NO171643B - ANTIBODIES WITH DOUBLE SPECIFICATIONS, THEIR PREPARATION AND USE - Google Patents

Description

The present invention relates to a hybrid monoclonal antibody for use in vitro, as the antibody has double specificity and is produced from a polydom, and the distinctive feature of the antibody according to the invention is that said hybrid antibody is produced by fusion of two hybridomas, or a hybridoma and a spleen cell (B-lymphocyte), or prepared by removing the nucleus from a first hybridoma and introducing it into the cytoplasm of another hybridoma, the antibody being a component of a mixture of antibodies comprising the hybrid antibody and two species of mono-specific antibodies in a ratio of approximately 2:1:1.

The invention also relates to the use of the aforementioned monoclonal antibody for immunochemical analyses.

These and other features of the invention appear in the patent claims.

The antigen-antibody reaction is already routinely exploited in a number of practical applications and is being intensively investigated to establish its value for other hitherto unproven applications. E.g. serum antibodies produced by a host's immune response to an immunogen can be used in affinity purification processes to isolate the immunogen from solutions in which it is present only in small amounts.

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 methods. E.g. detection of HBsAg using a radioimmunometric technique is the currently chosen method. On another front, serum antibodies to ferritin, obtained from New Zealand white rabbits and labeled with <131>I, have been reported as promising for the treatment of liver tumors (see Order at al., International Journal of Radiation Oncology, Biology and Physics, 6 , 703 (1980)). Serum antibodies, e.g. those obtained from rabbits, murine species or other mammals are "polyclonal" in nature as the host's immune system is stimulated to produce a mixture of specific antibodies directed at the various antigenic determinants or epitopes on the immunogen to which the host responds. The individual antibodies that make up the mixture are all the product of a B-cell clone; furthermore, each B cell secretes only one special antibody. The antibody produced by one clone differs from an antibody to the same antigen produced by another clone in that it has at least a subtle difference between its peptide sequence and the peptide sequence of the other antibody. Each antibody species is therefore actually a distinct molecule and the differences in peptide sequence between different species affect their general specificities as well as the particular epitopes that they recognize and their affinities for the antigen.

An individual B cell cannot be cultured indefinitely using currently available tissue culture techniques to obtain the particular antibody 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 secretion product of a hybrid cell referred to as a "hybridoma". (G. Kohler and C. Milstein, Nature, 256, 495 (1975)). Basically, the process involves fusing spleen cells taken from an immunized mouse with mouse myeloma cells to form the hybridoma. Myeloma cells that 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 then be easily obtained as a pure chemical compound. This is in contrast to the conventional antibody preparations obtained e.g. from serum, in which the individual antibody is only one of the components of a substantially non-separable antibody mixture of related, yet distinct chemical compounds.

Being 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 processed to select those that produce the monoclonal antibody with the most desirable properties for a given application. The immortality of the hybridoma guarantees an almost unlimited supply of the antibody it secretes and eliminates problems associated with variance in antibody titer and overall affinity from animal to animal used for the production of serum antibodies. Monoclonal antibodies obtained from hybridomas have e.g. been practically used in diagnostic kits.

An antibody molecule can generally be considered to express a single specificity that is exhibited towards the immunogen to which the host's immune system reacted in producing the antibody. The antibody is composed of two identical halves, each of which comprises a pair of heavy and light chains, and each of which recognizes the same antigenic determinant as the other.

A -S-S- disulfide bridge linking the two heavy chains at the site of the cysteine moieties can usually be selectively cleaved in vitro by careful reduction and the two half molecules dissociated by subsequent acidification. The half molecules can then be recombined (re-natured), also in vitro, at neutral pH, as the re-association takes place by non-covalent interaction.

If antibodies of different specificities are subjected to selective cleavage of the disulfide bridges between the heavy chains and conditions leading to renaturation are then established, reassociation between half molecules can occur randomly to produce a population of antibodies, at least some of which are hybrids by one half of an antibody molecule with one specificity combines with one half of an antibody molecule with a different specificity. E.g. describes Nisonoff et al. "The Antibody Molecule", Academic Press, New York (1975) at pages 260-261 an in vitro production of a polyclonal antibody hybrid of rabbit antiovalbumin and anti-BGG antibodies. Hybrid monoclonal antibodies have also been obtained using an analogous process. See D.M. Kranz et al., Proe. Nati. Acad. Pollock. USA, 78, 5807 (1981). At least theoretically, the hybrid antibody will exhibit a dual specificity in that one half of the antibody will recognize and bind to an antigenic determinant or epitope, while the other half will recognize a different epitope on the same or a different antigen.

Although hybrid antibodies can be obtained as is

described in the preceding, the yields are often very low, the reactions used to prepare them are difficult to reproduce and the hybrid antibodies are usually exposed to clear, irreversible denaturation. Such denaturation can

reduce immuno-reactivity and would be expected to result in different metabolic properties in vivo. As a result, the hybrid antibody has thus far largely remained a laboratory curiosity that is difficult to obtain.

Antibodies with dual specificities can also be prepared by conjugating pairs of intact antibodies, monoclonal or not, using a variety of coupling or cross-linking agents such as protein A (from Staphylococcus aureus), carbodiimide and bifunctional compounds such as N-succinimidyl-3-(2-pyridyldithio)propionate to obtain dimers and higher antibody multimers to which each member of the antibody pair contributes its specificity. E.g. have Mandoche et al. reported the formation of multivalent antibodies with dual specificities by means of 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, 42, 355, (1981). In this method, antibodies with one specificity bind to the surface antigen and the others to a proportion that allows detection.

The synthesis of antibodies with double specificity by means of the preceding methods is complicated and no commercial practice of them has been carried out to date.

The hybrid monoclonal antibodies according to the invention can be produced in good yields without denaturation. In this presentation, the term "hybrid antibody" will be used to denote a single antibody molecule with two different specificities. The individual specificities can be to antigenic determinants on two different antigens or to different antigenic determinants (epitopes) on the same antigen. Furthermore, the term "antigen" also includes haptens if not stated otherwise.

The hybrid antibodies with a double specificity according to the invention are obtained by fusion of a hybridoma, preferably a selectively destructible hybridoma, which secretes an antibody against a preselected antigenic determinant with a fusionable B-lymphocyte or another hybridoma, the B-lymphocyte or the another hybridoma secretes a different antibody against a different antigenic determinant, to form a second generation hybridoma (hereinafter "polydom"). As used herein, the term "selectively destructible hybridoma" means a hybridoma that lacks or at least substantially lacks the ability to survive in the medium in which the polydome is grown. It has unexpectedly been found that, unlike the parent hybridoma or the B-lymphocyte from which it is derived, each of which secretes a population of identical antibodies with a single specificity, the polydoma additionally secretes a high percentage of a monoclonal hybrid antibody with a dual specificity, ie, an ability to bind with any of the antigenic determinants recognized by the individual antibodies produced by the cells of origin or with both determinants simultaneously. The hybrid monoclonal antibody obtained in this way has not been subjected to the undesirable denaturation that characterizes hybrids obtained from the process of chemical recombination of antibody half-molecules. Furthermore, the method used here allows the hybrid to be obtained safely and in large quantities.

The hybrid monoclonal antibodies according to the invention can be used for immunodiagnosis. In general, such processes employ a monoclonal antibody or polyclonal antibodies with a first specificity against a target antigen and a second specificity against a substance, e.g. another antigen or hapten, which allows a diagnosis to be made for the target antigen or which allows the provision of or is itself an agent lethal to the target antigen or the tissue with which it is associated.

By a suitable selection of cells of origin, a polydom can thus be obtained which will secrete an antibody with a specificity for a target antigen and another specificity for a proportion useful in diagnosis. Alternatively, antibody half-molecules can be recombined using in vitro chemical agents or individual intact monospecific antibodies can be linked or cross-linked by death? of chemical means to provide antibody multimers (which may be a dimer, trimer or higher multimer) with a dual specificity and with the same or a similar utility as the hybrid monoclonal antibody with the same dual specificity according to the present invention.

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

Accordingly, an object of the present invention is hybrid monoclonal antibodies which have not been denatured by the manufacturing process. Applicable production processes will be apparent from the following together with examples of applications of the hybrid monoclonal antibodies according to the invention.

The way in which these and other purposes can be achieved will become apparent when considering the following description of preferred embodiments.

As stated above, the method for producing a hybrid monoclonal antibody according to the invention requires as a source cell a hybridoma, and preferably a selectively destructible hybridoma, which secretes a monoclonal antibody against a preselected antigenic determinant or epitope. The use of a selectively destructible hybridoma as a cell of origin has the advantage that it prevents the cells obtained by fusion of the selectively destructible hybridoma with a B-lymphocyte or another hybridoma, i.e. the polydoma, from being displaced by a population of the parent hybridoma when the cells obtained at the fusion process are cultured and to provide a means by which the polydom cells can be isolated from the parent hybridoma cells.

Selectively destructible hybridomas can be obtained from hybridomas that secrete an antibody with one of the desired specificities produced by the classic Kohler-Milstein process, i.e. hybridomas obtained by fusion of a myeloma cell and a B-lymphocyte found in the spleen cells of mice. In one embodiment, such a hybridoma is subjected to a backselection process to obtain a hybridoma which is selectively destructible.

In general, selective destructibility can be achieved by backselection to a hybridoma lacking a genetic component necessary for its survival in a selected medium in which the polydome produced by the fusion can be grown due to a genetic contribution from the fusion partner of the selectively destructible hybridoma, i.e. B- the lymphocyte or the other hybridoma.

The heretofore preferred backselection process involves culturing a hybridoma secreting an antibody with one of the desired specificities for incorporation into the hybrid antibody in a culture medium containing 8-azaguanine. In such a medium, any cell that incorporates 8-azaguanine and therefore can grow in the medium are those cells lacking the enzyme hypoxanthine phosphoribosyltransferase (HPRT). Clones of cells lacking this enzyme cannot grow in medium containing hypoxanthineamineopterinthymidine (HAT). They can thus be selectively destroyed in this medium.

A very similar process of backselection involves growing the hybridomas that secrete the desired antibody in a medium containing 6-thioguanine, another analog of guanine that is toxic to cells if incorporated into DNA. Here, too, certain cells that will grow in this medium lack the HPRT enzyme, and clones of these cells will necessarily be sensitive to HAT medium.

Yet another backselection process that can be used involves culturing cells of the selected hybridoma cell line in a medium containing either of the thymine analogs 5-bromouracildecsyribose (BUdR) 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 method for obtaining a selectively destructible parent hybridoma involves irreversible enzyme inhibition using metabolic inhibitors. Among these, the so-called Kcat inhibitors are preferred. These inhibitors are analogs of an enzyme's substrate that is converted by the target enzyme into a highly reactive molecule that reacts with the enzyme at its active site and results in irreversible inhibition of the enzyme. E.g. treatment of the selected hybridoma with an analogue of glutamine such as azaserine or 5-diazo-5-oxa-L-norleucine (DON) will irreversibly inhibit the enzyme formylglycinamide ribonucleotide amido-transferase by forming a covalent bond with a cysteine residue at the enzyme's active site. This inhibition will eventually result in cell death. However, the hybridoma can be saved by fusion with the other parent of the polydom that provides 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 that has been previously immunized with an antigen, which may be a hapten bound to a carrier protein, selected to cause the host to generating an immune reaction that produces antibodies of the other specificity desired in the hybrid antibody. The host is usually a mouse, but species of rabbits, humans or other animals can also be used, although the interspecies function may exhibit a low degree of stability. The procedure for immunization of 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 give the polydoma can be accomplished by combining the two groups of cells in a medium containing an agent 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 cultivation. The B-lymphocytes will survive only for a short time and the parent hybridoma cells cannot grow in this medium. The population of polydomes formed as a result of the fusion, however, can be grown in this medium, due to the complementation of the parent hybridoma with e.g. The B-lymphocyte, by means of a genetic contribution of the ability to form a missing enzyme such as HPRT or TK or by a direct contribution of an enzyme that is inhibited in the parent hybridoma. Clones of individual polydomes are cultured and those that secrete antibodies with the desired dual specificity are selected. Clones of polydomes with antibodies exhibiting the desired dual specificity for selection of those having the other specificity, ie that obtained from the B-lymphocytes, and affinity are highly desirable.

In a further embodiment, the polydom is obtained by fusing the selectively destructible hybridoma using a suitable fusion agent with another hybridoma which is also selectively destructible. The second parental hybridoma is obtained in the same way as the first, ie by a process of backselection, irreversible enzyme inhibition or by any other suitable technique. In such a case, the second hybridoma must be able to complement the first. If e.g. the first selectively destructible hybridoma lacks the enzyme HPRT, the second must be able to contribute to the polydome a gene that will enable the polydome to express HPRT. Similarly, if the other selectively destructible hybridoma lacks the enzyme TK

must first contribute a gene for TK to the polydom. Similar complementarity between the two hybridomas must be present if irreversible inhibition of an enzyme has been carried out to confer selective destructibility to them. It is also possible to use as one of the hybridoma parents a hybridoma that has been subjected to a backselection process, and as the other a hybridoma that has been subjected to a process of enzyme inhibition.

The use of complementary selectively destructible hybridomas as the origin of the polydom has the advantage that both origins can be selected on the basis of specificities and affinities of the monoclonal antibody they produce, whereas in the case of fusion of a single hybridoma with B lymphocytes no prefusion selection can among the B-lymphocytes is carried out to obtain those that produce an antibody with the desired specificity and affinity. Fusion of the two selectively destructible hybridomas can be carried out using polyethylene glycol or by using other fusion agents, also here using known methods. After fusion, the cells are transferred to a culture medium in which the two progenitors can grow, but in which the polydomes resulting from the fusion are capable of growth due to the complementary contributions of the progenitors.

Heretofore, selection processes for obtaining selectively destructible hybridomas for use as both hybridoma partners in a hybridoma-hybridoma fusion to form a polydom have been discussed. The necessity for the second of the hybridoma parents to be selectively destructible can, however, be avoided by imparting both a dominant and a recessive marker to the first of the hybridoma parents. A hitherto preferred method is HAT-ouabain selection. Ouabain is a specific inhibitor of the Na<+->K<+> activated ATPase in the plasma membrane. This enzyme is responsible for the introduction of K<+> into a cell and the removal of Na<+> from the cell.

Cells of a hybridoma that have previously been back-selected for e.g. to confer selective destructibility, HAT sensitivity, are cultured in ouabain medium for selection of ouabain-resistant cells. Clones of these cells will be HAT-sensitive but ouabain-resistant. In contrast, the hybridoma selected for fusion with this will be ouabain sensitive, but can survive and grow in HAT. Alternatively, selection for ouabain resistance can be made first either on the parent myeloma line or the hybridoma derived therefrom, followed by back-selection or other technique to confer selective indestructibility.

Cells obtained by fusion of the two hybridomas in polyethylene glycol or other fusion agent are transferred to HAT medium containing ouabain in a concentration that is lethal to the other of the hybridoma originators. The selectively destructible of the hybridoma parents cannot survive in HAT medium which is either e.g. lacking HPRT or other enzyme, although ouabain resistant. However, the polydom cells can grow in the medium as they will have the enzymes and ouabain resistance necessary for survival. The preceding method has the advantage that it is possible to obtain a polydom by direct fusion of a selectively destructible of the hybridoma parents secreting an antibody with one of the specificities desired in the hybrid with another "available" hybridoma secreting an antibody with the other specificity desired in the hybrid, and no use of technique to impart selective destructibility to the other of the hybridoma parents is then necessary.

Yet another method of obtaining a polydoma using a universal type of origin, i.e. one having both a positive and a negative marker, which can be fused to any "available" hybridoma, involves the use of recombinant DNA vectors carrying different drug resistance markers. E.g. SV40 carrying a gene for neomycin resistance can be used.

A hitherto preferred universal origin type is that which is HAT sensitive-neomycin resistant. The selected parent type is back-selected for HAT sensitivity and then transfected with SV40 vector carrying a gene for neomycin resistance. This method can also be reversed as transfection is carried out 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. Viable hybridomas, however, grow in HAT but die in the presence of neomycin.

Products from the fusion of the origins therefore survive in the presence of HAT and neomycin. While the use of vectors to impart resistance to neomycin is preferred heretofore, vectors carrying genes which will impart resistance to mammalian cells to other drugs may also be used.

Although it has hitherto been preferred, it is not essential for the present method for obtaining polydomes from pairs of hybridomas that at least one of the parent cell lines is selectively destructible. It is within the scope of this technique to fuse a pair of hybridomas, neither of which is selectively destructible, but secretes an antibody with one of the specificities desired in the hybrid antibody, in the presence of a suitable fusion agent followed by subcloning of all cells before the population of unfused parent hybridomas increases to such an extent that selection of the subclones for identification of polydomes is not practical. The subclones are then selected to determine which secreted antibodies have a dual specificity.

This process is best suited for obtaining polydomes when the fusion frequency of the parent cell lines is high. In any case, and especially when the cell fusion frequency is low, the cells obtained from the fusion of hybridomas with monoclonal antibodies to different antigens can be selected using a cytofluorograph for polydomain identification. To achieve this, samples of the two antigens are labeled with different fluorescent moieties whose fluorescence appears at different wavelengths. E.g. one antigen can be marked with fluorescein and the other antigen with rhodamine. The population of cells from the fusion, which preferably have been cultured overnight or for another suitable period of time to increase their numbers, are incubated with the two labeled antigens. The cells are then selected using the cytofluorograph. The cells that fluoresce at only one of the two wavelengths will be from the cell lines of the original hybridomas: cells that exhibit fluorescence at both wavelengths, however, will be polydomes that can be isolated and subcloned.

In yet another embodiment, a polydoma can be obtained directly by removing the nucleus from a first hybridoma secreting a monoclonal antibody with one of the specificities desired in the hybrid and introducing this into the cytoplasm of another hybridoma secreting a monoclonal antibody with the other desired specificity. Of course, none of the original hybridomas need to be selectively destructible in order to be used in this process. After introduction of the nuclear material, the cell is cloned to obtain a population of polydomes.

It has been found that unlike the parent hybridomas or B-lymphocytes which secrete a single antibody, polydom cells obtained by the above methods secrete a mixture of antibodies, at least one of which is a hybrid antibody with a dual specificity. Relatively smaller amounts of antibodies with the same specificity as those produced by the parent cells used to obtain the polydomain are also produced from the polydomain. The ratio between hybrid and monospecific antibodies appears to be approximately 2:1:1, which is the ratio expected if the polydome produces equal amounts of all the possible (H) chains synthesized by the cells of origin that are randomly combined in the polydome itself.

The polydomains can be cultured in vitro or grown in vivo in any genetically compatible animal or hairless mouse to yield larger amounts of the hybrid antibody recovered from the culture medium or ascites fluid of the animal using known procedures. See e.g. the protocols in "Monoclonal Antibodies", published by Kennett et al., Plenum Press, New York (1980) pages 336-418.

The mixture of antibodies produced by the polydomet can be separated to give the hybrid. E.g. allows sequential affinity chromatography against first one and then the other antigen for which the hybrid is specific, its separation from the monospecific antibody contaminants. It has also been found that simple ion exchange chromatography and electrophoretic methods can also be used at least under certain conditions. If necessary, the charge difference for ion exchange can be one of the properties of the antibody taken into account when selecting the lines of origin.

Example 1

A hybrid monoclonal antibody according to the invention with dual specificity for hepatitis B surface antigen (HBsAg) and prostatic acid phosphatase (PAP) was prepared as follows: A hybridoma secreting a monoclonal antibody to PAP was cultured in HAT medium for 1 week and then transferred to and cultured in a non-selective medium. After various lengths of growth time under non-selective conditions, 2 ml portions of cells were placed in medium containing 10~<4> M 8-azaguanine which prevents the cells from growing by incorporating 8-azaguanine into their DNA in place of guanine. The cells lacking the HPRT enzyme survived and grew in this medium and these cells did not necessarily survive in HAT.

Clones grown in the medium containing 8-azaguanine were tested for sensitivity to HAT and anti-PAP production. A clone that still produced anti-PAP and exhibited HAT sensitivity with a reversion frequency of less than 4 x 10 -^ was subcloned. All subclones behaved similarly to the parent clone.

Cells from one of the HAT-sensitive subclones were fused in polyethylene glycol with spleen cells obtained from Balb/c mice hyperimmunized with HBsAg to give polydomes using the fusion technique of Gerhard. See "Monoclonal Antibodies", supra, page 370. The fusion produced 220 polydoms which were selected to determine which secreted antibodies with specificity for both PAP and HBsAg. Clones of two such polydomes were found to produce antibody and could be recovered from ascites and exhibited both specificities.

Subclones of both polydomes continued to produce ascites exhibiting both specificities and yielding triple bands on Orstein-Davis PAGE similar to those corresponding to the parental clone. The ascites from both clones were found to bind <125>I-HBsAg and 12 5I_PAp by radioimmunoassay and gave Ka values of approximately 10^ for each, thus suggesting formation of antibodies with two specificities.

Data in Table I below show the results obtained by immunoassay using ascites obtained from a clone of one of the polydomains compared with ascites obtained from hybridomas secreting monoclonal antibodies, respectively against IgE (used as a control), PAP and HBsAg using of immobilized HBsAg as a solid phase and a variety of radiolabeled antigens as a solution phase.

A 200 microliter sample of ascites from the polydom and each of the three hybridomas was each incubated overnight with 12 polystyrene beads to which HBsAg was bound. The HBsAg beads were obtained from Abbott Laboratories, North Chicago, Illinois. After washing, triplicate samples were incubated for 4 hours with 100,000 cm of the indicated <125>i labeled antigen. After a second wash, the beads were counted to determine the amount of labeled antigen bound to the beads. In one set of tests using radiolabeled PAP as the solution phase antigen, anti-PAP was added to the antigen prior to incubation with the bead. The antibody to PAP used for this purpose is the monoclonal antibody produced by the parent hybridoma used to produce the polydom and is therefore against the same PAP epitope as that expected to be displayed by the hybrid antibody.

Data in Table I indicate that only the radiation expected from non-specific binding is measured for anti-PAP ascites when compared to the radiation for the IgE control.

The ascites containing the HBsAg antibody, on the other hand, bound the labeled HBsAg antigen as expected, but showed non-specific binding when the other labeled antigens were tested. However, the ascites from polydom-klonec bound both labeled HBsAg and labeled PAP, the former being due to the presence of some non-hybrid, monospecific antibody to KBsAg in the ascites and the latter attributable to a hybrid that can bind and bridge HBsAg on the bead and the trace-labeled PAP in the ascites -solution. The experiment using a mixture of labeled PAP and anti-PAP from the parent hybridoma confirms that the anti-PAP specificity of the hybrid is for the same epitope as the antibody secreted by the parent hybridoma, as only background radiation is observed upon inhibition of binding of the labeled PAP to the hybrid antibody due to the parent antibody.

Analysis of the ascites from the polydomin clone used in the comparative radiojudgment was carried out using polyacrylamide gel electrophoresis (PAGE) and immunoelectrophoresis (IEP). Both indicated the presence of at least three antibody species.

Preparative-scale DEAE ion-exchange chromatography gave three well-separated peaks, the middle one of which had a shoulder. Each of the three peaks was homogeneous when 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 HBsAg and PAP. The first peak bound HBsAg but not PAP. The middle peak and its shoulder bound to both HBsAg and PAP and the last peak bound only PAP. The middle peak is thus a hybrid antibody with a double specificity to HBsAg cg PAP consisting of at least two subspecies.

The hybrid antibody obtained as the middle peak by DE AS chromatography was radiolabeled with <125>j> After labeling, 85% of the labeled antibody would bind to PAP and 88 would bind to HEsAg. The affinity of the hybrid for PAP was found to be somewhat less than that of the monoclonal antibody to PAP produced by the parent line. This difference in affinity was about the same as observed between a monoclonal antibody and its Fab fragment.

DEAE chromatography indicates that hybrid antibody comprises more than 50% of the antibodies produced by the polydome and constitutes approximately the 2:1:1 ratio predicted on a statistical basis if the polydome were to synthesize all possible antibody heavy chains, i.e. those displaying either PAP or HBsAg specificity, which are combined inside the cell on a random basis to form hybrid antibody mixed with smaller amounts of ce two monospecific antibodies with the same specificity as those produced by the cells of origin. The existence of hybrid antibody subspecies suggests that they may differ with respect to their light chain conformation.

Example 2

Hybrid monoclonal antibodies according to the invention with dual specificity for human IgD and prolactin were produced by the fusion of two hybridomas, one of which was constructed to contain two selectable genetic markers: sensitivity to HAT medium and resistance to ouabain. This doubly marked hybridoma or so-called "universal origin hybridoma" could then be fused to any other hybridoma. The resulting polydomes grow in the presence of HAT and ouabain, while any unfused progenitor cells die. The advantages of using "universal origins" are described elsewhere herein.

For the construction of such universal origins, both selectable markers were introduced during the initial construction of the hybridoma. To obtain this lineage, the readily available HAT-sensitive mouse myeloma p3.653 was selected for another genetic marker, ouabain resistance, by introducing 1 mM ouabain into the culture medium. While most cells died, about 1/100,000 cells had by chance mutation acquired resistance to the aforementioned substance and thus survived and proliferated to form the new myeloma population which was HAT-sensitive and ouabain-resistant.

This HAT-sensitive ouabain-resistant myeloma was then fused with spleen cells obtained from Balb/c mice hyperimmunized with IgD using the previously stated technique of Gerhard. Hybrids were selected in HAT medium (without ouabain) and clones were selected for production of monoclonal antibody directed against IgD. From the positive clones, the one that produced an IgG against IgD was selected for further study. This clone was tested for retention of the ouabain resistance trait by adding 1 mM ouabain to the culture medium. About 1/3 of the cells retained this genetic marker. When the culture was grown exponentially in ouabain, the cells were subcloned. Ouabain-resistant subclones were tested for continued production of the monoclonal anti-IgD antibody. One of the subclones was further backselected by the method in example 1 to give a population of cells sensitive to HAT. This subclone was grown for 2 weeks under non-selective conditions and then placed in medium containing 6-thioguanine. As previously noted, 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 will not utilize 6-thioguanine from the medium and can therefore grow, but are consequently sensitive to HAT. This population of the back-selected subclone was then itself subcloned in 6-thioguanine and ouabain-containing medium. Subclones were assessed for continued production of the monoclonal anti-IgD antibody. A clone that showed all the desired properties - growth in ouabain and 6-thioguanine as well as production of monoclonal anti-IgD, was selected as so-called "universal origin". These universal origins could then be fused to any other HAT-resistant ouabain-sensitive hybdidom to form a polydom that would express a hybrid antibody and one specificity of which would be anti-IgD. For this purpose, a mouse hybridoma that secretes a monoclonal antibody directed against prolactin was initially selected. The anti-prolactin monoclonal antibody is of the same subclass (IgGi) as the anti-IgD expressed by the parent line and is easily separated from the antibody on Ornstein-Davis gels. Such a separation indicates very different charges on the antibodies and should thus allow easy isolation of a hybrid antibody by means of DEAE-Sephadex chromatography.

IO<7> cells of the HAT-sensitive, ouabain-resistant cell line were fused in polyethylene glycol with IO<7> cells that produce antiprolactin. The fused cells were first cultured for 3 days and finally placed again in HAT medium. More than 600 clones arose from this fusion: 66 clones were randomly selected for analysis. Of these clones, 36 exhibited both anti-IgD and antiprolactin activity.

These clone supernatants were assessed for the presence of hybrid antibody using the following assay. A polystyrene bead coated with another antiprolactin monoclonal antibody was incubated for 5 hours with 200 microliters of a 100 ng/ml prolactin solution. The antibody used binds prolactin at a distinct site compared to the corresponding antibody produced by the fused hybridor cell line. The pellet was washed and then incubated overnight with the clone supernatants. The next day after several washings, <!25>i labeled IgD was added. Hybrid antibody bound to the bead with a functional bond could bind the radiolabeled IgD with the free anti-IgD functionality, while neither parent antibody IgD-IgD nor prolactin-prolactin could form this bridge between the prolactin bead and <125>i IgD tracer. Results of a typical evaluation for hybrid bifunctional antibody-producing clones are shown in the following table 2. 21 of 36 clones showed significant bifunctional activity in this evaluation. Ascites developed from 2 hitherto available clones has been shown to respond in the bifunctional assessment. These ascites contain antibodies that separate the three distinct bands on Orstein-Davis gels: Two bands coincide exactly with antibodies produced by the parent hybridomas (Anti-IgD and antiprolactin). The third band migrates midway between the parent monoclonal antibody bands as expected for the hybrid antibody.

That a hybrid monoclonal antibody against IgD and prolactin exhibits a dose response when the amount of prolactin is varied in the assay used to develop the data in Table 2 is shown by the data in Table 3 using the antibody in clone #2 and, as controls, antibodies from the cell line of origin (anti-IgD and antiprolactin).

These data show that the amount of prolactin bound by the hybrid antibody is dose-responsive in that the amount of labeled IgD that is bound is increased when the dose of prolactin is increased. In contrast, no dose response is observed using the parent antibodies against IgD and prolactin as they cannot form the bridge between prolactin bound to the bead and the labeled IgD. The hybrid antibody can thus be used as a component in an assessment of prolactin. Tailoring other hybrid antibodies presents similar opportunities for other assessments.

Example 3

By a similar technique to example 2, a universal origin hybridoma was developed which secretes monoclonal antibody directed against the haptenarsenate-arsenate dimer and which is both resistant to ouabain and sensitive to HAT. This hybridoma was fused into a hybridoma secreting a monoclonal antibody with specificity for carcinoembryonic antigen (CEA), an antigen expressed both by embryonic tissues and several types of carcinoma. Here too, more than 600 clones resulted from the fusion. Of 72 clones tested for the ability to bind both CEA and arsenate, 69 had both binding activities. The clones that showed the greatest binding were selected for enzyme-linked immunosorbent assay (ELISA) of hybrid antibody. For each assay, CEA solution (600 ng or 250 ng) was allowed to absorb overnight in each well of a 96-well plastic microtiter plate. The next day, unabsorbed material was washed out of the wells with PBS-Tween 20. Clone supernatants were added and incubated for 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 absorbed antigen. The second antigen, arsenyl acid linked to the enzyme alkaline phosphatase, was added to the wells for 3 hours at 35°C. After a further wash with PSB-Tween, the chromagen substrate for alkaline phosphatase, paranitrophenyl phosphate, was added to the wells and color developed for 48 hours. Adsorption in each well was measured at 410 nm. When present in a clone supernatant, the hybrid antibody bound the adsorbed CEA on the plate at one functionality and left the other free to bind arsenylic acid coupled to alkaline phosphatase. Color from the chromagen substrate developed only where the alkaline phosphatase coupled to the arsenylic acid had been bound. In this assay, 3 of 12 supernatants exhibited hybrid antibody activity as indicated in Table 4.

The present invention also enables immunodiagnosis

. using antibodies with a dual specificity, e.g. hybrid antibodies obtained as described above and

from antibody half-molecules using the conventional technique of Nisonoff et al., supra, or antibody multimers obtained by linking or crosslinking individual monospecific antibodies. Preferably, the dual specificity antibody used in these methods is a hybrid antibody prepared in a safe manner and obtained as a substantially pure compound which has not been subjected to 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 other dual specificity antibody will be against the target antigen whose detection is desired and the other will be against another antigen, which may be a hapten or other molecular species, which allows the diagnosis. E.g. will an antibody useful in immunohistology have a first specificity for a putative antigen, e.g. a tumor associated antigen such as CEA, PAP or ferritin, and another specificity towards a hapten or antigen that will participate in a color reaction such as e.g. an enzyme that causes a color reaction in the presence of a suitable substrate. Among suitable enzymes to which the second specificity of the antibody can be directed are prostatic acid phosphatase (PAP) horseradish peroxidase, glucose oxidase and alkaline phosphatase.

To carry out the histological examination, a tissue section is first treated with the antibody with double specificity. Before this is done, the hybrid may have already been allowed to bind to the enzyme that catalyzes the color reaction. If not, the section is then treated with another solution containing the enzyme and cleared 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 the substrate in the tissue sample is a positive indication of the presence in the tissue of the target antigen. The hybrid antibody against HBsAg and PAP, the preparation of which is described herein, has been found to bind to HBsAg on a test substrate (polystyrene beads) and to PAP in a simulated color experiment using p-nitrophenyl phosphate as enzyme substrate. After incubation of the hybrid antibody with PAP and HBsAg, the addition of the p-nitrophenyl phosphate resulted in the beads undergoing the characteristic color change from yellow to brown.

As noted in Example 2, an antibody with dual specificity can also be used in immunoassays and immunometric analyses. Using the hybrid antibody against HBsAg and PAP whose preparation is described above, an immunometric assay for HBsAg can be carried out using an immobilized monoclonal antibody for HBsAg in a solid phase to extract HBsAg from a serum or other liquid sample which possibly containing the antigen. The sample is incubated with a bead, bead, test tube or other substrate that has anti-HBsAg bound or coated on the surface. The incubation with the serum sample can be followed by, carried out at the same time or carried out after an incubation with a solution of the hybrid. In any case, if HBsAg is present in the sample, the result will be the formation of a sandwich of the immobilized antibody, HBsAg, if present in the sample, and the hybrid antibody. As part of the assessment, PAP is allowed to bind to the hybrid antibody. This can be done during or after formation of the sandwich, or alternatively the antibody-PAP complex can be preformed. After formation of the sandwich, the solid phase is washed to remove sample residues and unbound hybrid antibody and then brought into contact with a solution containing a substrate such as e.g. p-nitrophenyl phosphate or α-naphthol phosphate which undergoes a color change in the presence of PAP. Occurrence of a color change confirms the presence of the target antigen in the sample.

In such an evaluation using the polyclonal anti-HBsAg bead in a commercial kit for the diagnosis of HBsAg manufactured by Abbott Laboratories, North Chicago, Illinois (marketed under the name "Aushia"), samples containing different amounts of HBsAg were incubated with the beads. The samples used were positive and negative controls from the commercial kit and two samples obtained by diluting the positive control with the negative control in the ratios of either one 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 HBsAg, the bead was washed and incubated with the hybrid antibody reactive to both HBsAg and PAP. This enabled the formation of a sandwich of immobilized antibodies: antigen: and hybrid antibody. The pellet was again washed and incubated with a solution of PAP. This incubation was followed by a further wash and the grain incubated with a substrate α-naphthol phosphate. PAP removes enzymatic phosphate. After an appropriate incubation, the substrate was removed from the grain and an indicator Fast Garnet GBC salt was added which changes from colorless to reddish purple in the presence of the product of the enzymatic reaction. A color change was observed to confirm the presence of HBsAg in the samples. Absorption at 570 nm was measured for the samples and these data are shown in the following table 5.

These data show a dose response with variation in HBsAg concentration that would be expected if the hybrid antibody forms a bridge between HBsAg bound to the bead and PAP. This further demonstrates the utility of a hybrid antibody in an immunoassay.

Other means of detection than enzymatically catalyzed reactions are also possible. E.g. the other specificity of the hybrid or another antibody with a dual specificity may be directed against a hapten or antigen which is radiolabelled or which is fluorescent or which can be detected in the sandwich by any suitable means.

A preferred process using a hybrid antibody or another antibody with a dual specificity in an immunoassay utilizes the phenomenon of fluorescence quenching. In such an assessment, one specificity of the antibody is directed against a target antigen and the other against e.g. a hapten carrying a fluorescent chromophore. The chromophore is either bound to

the hapten or in appropriate cases can be the hapten itself.

The analysis is carried out by incubating the antibody with serum or another sample which may contain the target antigen for which . is added a predetermined amount of the target antigen labeled with a quenching chromophore. The labeled antigen competes with any target antigen in the sample for the antibody's binding site which is specific for the target antigen. Before, during or after this incubation, a quantity of the hapten carrying the fluorescent chromophore is incubated with the antibody and binds at the second binding site.

The two chromophores are selected so that the first cv them fluoresces at a wavelength that can be absorbed (extinguished) by the other if they are placed sufficiently close to each other so that the photon that is emitted from there. fluorescent chromophore can be captured by the quencher. To do this, the two chromophores should be within about 100 Angstroms of each other and preferably within about 50 Angstroms of each other. This positioning will occur when the fluorescent chromophore binds at one antibody binding site and the quenching chromophore binds to the added antigen at the other. A suitable pair of chromophores includes fluorescein as the fluorescent chromophore and rhodamine as the quenching chromophore.

The measured fluorescence will vary inversely with the amount of native antigen in the sample since, in the absence of native antigen, all antigen bound to the antibody will be labeled with the quenching chromophore and be in a position to absorb fluorescence at the chromophore carried by the hapten. Comparison of the measured fluorescence with the fluorescence of a control sample containing a known amount of antigen allows a qualitative and quantitative determination of the presence of antigen in the sample.

This type of immunoassay can e.g. used to determine the serum content of drugs such as dilantin which must be closely monitored. In such an assessment, the target antigen should naturally be dilantin. It will be clear to the expert in the field that this process can be used to detect other antigens and especially including tumor-associated antigens.

Another preferred process using a hybrid antibody or another antibody with a dual specificity in an immunoassay uses an enzymatic reaction. In a hitherto preferred process, one of the antibody specificities is of course directed to the target antigen and the other to an enzyme or a hapten to which an enzyme is bound.

The assessment is carried out by incubating the antibody with a sample which may contain the target antigen to which has been added a predetermined amount of the target antigen which has been modified by binding to a substance which interacts with the enzyme to produce either a detectable substance or in another way allow the detection of formation of the antigen-antibody complex. The detection can e.g. happen by fluorimetry, luminescence, spectrophotometry etc.

In an alternative process, the added target antigen can be bound to the enzyme and in this case the antibody has one of its specificities directed at the substance that interacts with the enzyme or at a hapten to which the substance is bound. The substance that interacts with the enzyme may itself be another enzyme. In such a case, one of the enzymes catalyzes the production of a product required by the other. Thus, when the antibody binds both the added target antigen, to which one of the enzymes is bound, and the other enzyme, the product from the first enzymatic reaction is formed in the vicinity of the second enzyme before significant diffusion of the product into the surrounding medium can take place.

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

To utilize 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 that carries it). The sample is, in addition to the hybrid antibody and the predetermined amount of enzyme-labeled antigen, added glucose, ATP and the coenzyme NAD<+>. The hybrid antibody has preferred the other enzyme already bound to it. Alternatively, this enzyme can be added to the sample together with the other reagents. During the incubation, the enzyme-labeled antigen will compete with any native antigen in the sample for one of the binding sites in the hybrid antibody. The other enzyme is or will be bound to another binding site. This allows formation of glucose-6-phosphate catalyzed by HK to occur in close proximity to G-6-PDH. The latter converts glucose-6-phosphate to gluconolactone-6-phosphate, a result which is followed by the reduction of NAD<+> to NADH. NADH absorbs strongly at 340 nm and can therefore be detected spectrophotometrically. The amount of NADH formed varies inversely with the amount of native antigen in the sample, ie its maximum production occurs when there is no target antigen in the sample being assessed. Comparison of the amount of NADH formed with a control sample allows a qualitative and quantitative determination of the presence of antigen in the sample.

This type of determination can be used to monitor the amount of dilantin or other drugs in the serum. In such a case, the drug is the target antigen. However, such a determination can also be used to detect other serum antigens such as e.g. those associated with tumors or other diseases.

Claims (8)

1. A hybrid monoclonal antibody for use in vitro, the antibody having dual specificity and being prepared from a polydom, characterized in that said hybrid antibody is produced by fusion of two hybridomas, or a hybridoma and a spleen cell (B lymphocyte), or produced by removing the nucleus from a first hybridoma and introducing it into the cytoplasm of another hybridoma, the antibody being a component of a mixture of antibodies comprising the hybrid antibody and species of mono-specific antibodies in a ratio of approximately 2:1:1. 2. Hybrid monoclonal antibody as stated in claim 1, characterized in that one of the dual specificities is against a target antigen and the other against a substance that allows diagnosis of the target antigen. 3. Hybrid monoclonal antibody as stated in claim 2, characterized in that the substance that allows diagnosis is labeled with a radionuclide, a fluorescent substance or an enzyme. 4. Hybrid monoclonal antibody as stated in claim 1, characterized in that one of the double specificities of the antibody is against a target antigen and the other against a hapten which is, or is attached to, an agent which is lethal to the antigen or associated tissue. 5. Hybrid monoclonal antibody as stated in claim 4, characterized in that the lethal agent is a radionuclide or a tissue toxin. 6. Hybrid monoclonal antibody as stated in claim 1, characterized in that said hybrid antibody is specific for hepatitis B surface antigen, carcinoembryonic antigen, prostatic acid phosphatase or ferritin. 7. Hybrid monoclonal antibody as stated in claim 1, characterized in that said hybrid antibody has one specificity for a tumor-associated antigen and the other specificity for gelonin, α-amanitin, k diphtheria toxin A, methotrexate, dichlormethotrexate, duanomycin or chloronbucil. 8. Use of the monoclonal antibody as stated in claims 1-7 for immunochemical analyses.