GB2169921A - Antibodies having dual specificities, their preparation and uses therefor - Google Patents

Description

1 GB 2 169921 A 1

SPECIFICATION

Antibodies Having Dual Specificities, Their Preparation and Uses Therefor Field of the Invention

This invention relates to antibodies having dual specificities In another aspect it relates to immunodiagnostic and immunotherapeutic processes In yet another aspect it relates to hybridomas and 5 related monoclonal antibody technology.

Background of the Invention

The antigen-antibody reaction is already routinely exploited in a variety of practical applications and is being widely investigated to establish its value in other, as yet unproven, utilities For example, serum antibodies produced by a host animal's immune response to an immunogen can be used in affinity 10 purification procedures to isolate the immunogen from solutions in which it is present in only minute quantities.

In other circumstances, if the immunogen is a disease associated antigen, its presence in a patient's serum or other body fluid can be detected using immunoassay or immunometric techniques For example, 1 5 detection of H Bs Ag using a radioimmunometric technique is the current method of choice On another 1 5 front, serum antibodies to ferritin, obtained from New Zealand white rabbits and labeled with 1311, have been reported as showing promise for the treatment of liver tumors (See Order et al, International Journal of Radiation Oncology, Biology and Physics, 6,703 ( 1980)).

Serum antibodies, for example, those obtained from rabbits, murine species or other mammals are "polyclonal" in nature since the immune system of the host is stimulated to produce a mixture of specific 20 antibodies directed to the different antigenic determinants or epitopes on the immunogen to which the host is responding The individual antibodies making up the mixture are each the product of a B-cell clone; furthermore each B-cell secretes only one antibody specie The antibody produced by one clone differs from an antibody against the same antigen produced by another clone by having at least a subtle difference between its peptide sequence and that of the other antibody In effect, therefore, each antibody specie is a 25 distinct molecule and the differences in peptide sequence between different species affect their general specificities as well as the particular epitopes they recognize and their affinities for the antigen.

An individual B cell cannot be grown indefinitely using presently available tissue culture techniques to obtain the antibody specie it secretes as a pure compound Relatively recently, Kohler and Milstein discovered and reported a process by which a monoclonal antibody can be conveniently obtained as the 30 secretion product of a hybrid cell referred to as a "hybridoma" (G Kohler and C Milstein, Nature, 256, 495 ( 1975)) Basically, the process involves the fusion of spleen cells taken from an immunized mouse with mouse myeloma cells to form the hybridoma Myeloma cells which do not produce, or at least do not secrete their own immunoglobulin or parts thereof are preferred Cultures of cells obtained by cloning a single hybridoma will secrete identical antibody molecules which can subsequently be obtained readily as a 35 pure chemical compound This is in contrast to the conventional antibody preparation obtained, for example, from serum, in which any one antibody is but one of the components of a substantially unresolvable antibody mixture of related, yet distinct chemical compounds.

Since it is a pure compound, a monoclonal antibody will have a constant specificity for a single site on the antigen molecules and a well defined affinity Thus, clones of different hybridomas can be screened to 40 select the one producing the monoclonal antibody with the most desireable properties for a given application The immortality of the hybridoma guarantees an almost unlimited supply of the antibody it secretes and alleviates problems associated with variance in antibody titer and overall affinity from animal to animal used to produce serum antibodies Monoclonal antibodies obtained from hybridomas have, for example, been put to practical application in diagnostic kits A selection of such kits is available from 45 Hybritech, Inc, assignee of this application.

An antibody molecule can generally be considered to express a single specificity which is exhibited towards the immunogen to which the host's immune system responded by production of the antibody The antibody is composed of two identical halves, each of which is comprised of a heavy and light chain pair and each of which recognizes the same antigenic determinant as the other The following is a 50 representation of the arrangement of heavy (H) and light (L) chains in an antibody molecule:

The -S-S disulfide bridges which link the two (H) chains together at the location of cysteine moieties can usually be cleaved selectively in vitro by a mild reduction, and the half molecules disassociated by subsequent acidification The half molecules can then be recombined (renatured), again in vitro, at neutral p H, the reassociation taking place through noncovalent interaction 55 If antibodies of different specificities are subjected to a selective cleavage of the disulfide bridges between the heavy chains and conditions conducive to renaturation subsequently established, reassociation between half molecules may occur randomly to produce a population of antibodies, at least some of which are hybrids in that one half of an antibody molecule of one specificity combines with one half of an antibody molecule of a different specificity For example, in Nisonoff et al, "The Antibody Molecule", 60 Academic Press, New York ( 1975), at pages 260-261, is described an in vitro production of a polyclonal antibody hybrid of rabbit antiovalbumin and anti-BGG antibodies Hybrid monoclonal antibodies have also been pbtained using an analogous process See D M Kranz et al, Proc Natl Acad Sci USA, 78, 5807 GB 2 169 921 A 1 GB 2 169 921 A 2 ( 1981) Theoretically at least, the hybrid antibody will exhibit a dual specificity in that one half of the antibody will recognize and bind to one antigenic determinant or epitope, whereas the other half will recognize a different epitope on the same or a different antigen.

Although hybrid antibodies can be obtained in the manner described above, the yields are often very low, the reactions used to make them difficult to reproduce and the hybrid antibodies usually suffer 5 significant, irreversible denaturation Such denaturation can reduce immunoreactivity and would be expected to result in different metabolic characteristics in vivo As a result, the hybrid antibody today remains largely a laboratory curiosity which is difficult to obtain.

Antibodies having dual specificities may also be prepared by conjugating pairs of intact antibodies, monoclonal or otherwise, using a variety of coupling or crosslinking agents such as protein A (from 10 Staphylococcus aureus), carbodiimide and bifunctional compounds such as Nsuccinimidyl-3-( 2- pyridyidithio) propionate to obtain dimeric and higher antibody multimers to which each member of the antibody pair contributes its specificity For example, Mandoche et al have reported the formation of multivalent antibodies having dual specificities, by a sequential reaction of antibodies with protein A, which have been shown to be capable of detecting cell surface antigens in vitro See J Immunological Methods, 15 42,355, ( 1981) According to their method, antibodies of one specificity bind to the surface antigen and the others to a moiety which permits detection.

The synthesis of dual specificity antibodies by the foregoing techniques is complicated and thus far no commercial application of them has been made.

Summary of the Invention 20

The present invention provides, among other things, a novel, completely biological method for reliably obtaining hybrid monoclonal antibodies in good yields without denaturation Throughout this specification, the term "hybrid antibody" will be used to designate a single antibody molecule having two different specificities The individual specificities may be to antigenic determinants on two different antigens or to different antigenic determinants (epitopes) on the same antigen Furthermore, unless otherwise indicated, 25 the term "antigen" also embraces haptens.

According to the method of the present invention, hybrid antibodies having a dual specificity are obtained by fusion of a hybridoma, preferably a selectively destructible hybridoma, which secretes an antibody against a preselected antigenic determinant with a fusible B- lymphocyte or a second hybridoma, the B-lymphocyte or second hybridoma secreting a second antibody against a different antigenic 30 determinant, to form a second generation hybridoma (hereinafter "polydoma") As used herein, the term "selectively destructible hybridoma" means a hybridoma which lacks, or at least substantially lacks, the capability of surviving in the medium in which the polydoma is cultured We have unexpectedly found that, unlike the parent hybridoma or the B-lymphocyte from which it is derived, each of which secretes a population of identical antibodies having a single specificity, the polydoma in addition secretes a high 35 percentage of a monoclonal hybrid antibody having a dual specificity, i e, a capability to bind with either of the antigenic determinants recognized by the individual antibodies produced by the parent cells or with both determinants at the same time The hybrid monoclonal antibody obtained in this way has not suffered the undesireable denaturation which characterizes hybrids obtained from the process of chemical recombination of antibody half molecules Furthermore, the process of the invention permits the hybrid to 40 be obtained reliably and in large amounts.

Also according to the present invention there are provided processes for immunodiagnosis and immunotherapy employing antibodies having a dual specificity Generally these processes employ a monoclonal antibody or polyclonal antibodies having a first specificity against a target antigen and a second specificity against a substance, for example, another antigen or hapten, which permits a diagnosis 45 to be made of the target antigen or which permits delivery of, or is itself, an agent which is lethal to the target antigen or the tissue with which it is associated.

Thus, by an appropriate selection of parent cells, a polydoma can be obtained according to the present invention which will secrete an antibody having one specificity for a target antigen and a second specificity for a moiety useful in diagnosis or therapy Alternatively, antibody half molecules can be recombined using 50 in vitro chemical means or individual intact mono-specific antibodies can be coupled or crosslinked by chemical means to obtain antibody multimers (which may be a dimer, trimer or higher multimer) having a dual specificity and having the same or a similar utility as a hybrid monoclonal antibody having the same dual specificity made according to the present invention.

As used herein, the terms "antibody" includes antibody fragments having immunochemical properties 55 such as Fab or F(ab)2 fragments.

Accordingly, an object of the present invention is to obtain hybrid monoclonal antibodies reliably and in good yield that have not been denatured in the process of their preparation.

Another object of the present invention is an improved process for obtaining hybrid monoclonal antibodies 60 Yet another object of this invention is to provide immunodiagnostic and immunotherapeutic processes which employ antibodies having a dual specificity.

The manner in which these and other objects can be obtained will be apparent from a consideration of the following description of preferred embodiments.

3 GB 2169921 A 3 Description of Preferred Embodiments

As indicated above, the process for obtaining a hybrid monoclonal antibody according to the present invention requires, as one parent, a hybridoma, and preferably a selectively destructible hybridoma, which secretes a monoclonal antibody against a preselected antigenic determinant or epitope The use of a selective destructible hybridoma as a parent has the advantage that it prevents the cells obtained by fusion 5 of the selectively destructible hybridoma with a B-lymphocyte or a second hybridoma, i e, the polydoma, from being overgrown by a population of the parent hybridoma when the cells obtained in the fusion process are cultured and to provide a means by which the polydoma cells can be isolated from parental hybridoma cells.

We have found that selectively destructible hybridomas useful in our invention can be obtained from 10 hybridomas secreting an antibody having one of the desired specificities made by the classic Kohler-Milstein process, i e, hybridomas obtained by fusion of a myeloma cell and a B-lymphocyte such as that found in the spleen cells of a mouse According to one embodiment of the invention, such a hybridoma is subjected to a back selection process to obtain the hybridoma which is selectively destructible.

1 5 Generally, selective destructibility can be obtained by back selection to a hybridoma which lacks a 15 genetic component which is necessary to its survival in a medium of choice in which the polydoma produced by the fusion can be cultured because of a genetic contribution from the fusion partner of the selectively destructible hybridoma, i e, the B-lymphocyte or second hybridoma.

The presently preferred back selection process involves culturing a hybridoma which secretes an antibody having one of the desired specificities to be incorporated into the hybrid antibody in a growth 20 medium containing 8-azaquanine In such a medium, any cell which incorporates 8-azaguanine and can, therefore, grow in the medium are ones which lack the enzyme hypoxanthine- guanine phosphoribosyl transferase (HPRT) Clones of cells which lack this enzyme cannot grow in medium containing hypoxanthine aminopterin thymidine (HAT) Thus, they can now be selectively destroyed in that medium.

A very similar process for back selection involves growing the hybridomas secreting the desired 25 antibody in a medium containing 6-thioguanine, another analog of guanine toxic to the cell if incorporated into the DNA Again, certain cells which will grow in this medium lack the HPRT enzyme and clones of these cells will necessarily be sensitive to HAT medium.

Yet another process for back selection which can be used in the invention involves growing cells of the selected hybridoma cell line in a medium containing either of the thymine analogs 5-bromouracyl 30 deoxyribose (B Ud R) or 2-aminopurine Only those cells lacking the enzyme thymidine kinase (TK) can grow in a medium containing either of these two inhibitors As in the case of cells lacking the enzyme HPRT, cells lacking TK will not grow in HAT medium.

A different process for obtaining a selectively destructible parent hybridoma involves irreversible enzyme inhibition using metabolic inhibitors Among these, the so called Kcat inhibitors are preferred These 35 inhibitors are analogs of an enzyme's substrate which are converted by the target enzyme into a highly reactive molecule which reacts with the enzyme at its active site resulting in irreversible inhibition of the enzyme For example, treatment of the selected hybridoma with an analog of glutamine such as azaserine or 5-diazo-5-oxa-L-norleucine (DON) irreversibly inhibits the enzyme formylglycinamide ribonucleotide amidotransferase by formation of a covalent bond with a cysteine residue at the enzyme's active site This 40 inhibition will ultimately result in cell death However, the hybridoma can be rescued by fusion with the second parent of the polydoma which supplies the necessary enzyme.

In a preferred embodiment, the selectively destructible hybridoma is fused with complementary B-lymphocytes, typically obtained as spleen cells taken from a host which has previously been immunized with an antigen, which may be a hapten bound to a carrier protein, selected to cause the host to generate an 45 immune response which produces antibodies having the second specificity desired in the hybrid antibody.

The host is usually a mouse but species of rabbits, humans and other animals may also be used although interspecies fusion may exhibit a low order of stability The process for immunizing such a host is, of course, well known and details need not be given here.

Fusion of the selectively destructible hybridoma with the B-lymphocytes to obtain the polydoma can be 50 accomplished by combining the two groups of cells in a medium containing an agent known to promote cell fusion such as polyethylene glycol or Sendi virus according to known methods.

After fusion, the cells are transferred to a medium such as HAT medium for culturing The B-lymphocytes will survive for only a brief period of time and the parent hybridoma cells cannot grow in the medium However, the population of polydomas formed as a result of the fusion, because of 55 complementation of the parent hybridoma by the B-lymphocyte, for example, by a genetic contribution of the ability to make a missing enzyme such as HPRT or TK or by a direct contribution of an enzyme inhibited in the parent hybridoma, can be grown in the medium Clones of individual polydomas are cultured and screened to select those which secrete antibodies having the desired dual specificity Clones of polydomas whose antibodies exhibit the desired dual specificity are further screened to select those whose second 60 specificity, i e, that obtained from the B-lymphocytes, and affinity are most desireable.

In another embodiment, the polydoma is obtained by fusing the selectively destructible hybridoma using a suitable fusion agent with a second hybridoma which is also selectively destructible The second parent hybridoma is obtained in the same manner as the first, i e, by a process of back selection, irreversible enzyme inhibition or by any other suitable technique In such a case, the second hybridoma 65 GB 2 169 921 A 3 4 GB 2169921 A 4 must be able to complement the first For example, if the first selectively destructible hybridoma lacks the enzyme HPRT, the second must be capable of contributing to the polydoma a gene which will enable the polydoma to express HPRT Similarly, if the second selectively destructible hybridoma lacks the enzyme TK, the first must contribute a gene for TK to the polydoma Similar complementarity between the two hybridomas must be present if irreversible inhibition of an enzyme has been accomplished to confer 5 selective destructibility on them It is also possible to use, as one hybridoma parent, a hybridoma which has been subjected to a back selection process, and, as the other, a hybridoma which has been subjected to a process of enzyme inhibition.

The use of complementary selectively destructible hybridomas as parents for the polydoma has the advantage that both parents can be selected on the basis of the specificities and affinities of the monoclonal 10 antibody they produce whereas, in the case of fusion of a single hybridoma with B-lymphocytes, no pre-fusion selection among the B-lymphocytes to obtain those producing an antibody of the desired specificity and affinity can be made.

Fusion of the two selectively destructible hybridomas can be accomplished using polyethylene glycol or using otherfusing agents, again according to known methods After fusion, the cells are transferred to a 15 growth medium in which the two parents cannot grow, but in which the polydomas resulting from the fusion are capable of growth because of the complementary contributions of the parents.

Heretofore, we have discussed selection processes to obtain selectively destructible hybridomas for use as both of the hybridoma partners in a hybridoma-hybridoma fusion to form a polydoma However, the necessity for the second hybridoma parent to be selectively destructible can be avoided by conferring both 20 a dominant and a recessive marker on the first hybridoma parent A presently preferred method is HAT-ouabain selection The drug ouabain is a specific inhibitor of the Na'- K' activated AT Pase of the plasma membrane That enzyme is responsible for the importation of K+ into a cell and the export of Na' from the cell Cells of a hybridoma previously back selected to confer selective destructibility, for example, HAT sensitivity, are grown in ouabain medium to select for ouabain resistant cells Clones of these cells will 25 be HAT sensitive but ouabain resistant By contrast, the hybridoma selected forfusion with itwill be ouabain sensitive but can survive and grow in HAT Alternatively, selection for ouabain resistance can be done first either on the parental myeloma line or the hybridoma derived therefrom, followed by back selection or othertechnique to confer selective destructibility.

Cells obtained by fusion of the two hybridomas in polyethylene glycol or other fusion agent are 30 transferred to HAT medium containing ouabain in a concentration lethal to the second hybridoma parent.

The selectively destructible hybridoma parent cannot survive in the HAT medium either, lacking, for example, the HPRT or other enzyme, even though ouabain resistant However, the polydoma cells can grow in the medium since they will possess the enzymes and ouabain resistance necessary for survival The foregoing method has the advantage that it is possible to obtain a polydoma by directly fusing a selectively 35 destructible hybridoma parent secreting an antibody having one of the specificities desired in the hybrid with a second, "off the shelf' hybridoma secreting an antibody having the other specificity desired in the hybrid and no use of techniques for conferring selective destructibility on the second hybridoma parent is necessary.

Yet another technique for obtaining a polydoma which employs a universal parent, i e, one which has 40 both a positive and a negative marker, which can be fused with any "off the shelf" hybridoma, involves the use of recombinant DNA vectors carrying various drug resistance markers For example, SV 40 carrying a gene for neomycin resistance can be used.

A presently preferred universal parent is one that is HAT sensitiveneomycin resistant The chosen parent is back-selected to HAT sensitivity and then transfected with SV 40 vector carrying a gene for 45 neomycin resistance This procedure can also be reversed with transfection being done first The resulting hybridoma can grow in the presence of neomycin, which is normally toxic to mammalian cells, but will die in the presence of HAT Off-the-shelf hybridomas, however, grow in HAT but die in the presence of neomycin Products of the fusion of the parents, therefore, survive in the presence of HAT and neomycin.

Whilethe use of vectors to convey resistance to neomycin is presently preferred, vectors carrying genes 50 which will confer resistance on mammalian cells to other drugs can also be used.

Even though presently preferred, it is not essential to our process for obtaining polydomas from pairs of hybridomas that at least one of the parent cell lines be selectively destructible It is within the scope of our invention to fuse a pair of hybridomas, neither of which is selectively destructible but which secretes an antibody having one the specificities desired in the hybrid, in the presence of a suitable fusion agent 55 followed by the subcloning of all cells before the population of unfused parent hybridomas increases to an extentthat screening the subclones to identify polydomas is not practical The subclones are subsequently screened to establish which secrete antibodies having a dual specificity.

This process is best suited to obtaining polydomas when the fusion frequency of the parent cell lines is high In any case, and particularly when the cell fusion frequency is low, the cells obtained from the fusion 60 of hybridomas whose monoclonal antibodies are against different antigens can be screened using a cytofluorograph to identify the polydomas To accomplish this, samples of the two antigens are tagged with different fluorescing moieties whose fluorescence occurs at different wavelengths For example, one can be tagged with fluorescein and the other rhodamine The population of cells from the fusion, which have preferably been cultured overnight or for any other suitable period to increase their numbers, are incubated 65 GB 2 169 921 A 4 GB 2169921 A 5 with the two tagged antigens The cells are then screened using the cytofluorograph Those cells which fluoresce at only one of the two wavelengths will be from the cell lines of the parent hybridomas However, cells which exhibit fluorescence at both wavelengths will be polydomas which can be isolated and subcloned.

In yet another embodiment, a polydoma can be obtained directly by removing the nucleus from a first 5 hybridoma which secretes a monoclonal antibody having one of the specificities desired in the hybrid and inserting it into the cytoplasm of a second hybridoma which secretes a monoclonal antibody having the second desired specificity Of course, neither of the parent hybridomas needs to be selectively destructible in order to be used in this process After insertion of the nuclear material, the cell is cloned to obtain a population of the polydoma 10 We have found that, unlike the parent hybridomas or B-lymphocytes which secrete a single antibody, polydoma cells obtained according to our invention secrete a mixture of antibodies, at least one of which is a hybrid antibody having a dual specificity Also produced by the polydoma are relatively smaller amounts of antibodies of the same specificity as those produced by the parent cells used to obtain the polydoma The 1 5 ratio of hybrid to mono-specific antibodies appears to be about 2:1:1 which is that expected if the 1 5 polydomas produces equal amounts of all the possible (H) chains synthesized by the parent cells which are randomly combined in the polydoma itself.

The polydomas can be cultured in vitro or grown in vivo in either genetically compatible animals or nude mice to obtain large quantities of the hybrid antibody which is recovered from the culture medium or ascitic fluid of the animal using known processes See, for example, the protocols in "Monoclonal 20 Antibodies", Edited by Kennett et al, Plenum Press, New York ( 1980) at pp 363-418.

The mixture of antibodies produced by the polydoma can be resolved to obtain the hybrid For example, sequential affinity chromatography against first one and then the other antigen for which the hybrid is specific permits its separation from the mono-specific antibody contaminants We have also fou'nd that simple ion exchange chromatography and electrophoretic techniques can be employed as well in at 25 least certain circumstances If required, the charge difference for ion exchange could be one of the characteristics of the antibody considered in selecting the parental lines.

EXAMPLE 1

A hybrid monoclonal antibody having a dual specificity for hepatitis B surface antigen (H Bs Ag) and prostatic acid phosphatase (PAP) was made in accordance with the present invention in the following 30 manner:

A hybridoma secreting a monoclonal antibody to PAP was grown in HAT medium for one week and then transferred to and grown in a non-selective medium After various lengths of time of growth under non-selective conditions, 2 ml aliquots of cells were placed in medium containing 10-4 M 8-azaquanine which prevented cells from growing by incorporating 8-azaguaine in their DNA instead of guanine Cells 35 lacking the HPRT enzyme survived and grew in this medium and these cells necessarily did not survive in HAT.

Clones that grew in the medium containing 8-azaguanine were tested for sensitivity to HAT and anti-PAP production One clone which still produced anti-PAP and exhibited HAT sensitivity with a reversion frequency of less than 4 x 1 o-8 was subcloned All of the subclones behaved like the parental clone 40 Cells from one of the HAT sensitive subclones were fused in polyethylene glycol with spleen cells obtained from Balb/c mice hyperimmunized with H Bs Ag to obtain polydomas using the fusion techniqueof Gerhard See "Monoclonal Antibodies", supra, at p 370 The fusion produced 220 polydomas which were screened to determine which secreted antibodies exhibiting specificity for both PAP and H Bs Ag Clones of two such polydomas were determined to produce antibody and, subsequently, ascites which exhibited both 45 specificities.

Subclones of both polydomas continued to produce ascites exhibiting both specificities and yielded triple bands on Orstein-Davis PAGE like those of the parent clone The ascites from both clones were shown to bind 1251-H Bs Ag and '25 I-PAP in radioimmunoassays and yielded Ka values of approximately 1 9 for each, thus suggesting the formation of antibodies having two specificities 50 The data in Table I below show the results obtained in immunoassays using the ascites obtained from a clone of one of the polydomas compared with ascites obtained from hybridomas secreting monoclonal antibodies, respectively, against Ig E (used as a control), PAP and H Bs Ag using immobilized H Bs Ag as a solid phase and a variety of radiolabeled antigens as the solution phase.

A 200 p 1 sample of the ascites from the polydoma and each of the three hybridomas were each 55 incubated overnight with 12 polystyrene balls to which was bound H Bs Ag The H Bs Ag balls were obtained from Abbott Laboratories, North Chicago, Illinois After washing, triplicate samples were incubated for 4 hours with 100,000 cpm of the indicated 1251 labeled antigen After a second washing, the balls were counted to determine the amount of labeled antigen bound to the balls In one set of tests using radiolabeled PAP as the solution phase antigen, anti-PAP was added to the antigen before it was incubated with the ball The 60 antibody to PAP used for this purpose is the monoclonal antibody produced by the parental hybridoma used to make the polydoma and, therefore, is against the same PAP epitope as that expected to be exhibited by the hybrid antibody.

GB 2 169 921 A 5 6 GB 2169921 A 6 TABLE I

Results of Radioassays Demonstrating Presence of Hybrid Antibody Having Dual Specificity in Polydoma Ascites cpm Specificity cpm cpm PAP+ cpm of Ascites H Bs Ag PAP anti-PAP Ig E 5 anti-lg E 15,340 2,145 2,930 3,290 anti-PAP 16,280 2,956 3,128 3,180 anti-H Bs Ag 73,020 2,973 2,870 3,330 suspected dual 78,900 82,533 2,936 3,143 specificity 10 1251 labeled antigen The data in Table I indicate that oniy the radiation expected from non- specific binding is measured for the anti-PAP ascites when compared to that for the Ig E control The ascites containing the H Bs Ag antibody, on the other hand, bound the labeled H Bs Ag antigen as expected but exhibited non-specific binding when 1 5 the other labeled antigens were tested The ascites from the polydoma clone, however, bound both labeled 1 5 H Bs Ag and labeled PAP, the former attributable to the presence of some non-hybrid, mono-specific antibody to H Bs Ag in the ascites and the latter attributable to a hybrid that can bind and bridge the H Bs Ag on the ball and the trace labeled PAP in solution The experiment using a mixture of labeled PAP and anti-PAP from the parental hybridoma confirms that the anti-PAP specificity of the hybrid is for the same epitope as the antibody secreted by the parent since only background radiation is observed due to 20 inhibition bythe parental antibody of binding of labeled PAP to the hybrid antibody.

Analysis of the ascites from the polydoma clone used in the comparative radioassays was performed using polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis (IEP) Both indicated the presence of at least three antibody species Preparative scale DEAE ion exchange chromatography yielded three well separated peaks, the middle one of which had a shoulder Each of the peaks was homogenous as 25 analyzed by PAGE and IEP, and each corresponded to one of the bands in the original ascites.

Material representing each of the DEAE peaks was tested for antigen binding using radiolabeled H Bs Ag and PAP The first peak bound H Bs Ag but not PAP The middle peak and its shoulder bound to both H Bs Ag and PAP and the last peak bound only PAP Thus, the middle peak is hybrid antibody having a dual specificity to H Bs Ag and PAP comprised of at least two subspecies 30 The hybrid antibody obtained as the middle peak of the DEAE chromatography was radiolabeled with j After labeling, 85 % of the labeled antibody would bind to PAP and 88 % would bind to H Bs Ag The affinity of the hybrid for PAP was found to be slightly lower than that of the monoclonal antibody to PAP produced by the parental line This difference in affinity was about the same as that observed by us between a monoclonal antibody and its Fab fragment 35 DEAE chromatography indicates that hybrid antibody comprises more than 50 % of the antibodies produced bythe polydoma and roughly approximates the ratio 2:1:1 predicted on statistical grounds if the polydoma were to synthesize all the possible antibody heavy chains, i e, those exhibiting either PAP or H Bs Ag specificity, which are combined within the cell on a random basis to form hybrid antibody admixed with lesser amounts of the two mono-specific antibodies having the same specificity as those produced by 40 the parent cells The existence of subspecies of hybrid antibody suggests that they may differ in their light chain composition.

EXAMPLE 2

Hybrid monoclonal antibodies having dual specificity for human Ig D and prolactin were made in accordance with the present invention by the fusion of two hybridomas, one of which was constructed to 45 contain two selectible genetic markers: sensitivity to HAT medium and resistance to ouabain This doubly-marked hybridoma or so-called "universal parent" could then be fused to any other hybridoma The resulting polydomas grow in the presence of HAT and ouabain, while any unfused parent cells die The advantages of using such a "universal parent" have been described elsewhere herein.

To construct such a universal parent, both selectible markers were introduced during initial 50 construction of the hybridoma To obtain this parent cell line, the widely available HAT-sensitive mouse myeloma P 3 653 was selected for a second genetic marker, ouabain resistance, by introducing 1 m M ouabain into the growth medium While most cells died, approximately 1/100, 000 cells had by random mutation acquired resistance to the drug and so survived and multiplied to form the new myeloma population which was HAT-sensitive and ouabain resistant 55 This HAT-sensitive, ouabain-resistant myeloma was then fused with spleen cells obtained from Balb/c mice hyperimmunized with Ig D using the previously cited technique of Gerhard Hybrids were selected in G B 2 169 921 A 6 GB 2 169 921 A 7 HAT medium (without ouabain) and clones were screened for production of monoclonal antibody directed against Ig D From among the positive clones, one which produced an Ig G against Ig D was selected for further study This clone was tested for retention of the trait of ouabain resistance by adding 1 m M ouabain to the growth medium Approximately one-third of the cells retained this genetic marker When the culture was growing exponentially in ouabain, the cells were subcloned Ouabainresistant subclones were tested 5 for continued production of the monoclonal anti-lg D antibody One of the subclones was further backselected by the procedure of Example 1 to obtain a population of cells sensitive to HAT This subclone was grown fortwo weeks under non-selective conditions and then placed into medium containing 6-thioguanine As noted above, the mechanism of action of 6-thioguanine is similar to that for 8-azaguanine Cells which incorporate 6-thioguanine into their DNA instead of guanine will not grow Cells 10 lacking HPRT enzyme will not utilize 6-thioguanine from the medium and therefore can grow but are consequently sensitive to HAT This population of the backselected subclone was then itself subcloned in 6-thioguanine and ouabain-containing medium Subclones were assayed for continued production of the monoclonal anti-lg D antibody One clone which showed all the desired characteristics-growth in ouabain 1 5 and 6-thioguanine as well as production of monoclonal anti-lg D was selected to be a so-called "universal 1 5 parent" This universal parent could then be fused to any other HAT- resistant, ouabain-sensitive hybridoma to produce a polydoma which would express a hybrid antibody, one specificity of which would be anti-lg D.

For this purpose, we initially selected a mouse hybridoma which secretes a monoclonal antibody directed against prolactin The antiprolactin monoclonal antibody is of the same subclass (Ig G 1) as the anti-lg D expressed by the parent line and it is easily separated from that antibody on Ornstein-Davis gels Such a 20 separation is indicative of greatly different charge on the antibodies and so should allow easy isolation of a hybrid antibody by DEAE-Sephadex chromatography.

107 cells of the HAT sensitive, ouabain-resistant cell line were fused in polyethylene glycol with 107 O cells producing anti-prolactin The fused cells were first grown for three days in HAT medium, then refed with HAT+ouabain medium for three days and finally placed again in HAT medium Greater than 600 clones 25 arose from this fusion: 66 clones were randomly selected for analysis Of these clones 36 exhibited both anti-lg D and anti-prolactin activity.

These clone supernatants were assayed for the presence of hybrid antibody by the following assay A polystyrene bead coated with another anti-prolactin monoclonal antibody was incubated 5 hours with 200 p 1 of a 100 ng/ml prolaction solution The antibody used binds prolactin at a distinct site from that of the 30 antibody produced by the fused hybridoma cell line The bead was washed, then incubated overnight with the clone supernatants The next day, following several washes, 1251 labeled Ig D was added Hybrid antibody bound to the bead by one functional arm could bind the radiolabeled Ig D with the free anti-lg D functionality whereas neither parental type antibody Ig D-Ig D or Prolactin-Prolactin could form this bridge between the Prolactin bead and 1251-lg D tracer Results of a typical assay for clones producing hybrid, 35 bifunctional antibody are presented in Table 2 below:

TABLE 2

Results of immunometric assays demonstrating the presence of hybrid antibody having dual specificity for Ig D and prolactin in selected polydoma supernatants and ascites.

Clonate # cpm 121-lg D Tracer Bound 40 1 15339 2 16337 3 22886 4 23356 5 24434 45 Anti-lg D 9357 Anti-Prolactin 8721 21 to 36 clones exhibited significant bifunctional activity by this assay Ascites generated from 2 clones available to date have been shown to react in the bifunctional assay These ascites contain antibodies which separate into three distinct bands on Ornstein-Davis gels: two bands coincide exactly with antibodies 50 produced by the parent hybridomas (anti-lg D and anti-prolactin) The third band migrates midway between the parental monoclonal antibody bands as expected of the hybrid antibody.

That a hybrid monoclonal antibody against Ig D and prolactin exhibits a dose response when the amount of prolactin is varied in the assay used to generate the data of Table 2 is shown by the data of Table GB 2 169 921 A 8 3 using the antibody of clonate #2 and, as controls, antibodies from the parent cell lines (anti-lg D and antiprolactin).

TABLE 3

Results of Assays Using Hybrid Antibody and Varying Amounts of Prolactin Prolactin Hybrid Anti 5 ng/ml Antibody' Anti-lg D' Prolactin' 0 8010 6847 8020 9169 7982 7405 13558 7783 8314 100 17599 7654 7844 10 cpm of 1251-1 ge bound These data demonstratethatthe amount of prolactin bound bythe hybrid antibody is dose responsive as the amount of labeled Ig D bound increases as the dose of prolactin is increased By contrast, no dose response is observed using the parent antibodies against Ig D and prolactin as they cannot form the bridge 1 5 between prolactin bound to the bead and the labeled Ig D Thus, the hybrid antibody can be used as a 15 component of an assay for prolactin Tailor-making other hybrid antibodies offers similar opportunities for other assays.

EXAMPLE 3

By a similar technique to that of Example 2, a universal parent hybridoma was generated which secretes monoclonal antibody directed against the hapten arsenate- arsenate dimer and us both resistant to 20 ouabain and sensitive to HAT This hybridoma was fused to a hybridoma which secretes a monoclonal antibody with specificity for carcinoembryonic antigen (CEA), an antigen expressed by both embryonic tissues and several types of carcinoma Once again greaterthan 600 clones resulted from the fusion Of 72 clones tested for the ability to bind both CEA and arsenate, 69 had both binding activities Those clones exhibiting the greatest binding were selected for enzyme-linked immunosorbent (ELISA) assay of hybrid 25 antibody For each assay, a CEA solution ( 600 ng or 250 ng) was allowed to adsorb overnight to each well of a plastic 96-well microtiter plate The next day, unadsorbed material was washed out of the wells with PBS-Tween 20 Clone supernatants were added and incubated 2-1/2 hours at 35 C and then washed off the plate CEA-CEA and CEA-arsenate antibody would remain attached to the plate via the adsorbed antigen.

The second antigen, arsenylic acid coupled to the enzyme alkaline phosphatase, was added to Ihe wells for 30 3 hours at 35 C After another wash with PSB-Tween, a chromagen substrate for alkaline phosphatase; para-nitrophenylphosphate, was added to the wells and color developed for 48 hours Absorbance in each well was measured at 410 nm If present in a clone supernatant, hybrid antibody bound the adsorbed CEA on the plate through one functionality, leaving the other free to bind arsenylic acid coupled to alkaline phosphatase Colorfrom the chromagen substrate developed only where the alkaline phosphatase coupled 35 to arsenylic acid had been bound In this assay 3 of 12 supernatants exhibited hybrid antibody activity as indicated in Table 4.

TABLE 4

Demonstration of Hybrid Antibody with Dual Specificity by Enzymelinked Immunosorbance (ELISA) Assay 40 Absorbance at 490 nm Absorbance 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 45 anti-arsenate 0 010, 0 006 0 006, 0 005 The present invention also provides methods for immunodiagnosis and immunotherapy using antibodies having a dual specificity, for example, hybrid antibodies obtained as described above orfrom antibody half molecules by the conventional technique of Nisonoff et al, supra, or antibody multimers 9 G 82 169921 A 9 obtained by coupling or crosslinking individual monospecific antibodies Preferably, the antibody having a dual specificity used in these methods is a hybrid antibody prepared according to the present invention as such as antibody can be reliably obtained as a substantially pure compound which has not suffered denaturation and which has a uniform specificity and affinity for the antigen.

In the case of immunodiagnostic applications, one of the two specificities exhibited by the hybrid or 5 other antibody of dual specificity will be against the target antigen whose detection is desired and the other against another antigen, which may be a hapten or other molecular species, which permits the diagnosis.

For example, an antibody useful in immunohistology would have a first specificity for a suspect antigen, for example, a tumor associated antigen such as CEA, PAP or ferritin, and a second specificity against a hapten or antigen which will participate in a color reaction such as an enzyme which causes a color reaction in the 10 presence of a suitable substrate Among suitable enzymes to which the second specificity of the antibody may be directed is prostatic acid phosphatase (PAP), horse radish peroxidase, glucose oxidase, and alkaline phosphatase.

To perform the histological examination, a tissue section is first treated with the antibody of dual 1 5 specificity Prior to doing so, the hybrid can have already been allowed to bind the enzyme which catalyzes 15 the staining reaction If not, the section is then treated with a second solution containing the enzyme and rinsed after an appropriate incubation and then treated with the substrate which undergoes a color change in the presence of the enzyme The formation of the color produced by the enzyme and substrate in the tissue sample is a positive indication of the presence in the tissue of the target antigen The hybrid antibody against H Bs Ag and PAP whose preparation is described herein, has been found to bind to H Bs Ag on a test 20 substrate (polystyrene balls) and to PAP in a simulated staining experiment using p-nitrophenyl phosphate as the enzyme substrate After incubation of the hybrid antibody with PAP and the H Bs Ag, the addition of the p-nitrophenyl phosphate resulted in the balls undergoing the characteristic yellow to brown color change.

As noted in Example 2, an antibody of dual specificity can also be used in immunoassays and 25 immunometric assays Using the hybrid antibody against H Bs Ag and PAP whose preparation is described above, an immunometric assay for H Bs Ag can be performed using an immobilized monoclonal antibody to H Bs Ag as a solid phase to extract H-l Bs Ag from a serum or other liquid sample suspected of containing the antigen The sample is incubated with a ball, beads, test tube or other substrate which has the anti-H Bs Ag bound or coated on its surface The incubation with the serum sample can be followed by, done 30 simultaneously with or preceded by an incubation with a solution of the hybrid In any case, if H Bs Ag is present in the sample, the result will be the formation of a sandwich of the immobilized antibody, H Bs Ag if present in the sample, and the hybrid antibody As part of the assay, PAP is permitted to bind with the hybrid antibody This can be done during or after formation of the sandwich, or in the alternative, the antibody-PAP complex can be preformed After formation of the sandwich, the solid phase is washed to 35 remove sample residue and unbound hybrid antibody and then contacted with a solution containing a substrate such as p-nitrophenyl phosphate or a-napthol phosphate which undergoes a color change in the presence of PAP Occurrence of the color change confirms the presence of target antigen in the sample.

In such an assay, using the polyclonal anti-H Bs Ag bead of a commercial kit for diagnosis of H Bs Ag manufactured by Abbott Laboratories of North Chicago, Illinois (sold under the name "Aushia"), samples 40 containing various amounts of H Bs Ag were incubated with the bead The samples used were the positive and negative controls from the commercial kit and two samples obtained by diluting the positive control with negative control in the ratios of either 1 part negative control:2 parts positive control or 2 parts negative control:1 part positive control.

After incubation of the samples with the bead to bind H Bs Ag, the bead was washed and incubated with 45 the hybrid antibody reactive to both H Bs Ag and PAP This allowed a sandwich of immobilized antibodies:antigen:and hybrid antibodyto form The bead was washed again and incubated with a solution of PAP This incubation was followed by another wash and the bead incubated with a substrate a-napthol phosphate PAP enzymatically removes phosphate After an appropriate incubation, the substrate was removed from the bead and to it was added an indicator Fast Garnet GBC salt which turns from clear to a 50 reddish purple in the presence of the product of the enzymatic reaction A color change was observed to confirm the presence of H Bs Ag in the samples Absorbance at 570 nm was measured for the samples and these data are shown in Table 5 below.

TABLE 5

H Bs Ag Concentration Absorbance 55 (% of Positive Control) 570 nm 0 031 34 166 67 243 379 GB 2 169 921 A 9 GB 2169921 A 10 These data show a dose response with variation in H Bs Ag concentration which would be expected if the hybrid antibody forms a bridge between H Bs Ag bound to the ball and PAP This further demonstrates the utility of a hybrid antibody in an immunoassay.

Detection means other than enzymatically catalyzed reactions are also possible For example, the second specificity of the hybrid or other antibody having a dual specificity can be directed against a hapten 5 or antigen which is radiolabeled or which is fluorescent or which is detectable in the sandwich by any other suitable means.

A preferred process which utilizes a hybrid antibody or other antibody having a dual specificity in an immunoassay exploits the phenomenon of fluorescence quenching In such an assay, one specificity of the antibody is directed against a target antigen and the other against, for example, a hapten bearing a 10 fluorescing chromophore The chromophore is either bound to the hapten or, in appropriate cases, may be the hapten itself.

The assay is conducted by incubating the antibody with serum or other sample suspected of containing the target antigen to which has been added a predetermined quantity of target antigen labeled with a quenching chromophore The labeled antigen competes with target antigen in the sample, if any, for the 1 5 antibody binding site specific for the target antigen Before, during or after this incubation, a quantity of the hapten bearing the fluorescing chromophore is incubated with the antibody and binds at the other binding site.

The two chromophores are selected so that the first of them fluoresces at a wavelength which can be absorbed (quenched) by the other if they are positioned closely enough together so that the photon emitted 20 by the fluorescer can be captured by the quencher To do this, the two chromophores should be within about 100 angstroms and, preferably, within about 50 angstroms of each other This positioning will occur when the fluorescing chromophore is bound at one antibody binding site and the quenching chromophore is bound to added antigen at the other A suitable pair of chromophores includes fluorescein as the fluorescing chromophore and rhodamine as the quenching chromophore 25 The measured fluorescence will vary inversely with the amount of native antigen in the sample since, in the absence of native antigen, all of the antigen bound to the antibody will be labeled with the quenching chromophore and be positioned to absorb fluorescence by the chromophore carried by the hapten.

Comparison of the measured fluorescence with that of a control sample containing a known amount of antigen permits a qualitative and quantitative determination of the presence of antigen in the sample 30 This kind of immunoassay can, for example, be used to determine the levels in serum of drugs such as dilantin which must be closely monitored In such an assay, the target antigen would, of course, be dilantin.

It will be apparent to those skilled in the art that this process can be used to detect other antigens as well including, in particular, tumor associated antigens.

Another preferred process which utilizes a hybrid antibody or other antibody having a dual specificity in 35 an immunoassay relies upon an enzymatic reaction In a presently preferred process, one of the antibody specificities is directed, of course, to the target antigen and the other to an enzyme or a hapten to which is bound an enzyme.

The assay is conducted by incubating the antibody with a sample suspected of containing the target antigen to which has been added a predetermined quantity of the target antigen that has been modified by 40 binding to it a substance that interacts with the enzyme to produce either a detectable substance or in some otherway to permit detection of formation of the antigen-antibody complex Detection may be, for example, byfluorimetry, luminescence, spectrophotometry or the like.

In an alternative process, the added target antigen may have the enzyme bound to it in which case the antibody has one of its specificities directed against the substance which interacts with the enzyme or 45 against a hapten to which the substance is bound.

The substance which interacts with the enzyme can itself be another enzyme In such a case, one of the enzymes catalyzes production of a product required by the other Thus, when the antibody binds both the added target antigen, to which is bound one of the enzymes, and the other enzyme, the product of the first enzymatic reaction isformed in proximity to the second enzyme and can undergo a reaction catalyzed by 50 the latter enzyme before significant diffusion of the product into the surrounding medium can occur.

An example of such a process utilizes the two enzymes hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) in the following reaction scheme.

HK ( 1) adenosine triphosphate+glucose adenosine diphosphate+glucose-6phosphate (ATP) (ADP) ( 2) glucose-6-phosphate+nicotinamide adenine dinucleotide 55 (NAD+) 6-6-PDH gluconolactone-6-phosphate+dihydronicotinamide adenine dinucleotide (NDH) GB 2 169 921 A 11 To exploit this reaction scheme, the added target antigen will have either HK or G-6-PDH bound to it and the hybrid antibody will have one of its specificities directed against the other (or a hapten bearing it) The sample has added to it, in addition to the hybrid antibody and the predetermined amount of enzyme labeled antigen, glucose, ATP and the coenzyme NAD+) The hybrid antibody preferably has it the other enzyme already bound to it Alternatively, this enzyme can be added to the sample with the other reagents 5 During the incubation, the enzyme labeled antigen will compete with native antigen in the sample, if any, for one of the hybrid antibody binding sites The other enzyme is or will be, bound to the second binding site This permits the formation of glucose-6-phosphate catalyzed by HK to occur in close proximity to G-6-PDH The latter converts the glucose-6-phosphate to gluconolactone- 6-phosphate, a result which is accompanied by the reduction of NAD+ to NADH The NADH absorbs strongly at 340 nm and, therefore, can 10 be detected spectrophotometrically The amount of NADH formed varies inversely with the amount of native antigen in the sample, i e, its maximum production occurs when there is no target antigen in the sample being assayed Comparison of the amount of the NADH formed with a control sample permits a qualitative and quantitative determination of the presence of antigen in the sample.

This kind of assay can be used to monitor the level of dilantin or other drugs in serum In such a case, 15 the drug is the target antigen However, such an assay can also be used to detect other serum antigens such as those associated with tumors or other diseases.

In vivo immunodiagnosis can also be performed using a hybrid or other dual specificity antibody The antibody, having one specificity against a target antigen such as a tumor associated antigen and the second against a hapten to which is bound a suitable radionuclide, preferably one which emits y-radiation, is first 20 administered to the host After a sufficient time has passed during which the antibody has localized at the target site and unbound antibody has been permitted to clear from healthy tissue in the host, the hapten bearing the radionuclide is administered and binds to the localized antibody After a suitable interval to permit unbound hapten to clear the host, scanning of the host with a suitable camera is conducted to determine whether there are areas in which radiation has been concentrated If there are any, the presence 25 of the target antigen in the host is confirmed and its position determined.

This process has several advantages over that using monospecific antibody directed against the target antigen to which the radionuclide is directly bound In such cases, the radionuclide must have a long enough half life that a sufficient quantity remains afterthe time necessary for substantial localization of the antibody at the target site has elapsed Furthermore, during this process the antibody may be retained for a 30 period of time in the liver or other non-target tissues which are then subjected to theradiation carried by the antibody The present invention, on the other hand, permits the use of radionuclides having shorter half-lives than those used with monospecific antibodies Being a relatively small particle, the radionuclide bearing hapten has a high mobility in vivo and will travel rapidly through the host and either bind to the antibody which has localized at the target site or clear the body without spending appreciable time in 35 non-target tissue For this reason, isotopes of short half-life can be administered in quantities which pose the minimum risk to healthy tissue even though administered in substantial excess.

Preferably, the hapten is an agent to which the radionuclide is directly bound or which will complex with the radionuclide A chelating agent for the radionuclide bound to a hapten may be used for the latter purpose Those skilled in the art will appreciate that a wide variety of chelating agents and radionuclides are 40 suited for this purpose Phenylarsenate to which ethylenediaminetetraacetic acid (EDTA) is bound as a chelating agent is a suitable hapten A radionuclide suited for use with this hapten is "In.

The antibody of dual specificity can also be used in immunotherapy by constructing it to have one specificity against a disease associated antigen and the other against a hapten which is, or to which is bound, an agent lethal to the antigen or diseased tissue with which the antigen is associated and which it is 45 desired to destroy For example, the antibody may have one specificity against a tumor associated antigen such as PAP, carcinoembryonic antigen (CEA), ferritin, or other such antigen and a second specificity directed to a hapten to which is bound a radionuclide, preferably one which emits or radiation, or is comprised of a ricin A chain or other toxin or dug Among such drugs may be mentioned gelonin, a-amanitin, diphtheria toxin A, methotrexate, dichloromethatrexate, dounomycin and chlorombucil Of 50 course, if the toxin or drug can itself function as a hapten, it need not be bound to any other moiety.

In the case where a radionuclide is to be used as the lethal agent, just as in the situation where they are used for in vivo immunodiagnosis, the hapten can have the radionuclide bound directly to it or the hapten can be, or have bound to it, an agent such as a chelating agent which will form a complex with the radionuclide In such a case, the hybrid or other antibody of dual specificity is administered to the diseased 55 host and allowed to localize at the site of the affected tissue and any excess allowed to clear the host, followed by administration of the hapten which is bound by the antibody wherever it has localized This permits the use of a radionuclide having a short half-life which minimizes the risk of injury to healthy tissue even though the radionuclide bearing hapten is administered in substantial excess since that excess will rapidly clear the body and not localize in substantial quantities in healthy tissue because of the hapten's 60 relatively small size It also eliminates or reduces the possibility that circulating target antigen will bind antibody bearing a substance lethal to tissue and deliver it to healthy tissue as can occur when the lethal agent is bound directly to a monospecific antibody directed against the target antigen.

An example of a hapten to which a radionuclide is directly bound is 6-2 "At-astato-2-methyl-1,4- napthoquinol bis(disodium phosphate) which is described in "International Journal of Applied Radiation 65 1 1 GB 2 169921 A 12 and Isotopes", 33,75 ( 1982) The 211 At is an emitter of a-radiation Those skilled in the art will appreciate that there are numerous suitable radionuclides which can be bound directly to haptens or complexed with a hapten by means of any of a wide variety of chelating agents.

The foregoing description of the invention is of presently preferred embodiments Variations are possible without departure from the scope of the invention which is limited only by the appended claims 5

Claims (1)

1: A process for producing a polydoma which secretes a hybrid monoclonal antibody having a dual specificity which comprises removing the nucleus from a first hybridoma cell which produces a monoclonal antibody against a first antigenic determinant and inserting the nucleus into the cytoplasm of a second hybridoma which produces a monoclonal antibody against a second antigenic determinant 10 2 A polydoma which produces a hybrid monoclonal antibody having a dual specificity.

3 A polydoma according to Claim 2 which produces the hybrid antibody as a component of a mixture of antibodies.

4 A polydoma according to Claim 3 wherein the mixture of antibodies comprises the hybrid antibody and two species of mono-specific antibody 15 A polydoma according to Claim 4 wherein the hybrid antibody is comprised of two subspecies.

6 A polydoma according to Claim 5 or 6 wherein the specificity of one of the mono-specific antibodies is the same as one of the dual specificities of the hybrid and the specificity of the other mono-specific antibody is the same as the other specificity of the hybrid antibody.

7 A hybrid monoclonal antibody having a dual specificity produced by a polydoma 20 8 A hybrid monoclonal antibody according to Claim 7 wherein one of the dual specificities is against a target antigen and the other against a substance which permits diagnosis of the target antigen.

9 A hybrid monoclonal antibody according to Claim 8 wherein the substance permitting diagnosis is radiolabelled.

10 A hybrid antibody according to Claim 9 wherein the substance permitting diagnosis is a hapten 25 11 A hybrid antibody according to Claim 10 wherein the radiolabel is a radionuclide bound directly to the hapten.

12 A hybrid antibody according to Claim 10 wherein the radionuclide is bound to the hapten by a chelating agent.

13 A hybrid monoclonal antibody according to Claims 9, 10, 11 and 12 wherein the radiolabel is an 30 emitter of y-radiation.

14 A hybrid monoclonal antibody according to Claim 9 wherein the substance is fluorescent.

A hybrid monoclonal antibody according to Claim 10 wherein the hapten is fluorescent.

16 A hybrid monoclonal antibody according to Claim 10 wherein a fluorescent moiety is bound to the hapten 35 17 A hybrid monoclonal antibody according to Claim 9 wherein the substance permitting diagnosis is an enzyme.

18 A hybrid monoclonal antibody according to Claim 10 wherein an enzyme is bound to the hapten.

19 A hybrid monoclonal antibody according to Claim 8,9,10,11,12,13,14,15, 16,17 or 18 wherein the target antigen is a disease associated antigen 40 A hybrid monoclonal antibody according to Claim 19 wherein the antigen is a tumor associated antigen.

21 A hybrid monoclonal antibody according to Claim 7 wherein one of the dual specificities of the antibody is against a target antigen and the other against a hapten which is, or to which is bound, an agent lethal to the antigen or associated tissue 45 22 A hybrid monoclonal antibody according to Claim 21 wherein the lethal agent is a radionuclide.

23 A hybrid monoclonal antibody according to Claim 22 wherein the radionuclide is bound directly to the hapten.

24 A hybrid monoclonal antibody according to Claim 22 wherein the radionuclide is bound to the hapten by a chelating agent 50 A hybrid monoclonal antibody according to Claims 22,23 or 24 wherein the radionuclide is an emitter of a-radiation or,B-radiation.

26 A hybrid monoclonal antibody according to Claim 21 wherein the lethal agent is a tissue toxin.

27 A hybrid monoclonal antibody according to Claim 26 wherein the tissue toxin is comprised of a ricin A chain 55 28 A hybrid monoclonal antibody according to Claims 21,22,23, 24,25 or 26 wherein the target antigen is a disease associated antigen.

29 A hybrid monoclonal antibody according to Claim 28 wherein the antigen is a tumor associated antigen.

30 A process for producing a polydoma which produces a hybrid monoclonal antibody having a dual 60 specificity which comprises fusing a hybridoma which produces a monoclonal antibody against a first antigenic determinant with a B-lymphocyte which secretes a monoclonal antibody against a second antigenic determinant in the presence of a fusion promoting agent.

GB 2 169 921 A 13 31 A process according to Claim 30 wherein the hybridoma is selectively destructible.

32 A process according to Claim 31 wherein the hybridoma has been back selected to obtain cells that are sensitive to a medium in which the polydoma can be cultured.

33 A process according to Claim 32 wherein back selection is accomplished by culturing cells of the hybridoma in a medium comprising a member of the group consisting of 8- azaguanine, 6-thioguanine, 5 5-bromouracyl deoxyribose or 2-aminopurine whereby hybridoma cells sensitive to medium containing hypoxanthine aminopterin thymidine are obtained.

34 A process according to Claim 31 wherein said selectively destructible hybridoma is obtained by irreversible enzyme inhibition.

35 A process according to Claim 34 wherein the inhibition is obtained using a metabolic inhibitor 10 36 A process according to Claim 35 wherein the inhibitor is a Kcat inhibitor ' 37 A process according to Claim 36 wherein the Koat inhibitor is selected from azaserine or 5-diazo-5-oxa-L-norleucine.

38 A process according to Claims 30,31,32,33,34,35,36 or 37 wherein the Blymphocyte is a mammalian spleen cell 15 39 A process according to Claim 38 wherein the spleen cell is a spleen cell of a murine specie.

A process for producing a polydoma which secretes a hybrid monoclonal antibody having a dual specificity which comprises fusing a first hybridoma which secretes a monoclonal antibody against a first antigenic determinant with a second hybridoma which secretes a monoclonal antibody against a second antigenic determinant in the presence of a fusion promoter 20 41 A process according to Claim 40 wherein the first and second hybridomas are selectively destructible.

42 A process according to Claim 41 wherein at least one of the hybridomas has been back selected to obtain cells that are sensitive to a medium in which the polydoma can be cultured.

43 A process according to Claim 42 wherein both of the hybridomas have been back selected 25 44 A process according to Claim 41 wherein back selection is accomplished by culturing cells of the hybridoma in a medium comprising 8-azaguanine, 6-thiguanine, 5- bromouracyl or 2-aminopurine whereby hybridoma cells sensitive to medium containing hypoxanthine aminopterin thymidine are obtained.

A process according to Claim 40 wherein said selectively destructible hybridomas are obtained by irreversible enzyme inhibition 30 46 A process according to Claim 45 wherein the inhibition is obtained using a metabolic inhibitor.

47 A process according to Claim 46 wherein the inhibitor is a Kcat inhibitor.

48 A process according to Claim 47 wherein the inhibitor is selected from azaserine or 5-diazo-5-oxa-L- norleucine.

49 A process according to Claim 40 wherein only the first hybridoma is selectively destructible 35 A process according to Claim 49 wherein the selectively destructible hybridoma is further selected to confer the ability for it to survive in a medium which is lethal to the other hybridoma.

51 A process according to Claim 50 wherein the selectively destructible hybridoma is resistant to medium containing ouabain.

52 A process according to Claim 51 wherein the hybridoma is back selected to obtain cells that are 40 sensitive to a medium in which the polydoma can be cultured.

53 A process according to Claim 52 wherein back selection is accomplished by culturing cells of the hybridoma in a medium comprising a member selected from the group consisting of 8-azaguanine, 6-thioguanine, 5-bromouracyl deoxyribose or 2-aminopurine whereby hybridoma cells sensitive to medium containing hypoxanthine aminopterin thymidine are obtained 45 54 A process according to Claim 51 wherein the selectively destructible hybridoma is obtained by irreversible enzyme inhibition.

A process according to Claim 54 wherein the inhibition is obtained using a metabolic inhibitor.

56 A process according to Claim 54 wherein the inhibitor is a Kcat inhibitor.

57 A process for producing a hybrid monoclonal antibody having a dual specificity comprising 50 isolating the antibody from cells of a polydoma produced according to the process of Claims 1,30,31,32, 33,34,35,36,39,40,41, 42,43,44,45,46,47,48,49,50, 51, 52, 53, 54, 55 or 56.

58 An immunometric process comprising:

a) administering to a host an antibody having a dual specificity, one specificity of which is directed against a disease associated antigen and the other against a hapten, the hapten being an agent lethal to the 55 antigen or associated tissue or having a lethal agent bound to it; and b) administering the hapten after sufficient time has elapsed to permit the antibody to bind to the disease associated antigen.

59 A process according to Claim 58 wherein the lethal agent is a radionuclide.

60 A process according to Claim 59 wherein the radionuclide is an emitter of a-radiation or,3-radiation 60 61 A process according to Claim 58 wherein the lethal agent is a tissue toxin.

62 A process according to Claim 61 wherein the toxin is comprised of a ricin A chain.

63 A process according to Claims 58, 59,60, 61 or 62 wherein the antigen is a tumor associated antigen.

64 A process according to Claims 58, 59,60,61 or 62 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma 65 14 GB 2169921 A 14 A process according to Claim 63 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma.

66 A process according to Claims 58, 59,60,61 or 62 wherein the antibody is a hybrid antibody produced by the reassociation of antibody half molecules obtained by the selective cleavage of a mono-specific antibody against the disease associated antigen and a mono- specific antibody against the 5 hapten.

67 A process according to Claim 66 wherein the selectively cleaved antibodies are monoclonal antibodies.

68 A process according to Claim 66 wherein the selectively cleaved antibodies are polyclonal antibodies 10 69 A process according to Claims 58, 59,60,61 or 62 wherein the antibody is a multimer of a pair of intact mono-specific antibodies, one of said mono-specific antibodies being against the disease associated antigen and the other against the hapten.

A process according to Claim 69 wherein the mono-specific antibodies are monoclonal antibodies.

1 5 71 A process according to Claim 69 wherein the mono-specific antibodies are poly clonal antibodies 15 72 An in vivo immunodiagnostic process comprising:

a) administering to a host an antibody having a dual specificity, one specificity of which is directed against a disease associated antigen and the other against a hapten bearing a radionuclide; b) administering the hapten after sufficient time has elapsed to permit the antibody to bind to the disease associated antigen; and 20 c) scanning the host to detectthe location of radiation emitted by the radionuclide.

73 A process according to Claim 72 wherein the radionuclide is an emitter of y-radiation.

74 A process according to Claim 72 wherein the radionuclide is bound directly to the hapten.

A process according to Claim 72 wherein the radionuclide is bound to the hapten by a chelating agent 25 76 A process according to Claims 72,73,74 or 75 wherein the antigen is a tumor associated antigen.

77 A process according to Claims 72,73,74 or 75 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma.

78 A process according to Claim 76 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma 30 79 A process according to Claims 72,73,74 or 75 wherein the antibody is a hybrid antibody produced by the reassociation of antibody half molecules obtained by the selective cleavage of a mono-specific antibody against the disease associated antigen and a mono-specific antibody against the hapten.

A process according to Claim 79 wherein the selectively cleaved antibodies are monoclonal antibodies 35 81 A process according to Claim 79 wherein the selectively cleaved antibodies are polyclonal antibodies.

82 A process according to Claims 72,73,74 or 75 wherein the antibody is a multimer of a pair of intact mono-specific antibodies, one of said mono-specific antibodies being against the disease associated antigen and the other againstthe hapten 40 83 A process according to Claim 82 wherein the mono-specific antibodies are monoclonal antibodies.

84 A process according to Claim 82 wherein the mono-specific antibodies are polyclonal antibodies.

An immunoassay process comprising:

a) adding to a sample suspected of containing a target antigen a predetermined amount of the target antigen to which is fixed a chromophore; 45 b) adding to the sample an antibody having a dual specificity, one specificity of which is directed against the target antigen and the other against a hapten which is, or to which is bound, a fluorescing chromophore which chromophore fluoresces at a wavelength that is absorbable by the chromophore on the target antigen when said chromophores are within about 100 A of each other; c) binding the hapten to the antibody; 50 d) measuring the fluorescence of the sample after a period of incubation; e) comparing the intensity of fluorescence of the sample with that of a control sample containing a known amount of target antigen.

86 A process according to Claim 85 wherein the antigen is a tumor associated antigen.

87 A process according to Claim 85 wherein the hapten is bound to the antibody before the antibody is 55 added to the sample.

88 A process according to Claim 86 wherein the hapten is bound to the antibody before the antibody is added to the sample.

89 A process according to Claims 85,86,87 or 88 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma 60 A process according to Claims 85,86,87 or 88 wherein the antibody is a hybrid antibody produced by the reassociation of antibody half molecules obtained by the selective cleavage of a mono-specific antibody against the disease associated antigen and a mono-specific antibody against the hapten.

91 A process according to Claim 90 wherein the selectively cleaved antibodies are monoclonal antibodies 65 GB 2 169 921 A 14 GB 2 169 921 A 15 92 A process according to Claim 90 wherein the selectively cleaved antibodies are polyclonal antibodies.

93 A process according to Claims 85,86,87 or 88 wherein the antibody is a multimer of a pair of intact mono-specific antibodies, one of said mono-specific antibodies being against the target antigen and the other against the hapten 5 94 A process according to Claim 93 wherein the mono-specific antibodies are monoclonal antibodies.

A process according to Claims 85,86,87 or 88 wherein the fluorescing chromophore is fluorescein and the quenching chromophore is rhodamine.

96 An immunoassay process comprising:

a) adding to a sample suspected of containing a target antigen a predetermined amount of the target 10 antigen to which is bound a substance capable of interacting with an enzyme to produce a detectable product; b) adding to the sample an antibody having a dual specificity, one specificity of which is directed against the target antigen and the other against the enzyme or a hapten to which the enzyme is bound; c) binding the hapten to the antibody; 15 d) measuring the formation of the detectable substance after a period of incubation; e) comparing the formation of the detectable substance with that of a control sample containing a known amount of target antigen.

97 A process according to Claim 86 wherein the enzyme is bound to the antibody before the antibody is added to the sample 20 98 A process according to Claim 91 wherein the antigen is a tumor associated antigen.

99 A process according to Claim 97 wherein the antigen is a tumor associated antigen.

A process according to Claims 96,97, 98 or 99 wherein the antibody is a hybrid monoclonal antibody produced by a polydoma.

101 A process according to Claims 96,97,98 or 99 whereintheantibody is a hybrid antibody produced 25 by the reassociation of antibody half molecules obtained by the selective cleavage of a mono-specific antibody against the target associated antigen and a mono-specific antibody against the hapten.

102 A process according to Claim 101 wherein the selectively cleaved antibodies are monoclonal antibodies.

103 A process according to Claim 101 wherein the selectively cleaved antibodies are polyclonal 30 antibodies.

104 A process according to Claims 96, 97, 98 or 99 wherein the antibody is a multimer of a pair of intact mono-specific antibodies, one of said mono-specific antibodies being against the disease associated antigen and the other against the hapten.

105 A process according to Claim 104 wherein the mono-specific antibodies are monoclonal 35 antibodies.

106 A process according to Claims 96,97,98 or 99 wherein the substance bound to the target antigen is a second antigen and wherein one enzyme catalyzes the formation of a product which interacts with the other enzyme to produce a detectable substance.

107 A process according to Claim 106 wherein the detectable substance is detected by its fluorescence, 40 luminescence or spectroscopically.

108 A polydoma according to Claim 2 substantially as hereinbefore described with reference to the Examples.

109 A hybrid monoclonal antibody according to Claim 7 substantially as hereinbefore described with reference to the Examples 45 A process according to Claim 30 substantially as hereinbefore described with reference to any one of the Examples.

111 A process according to Claim 40 substantially as hereinbefore described with reference to the Examples.

112 A process according to Claim 1 substantially as hereinbefore described with reference to the 50 Examples.

113 A process according to Claim 58 substantially as hereinbefore described with reference to the Examples.

114 A process according to Claim 72 substantially as hereinbefore described with reference to the Examples 55 A process according to Claim 85 substantially as hereinbefore described with reference to the Examples.

116 A process according to Claim 96 substantially as hereinbefore described with reference to the Examples.

Printed for Her Majesty's Stationery Office by Courier Press, Leamington Spa 7/1986 Demand No 8817356.

Published by the Patent Office, 25 Southampton Buildings, London, WC 2 A 1 AY, from which copies may be obtained.