357 Anti-Prolactin 8721

Twenty-one of the 36 clones showed clear bifunctional activity by this analysis. Ascites fluid generated from two clones available to date has been shown to react in this bifunctional assay. These ascites fluids contain the antibody in three distinct bands on an Orstein-Davis gel. The two bands closely match the antibodies produced by the parental hybridomas (anti-IgD and anti-prolactin). The third band, as expected, shifts to the middle of the band of the parent monoclonal antibody of the hybrid antibody. It can be seen from the data in Table 3 that the hybrid monoclonal antibodies to IgD and prolactin show a dose response when the amount of prolactin was changed in the assay used to generate the data in Table 2. Table 3
Uses antibodies of clone # 2 and, as controls, antibodies from the parental cell line (anti-IgD and anti-prolactin).

[0036]

[Table 3] Table 3 Results of analysis using hybrid antibody and various amounts of prolactin Prolactin hybrid anti-IgD ¹ anti-prolactin ¹ ng / ml antibody ¹ 0 8010 6847 8020 10 9169 7982 7405 50 13558 7783 8314 100 17599 7654 7844 1. Bound ¹²⁵ I-IgE cpm

These data demonstrate that the amount of prolactin bound by the hybrid antibody is dose responsive such that the amount of labeled IgD bound increases with increasing amount of prolactin. In contrast, the use of parental antibodies to IgD and prolactin labeled them as bead-bound prolactin.
There is no dose response due to the inability to form a bridge with IgD. Thus, the hybrid antibody can be used as a component of a prolactin assay. Other hybrid antibodies made specifically tailored offer similar opportunities for other analyses.

EXAMPLE 3 By a method similar to that of Example 2, a monoclonal antibody against the hapten arsenate-arsenate dimer was secreted and was resistant to ouabain and to HAT. And formed a versatile parental hybridoma. This hybridoma was fused to a hybridoma that secretes a monoclonal antigen with specificity for carcinogenic antigen (CEA), embryonic tissue and antigens produced by any of several types of cancer. Once again,
Over 600 clones resulted from this fusion. CAE
Of the 72 clones tested for their ability to bind both arginine and arsenate, 69 had both binding abilities. The clone showing the highest binding was selected for the enzyme linked immunosorbent assay (ELISA) of the hybrid antibody. For each analysis, CEA solution (600 ng or 2
50 ng) was adsorbed to each well of a plastic 96-well microtiter plate overnight. The next day, the non-adsorbed material was washed out of the wells with PSB-Tween 20. Add the supernatant of the clone and mix at 35 ° C 2.
After incubating for 5 hours, the plate was washed off.

The CEA-CEA and CEA-arsenate antibodies remain bound to the plate via the adsorbed antigen. A second antigen, arsenic acid linked to the enzyme alkaline phosphatase, was added to the wells at 35 ° C for 3 hours.
After washing again with PBS-Tween, a chromagen substrate for alkaline phosphatase, para-nitrophenyl phosphate, was added to the wells and color was developed for 48 hours. The absorption of each well was measured at 410 nm. When present in the supernatant of the clone, the hybrid antibody is free to bind to the adsorbed CEA on the plate by one functionality and the other functionality with the arsenic acid bound to alkaline phosphatase. I left it. Color was developed from the chromagen substrate only where alkaline phosphatase bound to arsenyl acid was bound. In this analysis, 3 out of 12 supernatants showed hybrid antibody activity as shown in Table 4.

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[Table 4] Table 4 Demonstration of bispecific hybrid antibody by enzyme-linked immunosorbent assay (ELISA) Absorption at 490 nm Absorption at 490 nm Clone 600 ng CEA / well 250 ng CEA / well 1 0.087, 0.105 0.022, 0.060 2 0.082, 0.054 0.023, 0.033 3 0.011, 0.017 0.041, 0.036 Antiarsenate 0.010, 0.006 0.006, 0.005

The present invention was obtained as described above with antibodies having bispecificity. Or Nisonov (Ni
sonoff) Immunodiagnostic method using hybrid antibodies obtained from antibody half-molecules by conventional techniques (see above) or antibody multimers obtained by binding or cross-linking individual monospecific antibodies And immunotherapy methods are also provided. Preferably, the bispecific antibody used in these methods has not been denatured, ensuring that the antibody is obtained as a substantially pure compound with uniform specificity and affinity for the antigen. Is a hybrid antibody produced according to the present invention. For immunodiagnostic applications, one of the two specificities exhibited by the bispecific hybrid or other antibody will be for the target antigen that needs to be detected and the other for the other antigen. This other antigen may be a hapten or other molecular species,
Allows diagnosis. For example, an immunohistologically useful antibody has a first specificity for a suspected antigen, eg, a cancer-associated antigen such as CEA, PAP or Pheritin, and an enzyme that produces a chromogenic reaction in the presence of a suitable substrate. like,
Second for haptens or antigens involved in color reaction
Has specificity. Among the suitable enzymes to which the second specificity of this antigen may be directed are prostatic acid phosphatase (P
AP), horseradish peroxidase, glucose oxidase and alkaline phosphatase.

To perform histological examination, tissue sections are first treated with bispecific antibody. Before doing so, the enzyme that catalyzes the contamination reaction with the hybrid can be pre-sealed. If not, then the sections are treated after a suitable incubation with a second solution containing the enzyme, rinsed and then treated with a substrate that changes color in the presence of the enzyme. The color developed by the enzyme and substrate in the tissue sample is a positive sign that the target antigen is present in the tissue. HBsAg whose manufacturing method is described in this specification
The hybrid antibody against PAP and PAP was HB on a test substrate (polystyrene ball) in a simulated contamination experiment using p-nitro-phenyl phosphate as an enzyme substrate.
It was found to bind to sAg and PAP. After incubating this hybrid antibody with PAP and HBsAg, p-nitrophenyl phosphate was added, resulting in the balls turning from their characteristic yellow color to brown.

As mentioned in Example 2, bispecific antibodies can also be used in immunoassays and immunometric assays. Using the hybrid antibody against HBsAg and PAP as described in the above-mentioned method, an immunomeric assay for HBsAg can be performed using a solid antibody for extracting HBsAg from an immobilized monoclonal antibody against HBsAg from serum or another liquid sample suspected of containing an antigen. It can be performed using as a phase. The sample is incubated with balls, beads, test tubes or other substrates with anti-HBsAg bound or coated on the surface. Incubation with the serum sample can be followed by, simultaneously with, or prior to incubation with the solution of the hybrid. In either case, the result will be a sandwich of immobilized antibody, HBsAg (if present in the sample) and hybrid antibody when HBsAg is present in the sample. As part of the analysis, the binding of PAP to the hybrid antibody is allowed. This may be done during or after the formation of the sandwich, or instead
The antibody-PAP complex may be pre-formed. After formation of the sandwich, the solid phase is washed to remove hybrid antibody not bound to sample residue, and then PAP
It is contacted with a solution containing a substrate such as p-nitrophenyl phosphate or α-naphthol phosphate which undergoes a color change in the presence. The occurrence of discoloration confirms the presence of the target antigen in the sample.

In such an analysis, Abbott L, located in North Chicago, Illinois,
Samples containing varying amounts of HBsAg were incubated with the beads using the polyclonal anti-HBsAg beads of a commercial HBsAg diagnostic kit (sold under the name "Aushia") manufactured by Aboratories. The sample used was a positive control and a negative control of a commercial kit, and a positive control was a negative control diluted with either a negative control 1 part: positive control 2 parts or a negative control 2 parts: positive control 1 part. Two samples were obtained.

After incubating the sample with beads to bind HBsAg, the beads are washed,
Incubated with a hybrid antibody reactive with both HBsAg and PAP. This forms a sandwich of immobilized antibody: antigen: and hybrid antibody. The beads were washed again and incubated with a solution of PAP. Following this incubation, it was washed once more and the beads were incubated with the substrate α-naphthol phosphate. After a suitable incubation in which PAP enzymatically removes the phosphate, the substrate was removed from the beads and to this was added the indicator Farst Garnet GBC salt, which turned clear to magenta in the presence of the product of the enzymatic reaction. . Discoloration confirming the presence of HBsAg was observed in the sample. The absorption at 570 nm was measured for each sample and the data are shown in Table 5 below.

[0046]

[Table 5] Table 5 HBsAg concentration Absorption positive control% 570 nm 0. 033 34. 166 67. 243 100. 379

These data show a dose response for changes in HBsAg concentration, which would be expected if the hybrid antibody formed a bridge between sphere-bound HBsAg and PAP. This further demonstrates that the hybrid antibody can be used in immunoassays. Detection means other than enzymatically catalyzed reactions are possible. For example, a hapten in which the second specificity of a hybrid antibody or other antibody with bispecificity is radiolabeled or fluorescent, or is detectable in a sandwich by any other suitable means. Alternatively, it can be directed against the antibody.

One preferred method of utilizing hybrid or other antibodies with bispecificity for immunoassays is to utilize reduced fluorescence quenching. In such an assay, one specificity of the antibody is directed against the target antigen and the other specificity is directed towards, for example, a hapten bearing a fluorophore. The chromophore may be bound to the hapten or, where appropriate, the hapten itself. The assay is performed by incubating the antibody with a serum or other sample suspected of containing the target antigen, to which is added a predetermined amount of the target antigen labeled with a quenching chromophore. When the target antigen is present, the labeled antigen in the sample competes with the target antigen for the binding point of the antibody specific for the target antigen. Prior to, during, or after this incubation, an aliquot of the hapten bearing fluorophore is incubated with the antibody and bound at the other junction.

When the two chromophores are located close enough together that the photon emitted by the fluorescing side can be captured by the quenching side, the other at a wavelength that the quenching side can absorb (quenching) The two chromophores are selected to emit To this end, the two chromophores are within about 100 Å of each other, preferably about 50 Å.
Should be within Angstrom. This arrangement occurs when the fluorescent chromophore is attached to one antibody attachment point and the quencher chromophore is attached to the added antibody at the other attachment point. Suitable chromophores include fluorescein as a fluorescent chromophore and rhodamine as a quenching chromophore. The measured fluorescence varies inversely with the amount of natural antigen in the sample. In the absence of natural antigen, all of the antibody-bound antigen is labeled with a quenching chromophore and is positioned to absorb the fluorescence of the hapten-supported chromophore. Comparing the measured fluorescence with the fluorescence of a control sample containing a known amount of antigen,
Qualitative and quantitative measurements of the presence of antigen in the sample can be made. This type of immunoassay can be used, for example, to determine the serum concentration of agents such as dilantin which must be closely monitored. In such an assay, the target antigen is, of course, dilantin. It will be apparent to those skilled in the art that this method can be used for the detection of other antigens as well, especially those associated with tumors.

Another preferred method for utilizing bispecific hybrid antibodies or other antibodies in immunoassays relies on enzymatic reactions. In the currently preferred method,
One of the antibody's specificities is, of course, directed against the target antigen and the other towards the enzyme or hapten to which the enzyme is attached. The target antigen has been modified by binding it to a target antigen that interacts with the enzyme to produce a detectable substance or otherwise allows the formation of the antigen-antibody complex to be detected. . The assay is performed by incubating the antibody with a predetermined amount of modified target antigen added to a sample suspected of containing the target antigen. The detection may be performed, for example, by fluorescence measurement, luminescence, spectroscopic measurement, or the like. In an alternative method, the target antigen added may have the enzyme bound to it, in which case it is directed to the substance that interacts with the enzyme or to the hapten to which it is bound. Antibodies have one of the specificities. The substance that interacts with the enzyme may itself be another enzyme. In such cases, one of the enzymes catalyzes the production of the product required by the other. That is, when the antibody binds both the added target antigen (which has one of the enzymes attached) and the other enzyme, the product of the first enzymatic reaction is produced in close proximity to the second enzyme. , The latter can undergo a reaction mediated by the latter enzyme before a clear diffusion into the surrounding medium occurs.

One example of such a method is to use two enzymes, hexokinase (HK) and glucose-6 in the following reaction system.
-Phosphate dehydrogenase (G-6-PDH)
To use.

To utilize this reaction system, the added target antigen has either HK or G-6-PDH attached to it, and the hybrid antibody is directed against the other (or the hapten supporting it). Will have one of the peculiarities. The sample contains glucose, in addition to the hybrid antibody and a predetermined amount of enzyme-labeled antigen.
ATP and coenzyme NAD ⁺ were added to it. It is preferred that the hybrid antibody already has the other enzyme attached to it. Alternatively, the enzyme can be added to the sample along with other reagents. During this incubation
If the natural antigen is present in the sample, the enzyme-labeled antigen competes with the natural antigen in the sample for one of the hybrid antibody binding points. The other enzyme is or will be attached to the second attachment point. This causes the production of glucose-6-phosphate catalyzed by HK to be G-6.
-Proximity to PDH. The latter converts glucose-6-phosphate to gluconolactone-6-phosphate with the consequent reduction of NAD ⁺ to NADH.
This NADH absorbs strongly at 340 nm and can be detected by spectroscopic measurement. The amount of NADH produced varies inversely with the amount of natural antigen in the sample, ie its maximal production occurs when no target antigen is detected in the sample. Comparing the amount of NADH produced with a control sample provides a qualitative and quantitative measure of the presence of antigen in the sample. This type of assay can be used to monitor the concentration of dilantin or other drug in serum.
In such cases, the drug is the target antigen. However, such assays can also be used to detect other serum antigens such as those associated with tumors and other diseases.

In vivo immunodiagnosis can also be performed using hybrid antibodies or other bispecific antibodies. First against a target antigen, such as a tumor-associated antigen
And a second specificity for a hapten to which is attached a suitable radionuclide, preferably one that emits gamma radiation, is first administered to the host. After sufficient time for the antibody to localize to the target site and for the unbound antibody to leave the host's healthy tissue, the radionuclide hapten is administered and binds to the localized antibody. After a suitable gap to allow the unbound hapten to leave the host, the host is probed with a suitable camera to determine if there is a high concentration of radiation. If present, the presence of the target antigen in the host is confirmed and its location determined. This method has several advantages over methods that use monospecific antibodies directed against the target antigen to which the radionuclide is directly bound. In such cases, the radionuclide must have a sufficiently long half-life that it remains in sufficient quantity after the time required for the antibody to substantially localize at the target site. further,
During this process, the antibody may be retained in the liver or other non-target tissue for a period of time, which tissue will be exposed to the radiation carried by the antibody. On the other hand, the present invention allows the use of radionuclides that have a shorter half-life than the radionuclide used with monospecific antibodies. Since it is a relatively small particle, the hapten carrying the radionuclide has high mobility in vivo and moves rapidly in the chamber to bind to the antibody localized at the target site or Get out of the body without spending time in question in non-target tissues. For this reason, short half-life isotopes can be administered in an amount that poses a minimal risk to healthy tissue, even when administered in excess.

Preferably, the hapten is a drug to which the radionuclide is directly bound or complexed with the radionuclide. Chelating agents for hapten-bound radionuclides may be used for the latter purpose. Those skilled in the art will appreciate that a wide variety of chelating agents and radionuclides are suitable for this purpose. Phenyl arsenate with ethylenediaminetetraacetic acid (EDTA) attached as a chelating agent is a suitable hapten.
A suitable radionuclide for use with this hapten is ″ ″ _n .

Are Bispecific Antibodies a Deadly Agent for Diseased Tissues Having One Specificity for Disease-Associated Antigens and where it is desired that the antigen or antigens be involved and destroyed? Alternatively, it can also be used in immunotherapy by configuring this bispecific antibody to have the other specificity for the hapten bound to the drug. For example, the antibody has one specificity for a tumor-associated antigen such as PAP, carcinogenic antigen (CEA), ferritin, or other such antigen, and is radionuclide, preferably alpha or beta radiation. May be bound or have a second specificity directed against a hapten consisting of ritin A chain or other toxins or drugs. Such agents include gelonin, α-amantine, diphtheria toxin A, methotrexate, dichlorometatrexate, dounomycin and chlorombucil. Of course, if the toxin or drug itself can function as a hapten, it need not be attached to any other moiety.

If the radionuclide is to be used as a lethal agent, as in the situation used for in vivo immunodiagnostics, the hapten can have the radionuclide bound directly to it, or the hapten forms a complex with the radionuclide. Can be a chelating agent or have the agent attached to it. In such cases, the bispecific hybrid antibody or other antibody is administered to the diseased host, localized at the site of the affected tissue, and any excess exits the host. Then the hapten is given,
It binds to the antibody wherever the antibody is localized. This allows the use of short half-life radionuclides that minimize the risk of harming healthy tissue, even if the hapten bearing the radionuclide is effectively overdosed. The reason is that the hapten is of relatively small size, so that the excess exits the body rapidly and is not localized in healthy tissue in substantial amounts. Also, as occurs when the lethal agent binds directly to the monospecific antibody directed against the target antigen, the circulating target antigen binds to the antibody carrying the substance fatal to the tissue. The possibility of delivering this to healthy tissues is eliminated or diminished. An example of a hapten to which a radionuclide is directly bound is “Internatio
nal Journal of Applied Radiation and Isotopes ”,
33, 75 (1982) described the 6- ^{21 1} At- Asutato-2-methyl-1,4-Nafutokinoru bis (2
Sodium phosphate). ²¹¹ At is an α-ray emitter. One of ordinary skill in the art will appreciate that there are many suitable radionuclides that can be attached directly to the hapten or can be complexed with the hapten by any of a wide variety of chelating agents.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location G01N 33/577 A61K 39/395 H // A61K 39/395 45/00 45/00 9162-4B C12N 15/00 C 51/00 A61K 43/00 (C12P 21/08 C12R 1:91) (72) Inventor David Garry Ees USA California 92307 La Jolla Pool Street 9477 (72) Inventor Adams Thomas H. USA California 92024 Lucadia Neptune 860 (72) Inventor Flinke James M USA California 92705 Solana Beach Shoemaker Rain 339 (56) Reference JP-A-58-59994 (JP, A) PROC. NATL. ACAD. SCI. USA, 78-9! (1981) P. 5807 −5811

Claims (1)

(57) [Claims] 1. A hybrid monoclonal antibody having bispecificity as a component of an antibody mixture comprising the hybrid monoclonal antibody and two monospecific antibodies in a ratio of about 2: 1: 1, and these A hybrid monoclonal antibody having bispecificity, characterized in that it is obtained from a polydoma produced so that there is a difference in the amount of charge sufficient for separating the antibody by an ion exchange method.