WO1983003679A1 - Antibodies having dual specificities, their preparation and uses therefor - Google Patents

Antibodies having dual specificities, their preparation and uses therefor

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Publication number
WO1983003679A1
WO1983003679A1 PCT/US1983/000525 US8300525W WO8303679A1 WO 1983003679 A1 WO1983003679 A1 WO 1983003679A1 US 8300525 W US8300525 W US 8300525W WO 8303679 A1 WO8303679 A1 WO 8303679A1
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WO
Grant status
Application
Patent type
Prior art keywords
antibody
hybrid
antigen
process
according
Prior art date
Application number
PCT/US1983/000525
Other languages
French (fr)
Inventor
Incorporated Hybritech
Joanne Martinis
Richard M. Bartholomew
Gary S. David
Thomas H. Adams
James M. Frincke
Original Assignee
Hybritech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46Hybrid immunoglobulins
    • C07K16/468Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04Organic compounds
    • A61K51/08Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody or an immunoglobulin, or a fragment thereof, e.g. a camelised human single domain antibody, or the Fc fragment of an antibody
    • A61K51/1003Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody or an immunoglobulin, or a fragment thereof, e.g. a camelised human single domain antibody, or the Fc fragment of an antibody not used, see subgroups
    • A61K51/1084Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody or an immunoglobulin, or a fragment thereof, e.g. a camelised human single domain antibody, or the Fc fragment of an antibody not used, see subgroups the antibody being a hybrid immunoglobulin
    • A61K51/109Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody or an immunoglobulin, or a fragment thereof, e.g. a camelised human single domain antibody, or the Fc fragment of an antibody not used, see subgroups the antibody being a hybrid immunoglobulin immunoglobulins having two or more different antigen-binding sites, multifunctional antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K2123/00Preparations for testing in vivo

Abstract

Les "polydomas" ci-décrits sont le produit de la fusion d'un hybridoma avec un B-lymphocyte ou un autre hybridoma. The "polydomas" described below are the product of fusion of a hybridoma with a B-lymphocyte or another hybridoma. Les polydomas produisent un anticorps hybride monoclonal ayant une double spécificité contre deux déterminants antigènes différents. The polydomas produce a hybrid monoclonal antibody having a dual specificity determinants against two different antigens. Des procédés immunodiagnostics et immunothérapeutiques sont également décrits, lesquels utilisent les anticorps hybrides monoclonaux ou autres anticorps ayant une double spécificité. Of immunodiagnostic and immunotherapeutic processes are also described which utilize hybrid monoclonal antibodies or other antibody having a dual specificity. Dans ces procédés, une spécificité de l'anticorps est dirigée contre un antigène cible et l'autre contre une moitié, ce qui permet d'effectuer un diagnostic ou d'envoyer un agent mortel à l'antigène cible ou aux tissus associés. In these methods, specificity of the antibody is directed against a target antigen and the other against a half, which allows to perform a diagnostic or send an agent lethal to the target antigen or associated tissues.

Description

DESCRIPTION

ANTIBODIES HAVING DUAL SPECIFICITIES, THEIR PREPARATION AND USES THEREFOR

Field of the Invention

This invention relates to antibodies having du specificities. In another aspect it relates to immun diagnostic and immunotherapeutic processes. In y another aspect it relates to hybridomas and relat monoclonal antibody technology.

Background of the Invention

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

In other circumstances, if the immunogen is a diseas associated antigen, its presence in a patient's serum o other body fluid can be detected using immunoassay o immunometric techniques. For example, detection of HBs using a radioimmunometric technique is the current metho of choice. On another front, serum antibodies to ferri tin, obtained from New Zealand white rabbits and labele with 131l, have been reported as showing promise for th treatment of liver tumors. (See Order et al, Internation al Journal of Radiation Oncology, Biology and Physics, j 703 (1980)).

Serum antibodies, for example, those obtained fro rabbits, urine species or other mammals are "polyclonal in nature since the immune system of the host is stimula ted to produce a mixture of specific antibodies directe

OMPI to the different antigenic determinants or epitopes on t immunogen to which the host is responding. The individu antibodies making up the mixture are each the product of B-cell clone; furthermore each B-cell secretes only o antibody specie. The antibody produced by one clon differs from an antibody against the same antigen produc by another clone by having at least a subtle differenc between its peptide sequence and that of the other anti body. In effect, therefore, each antibody specie is distinct molecule and the differences in peptide sequen between different' species affect their general specifici ties as well as the particular epitopes they recognize an their affinities for the antigen.

An individual B cell cannot be grown indefinitel using presently available tissue culture techniques t obtain the antibody specie it secretes as a pure compound Relatively recently, Kohler and Milstein discovered an reported a process by which a monoclonal antibody can b conveniently obtained as the secretion product of a hybri cell referred to as a "hybridoma". (G. Kohler and C Milstein, Nature, 256, 495 (1975)). Basically, the proces involves the fusion -of spleen cells taken from an immu nized mouse with mouse myeloma cells to form the hybri doma. Myeloma cells which do not produce, or at least d not secrete their own immunoglobulin or parts thereof ar preferred. Cultures of cells obtained by cloning single hybridoma will secrete identical antibody molecule which can subsequently be obtained readily as a pur chemical compound. This is in contrast to the conven tional antibody preparation obtained, for example, fro serum, in which any one antibody is but one of the com ponents of a substantially unresolvable antibody mixtur of related, yet distinct chemical compounds.

Since it is a pure compound, a monoclonal antibod will have a constant specificity for a single site on th antigen molecules and a well defined affinity. Thus clones of different hybridomas can be screened to sele the one producing the monoclonal antibody with the mo desireable properties for a given application. T immortality of the hybridoma guarantees an almost u limited supply of the antibody it secretes and alleviat problems associated with variance in antibody titer a overall affinity from animal to animal used to produ serum antibodies. Monoclonal antibodies obtained fr hybridomas have, for example, been put to practic application in diagnostic kits. A selection of such ki is available from Hybritech, Inc., assignee of th application.

An antibody molecule can generally be considered express a single specificity which is exhibited towar the immunogen to which the host's immune system respond by production of the antibody. The antibody is compos of two identical halves, each of which is comprised of heavy and light chain pair and each of which recognize the same antigenic determinant as the other. The fol lowing is a 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 ca usually be cleaved selectively j^n vitro by a mild reduc tion, and the half molecules disassociated by subsequen acidification. The half molecules can then be recombine

OM (renatured), again in vitro, at neutral pH, the reassocia- tion taking place through non-covalent interaction. If antibodies of different specificities are sub¬ jected 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", Academic Press, New York (1975), at pages 260-261, is described an n vitro pro¬ duction of a polyclonal antibody hybrid of rabbit anti- ovalbumin and anti-BGG antibodies. Hybrid monoclonal antibodies have also been obtained using an analogous process. See D.M. Kranz et al, Proc. Natl". Acad. Sci. USA, 78, 5807 (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 significant, irreversible denaturation. Such denaturation can reduce immunore- activity and would be expected to result in different metabolic characteristics in vivo. As a result, the hybrid antibody today remains largely a laboratory curi¬ osity 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 Staphylococcus aureus), carbodiimide and bifunctional compounds such as N-succinimidyl-3-(2-pyridyldithio) propionate to obt dimeric and higher antibody multimers to which each mem of the antibody pair contributes its specificity. F example, Mandoche et al have reported the formation multivalent antibodies having dual specificities, by sequential reaction of antibodies with protein A, wh have been shown to be capable of detecting cell surf antigens jLn vitro. See J. Immunological Methods, 2_, 35 (1981). According to their method, antibodies of o specificity bind to the surface antigen and the others a moiety which permits detection.

The synthesis of >dual specificity antibodies by t foregoing techniques is complicated and thus far commercial application of them has been made.

Summary of the Invention

The present invention provides, among other things, novel, completely biological method for reliably obtaini hybrid monoclonal antibodies in good yields witho denaturation. Throughout this specification, the te "hybrid antibody" will be used to designate a sing antibody molecule having two different specificities. T individual specificities may be to antigenic determinan on two different antigens or to different antigen determinants (epitopes) on the same antigen. Furthermor unless otherwise indicated, the term "antigen" al embraces haptens.

According to the method of the present inventio hybrid antibodies having a dual specificity are obtain by fusion of a hybridoma, preferably a selectively d structible hybridoma, which secretes an antibody against preselected antigenic determinant with a fusible B-lymph cyte or a second hybridoma, the B-lymphocyte or secon hybridoma secreting a second antibody against a differe antigenic determinant, to form a second generation hybri doma (hereinafter "polydoma"). As used herein, the ter "selectively destructible hybridoma" means a hybridom which lacks, or at least substantially lacks, the capabi ity of surviving in the medium in which the polydoma i cultured. We have unexpectedly found that, unlike th parent hybridoma or the B-lymphocyte from which it i derived, each of which secretes a population of identic antibodies having a single specificity, the polydoma i addition secretes a high percentage of a monoclonal hybri antibody having a dual specificity, i.e., a capability t bind with either of the antigenic determinants recognize by the individual antibodies produced by the parent cell or with both determinants at the same time. The hybri monoclonal antibody obtained in this way has not suffere the undesireable denaturation which characterizes hybrid obtained from the process of chemical recombination o antibody half molecules. Furthermore, the process of th invention permits the hybrid to be obtained reliably a in large amounts.

Also according to the present invention there ar provided processes for immunodiagnosis and i munothera employing antibodies having a dual specificity. Generall these processes employ a monoclonal antibody or polyclona antibodies having a first specificity against a targe antigen and a second specificity against a substance, fo example, another antigen or hapten, which permits diagnosis to be made of the target antigen or whic permits delivery of, or is itself, an agent which i lethal to the target antigen or the tissue with which i is associated.

Thus, by an appropriate selection of parent cells, polydoma can be obtained according to the present inven tion which will secrete an antibody having one specificit for a target antigen and a second specificity for a moiet useful in diagnosis or therapy. Alternatively, antibod half molecules can be recombined using _in vitro chemica means or individual intact mono-specific antibodies can b coupled or crosslinked by chemical means to obtain anti body multi ers (which may be a dimer, trimer or highe multimer) having a dual specificity and having the same o a similar utility as a hybrid monoclonal antibody havin the same dual specificity made according to the presen invention. As used herein, the terms "antibody" includes anti¬ body fragments having immunochemical properties 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 im¬ proved process for obtaining hybrid monoclonal antibodies. 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.

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 of the selectively destructible hybridoma with a B-lymphocyte or a second hybridoma, i.e., the polydoma, from being over¬ grown 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 isola¬ ted from parental hybridoma cells.

O P " We have found that selectively destructible hybri¬ domas useful in our invention can be obtained from hybri¬ domas 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. Generally, selective destructibility can be obtained by back selection to a hybridoma which lacks a 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 medium containing 8-azaguanine. In such a medium, any cell which incorpora¬ tes 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 hypoxan- thine aminopterin thy idine (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 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 deoxyribose (BUdR) o 2-aminopurine. Only those cells lacking the enzym thymidine kinase (TK) can grow in a medium containin either of these two inhibitors. As in the case of cell lacking the enzyme HPRT, cells lacking TK will not grow i HAT medium.

A different process for obtaining a selectivel destructible parent hybridoma involves irreversible enzym inhibition using metabolic inhibitors. Among these, th so called Kcat. inhibitors are preferred. These inhibi tors are analogs of an enzyme's substrate which ar converted by the target enzyme into a highly reactiv molecule which reacts with the enzyme at its active sit resulting in irreversible inhibition of the enzyme. Fo example, treatment of the selected hybridoma with a analog of glutamine such as azaserine or 5-diazo-5-oxa-L norleucine (DON) irreversibly inhibits the enzyme formyl- glycinamide ribonucleotide amidotransferase by formatio of a covalent bond with a cysteine residue at the enzyme's active site. This 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 destruc- tible 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 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 wit the B-lymphocytes to obtain the polydoma can be accom plished by combining the two groups of cells in a mediu containing an agent known to promote cell fusion such a polyethylene glycol or Sendi virus according to know methods.

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

In another embodiment, the polydoma is obtained b fusing the selectively destructible hybridoma using suitable fusion agent with a second hybridoma which i also selectively destructible. The second parent hybri doma is obtained in the same manner as the first, i.e., b a process of back selection, irreversible enzyme inhibi tion or by any other suitable technique. In such a case the second hybridoma must be able to complement th first. For example, if the first selectively destructibl hybridoma lacks the enzyme HPRT, the second must b capable of contributing to the polydoma a gene which wil enable the polydoma to express HPRT. Similarly, if th second selectively destructible hybridoma lacks the enzym

OM TK, the first must contribute a gene for TK to the poly doma. Similar complementarity between the two hybridoma must be present if irreversible inhibition of an enzym has been accomplished to confer selective destructibilit on them.' It is also possible to use, as one hybridom parent, a hybridoma which has been subjected to a bac selection process, and, as the other, a hybridoma whic has been subjected to a process of enzyme inhibition The use of complementary selectively destructibl hybridomas as parents for the polydoma has the advantag that both parents can be selected on the basis of th specificities and affinities of the monoclonal antibod they produce whereas, in the case of fusion of a singl hybridoma with B-lymphocytes, no pre-fusion selectio among the B-lymphocytes to obtain those producing a antibody of the desired specificity and affinity can b made.

Fusion of the two selectively destructible hybridoma can be accomplished using polyethylene glycol or usin other fusing agents, again according to known methods. After fusion, the cells are transferred to a growth mediu in which the two parents cannot grow, but in which th polydomas resulting from the fusion are capable of growt because of the complementary contributions of the parents. Heretofore, we have discussed selection processes t 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 a dominant and a recessive marker on the first hybridoma parent. A presently pre¬ ferred method is HAT-ouabain selection. The drug ouabain is a specific inhibitor of the Na+-K+ activated ATPase 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 be HAT sensitive but ouabain resistant. By contrast, the hybridoma selected for fusion with it will 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 other technique to confer selective destructibility. 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 lethal to the second hybridoma parent. The selectively destruc¬ tible 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 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 both a positive and a negative marker, which can be fused with any "off the shelf" hybridoma, involves the use of recom- binant DNA vectors carrying various drug resistance markers. For example, SV40 carrying a gene for neomycin resistance can be used.

A presently preferred universal parent is one that is HAT sensitive-neomycin resistant. The chosen parent is back-selected to HAT sensitivity and then transfected with SV40 vector carrying a gene for neomycin resistance. This procedure can also be reversed with transfection bein done first. The resulting hybridoma can grow in th presence of neomycin, which is normally toxic to mammalia cells, but will die in the presence of HAT. Off-the-shel hybridomas, however, grow in HAT but die in the presenc of neomycin. Products of the fusion of the parents, therefore, survive in the presence of HAT and neomycin. While the use of vectors to convey resistance to neomyci is presently preferred, vectors carrying genes which wil confer resistance on mammalian cells to other drugs ca 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 followed by the subcloning of all cells before the population of unfused parent hybridomas increases to an extent that 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 of hybridomas whose monoclonal antibodies are against differ- ent 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 wave¬ lengths. 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 num bers, are incubated with the two tagged antigens. Th cells are then screened using the cytofluorograph. Thos cells which fluoresce at only one of the two wavelength 5. will be from the cell lines of the parent hybridomas However, cells which exhibit fluorescence at both wave lengths will be polydomas which can be isolated an subcloned.

In yet another embodiment, a polydoma can b 0 obtained directly by removing the nucleus from a firs hybridoma which secretes a monoclonal antibody having on of the specificities desired in the hybrid and insertin it into the cytoplasm of a second hybridoma which secrete a monoclonal antibody having the second desired speci 5 ficity. Of course, neither of the parent hybridomas need to be selectively destructible in order to be used in thi process. After insertion of the nuclear material, th cell is cloned to obtain a population of the polydoma

We have found that, unlike the parent hybridomas o B-lymphocytes which secrete a single antibody, polydom cells obtained according to our invention secrete mixture of antibodies, at least one of which is a hybri antibody having a dual specificity. Also produced by th polydoma are relatively smaller amounts of antibodies o the same specificity as those produced by the parent cell used to obtain the polydoma. The ratio of hybrid t mono-specific antibodies appears to be about 2:1:1 whic is that expected if the polydoma produces equal amount of all the possible (H) chains synthesized by the paren cells which are randomly combined in the polydoma itself

The polydomas can be cultured Ln vitro or grow in vivo in either genetically compatible animals or nud mice to obtain large quantities of the hybrid antibod which is recovered from the culture medium or asciti fluid of the animal using known processes. See, fo example, the protocols in "Monoclonal Antibodies", Edite

f OMPI

Wfa, IPO by Kennett et al. Plenum Press, New York (1980) at p 363-418.

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

Example 1 A hybrid monoclonal antibody having a dual spec ficity for hepatitis B surface antigen (HBsAg) and pr static acid phosphatase (PAP) was- made in accordance wi the present invention in the following manner:

A hybridoma secreting a monoclonal antibody to P was grown in HAT medium for one week and then transferr to and grown in a non-selective medium. After variou lengths of time of growth under non-selective conditions 2 ml aliquots of cells were placed in medium containin 10-4 M 8-azaguanine which prevented cells from growin by incorporating 8-azaguaine in their DNA instead o guanine. Cells lacking the HPRT enzyme survived and gre in this medium and these cells necessarily did not surviv in HAT.

Clones that grew in the medium' containing 8-aza guanine were tested for sensitivity to HAT and anti-PA production. One clone which still produced anti-PAP an exhibited HAT sensitivity with a reversion frequency o less than 4 x 10~"8 was subcloned. All of the subclone behaved like the parental clone. Cells from one of the HAT sensitive subclones wer fused in polyethylene glycol with spleen cells obtaine

O from Balb/c mice hyperimmunized with HBsAg to obtai polydomas using the fusion technique of Gerhard. Se "Monoclonal Antibodies", supra, at p. 370. The fusio produced 220 polydomas which were screened to determin which secreted antibodies exhibiting specificity for bot PAP and HBsAg. Clones of two such polydomas were deter mined to produce antibody and, subsequently, ascites whic exhibited both specificities.

Subclones of both polydomas continued to produc ascites exhibiting both specificities and yielded tripl bands on Orstein-Davis PAGE like those of the paren clone. The ascites from both clones were shown t bind 125j_HBs g and 125ι_ & ιn radioimmunoassays an yielded Ka values of approximately 109 for each, thu suggesting the formation of antibodies having two speci ficities.

The data in Table I below show the results obtaine in immunoassays using the ascites obtained from a clone o one of the polydomas compared with ascites obtained fro hybridomas secreting monoclonal antibodies, respectively against IgE (used as a control) , PAP and HBsAg usin immobilized HBsAg as a solid phase and a. variety o radiolabeled antigens as the solution phase.

A 200 sample of the ascites from the polydoma an each of the three hybridomas were each incubated overnigh with 12 polystyrene balls to which was bound HBsAg. Th HBsAg balls were obtained from Abbott Laboratories, Nort Chicago, Illinois. After washing, triplicate samples wer incubated for 4 hours with 100,000 cpm of the indicate 125ι labeled antigen. After a second washing, the ball were counted to determine the amount of labeled antige bound to the balls. In one set of tests using radiola beled PAP as the solution phase antigen, anti-PAP wa added to the antigen before it was incubated with th ball. The antibody to PAP used for this purpose is th monoclonal antibody produced by the parental hybridom used to make the polydoma and, therefore, is against th same PAP epitope as that expected to be exhibited by th hybrid antibody.

TABLE I Results of Radioassays Demonstrating Presence of Hybrid Antibody Having Dual Specificity in Polydoma Ascites cpra

Specificity cpm cpm *PAP + cpm of Ascites *HBsAg *PAP anti-PAP *IgE anti-IgE .15,340 2,145 2,930 3,290 anti-PAP 16,280 2,956 3,128 3,180 anti-HBsAg 73,020 2,973 2,870 3,330 suspected dual specificity 78,900 82,533 2,936 3,143 * 125j labeled antigen

The data in Table I indicate that only the radiatio expected from non-specific binding is measured for the anti-PAP ascites when compared to that for the IgE con¬ trol. The ascites containing the HBsAg antibody, on the other hand, bound the labeled HBsAg antigen as expected but exhibited non-specific binding when the other labeled antigens were tested. The ascites from the polydoma clone, however, bound both labeled HBsAg and labeled PAP, the former attributable to the presence of some non-hy- brid, mono-specific antibody to HbsAg in the ascites and the latter attributable to a hybrid that can bind and bridge the HBsAg 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 inhibition by the parental antibody of binding of labeled PAP to the hybrid antibody.

O P Analysis of the ascites from the polydoma clone used in the comparative radioassays was performed using poly- acrylamide gel electrophoresis (PAGE) and immunoelectro- phoresis (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 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. Thus, the middle peak is hybrid antibody having a dual specificity to HBsAg and PAP comprised of at least two subspecies.

The hybrid antibody obtained as the middle peak of the DEAE chromatography was radiolabeled with 125ι. After labeling, 85% of the labeled antibody would bind to PAP and 88% would bind to HBsAg. 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.

DEAE chromatography indicates that hybrid antibody comprises more than 50% of the antibodies produced by the polydoma and roughly approximates the ratio 2:1:1 pre- dieted on statistical grounds if the polydoma were to synthesize all the possible antibody heavy chains, i.e., those exhibiting either PAP or HBsAg 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 the parent cells. The existence of subspecies of hybrid antibody suggests that they ma differ in their light chain composition.

Example 2

Hybrid monoclonal antibodies having dual specificit for human IgD and prolactin were made in accordance wit the present invention by the fusion of two hybridomas, on of which was constructed to contain two selectible geneti markers: sensitivity to HAT medium and resistance t ouabain. This doubly-marked hybridoma or so-calle "universal parent" could then be fused to any othe hybridoma. The resulting polydomas grow in the pre sence of HAT and ouabain, while any unfused parent cell die. The advantages of using such a "universal parent have been described elsewhere herein. To construct such a universal parent, both selectibl markers were introduced during initial construction of th hybridoma. To obtain this parent cell line, the widel available HAT-sensitive mouse myeloma P3.653 was selecte for a second genetic marker, ouabain resistance, b introducing 1 mM ouabain into the growth medium. Whil most cells died, approximately 1/100,000 cells had b random mutation acquired resistance to the drug an so survived and multiplied to form the new myeloma popula tion 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 cited techni¬ que of Gerhard. Hybrids were selected in HAT medium (without ouabain) and clones were screened for production of monoclonal antibody directed against IgD. From among the positive clones, one which produced an IgG against IgD was selected for further study. This clone was tested for retention of the trait of ouabain resistance by adding 1 mM 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

OM 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 procedure of Example 1 to obtain a population of cells sensitive to HAT. This subclone was grown for two 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 lacking HPRT enzyme will not utilize 6-thioguanine from the medium and therefore can grow but are consequent¬ ly 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-IgD antibody. One clone which showed all the desired characteristics— growth in ouabain and 6-thioguanine as well as production of monoclonal anti-IgD was selected to be a so-called "universal 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- IgD. 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 (IgG i) as the anti-IgD ex¬ pressed by the parent line and it is easily separated from that antibody on Ornstein-Davis gels. Such a separation is indicative of greatly different charge on the anti¬ bodies and so should allow easy isolation of a hybrid antibody by DEAE-Sephadex chromatography.

10*7 cells of the HAT sensitive, ouabain-resistant cell line were fused in polyethylene glycol with 10? 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 arose fr this fusion: 66 clones were randomly selected for anal sis. Of these clones 36 exhibited both anti-IgD a anti-prolactin activity. These clone supernatants were assayed for the pre ence of hybrid antibody by the following assay. A pol styrene bead coated with another anti-prolactin monoclon antibody was incubated 5 hours with 200 U.1 of a 100 ng/ prolaction solution. The antibody used binds prolactin a distinct site from that of the antibody produced by t fused hybridoma cell line. The bead was washed, th incubated overnight with the clone supernatants. The ne day, following several washes, 125ι labeled IgD w added. Hybrid antibody bound to the bead by one functio al arm could bind the radiolabeled IgD with the fr anti-IgD functionality whereas neither parental ty antibody IgD-Ig.D or Prolactin-Prolactin could form th bridge between the Prolactin bead and 125ι-igD trace Results of a typical assay for clones producing hybri bifunctional antibody are presented in Table 2 belo

TABLE 2 Results of immunometric assays demonstrating th presence of hybrid antibody having dual specificity f IgD and prolactin in selected polydoma supernatants an ascites. donate # cpm 125ι-igp tracer bound

1 15339

2 16337

3 - 22886

4 23356 5 24434

Anti-IgD 9357

Anti-Prolactin 8721 21 of 36 clones exhibited significant bifunction activity by this assay. Ascites generated from 2 clon available to date have been shown to react in the bi functional assay. These ascites contain antibodies whi separate into three distinct bands on Ornstein-Davi gels: two bands coincide exactly with antibodies produce by the parent hybridomas (anti-IgD and anti-prolactin) The third band migrates midway between the parenta monoclonal antibody bands as expected of the hybri antibody.

That a hybrid monoclonal antibody against IgD an prolactin exhibits a dose response when the amount o prolactin is varied in the assay used to generate the dat of Table 2 is shown by the data of Table 3 using th antibody of donate #2 and, as controls, antibodies fro the parent cell lines (anti-IgD and antiprolactin)

TABLE 3 Results of Assays Using Hybrid Antibody and Varying Amounts of Prolactin

Prolactin Hybrid Anti- Anti ng/ml Antibodyl IgDl Prolactinl

0 8010 6847 8020

10 9169 7982 7405

50 13558 7783 8314

100 17599 7654 7844

1. cpm of 125ι-igE bound

These data demonstrate that the amount of prolacti bound by the hybrid antibody is dose responsive as th amount of labeled IgD bound increases as the dose o prolactin is increased. By contrast, no dose response i observed using the parent antibodies against IgD an prolactin as they cannot form the bridge between prolacti

O bound to the bead and the labeled IgD. Thus, the hybr antibody can be used as a component of an assay for pr lactin. Tailor-making other hybrid antibodies offe similar opportunities for other assays.

Example 3

By a similar technique to that of Example 2, universal parent hybridoma was generated which secret monoclonal antibody directed against the hapten arsenat arsenate dimer and is both resistant to ouabain a sensitive to HAT. This hybridoma was fused to a hybrido which secretes a monoclonal antibody with specificity f carcinoembryonic antigen (CEA) , an antigen expressed both embryonic tissues and several types of carcinom Once again greater than 600 clones resulted from th fusion. Of 72 clones tested for the ability to bind bo CEA and arsenate, 69 had both binding activities. Tho clones exhibiting the greatest binding were selected f enzyme-linked immunosorbent (ELISA) assay of hybri antibody. For each assay, a CEA solution (600 ng or 25 ng) was allowed to adsorb overnight to each well of plastic 96-well microtiter plate. The next day, unad sorbed material was washed out of the wells with PBS-Twee 20. Clone supernatants were added and incubated 2-1/ hours at 35βC and then washed off the plate. CEA-CEA an CEA-arsenate antibody would remain attached to the plat via the adsorbed antigen. The second antigen, arsenyli acid coupled to the enzyme alkaline phosphatase, was adde to the wells for 3 hours at 35°C. After another was with PSB-Tween, a chromagen substrate for alkaline phos phatase; para-nitrophenylphosphate, was added to the well and color developed for 48 hours. Absorbance in each wel was measured at 410 nm. If present in a clone super natant, hybrid antibody bound the adsorbed CEA on th plate through one functionality, leaving the other free t bind arsenylic acid coupled to alkaline phosphatase Color from the chromagen substrate developed only wher the alkaline phosphatase coupled 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.

Absorbance at 490 nm Absorbance at 490 n

Clone 600 ng CEA/well 250 ng CEA/well

1 0.087, 0.105 0.022, 0.060 2 0.Q82, 0.054 0.023, 0.033

3 0.011, 0.017 0.041, 0.036 anti-arsenate 0.010, 0.006 0.006, 0.005

The present invention also provides methods for immunodiagnosis and immunotherapy using antibodies having a dual specificity, for example, hybrid antibodies ob¬ tained as described above or from antibody half molecules by the conventional technique of Nisonoff et al, supra, or antibody multimers 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 an 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 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 enzym which causes a color reaction in the presence of suitable substrate. Among suitable enzymes to which th second specificity of the antibody may be directed i prostatic acid phosphatase (PAP) , horse radish peroxidase glucose oxidase, and alkaline phosphatase.

To perform the histological examination, a tissu section is first treated with the antibody of dual speci ficity. Prior to doing so, the hybrid can have alread been allowed to bind the enzyme which catalyzes th staining reaction. If not, the section is then treate with a second solution containing the enzyme and rinse after an appropriate incubation and then treated with th substrate which undergoes a color change in the presenc of the enzyme. The formation of the color produce by the enzyme and substrate in the tissue sample is positive indication of the presence in the tissue of th target antigen. The hybrid antibody against HBsAg and PA whose preparation is described herein, has been found t bind to HBsAg on a test substrate (polystyrene balls) and to PAP in a simulated staining experiment using p-nitro- phenyl phosphate as the enzyme substrate. After incuba¬ tion of the hybrid antibody with PAP and the HBsAg, 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 speci¬ ficity can also be used in immunoassays and immunometric assays. Using the hybrid antibody against HBsAg and PAP whose preparation is described above, an immunometric assay for HBsAg can be performed using an immobilized monoclonal antibody to HBsAg as a solid phase to extract HBsAg 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-HBsAg bound or coated on its surface. The incubation with the serum sample can be followed by, done simulta eously with or preceded by an incubation with a solutio of the hybrid. In any case, if HBsAg is present in t sample, the result will be the formation of a sandwich o the immobilized antibody, HBsAg if present in the sample and the hybrid antibody. As part of the assay, PAP i permitted to bind with the hybrid antibody. This can b done during or after formation of the sandwich, or in t alternative, the antibody-PAP complex can be preformed After formation of the sandwich, the solid phase is washe to remove sample residue and unbound hybrid antibody a then contacted with a solution containing a substrate suc as p-nitrophenyl phosphate or c^-napthol phosphate whi undergoes a color change in the presence of PAP. Occu rence of the color change confirms the presence of targe antigen in the sample.

In such an assay, using the polyclonal anti-HBsA bead of a commercial kit for diagnosis of HBsAg manufa tured by Abbott Laboratories of North Chicago, Illinoi (sold under the name "Aushia") , samples containing variou amounts of HBsAg were incubated with the bead. Th samples used were the positive and negative controls fro the commercial kit and two samples obtained by dilutin the positive control with negative control in the ratio of either 1 part negative control:2 parts positive co trol or 2 parts negative control:1 part positive control After incubation of the samples with the bead to bin HBsAg, the bead was washed and incubated with the hybri antibody reactive to both HBsAg and PAP. This allowed sandwich of immobilized antibodies:antigen:and hybri antibody to form. The bead was washed again and incubate with a solution of PAP. This incubation was followed b another wash and the bead incubated with a substrat p-napthol phosphate. PAP enzymatically removes phosphate After an appropriate incubation, the substrate wa removed from the bead and to it was added an indicato Fast Garnet GBC salt which turns from clear to a reddis purple in the presence of the product of the enzymati reaction. A color change was observed to confirm th presence of HBsAg in the samples. Absorbance at 570 n was measured for the samples and these data are shown i Table 5 below.

TABLE 5

HBsAg Concentration Absorbance

(% of Positive Control) 570nm

0 .031

34 .166

67 .243

100 .379

These data show a dose response with variation in HBsAg concentration which would be expected if the hybrid antibody forms a bridge between HBsAg 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 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 immuno¬ assay 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 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 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 by the fluores- cer 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. 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 chromo¬ phore 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. - 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 hybri antibody or other antibody having a dual specificity in a immunoassay relies upon an enzymatic reaction. In presently preferred process, one of the antibody speci ficities is directed, of course, to the target antigen an the other to an enzyme or a hapten to which is bound a enzyme.

The assay is conducted by incubating the antibod with a sample suspected of containing the target antige to which has been added a predetermined quantity of th target antigen that has been modified by binding to it substance that interacts with the enzyme to produce eithe a detectable substance or in some other way to permi detection of formation of the antigen-antibody complex Detection may be, for example, by fluorimetry, lumines cence, spectrophotometry or the like.

In an alternative process, the added target antige may have the enzyme bound to it in which case the antibod has one of its specificities directed against the sub stance which interacts with the enzyme or against a' hapte 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 is formed in proximity to the second enzyme and can undergo a reaction catalyzed by 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.

f O HK

(1) adenosine triphosphate + glucose

(ATP) adenosine diphosphate + glucose-6-phosphate (ADP)

(2) glucose-6-phosphate + nicotinamide adenine dinucleotid

(NAD+) 6-6-PDH gluconolactone-6-phosphate + dihydronico- tinamide adenine dinucleotide

(NADH)

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 di- rected 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.

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 cata¬ lyzed 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 reduc- tion of NAD+ to NADH. The NADH absorbs strongly at 340 nm and, therefore, can be detected spectrophotmetrically. The amount of NADH formed varies inversely with the amount of native antigen in the sample, i.e., its maximum produc¬ tion 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 antig in the sample.

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

In vivo immunodiagnosis can also be performed using hybrid or other dual specificity antibody. The antibod having one specificity against a target antigen such as tumor associated antigen and the second ' against a hapt to which is bound a suitable radionuclide, preferably o which emits o-radiation, is first administered to the hos After a sufficient time has passed during which t antibody has localized at the target site and unbou antibody has been permitted to clear from healthy tiss in the host, the hapten bearing the radionuclide i administered and binds to the localized antibody. After a suitable interval to permit unbound hapten t clear the host, scanning of the host with a suitabl camera is conducted to determine whether there are area in which radiation has been concentrated. If there ar any, the presence of the target antigen in the host i confirmed and its position determined. This process has several advantages over that . usin monospecific antibody directed against the target antige to which the radionuclide is directly bound. In suc cases, the radionuclide must have a long enough half lif that a sufficient quantity remains after the time neces sary for substantial localization of the antibody at th target site has elapsed. Furthermore, during this proces the antibody may be retained for a period of time in th liver or other non-target tissues which are then subjecte to the radiation carried by the antibody. The presen invention, on the other hand, permits the use of radio nuclides having shorter half-lives than those used wit monospecific antibodies. Being a relatively smal particle, the radionuclide bearing hapten has a hi mobility ^n vivo and will travel rapidly through the ho and either bind to the antibody which has localized at t target site or clear the body without spending appreciab time in non-target tissue. For this reason, isotopes short half-life can be administered in quantities whi pose the minimum risk to healthy tissue even thou administered in substantial excess.

Preferably, the hapten is an agent to which t radionuclide is directly bound or which will complex wi the radionuclide. A chelating agent for the radionucli bound to a hapten may be used for the latter purpos Those skilled in the art will appreciate that a wi variety of chelating agents and radionuclides are suit for this purpose. Phenylarsenate to which ethylen diaminetetraacetic acid (EDTA) is bound as a chelati agent is a suitable hapten. A radionuclide suit for use with this hapten is U ln.

The" antibody of dual specificity can also be used immunotherapy by constructing it to have one specifici against a disease associated antigen and the other again a hapten which is, or to which is bound, an agent leth to -the antigen or diseased tissue with which the antig is associated and which it is desired to destroy. Fo example, the antibody may have one specificity against tumor associated antigen such as PAP, carcinoembryoni antigen (CEA) , ferritin, or other such antigen and second specificity directed to a hapten to which is bou a radionuclide, preferably one which emits or -radi tion, or is comprised of a ricin A chain or other toxi or drug. Among such drugs may be mentioned gelonin

< -amanitin, diphtheria toxin A, methotrexate, dichlor methatrexate, dounomycin and chlorombucil. Of course, i the toxin or drug can itself function as a hapten, it ne not be bound to any other moiety. In the case where a radionuclide is to be used as t lethal agent, just as in the situation where they are us for iτ\ vivo immunodiagnosis, the hapten can have t radionuclide bound directly to it or the hapten can be, have bound to it, an agent such as a chelating agent whi will form a complex with the radionuclide. In such case, the hybrid or other antibody of dual specificity administered to the diseased host and allowed to locali at the site of the affected tissue and any excess allow to clear the host, followed by administration of t hapten which is bound. by the antibody wherever it h localized. This permits the use of a radionucli having a short half-life which minimizes the risk injury to healthy tissue even though the radionucli bearing hapten is administered in substantial excess sin that excess will rapidly clear the body and not locali in substantial quantities in healthy tissue because of t hapten' s relatively small size. It also eliminates reduces the possibility that circulating target antig will bind antibody bearing a substance lethal to tiss and deliver it to healthy tissue as can occur when t lethal agent is bound directly to a monospecific antibo directed against the target antigen.

An example of a hapten to. which a radionuclide directly bound is 6-211At-astato-2-methyl-l,4-napth quinol bis(disodium phosphate) which is described "International Journal of Applied Radiation and Isotopes" 33, 75 (1982). The 211&t is an emitter of < -radiatio Those skilled in the art will appreciate that there a numerous suitable radionuclides which can be bound di rectly to haptens or complexed with a hapten by means any of a wide variety of chelating agents.

The foregoing description of the invention is o presently preferred embodiments. Variations are possibl without departure from the scope of the invention which i limited only by the appended claims.

Claims

Claims:
1. A polydoma which produces a hybrid monoclona antibody having a dual specificity.
2. A polydoma according to Claim 1 which produces th hybrid antibody as a component of a mixture of antibodies
3. A polydoma according to Claim 2 wherein the mixtur of antibodies comprises the hybrid antibody and tw species of mono-specific .antibody.
4. A polydoma according to Claim 3 wherein the hybri antibody is comprised of two subspecies.
5. A polydoma according to Claim 4 or 5 wherein th specificity of one of the mono-specific antibodies is th same as one of the dual specificities of the hybrid an the specificity of the other mono-specific antibody is th same as the other specificity of the hybrid antibody
6. A hybrid monoclonal antibody having a dual speci ficity produced by a polydoma.
7. A hybrid monoclonal antibody according to Claim wherein one of the dual specificities is against a targe antigen and the other against a substance which permit diagnosis of the target antigen.
8. A hybrid monoclonal antibody according to Claim wherein the substance permitting diagnosis is radio labelled.
9. A hybrid antibody according to Claim 8 wherein th substance permitting diagnosis is a hapten.
10. A hybrid antibody according to Claim 9 wherein th radiolabel is a radionuclide bound directly to the hapten 11. A hybrid antibody according to Claim 9 wherein t radionuclide is bound to the hapten by a chelating agen
12. A hybrid monoclonal antibody according to Claims 9, 10 and 11 wherein the radiolabel is an emitter O-radiation.
13. A hybrid monoclonal antibody according to Claim wherein the substance is fluorescent.
14. A hybrid monoclonal antibody according to Claim wherein the hapten is fluorescent.
15. A hybrid monoclonal antibody according to Claim wherein a fluorescent moiety is bound to the hapten
16. A hybrid monoclonal antibody according to Claim wherein the substance permitting diagnosis is an enzyme
17. A hybrid monoclonal antibody according to Claim wherein an enzyme is bound to the hapten.
18. A hybrid monoclonal antibody according to Claims 7 8, 9, 10, 11, 13, 14, 15, 16 or 17 wherein the targe antigen is a disease associated antigen.
19. A hybrid monoclonal antibody according to Clai 18 wherein the antigen is a tumor associated antigen
20. A hybrid monoclonal antibody according to Clai 6 wherein one of the dual specificities of the antibody i against a target antigen and the other against a hapte which is, or to which is bound, an agent lethal to th antigen or associated tissue.
21. A hybrid monoclonal antibody according to Clai 20 wherein the lethal agent is a radionuclide. 22. A hybrid monoclonal antibody according to Clai 21 wherein the radionuclide is bound directly to th hapten.
23. A hybrid monoclonal antibody according to Clai 21 wherein the radionuclide is bound to the hapten by chelating agent.
24. A hybrid monoclonal antibody according to Claim 21, 22, or 23 wherein the radionuclide is an emitter o o -radiation or^-radiation.
25. A hybrid monoclonal antibody according to Clai 20 wherein the lethal agent is a tissue toxin.
26. A hybrid monoclonal antibody according to Clai 25 wherein the tissue toxin is comprised of a ricin chain.
27. A hybrid monoclonal antibody according to Claim 20, 21, 22, 23, 25 or 26 wherein the target antigen is disease associated antigen.
28. A hybrid monoclonal antibody according to Clai 27 wherein the antigen is a tumor associated antigen
29. A process for producing a polydoma which produce a hybrid monoclonal antibody having a dual specificit which comprises fusing a hybridoma which produces monoclonal antibody against a first antigenic determinan with a B-lymphocyte which secretes a monoclonal antibod against a second antigenic determinant in the presence o a fusion promoting agent.
30. A process according to Claim 29 wherein the hy bridoma is selectively destructible.
OMP 31. A process according to Claim 30 wherein th hybridoma has been back selected to obtain cells that ar sensitive to a medium in which the polydoma can be cul tured.
32. A process according to Claim 31 wherein bac selection is accomplished by . culturing cells of th hybridoma in a medium comprising a member of the grou consisting of 8-azaguanine, 6-thioguanine, 5-bromouracyl deoxyribose or 2-aminopurine whereby hybridoma cells sensitive to medium containing hypoxanthine aminopteri thymidine are obtained.
33. A process according to Claim 30 wher.ein said selectively destructible hybridoma is obtained by irre¬ versible enzyme inhibition.
34. A process according to Claim 33 wherein the in¬ hibition is obtained using a metabolic inhibitor.
35. A process according to Claim 34 wherein the inhi¬ bitor is a Kcat inhibitor.
36. A process according to Claim 35 wherein the Kca|- inhibitor is selected from azaserine or 5-diaz.α-5-oxa-L- norleucine.
37. A process according to Claims 29, 30, 31, 32, 33, 34, 35 or 36 wherein the B-lymphocyte is a mammalian spleen cell.
38. A process according to Claim 37 wherein the spleen cell is a spleen cell of a murine specie.
39. 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 s econd hybridoma which secretes a monoclonal antibody against a second antigenic determinant in the presence of a fusion promoter.
40. A process according to Claim 39 wherein the f irst and second hybridomas are select ively destructible .
41. A process according to Claim 40 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.
42. A process according to Claim 41 wherein both of the hybridomas have been back selected.
43. A process according to Claim 40 wherein back selection is accompl ished by culturing cells of the hybridoma in a med ium compris ing 8 -azaguanine , 6- thiguanine, 5-bromouracyl or 2-aminopurine whereby hybri¬ doma cells sensitive to medium containing hopyxanthine aminopterin thymidine are obtained.
44. A process according to Claim 39 wherein said selectively destructible hybridomas are obtained by irreversible enzyme inhibition.
45. A process according to Claim 44 wherein the in¬ hibition is obtained using a metabolic inhibitor.
46. A process according to Cl aim 45 wherein the inhibitor is a cat inhibitor.
47. A proce s s accord ing to Cl aim 46 where in the inhibitor is selected from azaserine or 5-diazo-5-oxa-L- norleucine.
OMPI 48. A process according to Claim 39 wherein onl the first hybridoma is selectively destructible.
49. A process according to Claim 48 wherein th selectively destructible hybridoma is further selected t confer the ability for it to survive in a medium which i lethal to the other hybridoma.
50. A process according to Claim 49 wherein th selectively destructible hybridoma is resistant to mediu containing ouabain.
51. A process according to Claim 50 wherein the hybridoma is back selected to obtain cells that are sensitive to a medium in which the polydoma can be cul¬ tured.
52. A process according to Claim 51 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 hybri¬ doma cells sensitive to medium containing hypoxanthine aminopterin thymidine are obtained.
53. A process according to Claim 50 wherein the selectively destructible hybridoma is obtained by irre¬ versible enzyme inhibition.
54. A process according to Cl'aim 53 wherein the inhibition is obtained using a metabolic inhibitor.
55. A process according to Claim 54 wherein the inhibitor is a Kcaj- inhibitor.
56. A process for producing a polydoma which secretes a hybrid monoclonal antibody having a dual specificity
O PI which comprises removing the nucleus from a first hybri doma cell which produces a monoclonal antibody against first antigenic determinant and inserting the nucleus int the cytoplasm of a second hybridoma which produces monoclonal antibody against a second antigenic deter minant.
57. A process for producing a hybrid monoclonal anti body having a dual specificity comprising isolating th antibody from cells of a polydoma produced according t the process of Claims 29, 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 havin a dual specificity, one specificity of which is directe against a disease associated antigen and the other, agains a hapten, the hapten being an agent lethal to the antige or associated tissue or having a lethal agent bound to it and b) administering the hapten after sufficien time has elapsed to permit the antibody to bind to th disease associated antigen.
59. A process according to Claim 58 wherein the letha agent is a radionuclide.
60. A process according to Claim 59 wherein the radio nuclide is an emitter of c^-radiation or ^-radiation
61. A process according to Claim 58 wherein the letha agent is a tissue toxin.
62. A process according to Claim 61 wherein the toxi is comprised of a ricin A chain. 63. A process according to Claims 58 , 59 , 60 , 61 62 wherein the antigen is a tumor associated antige
64. A process according to Claims 58 , 59 , 60 , 61 62 wherein the antibody is a hybrid monoclonal antibo produced by a polydoma.
65. A proces s accord ing to Cl aim 63 where in th antibody is a hybrid monoclonal antibody produced by polydoma.
66. A process according to Claims 58 , 59 , 60 , 61 o 62 wherein the antibody is a hybrid antibody produced b the reassociation of antibody half molecules obtained b the selective cleavage of a mono-specific antibody agains the disease associated antigen and a mono-specific anti body against the hapten.
67. A process accord ing to Cl aim 66 where in th selectively cleaved antibodies are monoclonal antibodies
68. A process accord ing to Cl aim 66 where in th selectively cleaved antibodies are polyclonal antibodies
69. A process according to Claims 58 , 59 , 60 , 6 or 62 wherein the antibody is a multimer of a pair o intact mono-specific antibodies, one of said mono-specifi antibodies being against the disease associated antige and the other against the hapten.
70. A process accord ing to Cl aim 69 where in th mono-specific ant ibodies are monoclonal antibodies .
71. A process according to Claim 69 wherein the mono specific antibodies are polyclonal antibodies.
72. An jLn vivo immunodiagnostic process comprising : a) administering to a host an antibody havi a dual specificity, one specificity of which is direct against a disease associated antigen and the other again a hapten bearing a radionuclide; b) administering the hapten after sufficie time has elapsed to permit the antibody to bind to t disease associated antigen; and c) scanning the host to detect the location radiation emitted by the radionuclide.
73. A process according to Claim 72 wherein t radionuclide is an emitter of -radiation.
74. A process according to Claim 72 wherein t radionuclide is bound directly to the hapten.
75. A process according to Claim 72 wherein t radionuclide is bound to the hapten by a chelating agen
76. A process according to Claims 72, 73, 74 75 wherein the antigen is a tumor associated antige
77. A process according to Claims 72, 73, 74
75 wherein the antibody is a hybrid monoclonal antibo produced by a polydoma.
78. A process according to Claim 76 wherein t antibody is a hybrid monoclonal antibody produced by polydoma.
79. A process according to Claims 72, 73, 74 75 wherein the antibody is a hybrid antibody produced the reassociation of antibody half molecules obtained the selective cleavage of a mono-specific antibody again the disease associated antigen and a mono-specific ant body against the hapten.
OMPI 80. A process according to Claim 79 wherein th selectively cleaved antibodies are monoclonal antibodies
81. A process according to Claim 79 wherein th selectively cleaved antibodies are polyclonal antibodies
82. A process according to Claims 72, 73, 74 o
75 wherein the antibody is a multimer of a pair of intac mono-specific antibodies, one of said mono-specifi antibodies being against the disease associated antige and the other against the hapten.
83. A process according to Claim 82 wherein th mono-specific antibodies are monoclonal antibodies.
84. A process according to Claim 82 wherein th mono-specific antibodies are polyclonal antibodies.
85. An immunoassay process comprising: a) adding to a sample suspected of containing a target antigen a predetermined amount of the targe antigen to which is fixed a chromophore; 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; 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 th antigen is a tumor associated antigen.
87. A process according to Claim 85 wherein th hapten is bound to the antibody before the antibody i added to the sample.
88. A process according to Claim 86 wherein th hapten is bound to the antibody before the antibody i added to the sample.
89. A process according to Claims 85, 86, 87 or 8 wherein the antibody is a hybrid monoclonal antibod produced by a polydoma.
90. A process according to Claims 85, 86, 87 or 8 wherein the antibody is a hybrid antibody produced by th reassociation of antibody half molecules obtained by th selective cleavage of a mono-specific antibody against th disease associated antigen and a mono-specific antibod against the hapten.
91. A process according to Claim 90 wherein' th selectively cleaved antibodies are monoclonal antibodies.
92. A process according to Claim 90 wherein th selectively cleaved antibodies are polyclonal antibodies.
93. A process according to Claims 85, 86, 87 o 88 wherein the antibody is a multimer of a pair of intac mono-specific antibodies, one of said mono-specifi antibodies being against the target antigen and the othe against the hapten.
94. A process according to Claim 93 wherein th mono-specific antibodies are monoclonal antibodies. 95. A process according to Claims 85, 86, 87 o 88 wherein the fluorescing chromophore is fluorescein an the quenching chromophore is rhodamine.
96. An immunoassay process comprising: a) adding to a sample suspected of containing target antigen a predetermined amount of the targe antigen to which is bound a substance capable of inter acting with an enzyme to produce a detectable product; b) adding to the sample an antibody having dual specificity, one specificity of which is directe against the target antigen and the other against th enzyme or a hapten to which the enzyme is bound; c) binding the hapten to the antibody; 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.
98. A process according to Claim 91 wherein the anti¬ gen is a tumor associated antigen.
99. A process according to Claim 97 wherein the anti- gen is a tumor associated antigen.
100. 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 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 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 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 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, luminescence or spectroscopically.
OMP
PCT/US1983/000525 1982-04-12 1983-04-12 Antibodies having dual specificities, their preparation and uses therefor WO1983003679A1 (en)

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US5217713A (en) * 1988-12-27 1993-06-08 Takeda Chemical Industries, Ltd. Cytotoxic bispecific monoclonal antibody, its production and use
US5141736A (en) * 1988-12-27 1992-08-25 Takeda Chemical Industries, Ltd. Bispecific monoclonal antibody, its production and use
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US7306921B2 (en) 2001-01-17 2007-12-11 Nevalainen Marja T Diagnostic and monitoring methods for cancer
US7642050B2 (en) 2001-01-17 2010-01-05 Nevalainen Marja T Method for predicting responsiveness of breast cancer to antiestrogen therapy
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