JPH0467959B2 - - Google Patents

Description

Claim 1: A first hybridoma cell producing a monoclonal antibody against a first antigenic determinant;
A method for producing a polydoma producing a bispecific hybrid monoclonal antibody, comprising fusing it with a second hybridoma producing a monoclonal antibody against an antigenic determinant in the presence of a fusion promoter, the method comprising: The first hybridoma cell is hypoxanthine aminopterthymidine sensitive and neomycin resistant, and the second
A production method characterized in that the hybridoma cells are hypoxanthine aminopterin thymidine insensitive and neomycin non-resistant. FIELD OF THE INVENTION The present invention relates to antibodies with bispecificity. In another aspect, the invention relates to immunodiagnostic and immunotherapeutic methods. In yet another aspect, the invention relates to hybridomas and related monoclonal antibody technology. BACKGROUND OF THE INVENTION Antigen-antibody reactions are routinely used in a variety of practical applications, and although they are not yet fully understood,
It has been widely studied to establish its value in other uses. For example, serum antibodies produced by a host animal's immune response to an immunogen can be used in affinity purification methods to separate the immunogen from solutions in which only small amounts of the immunogen are present. In other cases, if the immunogen is an antigen associated with the disease, its presence in the patient's serum or other body fluids can be determined using immunoassay or immunoassay techniques.
metric techniques). For example, using radioimmunoassay techniques
Detection of HBsAg is currently the method of choice. Serum antibodies to ferritin obtained from New Zealand white rabbits and labeled with I 131 have also been reported as showing promise in the treatment of hepatomegaly. (Order, etc.)
International Journal of Radiation Oncology,
(See Biology and Physics, 6 , 703 (1980)). Serum antibodies, such as those obtained from rabbits, rodents, or other mammals, are "polyclonal" in nature, meaning that once the host's immune system is stimulated, the immune system that the host is responding to is This is because it produces a mixture of specific antibodies directed against different antigenic determinants or epitopes on the source. Each individual antibody that makes up the mixture is the product of a B cell clone, and each B cell secretes only one antibody species. An antibody produced by one clone will have at least a subtle difference between its peptide order and the peptide order of the other antibody from an antibody directed against the same antigen produced by another clone. So they are different. In fact, therefore, each antibody species is a distinct molecule, and differences in the order of peptides between different antibody species affect not only the particular epitope they recognize and their affinity for that antigen, but also their overall specificity. Affect. It is not possible to grow individual B cells to obtain the antibody species that they secrete as pure compounds through the vague use of currently available tissue culture techniques. Relatively recently, Kohler and Milstein have discovered and reported a convenient method for obtaining monoclonal antibodies as secreted products of fused cells called "hybridomas." (G. Kohler and C. Milstein, Nature , 256, 495
(1975)). Basically, the method consists of fusing splenocytes taken from an immunized mouse with cells of the mouse's myeloma to create a hybridoma.
Preferably, the myeloma cells do not produce or at least secrete their own immunoglobulins or parts thereof. Cultures of cells obtained by cloning single hybridomas secrete identical antibody molecules, which can then be easily obtained as pure compounds. This is a major change from conventional antibody production from serum, for example. In this serum, each antibody is only one component of an almost inseparable antibody mixture of related but distinct compounds. Because monoclonal antibodies are pure compounds, they have certain specificity for a unique site on an antigen molecule and have a well-defined affinity. Thus, different hybridoma clones can be screened to select those that produce monoclonal antibodies with the most desirable properties for a given application. The permanence of the hybridoma ensures an almost unlimited supply of the antibodies it secretes, reducing problems associated with variations in antibody titer and overall affinity from animal to animal used to produce serum antibodies. Monoclonal antibodies obtained from hybridomas are practically applied, for example, to diagnostic kits. A selection of such kits is available from Hybritech, Inc., the assignee of this application. Antibodies are generally considered to exhibit only one specificity, which is directed against the immunogen to which the host's immune system responds by producing antibodies.
Antibodies consist of two identical halves, each with a pair of heavy and light chains, each of which recognizes the same antigenic determinant as the other. The -S-S-disulfide bridge linking the two (H) chains at the cysteine moiety can be selectively cleaved in vitro by mild reduction, and half of the molecule is released by subsequent acidification. Separated. These half molecules can then be recombined (regenerated) again in vitro at neutral PH, and this recombination occurs through non-covalent interactions. If antibodies of different specificity are subjected to conditions that lead to selective cleavage of the disulfide bridges between the heavy chains and subsequent refolding, recombination between the half molecules will occur randomly and the population of antibodies will form. may occur, at least some of which are hybrids in that half of the antibody molecules of one specificity are combined with half of the antibody molecules of a different specificity. For example, Nisonoff et al., “The Antibody Molecule”, Academic
Press, New York (1975), pages 260-261, describes the in vitro production of polyclonal antibody hybrids of rabbit anti-ovalbumin and anti-BGG antibodies. Hybrid monoclonal antibodies have also been obtained using similar methods. Outside of DM Kranz, Proc.Matl.Acad.Sci.USA ,
See 78, 5807 (1981). At least in theory, one half of an antibody recognizes and binds to one antibody determinant, or epitope, while the other half recognizes a different epitope on the same antigen or another antigen, resulting in dual properties. It will be shown. Hybrid antibodies can be obtained as described above, but the yields are often very low and the reactions used to produce them are difficult to reproduce.
The hybrid antibody usually undergoes significant and irreversible denaturation. Such denaturation may reduce immunoreactivity and is expected to exhibit other metabolic properties in vivo. As a result, hybrid antibodies remain of great experimental interest today. Antibodies with dual specificity are protein A
(from Staphylococcus aureus), carbodiimide and N-succinimidyl-3-(2
- each member of an antibody pair by joining pairs of complete antibodies, monoclonal or otherwise, using various coupling or cross-linking agents such as bifunctional compounds such as pyridyldithio)propionate; They can also be produced by obtaining dimeric or more multimeric antibodies, which confer their specificity. For example, Mondoche et al. has antibodies and protein A.
have reported the generation of bispecific multivalent antibodies through a series of reactions with 2000, which has been shown to be able to detect cell surface antigens in vitro. J.
See Immunological Methods, 42 , 355, (1981). According to these methods, antibodies of one specificity bind to the surface antigen and the other to a moiety that allows detection. Synthesis of bispecific antibodies by the techniques described above
It is complex and far removed from commercial applications. SUMMARY OF THE INVENTION The present invention provides, among other things, a novel and completely physiological method by which monoclonal hybrid antibodies can be reliably obtained in high yields without denaturation. Therefore, the present invention provides a first hybridoma cell that produces a monoclonal antibody against a first antigenic determinant and a second hybridoma cell that produces a monoclonal antibody against a second antigenic determinant in the presence of a fusion promoter. A method for producing a polydoma producing a bispecific hybrid monoclonal antibody, the first hybridoma cell being hypoxanthine aminopterthymidine sensitive and neomycin resistant, comprising: The present invention provides a production method characterized in that the second hybridoma cell is hypoxanthine aminopterynthymidine insensitive and neomycin non-resistant. Throughout this specification,
The term "hybrid antibody" is used to refer to a single antibody molecule with two different specificities. Individual specificity may be for antigenic determinants on two different antigens or for different antigenic determinants (epitopes) on the same antigen. Additionally, unless otherwise specified, the term "antigen" also includes haptens. The bispecific hybrid antibody is fused with a B-lymphocyte or a second hybridoma that is capable of fusion with a hybridoma, preferably a selectively destructible hybridoma, which secretes antibodies against preselected antigenic determinants. A second generation hybridoma (hereinafter referred to as a "Polydoma") is formed by the fusion of the following.
The B-lymphocyte, or second hybridoma, secretes another antigen against a different antigenic determinant. As used herein, the term "selectively destructible hybridoma" refers to a hybridoma that lacks, or at least largely lacks, the ability to survive in the medium in which it is cultured. do. We unexpectedly discovered the following: Unlike the parental hybridoma or B-lymphocytes from which it is derived, both of which secrete the same population of antigens with a single specificity, this polydoma also has a dual specificity. ie, secretes a high percentage of monoclonal hybrid antibodies that have the ability to simultaneously bind either or both antigenic determinants recognized by the individual antibodies produced by the parent cells. The monoclonal hybrid antibodies thus obtained are free from the undesirable denaturation characteristic of hybrid antibodies obtained by methods of chemical recombination of antibody half molecules. Furthermore, the method of the invention makes it possible to obtain hybrids reliably and in large quantities. The present invention also provides immunodiagnostic and immunotherapeutic methods using bispecific antibodies.
Generally, these methods use monoclonal or polyclonal antigens with a first specificity for a target antigen and a second specificity for some 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 an agent that is lethal to the target antigen or the tissue to which it binds, or it is itself capable of such a diagnosis. It is a very active substance. Thus, by appropriate selection of parent cells, polydomas can be obtained according to the invention that secrete antibodies with specificity for the target antigen and a second specificity for a diagnostically or therapeutically useful moiety. be able to. Alternatively, the antibody half molecules can be recombined using chemical methods in vitro, or individual complete monospecific antibodies can be combined or cross-linked by chemical methods, and can be produced by the present invention. Multimers of antibodies with the same bispecificity and the same or similar utility as monoclonal hybrid antibodies with bispecific
trimers or higher multimers). As used in the present invention, the term "antibody"
Includes antibody fragments with immunochemical properties such as Fab or F(ab) ₂ fragments. Therefore, an object of the present invention is to reliably obtain monoclonal hybrid antibodies that are not denatured during the manufacturing process and in high yields. Another object of the invention is an improved method of obtaining monoclonal hybrid antibodies. Yet another object of the invention is to provide immunodiagnostic and immunotherapeutic methods using antibodies with bispecificity. The manner in which these and also other objectives can be achieved will be apparent from the consideration of the following description of the preferred embodiments. DESCRIPTION OF PREFERRED EMBODIMENTS As mentioned above, the method for obtaining monoclonal hybrid antibodies according to the invention requires a hybridoma as one parent, preferably a selectively destructible hybridoma, which has preselected antigenic determinants. That is, it secretes monoclonal antibodies directed against the epitope. When a selectively destructible hybridoma is used as a parent, the cells obtained by fusion of the selectively destructible hybridoma (B-lymphocytes, i.e., the second hybridoma), i.e. polydomas, are obtained in the fusion process. This has the advantage that when the cells are cultured, it prevents overpopulation due to the population of parental hybridomas, and it also provides a method by which polydoma cells can be separated from the parental hybridomas. The selectively destructible hybridomas useful in the invention are produced by the classic Kohler-Milstein method, with one of the desired specificities.
It has been found that hybridomas secreting antibodies having the same antibody can be obtained from hybridomas obtained by fusion of myeloma 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 can be selectively destroyed.
selection process). In general, selective destructibility can be obtained by counter-selection against hybridomas lacking the genetic elements necessary for survival in a specifically chosen medium, where the medium is The polydomas produced by the fusion can be cultured therein with genetic contributions from the selectively destructible hybridoma's fusion partner, ie, B-lymphocytes, the second hybridoma. A currently preferred method of counterselection consists of culturing hybridomas secreting antibodies with one of the desired specificities to be included in the hybrid antibody in a growth medium containing 8-azaguanine. In such a medium, any cell that contains 8-azaguanine and therefore can grow in the medium is one that lacks the enzyme hypoxanthine-guanine phosphoribosyl transferase (HPRT).
Clones of cells lacking this enzyme are unable to grow in media containing hypoxanthine-aminopterin-thymidine (HAT). They are therefore selectively destroyed in this medium. A very similar method of counterselection involves growing hybridomas secreting the desired antibody in a medium containing 6-thioguanine (another analog of guanine that is toxic to cells if incorporated into DNA). consists of causing. Similarly, certain cells growing in this medium lack the HPRT enzyme, and clones of these cells are necessarily sensitive to HAT medium. Yet another method of counterselection that can be used in the present invention involves cultivating cells of a selected hybridoma line in a medium containing either the thymine analog 5-bromouracil deoxyribose (BUdR) or 2-aminopurine. Consists of growing. Only those cells lacking the enzyme thymidine kinase (TK) can grow in media containing either of these two inhibitors. enzyme
As is the case with cells lacking HPRT, cells lacking TK do not grow in HAT medium. Another method of obtaining selectively destructible parental hybridomas involves irreversible enzyme inhibition using metabolic inhibitors. Of these inhibitors,
So-called K _cat inhibitors are preferred. These inhibitors are analogs of the enzyme's substrate and 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) results in
It inhibits the enzyme formyl glycinamide ribonucleotide amidotransferase by forming a covalent bond with cysteine residues at the active site of the enzyme. This inhibition ultimately leads to cell death. However, this hybridoma can be rescued by fusing it with a second parent of the polydoma that provides the necessary enzymes. In a preferred embodiment, the selectively destructible hybridomas are fused with complementary B-lymphocytes, typically obtained as splenocytes taken from a host previously immunized with the antigen. be done. At this time, the antigen may be a hapten bound to a carrier protein, and the second antigen required in the hybrid antibody may be a hapten.
is selected such that its host mounts an immune response that produces antibodies with the specificity of.
The host is usually the mouse, but rabbits, humans, and other animal species can also be used, although interspecies fusions may exhibit reduced stability. Methods of immunizing such hosts are, of course, well known and need not be detailed here. The fusion of selectively destructible hybridomas with B-lymphocytes to obtain polydomas involves the fusion of the two groups of cells with polyethylene glycol or Sendai virus-like cells according to known methods. This can be done by combining known agents in a medium containing them. After performing the fusion, the cells are prepared for culture.
Transferred to a medium such as HAT medium. B-lymphocytes survive only for a short time and the parental hybridomas cannot grow in this medium. However, the number of polydomas formed as a result of fusion is
This is due to the supplementation of the parental hybridoma by lymphocytes, e.g. by genetic contribution of the ability to make lost enzymes such as HPRT or TK, or by the direct contribution of enzymes that were inhibited in the parental hybridoma. Can be grown in culture. Individual polydoma clones are cultured and screened to select those secreting antibodies with the desired bispecificity. Polydoma clones whose antibodies exhibit the desired bispecificity are further screened to select those whose second specificity, i.e., the specificity and affinity obtained for B-lymphocytes, is most favorable. . In another embodiment, polydomas are obtained by fusing a selectively destructible hybridoma with a second hybridoma that is also selectively destructible using a suitable fusion agent.
The second parental hybridoma is obtained in the same manner as the first, ie by the method of reverse selection, reversible 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,
We must be able to give polydomas a gene that allows them to produce HPRT. Similarly, if a second selectively destructible hybridoma lacks the enzyme TK, then the first
one must feed the polydoma with the gene for TK. If irreversible inhibition of the enzyme is used to confer selective destructibility, a similar complementarity must exist between the two hybridomas. It is also possible to use a hybridoma that has been subjected to a reverse selection method as the parent of one hybridoma, and a hybridoma that has been subjected to an enzyme inhibition method as the other hybridoma. The use of complementary selectively destructible hybridomas as parents for polydomas has the advantage that both parents can be selected based on the specificity and affinity of the monoclonal antibodies they produce, whereas single In the case of fusion of hybridomas with B-lymphocytes, it is not possible to carry out pre-fusion selection between B-lymphocytes to obtain those producing antibodies of the desired specificity and affinity. Fusion of two selectively destructible hybridomas can be performed using polyethylene glycol or other fusion agents, also according to known methods. After fusion, the cells are transferred to a growth medium in which the two parents cannot grow, but where the polydomas resulting from post-fusion fusion can grow due to the complementary contributions of the two parents. So far we have discussed selection methods for obtaining selectively destructible hybridomas for use as both partner hybridomas in hybridoma-hybridoma fusions to form polydomas. However, the need for a second hybridoma parent to be selectively destroyed can be eliminated by providing the first hybridoma parent with both dominant and recessive genetic markers. The currently preferred method is HAT-
This is the ouabain selection method. The drug ouabain is a specific inhibitor of the plasma membrane Na ⁺ -K ⁺ activated adenosine triphosphatease (Arpase).
This enzyme is responsible for the import of K ⁺ into the cell and the export of Na ⁺ from the cell. selective destructibility,
For example, hybridoma cells previously counter-selected to confer HAT sensitivity are grown in ouabain medium to select for ouabain-resistant cells. Clones of these cells are HAT sensitive but ouabain resistant. In contrast, hybridomas selected to fuse with
Although susceptible to ouabain, it can survive and grow in HAT. Alternatively, selection for ouabain resistance can be performed first on either the parental myeloma line or the hybridomas derived therefrom, followed by reverse selection or other methods that provide the possibility of selective destruction. can. Cells obtained by fusion of two hybridomas in polyethylene glycol or other fusion agents are transferred to HAT medium containing ouabain at a concentration lethal to the second, hybridoma parent. The selectively destructible hybridoma parent may be ouabain resistant, for example because it lacks HPRT or other enzymes.
Cannot survive even in HAT medium. However, polydoma cells can grow in this medium because they have the enzyme and the ouabain resistance necessary for survival. The method described above includes the use of a parent selectively destructible hybridoma that secretes an antibody with one of the desired specificities of the hybrid antibody and a second parent hybridoma that secretes an antibody with the other desired specificity of the hybrid antibody. “off the stock”
The advantage is that polydomas can be obtained by direct fusion with hybridomas and that there is no need to use any technique that confers selective destructibility on their second hybridoma parent. Another technique for obtaining polydomas is the use of a universal parent, a parent that has both positive and negative genetic markers and can be fused with any "off-the-shelf" hybridoma. Techniques for this include the use of recombinant DNA vectors carrying various drug resistance genetic markers. For example, SV ₄₀ carrying the gene for neomycin resistance can be used. Currently the preferred universal parent is HAT sensitive-neomycin resistant.The selected parents are:
After being counterselected for HAT sensitivity, they are transfected with the SV ₄₀ vector carrying the gene for neomycin resistance. This procedure can be reversed to allow transfection to occur first. The resulting hybridomas can grow in the presence of neomycin, which is normally toxic to mammalian cells, but die in the presence of HAT. However, off-the-shelf hybridomas grow in HAT but die in the presence of neomycin. Therefore the product of the fusion of these parents is
Survives in the presence of HAT and neomycin. Although it is currently preferred to use vectors to transfer resistance to neomycin, vectors carrying genes that confer resistance to other drugs in mammalian cells can also be used. Although preferred, it is not essential to the method of the invention to obtain polydomas from hybridoma pairs that at least one of the parental cell lines is selectively destructible. None of them can be selectively destroyed, but secretes antibodies that have one of the selectivities required in hybrid antibodies.
After fusion of paired hybridomas in the presence of a suitable fusion agent, all unfused parental hybridomas increase to the extent that it is impractical to screen subclones to confirm polydomas. It is within the scope of our invention to perform subcloning of cells.
Subclones were then screened and
Check which ones secrete antibodies with bispecificity. This method is optimal for obtaining polydomas when the parental cells have a high fusion frequency. Anyway, especially when the frequency of cell fusion is low, cells whose monoclonal antibodies are obtained from the fusion of hybridomas against various antigens can be screened using a cytofluorograph to identify polydomas. can. To do this, the two antigen samples are separated into separate fluorescent moieties that fluoresce at different wavelengths.
is labeled with. For example, one can be labeled with fluorescein and the other with rhodamine. The fused cells, preferably cultured overnight or for another suitable period of time to increase their number, are incubated with the two labeled antigens in their entirety. The cells are then screened using cytofluorography.
Cells that fluoresce at only one of the two wavelengths will be derived from the parental hybridoma cell line. On the other hand, cells that exhibit fluorescence at both wavelengths are polydomas and can be isolated 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 of the hybrid antibody, and a second cell that secretes a monoclonal antibody having a second required specificity is removed. Polydomas can be obtained directly by inserting it into the cytoplasm of hybridomas. Of course, neither parent hybridoma needs to be selectively destructible for use in this method. After insertion of the nuclear material, the cells are cloned to obtain a population of polydomas. Unlike the parental hybridoma or B-lymphocytes, which secrete a single antibody, the polydoma cells obtained by our invention secrete a mixture of antibodies, at least one of which is bispecific. We found that it is a hybrid antibody with The polydoma also produces relatively small amounts of antibodies of the same specificity as those produced by the parental cells used to obtain the polydoma. The ratio of hybridization to monospecific antibodies is approximately 2:1:1, which means that the parental polydoma contains all possible (H) chains synthesized by the parental cell and not the polydoma itself. This is the ratio that would be expected if equal amounts of those bound to the irregular side were produced. Polydomas can be cultured in vitro to obtain large quantities of hybrid antibodies or grown in adults either in genetically compatible animals or nude mice, which can be produced using known methods. It is recovered from the culture medium or ascites fluid of the animal. For example, “Monoclonal
Autibodies” (edited by Kenett et al.)
See Protocols, Plenum Press, New York (1980), pp. 363-418. The mixture of antibodies produced by polydomas can be separated to obtain hybrid antibodies. For example, by sequential affinity chromatography of the first one followed by another antigen for which the hybrid antibody is specific, its separation from monospecific antibody impurities is effected. be able to. We have also found that simple ion exchange chromatography and electrophoresis techniques can be used, at least in certain cases. If desired, differences in charge for ion exchange can be one of the antibody characteristics considered in selecting parental lines. Example A first hybridoma cell DMS-1 producing anti-human rIL-2 antibody (anti-human recombinant interleukin-2 antibody) was adapted to be sensitive to the drug HAT by growing in 8-azaguanine. I let it happen. 8-Azaguanine concentration is 0.96~
A dilution solution of 60 μg/ml was prepared. 24 for each concentration
1 ml of the solution was added to 3 wells of a well plate, and 1 ml of the medium was added to 3 wells as a control. These solutions were incubated at 37℃ and 5% _CO2 for 1 hour.
and allowed to equilibrate to this state. Using a pipette, 5 x 10 ⁵ hybridoma cells were placed in each well. After several days of growth, cells growing at the highest concentration of 8-azaguanine were collected and
The cells were washed, cultured again at the same concentration, and the concentration was increased stepwise to select a group of cells that could survive and grow at 30 μg/ml. The obtained group was made into subclones and tested for HAT sensitivity and high antibody production. Next, clones of DMS-1 hybridomas selected in the mid-logarithmic phase and with survival of 90% or more were
The selectable neogene PSV2/neo linearized using PVU restriction enzyme at 1 unit/ml was transfected by electroporation and transfected with G418 (Sigma Chemical, a drug containing a derivative of neomycin). ). More specifically, the number of cells is 15
Adjust to 20 × 10 ⁶ × 10 ⁶ and 25mM
Washed twice in HBSS containing HEPES, then
The cells were resuspended to 15×10 ⁶ cells/ml in HBSS containing HEPES and 6 mM glucose. When preparing electroporation, add 20 μg of PSV2/neo DNA in 330 μ and 25 μ of this cell suspension.
A solution containing 16μ of salmon sperm carrier DNA was placed in a sterile cube, mixed, and chilled on ice for 10 minutes. Electroporation was performed with a single pulse of 250V for 5 microseconds. The electroporated cells were cultured in a drug-free growth medium at a concentration of 2×10 ⁵ cells/ml. Three days later, the cells were counted and cultured again in a 24-well plate in 2 ml of medium containing G418 (1 mg/ml) to a total of 5 x 10 ⁴ viable cells. The transfected cells are screened by antibody production, subcloned,
It was named DMS-1.N1. Next, this DMS-1.N1 clone is used as a second clone that produces anti-NK1,1 antibody (anti-natural killer cell 1,1 antibody) using standard fusion technology.
hybridoma PK136 (ATCC (American
B16 tumor cell line obtained from Type Culture Collection). The fused cells were then selected by repeated selection techniques combining HAT and G418.
Only cells with both HAT resistance and neoresistance survived and produced antibodies with bispecificity.

[Table] Reference Example 1 A monoclonal hybrid antibody having bispecificity for hepatitis B surface antigen (HBsAg) and prostatic acid phosphatase (PAP) was produced according to the present invention as follows. Hybridomas secreting monoclonal antibodies against PAP were grown in HAT medium for one week and then transferred to non-selective medium for growth. After various times of growth under non-selective conditions, 2 ml aliquots of cells were placed in medium containing 10 ^-4 M 8-azaguanine. This 8-azaguanine inhibits cell growth by being included in their DNA instead of guanine.
Although cells lacking the HPRT enzyme survived and grew in this medium, these cells did not necessarily survive in the HAT. Clones grown in medium containing 8-azaguanine were tested for sensitivity to HAT and anti-PAP productivity. Still producing anti-PAP 4x
One clone that showed HAT sensitivity with a reversible frequency of less than 10 ⁻⁸ was subcloned. All subclones behaved like their parent clones. Cells from one of the HAT-sensitive subclones were fused in polyethylene glycol with cells from spleens obtained from Balb/c mice hyperimmunized with HBsAg using Gerhard's fusion technique to obtain polydomas. Ta. See “Monoclonal Antibodies”, supra, p. 370. This fusion produced 220 polydomas, which were screened for measurement and secreted antibodies showing specificity for both PAP and HBsAg. Two such polydoma clones were measured and found to produce antibodies followed by ascites exhibiting both specificities. Subclones of both polydomas produce ascites exhibiting both specificities and, like those of the parental clones, Orstein-Davis.
Three bands were generated on PAGE. Ascites from both clones was ¹²⁵ I− by radioimmunoassay.
It was found that HBSAg and ¹²⁵ I-PAP bind together.
A value of K of approximately 10 ⁹ was obtained for each. Thus, the formation of bispecific antibodies was suggested. The data in the table compares ascites from one clone of this polydoma with ascites from hybridomas secreting monoclonal antibodies against IgE (used as a control), PAP, and HBsAg, respectively. , fixed
Results obtained in an immunoassay using HBsAg as a solid phase and various radiolabeled antigens as a solution phase are shown. A 200μ sample of ascites from a polydoma and each of the three hybridomas were treated with HBsAg.
was incubated overnight with 12 bound polystyrene spheres. HBsAg spheres were obtained from Abbott Laboratovies, North Chicago, IL. After washing,
Triplicate samples are shown. ¹²⁵ I labeled antigen 100000cpm
and incubated for 4 hours. After the second wash,
The labeled antigen bound to the balls was determined by counting the balls. In a set of studies using radiolabeled PAP as the solution phase antigen, anti-PAP
was added to the antigen, which was then incubated with the balls. used for this purpose
The antibodies to PAP are monoclonal antibodies produced by the parental hybridomas used to make the polydomas, so they are directed against the same PAP epitope, as would be expected to be exhibited by a hybrid antibody.

Table: Bispecific * ¹²⁵ I Labeled Antigen The data in the table shows that only the radiation expected from non-specific binding is measured for anti-PAP when compared to the data for the IgE control. On the other hand, ascites containing HBsAg antibodies bound to labeled HBsAg antibodies as expected;
Other labeled antigens showed non-specific binding when tested. However, ascites from polydoma clones bound both labeled HBsAg and labeled PAP. The former is due to the presence of small amounts of nonhybrid monospecific antibodies against HbsAg in the ascites, while the latter is due to the presence of a hybrid that can bind and bridge HBsAg on the spheres with trace amounts of labeled PAP in solution. Attribute. Experiments using a mixture of labeled PAP and anti-PAP from the parental hybridoma confirm that the anti-PAP specificity of the hybrid is against the same epitope as the antibody secreted by the parent. This is because the binding of labeled PAP to the hybrid antibody is inhibited by the parent antibody, so only basal radiation is observed. Analysis of ascites from polydoma clones used in comparative radioimmunoassays was performed using polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis (IEP). Both showed the presence of at least three antibody species. preparative scale
DEAE ion-exchange chromatography yielded three well-separated peaks, one of which had a shoulder in the middle. Each of the peaks is PAGE
and homogeneous as analyzed by IEP,
Each corresponded to one of the bands in the initial ascitic fluid. The antigen binding properties of substances representing each of the DEAE peaks were tested using radiolabeled HBsAg and PAP. The first peak was combined with HBsAg, but
It did not bind to PAP. The middle peak and its shoulder bound both HBsAg and PAP, and the last peak bound only PAP. Therefore, the middle peak is a hybrid antibody that has dual specificity for HBsAg and PAP and consists of at least two subspecies. The hybrid antibody obtained as the middle peak of DEAE chromatography was radiolabeled with ^125I . After labeling, 85% of the labeled antibody binds to PAP and 88% to HBsAg. The affinity of the hybrid for PAP was found to be slightly lower than that of the monoclonal antibody against PAP produced by the parental strain. This difference in affinity was similar to that observed by us between monoclonal antibodies and their Fab fragments. DEAE chromatography shows that the hybrid antibody contains 50% more than the antibody produced by the polydoma, a ratio expected for statistical reasons of 2:
This shows that the ratio approaches 1:1. This 2:1:1 ratio is such that the polydomas exhibit all possible antibody heavy chains, either PAP or HBsAg specificity, and are randomly combined within the cell to combine with the hybrid antibody. This is expected on the basis of statistical reasons if a small amount of two monospecific antibodies are formed that have the same specificity as the antibody produced by the parent cell. It is something. The existence of hybrid antibody subspieces suggests that they differ in the composition of their light chains. Reference Example 2 A monoclonal hybrid antibody having dual specificity for human IgD and prolactin was produced by fusion of two hybridomas according to the present invention. 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 "universal parents" have been described elsewhere herein. This doubly labeled hybridoma, the so-called "universal parent", can then be fused with any other hybridoma. The resulting polydomas grow in the presence of HAT and ouabain, but both unfused parental cells die. To form such universal parents, both selectable labels were introduced during initial formation of the hybridoma. To obtain this parental cell line,
Widely available HAT-sensitive murine myeloma
P3.653 was selected for a second genetic marker, ouabain resistance, by introducing 1mM ouabain into the growth medium. Although most of the cells died, about 1/100,000 cells acquired resistance to the drug through uncontrolled mutations and survived and proliferated, creating a new myeloma population that was HAT-sensitive and aubain-resistant. Formed. This HAT-sensitive, Aubain-resistant myeloma was then fused with splenocytes obtained from Balb/c mice hyperimmunized with IgD using the method of Gerhard, cited above. Hybrids were selected on HAT medium (without ouabain) and clones were isolated using IgD.
screened for the production of monoclonal antibodies against. Among the positive clones, those that produced IgG against IgD were selected for further study. This clone was tested for maintenance of ouabain resistance by adding 1mM ouabain to the growth medium. About one-third of the cells retained this genetic mark. As this culture was growing 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. Example 1 of one of the subclones
Further counter-selection was performed to obtain a HAT-sensitive cell population. This subclone was grown under non-selective conditions for 2 weeks before being placed in medium containing 6-thioguanine. As mentioned 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 will not grow. Cells lacking the HPRT enzyme do not utilize 6-thioguanine from the medium and are therefore able to grow but are therefore susceptible to HAT. This population of counter-selected subclones was then itself subcloned in medium containing 6-thioguanine and ouabain. Subclones were isolated using monoclonal anti-
Continuous production of IgD antibodies was tested. One clone that showed all the desired properties - growth in ouabain and 6-thioguanine and production of monoclonal anti-IgD was selected as the so-called "universal parent". Then this all-purpose parent works with any other HAT
- Generated polydomas that could be fused with resistant, ouabain-sensitive hybridomas and generate hybrid antibodies, the monospecific of which is anti-IgD. For this purpose, we first selected mouse hybridomas secreting monoclonal antibodies against prolactin. Anti-prolactin monoclonal antibodies are of the same subclass (IgG ₁ ) as anti-IgD generated by new strains,
Ornstein-Davis Gell
Davis gels), it is easily separated from its antibodies.
Such separation suggests a very different burden on the antibodies. Therefore, separation of hybrid antibodies should be easily carried out by DEAE-Sephadex chromatography. 10 ⁷ of HAT-sensitive and ubain-resistant cell lines
7 cells were fused in polyethylene glycol with 10 ⁷ cells producing anti-prolactin.
The fused cells were first grown in HAT medium for 3 days, then modified with HAT+ouabain medium for 3 days, and finally placed back into HAT medium. Over 600 clones were generated from this fusion. Sixty-six clones were randomly selected for analysis. Of these clones, 36 showed anti-IgD and anti-prolactin activities. 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 200μ of a 100ng/ml prolactin solution for 5 hours. The antibody used binds prolactin at a site remote from that of the antibody produced by the fused hybridoma cell line. After washing the beads, they were incubated with the clone supernatant overnight. The next day, after several washes, ¹²⁵ I-labeled IgD was added. Hybrid antibodies bound to beads by one functional arm were able to bind radiolabeled IgD with free anti-IgD functionality, but new types of antibodies, IgD-IgD and prolactin-prolactin. Moga prolactin beads and ¹²⁵ I−
This bond could not be formed between IgD tracers. Typical analysis results for clones producing hybrid bifunctional antibodies are shown in Table 2 below.

[Table] Chin
21 out of 36 clones showed clear bifunctional activity by this analysis. 2 available so far
Ascites developed from two clones was shown to be reactive in this bifunctional assay. These ascitic fluids contain antibodies that are divided into three distinct bands in the form of an Orstein-Davis gel. The two bands correspond exactly to the antibodies produced by the parental hybridomas (anti-IgD and anti-prolactin). The third band shifts as expected between the bands of the hybrid antibody's parent monoclonal antibody. The data in Table 3 show that hybrid monoclonal antibodies to IgD and prolactin exhibit a dose response when the amount of prolactin is varied in the assay used to generate the data in Table 2. Table 3 uses antibodies from clonate #2 and antibodies from the parental cell line (anti-IgD and anti-prolactin) as controls.

Table: These data demonstrate that the amount of prolactin bound by the hybrid antibody is dose-responsive such that as the amount of prolactin increases, the amount of labeled IgD bound increases. There is. In contrast, when using parental antibodies against IgD and prolactin, no dose response is seen as these cannot form a bridge between the bead-bound prolactin and the labeled IgD. Thus, hybrid antibodies can be used as a component of prolactin assays. Other specifically tailored hybrid antibodies offer similar opportunities for other analyses. Reference Example 3 A monoclonal antibody against the hapten arsenate-arsenate dimer was secreted by a method similar to that of Example 2, and was resistant to ouabain and resistant to HAT. A hybridoma with a universally susceptible parent was formed. This hybridoma is known as carcinogenic antigen (CEA), an antigen produced from both embryonic tissue and several types of cancer.
fused to a hybridoma that secretes a monoclonal antigen with specificity for. Once again, over 600 clones resulted from this fusion.
Of the 72 clones tested for the ability to bind both CAE and arsenate, 69 clones had both binding abilities. Clones showing maximum binding were selected for hybrid antibody enzyme-linked immunosorbent assay (ELISA). For each analysis, CEA solution (600 ng or 250 ng) was adsorbed overnight to each well of a plastic 96-well microtiter plate. The next day, unadsorbed substances were washed out of the wells with PBS-Tween20. Add clone supernatant and incubate for 2.5 min at 35°C.
After incubation for an hour, the plates were washed off. CEA-CEA and CEA-arsenate antibodies remain bound to the plate via the adsorbed antigen. A second antigen, arsenyl acid coupled to the enzyme alkaline phosphatase, was added to the wells for 30 minutes at 35°C.
Added time. After another wash with PSB-Tween, chromagen substrate for alkaline phosphotase, para-nitrophenyl phosphate, was added to the wells and allowed to develop for 48 hours. absorption of each well.
Measured at 410nm. When present in the clone supernatant, the hybrid antibody binds with one functionality to the adsorbed CEA on the plate and the other functionality to the arsenyl acid bound to alkaline phosphotase. left free. Color was developed from the chromagen substrate only where alkaline phosphotase bound to arsenylic acid was bound. In this analysis, 3 out of 12 supernatants showed hybrid antibody activity as shown in Table 4.

[Table] The values listed in Table 4 are raw data from experiments conducted in duplicate. The present invention provides antibodies with bispecificity, e.g.
Hybrid antibodies obtained as described above or obtained from antibody half molecules by conventional techniques of Nisonoff et al. (cited above), or by combining or cross-linking individual monospecific antibodies. Immunodiagnostic and immunotherapeutic methods using the resulting antibody multimers are also provided. Preferably, the bispecific antibodies used in these methods are undenatured to ensure that the antibodies are obtained as substantially pure compounds with uniform specificity and affinity for the antigen. A hybrid antibody produced according to the present invention. In immunodiagnostic applications, the two specificities exhibited by a bispecific hybrid or other antibody will be one for the target antigen that needs to be detected and the other for another antigen. This other antigen may be a hapten or other molecular species, allowing diagnosis. For example, antibodies useful in immunohistology have primary specificity for the suspected antigen, e.g., cancer-associated antigens such as CEA, PAP, or ferritin, and contain enzymes that produce a color reaction in the presence of an appropriate substrate. It has a second specificity for the hapten or antigen involved in the color reaction, such as: Among the suitable enzymes to which the second specificity of the antibody can be directed are prosstate acid phosphotase (PAP), horseradish peroxidase, glucose oxidase and alkaline phosphatase. To perform histological examination, tissue sections are first treated with bispecific antibodies. Before doing so, the enzymes that catalyze the contaminating reaction can be preliminarily blocked by the hybrid. If this is not the case, then, after appropriate incubation, the sections are treated 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 development produced by the enzyme and substrate in the tissue sample is a positive indication of the presence of the target antigen in the tissue. The manufacturing method is described in this specification.
Hybrid antibodies against HBsAg and PAP
-Nitro-phenyl phosphate was found to bind to HBsAg and PAP on the test substrate (polystyrene balls) in simulated contamination experiments using it as enzyme substrate. This hybrid antibody
After incubation with PAP and HBsAg, addition of p-nitrophenyl phosphate resulted in the ball changing color from its characteristic yellow to brown. As mentioned in Reference Example 2, bispecific antibodies can also be used in immunoassays and immunometric assays. Using the hybrid antibody against HBsAg and PAP as described above, immunometric assays for HBsAg can be performed on HBsAg and PAP.
This can be done using immobilized monoclonal antibodies against HBsAg as a solid phase to extract HBsAg from serum or other liquid samples suspected of containing antigen. The sample is incubated with a ball, bead, test tube or other substrate that has anti-HBsAg bound or coated on its surface. Incubation with a solution of the hybrid can follow, simultaneously with, or before incubation with a serum sample. In either case, the result is that if HBsAg is present in the sample, a sandwich of immobilized antibody, HBsAg (if present in the sample), and hybrid antibody will be formed. As part of the assay, binding of PAP to hybrid antibodies is allowed. This can be done during or after the formation of the sanderch. Otherwise, an antibody-PAP complex may be preformed instead. After formation of the sandwich, the solid phase is washed to remove sample residue and unbound hybrid antibodies and then loaded with a substrate such as p-nitrophenyl phosphate or α-naphthol phosphate, which undergoes a color change in the presence of PAP. contact with the containing solution. The occurrence of a color change confirms the presence of the target antigen in the sample. In such an analysis, North Illinois
Manufactured by Abbott Laboratovies in Chicago
Polyclonal anti-HBsAg beads from a commercial kit for HBsAg diagnosis (sold under the name "Aushia") were used to incubate samples containing various amounts of HBsAg with the beads. The samples used were obtained by diluting the positive control, negative control, and positive control from a commercially available kit with the negative control at a ratio of either 1 part of the negative control: 2 parts of the positive control, or 2 parts of the negative control: 1 part of the positive control. There were two samples that were tested. After incubation of the sample with beads to bind HBsAg, the beads were washed and incubated with a hybrid antibody reactive against 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. This incubation was followed by another wash and the beads were incubated with the substrate alpha-naphthol phosphate. After a suitable incubation in which PAP removes the phosphate by enzymatic reaction,
The substrate was removed from the beads and an indicator, first garnet GBC salt, which changes color from clear to purplish red in the presence of the products of the enzymatic reaction, was added. A discoloration was observed confirming the presence of HBsAg in the sample.
The absorbance at 570 nm was measured for each sample and the data are shown in Table 5 below. Table 5 % of HBsAg concentration positive control Absorption 570nm 0. 031 34． 166 67． 243 100． 379 These data show a dose response to 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 hybrid antibodies can be used in immunoassays. Detection means other than enzymatically catalyzed reactions are also possible. For example, the second specificity of a bispecific hybrid antibody or other antibody can be detected with a hapten that is radiolabeled or fluorescent or by any other suitable means detectable in a sandwich. Or it can be directed against antibodies. One preferred method of utilizing bispecific hybrid antibodies or other antibodies in immunoassays is to utilize reduced fluorescence quenching. In such assays, one specificity of the antibody is directed against the target antigen and the other specificity is directed against, for example, a hapten with a fluorophore. This chromophore may be attached to the hapten or, if appropriate, the hapten itself. This assay is performed by incubating the antibody with serum or other sample suspected of containing the target antigen to which a predetermined amount of a quenching chromophore-labeled target antigen has been added. If a target antigen is present, the labeled antigen competes with the target antigen in the sample for the binding points of antibodies specific for that target antigen. Before, during, or after this incubation, an amount of a hapten with a fluorophore is incubated with the antibody and bound at the other point of attachment. The wavelength at which the quenching side can absorb (quench) if the two chromophores are located close enough together that a photon emitted by the fluorescing side can be captured by the quenching side. The two chromophores are selected such that the other fluoresces at . To this end, the two chromophores should be within about 100 angstroms of each other, preferably within about 50 angstroms. This configuration occurs when a fluorescent chromophore is attached to one antibody attachment point and a quenching chromophore is attached to the added antibody at the other attachment point. Suitable chromophore pairs include fluorescein as the fluorescent chromophore and rhodamine as the quenching chromophore. The measured fluorescence varies inversely with the amount of native antigen in the sample, because in the absence of native antigen, all of the antigen bound to the antibody is labeled with a quenching chromophore, and the fluorescence supported by the hapten. This is because it is positioned so as to absorb the fluorescent light from the group. Comparing the measured fluorescence to the fluorescence of a control sample containing a known amount of antigen provides a qualitative and quantitative determination of the presence of antigen in the sample. This type of immunoassay can be used, for example, to measure serum concentrations of drugs such as dilantin, which must be closely monitored. In such assays, the target antigen is of course dilantin. It will be clear to those skilled in the art that this method can be used to detect other antigens as well, including in particular antigens associated with tumors. Another preferred method of utilizing bispecific hybrid antibodies or other antibodies in immunoassays relies on enzymatic reactions. In the currently preferred method, one of the specificities of the antibody is of course directed against the target antigen and the other is directed against the enzyme or the hapten to which the enzyme is attached. modifying the target antigen by attaching to it a substance that interacts with an enzyme to produce a detectable substance or that in some other way allows detection of the formation of an antigen-antibody complex; I'll keep it. This assay is performed by incubating the antibody with a sample suspected of containing the target antigen plus a predetermined amount of the modified target antigen.
Detection may be by, for example, fluorometry, luminescence, spectrometry, etc. In an alternative method, the target antigen added may have an enzyme bound to it, in which case the target antigen is directed against a substance that interacts with the enzyme, or directed against a hapten to which that substance is bound. Antibodies have one type of specificity. 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 an antibody binds both the added target antigen (to which one of the enzymes is bound) and the other enzyme, the products of the first enzymatic reaction are produced in close proximity to the second enzyme. , can undergo a reaction mediated by the latter enzyme before an appreciable diffusion of this product into the surrounding medium takes place. An example of such a method is in the following reaction system:
The enzyme hexokinase (HK) and glucose
6-phosphate dehydrogenase (G-6-
PDH). (1) Adenosine triphosphate (ATP)
+ Glucose HK ---→ Adenosine diphosphate (ADP) + Glucose-6-phosphate (2) Glucose-6-phosphate + Nicotinamide (NAD ⁺ ) Adenine dinucleotide G-6-PDH --- ---→ Gluconolactone-6-phosphate + dihydronicotinamide adenine (NADH) dinucleotide To utilize this reaction system, the added target antigen is either HK or G-6-PDH bound to it. , the hybrid antibody will have one specificity directed against the other (or the supporting hapten). The sample contains glucose, ATP, and coenzymes in addition to the hybrid antibody and a predetermined amount of enzyme-labeled antigen.
Added NAD ⁺ to it. Preferably, the hybrid antibody already has the other enzyme bound to it. Alternatively, the enzyme can be added to the sample along with other reagents. During this incubation, the enzyme-labeled antigen competes with the native antigen in the sample for one of the binding points of the hybrid antibody, if present in the sample. The other enzyme is or will be attached to the second attachment point. By this,
The formation of glucose-6-phosphate catalyzed by HK occurs in close proximity to G-6-PDH.
The latter converts glucose-6-phosphate to gluconolactone-6-phosphate and converts NAD ⁺
Reduction to NADH ensues. This NADH
Because it absorbs strongly at 340 nm, it can be detected by spectrometry. The amount of NADH produced varies inversely with the amount of native antigen in the sample, ie its maximum production occurs when no target antigen is detected in the sample. Comparing the amount of NADH produced to a control sample provides a qualitative and quantitative determination of the presence of antigen in the sample. This type of assay can be used to monitor the concentration of dilantin or other drugs 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. An antibody having a first specificity for a target antigen, such as a tumor-associated antigen, and a second specificity for a hapten to which a suitable radionuclide, preferably one that emits gamma radiation, is conjugated is first introduced into the host. Administer. After sufficient time has elapsed for the antibody to localize to the target site and for unbound antibody to leave the host's healthy tissue, a hapten with a radionuclide is administered and binds to the localized antibody. After an appropriate interval to allow unbound haptens to leave the host,
The host is scanned with a suitable camera to determine if there are areas of high radiation concentration. 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 a target antigen to which a radionuclide is directly attached.
In such cases, the radionuclide must have a half-life long enough to remain in sufficient quantity after the time necessary for the antibody to substantially localize at the target site. Additionally, during this process the antibodies may be retained in the liver or other non-target tissues for a period of time, exposing these tissues to the radiation carried by the antibodies. On the other hand, the present invention allows the use of radionuclides that have a shorter half-life than the radionuclides used with monospecific antibodies. Being relatively small particles, haptens carrying radionuclides are highly mobile in vivo, moving rapidly within the chamber and binding to antibodies localized at target sites or It leaves the body without spending enough time in non-target tissue to cause problems. For this reason, short half-life isotopes are
It can be administered in an amount that poses minimal risk to healthy tissue even in the event of a substantial overdose. Preferably, the hapten is an agent to which a radionuclide is directly attached or complexed with a radionuclide. Chelating agents for radionuclides bound to haptens 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 coupled with ethylenediaminetetraacetic acid (EDTA) as a chelating agent is a suitable hapten. A suitable radionuclide for use with this hapten is ¹¹¹ _Io . Bispecific antibodies have one specificity for an antigen associated with a disease and are lethal agents for the antigen or the diseased tissue to which the antigen is associated and which it is desired to destroy; This bispecific antibody can also be used in immunotherapy by constructing it to have specificity for the hapten attached to the drug, but not the other. For example, the antibody has one specificity for a tumor-associated antigen, such as PAP, carcinogenic antigen (CEA), ferritin, or other such antigen, and a radionuclide, preferably α- or β - a second specificity directed against a hapten to which a radioactive substance is attached or which consists of lytin A chain or other toxins or drugs. Such agents may include gelonin, alpha-amantine, diphtheria toxin A, methotrexate, dichloromethatrexate, dounomycin, and chlorombucil. Of course, if the toxin or drug itself can function as a hapten, it need not be conjugated to any other moiety. When radionuclides are to be used as lethal agents, such as in situations where they are used for in vivo immunodiagnosis,
A hapten can have a radionuclide directly bound to it, or it can be or have an agent, such as a chelating agent, that complexes with a radionuclide or has an agent attached to it.
In such cases, bispecific hybrid antibodies or other antibodies are administered to the diseased host, localized to the affected tissue site, and any excess exits the host. The hapten is then administered;
It binds to antibodies wherever they are localized. This allows the use of short half-life radionuclides which minimizes the risk of harming healthy tissue even if the hapten carrying the radionuclide is administered in virtual excess. This is because, due to the relatively small size of haptens, excess quickly leaves the body and is not localized in substantial quantities in healthy tissues. Also, antibodies carrying circulating target antigens are lethal to tissues, as occurs when a lethal agent binds directly to a monospecific antibody directed against the target antigen. The possibility of combining with and transporting this to healthy tissue is eliminated or reduced. An example of a hapten with a radionuclide directly attached to it is
“International Journal of Applied Radiation
and Isotopes”, 33 , 75 (1982).
²¹¹ At-astat-2-methyl-1,4-naphthoquinol bis(disodium phosphate)
It is. ²¹¹ At is an emitter of alpha rays. Those skilled in the art will appreciate that there are a large number of suitable radionuclides that can be directly attached to a hapten or can be complexed with a hapten by any of a wide variety of chelating agents. The above description of the invention is of the presently preferred embodiments. Modifications may be made without departing from the scope of the invention, which is limited only by the appended claims.