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Hybrid hybridomas and the production of bi-specific monoclonal antibodies C. Milstein and A. C. Cuello* Cellfusion techniques can now be used to generate antibodies with two different specificities. Here C~sarMilstein andA. C. Cuello discuss the theoretical basis of the method they have devised and its first applications in histochemisoy and immunoassay. The idea of using the bivalency of antibodies to cross link two antigenic substances is not new. Hybrid antibody molecules have been made by chemical rec0nstitution experiments and used successfully in histochemistry ~ to visualize antigenic sites with ferritin. This method has, however, been little used due to the difficulties in obtaining such hybrid antibodies. Other immunoenzyme techniques such as those using a 'sandwich' method or enzyme-antibody have become increasingly popular. While the latter requires purified antibodies, in the unlabelled enzyme method two IgG antibodies are cross linked by a third antibody and there is often no need to purify the antibodies in question 2'3. With the advent of monoclonal antibodies, the purification step was largely overcome and the direct labelling method gained support. A more recent development has been the use of cell fusion techniques for the in-vivo production of bispecific antibodies. Hybrid hybridomas secreting antiperoxidase antisomatostatin bispecific molecules have been made and successfully applied in immunocytochemistry 4. The theoretical basis for the derivation of such cell lines was implicit in the derivation of monoclonal antibodies by the hybrid myeloma method. When two antibody producing cells are fused, the derived hybrid co-dominantly expresses the immunoglobulin chains of its parents 5'6. This fundamental property is at the root of the hybrid myeloma method, used for the derivation of permanent cell lines capable of producing monoclonal antibodies to a predefined antigen. Since antibody-producing cells have the V and C regions of each chain attached to a single transcriptional unit, the expression of each chain is not altered by the fusion, and remains in the 'cis' configuration. However, the individual light and heavy chains are themselves on separate transcriptional units. During transcription, chains are synthesized through the membrane of the rough endoplasmic reticulum and released in the cisternal space where they are assembled. When there is only one pair of heavy and light chains, a homogeneous antibody consisting of two identical pairs of heavy and light chains is formed. Under physiological conditions, the way to ensure the presence of two identical combining sites is by the phenomenon known as allelic exclusion, whereby individual cells normally express only one heavy and one light chain allele. Hence in a heterozygous individual, each antibody producing cell expresses either one or other Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK. *Neuroanatomy/Neuropharmacology Group, University Departments of Pharmacology and Human Anatomy, South Parks Road, OX1 3QT, UK.

of the two possible alleles. The production of hybrid cells capable of expression of both alleles was a clear demonstration that the phenomenon was not dominant in cells already committed to antibody production 5. W e now know that this so-called allelic exclusion is due to the correct integration of the V and C region D N A fragments in only one of the two alleles. This is why, under conditions in which two such cells are fused, the derived hybrid expresses the four chains, two heavy and two light from both alleles, which are then free to recombine in the cysternal space and be secreted as hybrid molecules. The expected molecular species resulting from random association of the two heavy (which we will refer to arbitrarily as G and H) and two light chains (referred to as K and L) are shown in Fig. 1. The intracellular assembly of chains depends on the preferential association of homologous v. heterologous pairs. In addition, there is likely to be a differential rate of synthesis of the different chains. In practice, therefore, the different forms are unlikely to occur randomly at t :1 : 1 : 1 ratio for the LH, LG, K H and K G heavy-light chain pairs, and of 1:2:1 for the H - H , H-G, G - G heavy chain (regardless of light chain attached to them) pairs. The heterologous association between 7 and/a chains has not been observed 7 and therefore no hybrid molecules of this type are likely. Different subclasses Of heavy chains, and different light chains, even originating from different species 8, can associate, though not necessarily in a random fashion. This will affect the yield of the desired bi-specific hybrid molecule. The expected concentrations of each molecular species is bound to be rather speculative because the different parameters are difficult to predict. An analysis of the products of hybrids of this type has shown in one example the expected random distribution of heavy chains 1 : 2:1 (Ref. 9). A more elaborate analysis of the different components of the antiperoxidase-antisomatostatin hybrid hybridoma, providing an interesting example of the type of problems discussed, is shown in Fig. 2. The presence of rat IgG2a, IgG1 and the IgG hybrid was (after allowing for small differences in overall production of chains) in a ratio of 1: 1: 1 rather than the randomly expected 1: 2:1. As a first approximation, this would result from a homologous v. heterologous heavy-heavy chain preference of 1:0.4. The overall yield ofbispecific molecules would thus be 1 : 12 of the total production, instead of the 1:8 ratio expected from a completely random distribution of heavy-light chain combinations. From this one might conclude that hybrids of a single subclass have an advantage in that they are the most likely to give best yields. However, the problem is more complex. Antibodies made up of two different subclasses of heavy chains may © 1984) Elsevier Science Publishe~ B.V., Amsterdam 0167 - 4919184/$02,00

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Bispecific

Monospecific

"Inactive"

Monovalent

KH

Bivalent

HK

Fig. 1. Molecular species in hybrid hybridomas expected to arise by random association of heavy and light chains. The two antibody specificitiesare defmed by the heavy-light chain combination GK (specificity of parental hybridoma) and LH (specificity selected from the immunized mouse cells). The' inactive' molecules are the artificial combination of heavy and light chains produced by non-homologous pairs. The term' inactive' should be taken cautiously, since some activity could be retained by the heavy (or light) chain in the artificialheavylight chain combination. culture 5a°. However, it is quite possible that under conditions of competitive assembly in vivo, there is often a preferential combination of the homologous light-hea W chain pairs. In-vitro studies have shown that under conditions of competitive reassociation, the homologous pair prevails in 80% of cases u. W e have recently studied another two examples of hybrid hybridomas and found that in both there was a very marked preference for the homologous pairs. T h u s in both cases the hybrid fraction on D E A E cellulose (equivalent to pool II in Fig. 2) was between 80-100% pure bivalent bi-specific molecules (unpublished results).

have special advantages due to the unique assymetric properties of their respective constant regions. In addition, the difference in chromatographic or electrophoretic behaviour of the assymetric hybrids is very advantageous for the fractionation of the different molecular species. This m a y more than compensate for the lower absolute yield of the bi-specific hybrid. For instance - in the peroxidase-somatostatin hybrid hybridoma, the three major species (antiperoxidase, antisomatostatin and bispecific antibody) could be separated by a single D E A E chromatography step (Fig. 2A), and the middle pool was 50% bivalent hybrid; this was because the heterologous pair ( K H ) was undetectable. W h e n all the molecules containing the K H combination are eliminated from the computation, the final yield of the bivalent hybrid is 1:8 of the total. Pool II of Fig. 2 should contain only the active bispecific L H G K hybrid and the hybrid molecule L H G L which is functionally a monovalent antisomatostatin antibody. This highlights the importance of the preferential association of homologous heavy-light chains. Examples ofheterologous 'artificial' heavy-light chain pairs, i n d u d ing rat-mouse combinations, are quite c o m m o n in cells in

Applications of b i s p e c i f i c h y b r i d h y b r i d o m a s Bispecific hybridomas can be used for the specific visualization, or quantitative estimation, of antigens - in more general terms, for situations specifically requiring the cross-linking of two antigens. T h e y could therefore be valuable alternatives in a variety of enzyme-linked immunoassays, immunocytochemistry, antibody-directed delivery of drugs, etc. It is still too early to say to what extent it will find great favour, especially taking into con-

TABLE I. Some advantages and disadvantages in the immunochemical methods used to cross link two substances. Chemical Direct Indirect methods Hybrid reconstitution labelling using anti IgG hybridoma Chemical treatment Purification of Ab Purification of one Ag Multiple steps Signal amplification Competing inactive species

+ + Needs special purification Good +

+ + + ±

+ + -

Needs special purification Good +

Probable shelf life Poor Good Can it cross link two antigens in ± a mixture? Stereochemical defmition Good Poor Very poor Good of reagent For reasons of simplicity, in all cases it is assumed that antibodies are monclonal. Other methods can be taken as a combination of the direct and indirect methods. For technicalreasonswe are unableto reproducethis figurein colourin this edition--seethe OctoberissueofImmunologyTodayfbr fullcolourillustration.

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Pool I I

II I

III

I

I

I

I

Anti St

1:1 000

1:1 000

< 1:100

Anti Po

< 1:100

1:5000

1:10000

Ratio Sypo

> 10

0.2

< 0.01

-50 0.5-

I

~x

o,i LLI

O

-25 0-

/ 1~)0

i 200

250

Eluate (ml)

Fig. 2. Analysis o f the a n t i b o d i e s from a hybrid hybridoma. (a) DEAE fractionation of the rat antisomatostatin antiperoxidase bi-specific antibody. (b) SDS-polyacrylamide (10%) gel electrophoresis of reduced chains. A: the 50% (NH4) 2SO4 cut from ascites applied to and pools I, II and III eluted from the DEAE column. G 9r -~- ~nd H or L indicate the position of the heavy and light chains from the antiperoxidase and the antisomatostatin monoclonal antibodies, respectively. (Takenfrom R~. 4.)

sideration that it will have to compete with well-established procedures. Table I describes some of the advantages and disadvantages of hybrid hybridomas. There are, however, certain characteristics of bispecific antibodies which are unique. For instance, chemical attachment of an enzyme to an antibody is often through lysine residues. Different lysine residues are usually involved and are likely to give different, undefined mixtures. The attachment through an antibody combining site, although likely to remain unknown in most cases, is well defined and reproducible. Another specific characteristic of bi-specific antibodies is their monovalency. The advantages of monovalent antibodies in certain circumtances have been noted elsewhere 12and these include a lower degree of antigenic modulation. Therefore monovalent bi-specific antibodies are potentially better for complement-dependent cytotoxicity. Monovalent-monoclonal antibodies have been prepared, using a light chain producing myeloma, to produce hybridomas of the H L K variety. However, separation of the monovalent from the other forms requires immunoaffinity purification by specific recognition of the two light chains. This is not easy, especially if the light chains are of the same isotype and allotype. The bi-specific hybrid hybridoma is a much simpler proposition, as illustrated in Fig. 2. Regardless of the nature of the mixture of Pool II, all their components are of necessity monovalent in nature.

A

I

II

HI

302

Immunoassays Bispecific hybridomas have the simplicity of the direct antibody-enzyme conjugate, but without the shortcomings of the chemical preparation of the reagent. For instance, the supernatant fluid of clone P4C 7, to which an excess ofperoxidase has been added, can be used to detect the presence of somatostatin bound to a solid support. The inhibition of this binding by competing free somatosatin can be used to measure its concentration (unpublished observations). The procedure is similar in execution to the use of a peroxidase-coupled somatostatin antibody, but it has the great advantage that it does not need chemical coupling. Chemically coupled enzymeantibodies not only require a skilled chemical step, but also tend to have a relatively short shelf life. Unfractionated preparations of hybrid hybridomas contain a mixture of competing components. When such complex mixtures are used, the monovalent hybrid molecules could be preferentially displaced by the symmetrical bivalent antibody, as the bivalent species could have a higher avidity than the monovalent ones. This is likely to depend on the distribution of antigenic sites. Bivalent molecules require antigens spaced sufficiently close to each other for the two antibody sites to attach. If only a fraction of them are so distributed, the effect is negligible. For example, bovine serum albumin-somatostatin conjugates do not show a significantly enhanced inhibition by the bivalent species, and for this reason the complete mixture could be used with some loss of sensitivity (unpublished results). In other cases the inhibition could be a major problem requiring separation of monovalent and bivalent species. For example, a mouse-rat hybrid molecule which recognizes a rat alloantigen on red cells gave a very low signal with a marked prozone, as would be expected if the bivalent competing species was binding more tightly under saturating conditions 13. In summary, then, when bispecific hybrid hybridomas are used for enzyme-linked binding reactions, a loss of sensitivity is expected when the bi-specific antibody is not free of competing bivalent or monovalent species. The inhibition can be very marked if the competing bivalent species has a better avidity than the monovalent form.

Immunohlstochemlstry The practical applications of the products of a hybrid hybridoma are at present restricted to the use of P4C 7 bispecific monoclonal antibody in immunocytochemistry ~. P4C7 binds peroxidase and somatostatin. The partially purified hybrid fraction (pool II, Fig. 2) contained, as discussed above, not only bi-specific antibodies but also monospecific (LH-GL) antisomatostatin. The only reagent required for the one-step immunocytochemistry procedure was a mixture of the antibody fraction and peroxidase. As expected 14'~5,somatostatin immunoreactive sites were readily visible in the hypothalamus, particularly in the pituitary stalk and the median eminence. Light microscopy revealed a consistently heavy immunoperoxidase reaction on varicose axons of the external layer of the median eminence, in close relation to portal vessels (Fig. 3A). The signal obtained with the bi-specific antibody (P4C7) was sharp and unequivocal and with a complete absence of background staining to the point that phase

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contrast microscopy was needed to identify tissue landmarks. This is in contrast to most immunoenzyme procedures, where some non-specific binding of antibodies is expected to occur, discretely revealing features of the tissues under study. At electron microscopical levels somatostatin immunoreactive sites were located on large, dense core vesicles within axons of the median eminence (Fig. 3B). The reaction products of the bi-specific antibodies were easily recognized, both in lead counterstained and in non-lead-stained, ultra-thin sections 4. The procedure was extremely simple as it obviated the lengthy incubations with link antibodies and enzyme-antibody complexes (for review of immunocytoehemistry by electron microscopy see Refs 16 and 17). The shortening of the enzyme staining protocol by days may help the allimportant ultrastructural preservation of complex structures. This and other related points, like the penetration of the bi-specific antibodies as compared with that of antibody-enzyme complexes, require further comparative studies. The importance of penetration is also suggested by the following observations. In our initial series of investigations we were unable to demonstrate extrahypothalamic somatostatin immunoreactive sites applying the bispecific antibody P4C7. We reasoned that this could be due to the fact that the bi-specific antibody-peroxidase complex has an increased molecular weight from approximately 150000 to 190000. This and other physico-chemical parameters probably further hinders antibody penetration through tissue barriers. Therefore we tried incubating extrahypothalamic tissue with the bispecific monoclonal antibody P4C7 alone in a first step, followed by the addition of peroxidase in a second step. This two-stage procedure allowed the demonstration of immunoreactive sites in the expected regions of the CNS, including sensory terminals in the dorsal horn of the spinal cord as illustrated in Figs. 3C and D. This result has important implications. The fraction we have used does not contain fully purified bi-specific antibodies because it also contains a monovalent L H G L impurity. Thus competition with a faster penetrating molecule that is not bi-speeifie may be a very important factor in lowering the sensitivity as observed by the one-step method. Although it is possible to further purify the bi-specific molecule by affinity columns, it may be better to add at the screening stage appropriate provision to select hybridomas with preferential expression of the homologous H L and GK pairs. As described above, this seems to be a common property of hybrid hybridomas which is only partly shown by the preferential H L (but not GK) combination in the hybrid hybridoma P4C7. The simultaneous localization of multiple antigenic sites requires primary antibodies made on different species or conjugated antibodies such as those prepared by covalent attachment of enzymes to monoclonal antibodies 18'19.They could be used for the simultaneous localization of two antigenic sites at the light microscope level 20 and the ultrastructural level. Autoradiography with internally radio-labelled antibodies combine effectively with immunoenzyme histoehemistry, because they produce different electron dense signals. Bi-speeific reagents may provide a better alternative to covalent

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Fig. 3 A. Light microscopy direct immunocytochemistry demonstration of somatostatin immunoreactive axons in the external layer of the rat median eminence with bi-specific monoclonal antibodies P4C7. Lateral aspects. Scale bar = 100 tam. Fig. 3 B. Low magnification electron micrograph from an area similar to that represented in (A). Asterisk denotes immunoreactive nerve profiles to P4C7. Scale bar = 1/am. Fig. 3 C. Combined direct radio-immunocytochemistry with internally labelled anti-substance P monoclonal antibody (3H-NC 1/34) (see Ref. 20) and direct immunoperoxidase with bi-specific antisomatostatin monoclonal antibodies (P4C 7) in the dorsal horn of the guinea pig spinal cord. Open arrow denotes silver grains (substance P sites) and solid arrow peroxidase immunoreaction (somatostatin sites). 0.5/am thick plastic embedded sections, counterstained with toluidine blue. Note that immunoreaction is present only in marginal layer and substantia gelatinosa and absent in myelinated axon profiles. Scale bar = 10/am. Fig. 3 D. Electron micrograph of the same preparation as in (C). Solid arrow indicates the presence of an irnrnunoreaction to the bi-specific antibody P4C7. Note peroxidase reaction of large granular resides in axon and varicosity. In the same fidd the dusters of silver grains denote the presence of a bouton immunoreactive to internally labelled NC 1/34 (substance P site, open arrow) in contact with a small, unstained dendritic profile (d). Scale bar = 1/xm.

304 enzyme conjugate. W e h a v e r e c e n t l y c o m b i n e d t h e bi-specific a n t i s o m a t o statin-antiperoxidase antibody with internally labelled anti-substance P (3H-NC1/34) monoclonal antibodies. T h e t w o a n t i g e n i c sites in t h e s u b s t a n t i a g e l a t i n o s a o f t h e s p i n a l c o r d largely r e p r e s e n t c e n t r a l t e r m i n a t i o n s o f a x o n s f r o m s e n s o r y e l e m e n t s b e l o n g i n g to d i f f e r e n t n e u r o n a l s u b s e t s 2~. T h e r e l a t i o n s h i p o f t h e s e t w o n e r v e t e r m i n a l s to e a c h o t h e r is u n k n o w n a n d it is i n t e r e s t i n g to i n t e r p r e t t h e i r function. Fig. 3 C a n d D r e p r e s e n t early a t t e m p t s to d e m o n s t r a t e s i m u l t a n e o u s l y t h e s e t w o sites. T h e results h i g h l i g h t t h e p o t e n t i a l o f a bi-specific m o n o c l o n a l a n t i b o d y for c o m b i n e d i m m u n o c y t o c h e m i c a l procedures. It c a n b e e x p e c t e d t h a t bi-specific m o n o c l o n a l antib o d i e s will b e valuable a l t e n a t i v e s in h i s t o p a t h o l o g i c a l d i a g n o s i s . T h e y are v e r y suitable for l a r g e n u m b e r s o f s a m p l e s , b e c a u s e t h e y c a n b e u s e d in o n e - s t e p i m m u n o c y t o c h e m i s t r y . T h e r e a g e n t s are m o r e easily s t a n d a r d i z e d because they do not require chemical modifications. The h y b r i d h y b r i d o m a s that h a v e p a r t i c u l a r v a l u e m a y b e t h o s e d i r e c t e d t o w a r d s easily d e t e c t a b l e a n t i g e n s o f medical relevance. Another area of potential which needs to b e e x p l o r e d is t h e q u a n t i t a t i o n o f a n t i g e n i c sites for diagnostic purposes, tT]

Acknowledgements A.C.C. would like to thank the Medical Research Council, the Wellcome Trust, and the Cephalosporin Fund for their financial support.

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References 1 H~mmerling, U., Aoki, T., de Harven, E., Boyse, E. A. and Old, C. J. (1968),)'. Exp~ Med. 128, 1461-1473 2 Sternberger, L. A. (1979) in Immunocytochemistry,John Wiley, New York 3 Vandesande, F. in Immunohistochemistry (Cuello, A. C., ed.), pp. 101-119, John Wiley, Chichester 4 Milstein, C. and Cuello, A. C. (1983) Nature (London) 305, 537-540 5 Cotton, R. G. H. and Milstein, C. (1979) Nature (London) 244, 42-43 6 K6hler, G. and Milstein, C. (1975) Nature (London) 256, 495-497 7 Nisonoff, A., Hopper, J. E. and Spring, S. B. (1975) in The Antibody Molecule, Academic Press, New York 8 Galfre, G., ButCher, G. W., Howard, J. C., Wilde, C. D. and Milstein, C. (1980) Trans. Proe. 12, 371-376 9 Martinis, J., Kull, J. F., Franz, G. and Bartholomew, R. M. (1984) in Colloquium of the Protidesof Biological Fluids, Vol. XXX (Peeters, H., ed.), pp. 311-316, Pergamon, Oxford 10 Milstein, C., Adetugbo, K., Cowan, N. J., K6hler, G., Secher, D. S. and Wilde, C. D. (1977) Cold Spring Harbor. Symp. Quant. Biol. 41, 793-803 11 De Preval, C. and Fougereau, M. (1976)J. MoL BioL 102, 657-678 12 Cobbold, S. P. and Waldmann, H. (1984)Nature (London)308, 460-462 13 Howard, J. C., Butcher, G. W., Galfre, G., Milstein, C. and Milstein, C. P. (1979) Immunol. Rev. 47, 139-173 14 Hokfeh, T., Efendic, S., Hellerstr6m, A., Johansson, O., Luft, R. and Arimura, A. (1975)Acta Endocrinol. (Copenhagen) Suppl. 200, 5-41 15 Krisch, B. (1978) Cell Tissue Res. 195, 499-513 16 Pickel, V. M. in Neuroanatomical Trueting TracingMethods (Heinuner, L. and Robards, M. J., eds), pp. 483-509, Plenum Press, New York 17 Priestley,J. V. and CueUo,A. C. (1983) in Immunohistoehemist~y)(Cuello, A. C., ed.), pp. 273-322, Wiley, Chichester 18 Boorsma, D. M., Cuello, A. C. and Van Leuwen, F. M. (1982),f Histochem. Cytochem. 30, 1211-1216 19 Charlton, H. M., Barclay, A. N. and Williams, A. F. (1983) Nature (London) 305,825~827 20 Boorsma, D. M. (1984) Histoehemist~y 80, 103-106 21 Cueno, A. C., Galfre, G. and Milstein, C. (1979) Proc. NatlAead. Sci. USA 76, 3532-3536 22 Hokfelt, T,, Elde, R., Johansson, O., Luft, R., Nilsson, G. and Arimura, A. (1976) Nenroseience 1, 131-136

Hybrid hybridomas and the production of bi-specific monoclonal antibodies.

Cell fusion techniques can now be used to generate antibodies with two different specificities. Here Cesar Milstein and A. C. Cuello discuss the theor...
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