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4 Richards, F. F., Konigsberg, W. H., Rosenstein, R. W. and Varga, J. M. (1975) Science 187, 130-137 5 Clevinger, B. L., Thomas, J., Davie, J. M., Schilling, J., Bond, M., Hood, L. and Kearney, J. (1981) in Immunoglobulin Idiotypes and Their Expression (Janeway, C., Sercarz, E. E., Wigzell, H. and Fox, C. F., eds), pp. 159-168, Academic Press 6 Nahm, M. H., Murray, P. R., Clevinger, B. L. and Davie, J. M. (1980)J. Clin. Microbiol. 12, 506-508 7 Sevier, E. D., David, G. S., Martinis, J., Desmond, W. J., Bartholomew, R. M. and Wang, R. (1981) Clin. Chem. 27, 1797-1806 8 Haaijman, J. J., Deen, C., Krose, J. M., Zylstra, J. J., Coolen, J. and Radl, J. (1984) Immunol. Today 5, 56-58 9 Davie, J. M. and Paul, W. E. (1973) J. Exp. Med. 137, 201-204 10 Charlish, P., ed. (1985) Clinica 134, 1-2 11 Reinherz, E., Strelkauskas, A., O'Brein, C. and Schlossman, S. (1979) J. Immunol. 123, 83-86 12 Talle, M. A., Allegar, N., Makowski, M., Rae, P. E., Mittler, R. S. and Goldstein, G. (1983)Diagn. Immunol. 1, 129-135 13 Reinherz, E. L., Meur, S. C. and Schlossman, S. F. (1984) Springer Semin. Immunopathol. 7, 9-18 14 Foon, K. A., Schroff, R. W. and Gale, R. P. (1982)Blood 60, 1-19

175 15 Schlom, J. and Weeks, M. O. (1985) in Important Advances in Oncology (DeVita, V. T., Hellman, S. and Rosenberg, S. A., eds), pp. 170-189, J. B. Lippincott Co. 16 Rattle, S. J., Purnell, D. R., Williams, P. I. M., Siddle, K. and Forrest, G. C. (1984) Clin. Chem. 30, 1457-1462 17 Maynard, Y., Roden, D. C., Tikkanen, M. J., Schonfeld, G. and Ladenson, J. H. (1984) Clin. Chem. 30, 1620-1625 18 Scott, M. G. and Nahm, M. H. (1984) J. Immunol. 135, 2454-2460 19 Staehlin, T., Takacs, B., Durrer, B., Schmidt, J., Stocker, J., Miggiano, V., Staehli, C., Hobbs, D. S., Kung, H-F., and Pestka, S. (1981) in Monoclonal Antibodies and Developments in Immunoassay (Albertini, A. and Ekins, R. eds.),

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Elsevier Biomedical Press Fantl, V. E. and Wang, D. Y. (1984) ft. Endocol. 100, 367-376 Haber, E. (1982) in MonoclonalAntibodies in Clinical Medicine (McMichael, A. J. and Fabre, J. W., eds), pp. 477-500, Academic Press Ngo, T. T. and Lenhoff, H. M. (1981) Appl. Biochem. Biotechnol. 6, 53-64 Zimmerman, U. (1982)Biochim. Biophys. Acta. 694, 227-277 Lo, M. M. S., Tian, Y. T., Conrad, M. K., Strittmatter, S. M., Hester, L. D. and Snyder, S. H. (1984) Nature 310, 792 794 Boyd, J. E., James, K. and McClelland,

Immunotoxins J. Michael Lord, Lynne M. Roberts, Philip E. Thorpe and Ellen S. Vitetta M a n y t y p e s o f t u m o u r cell express h i g h levels o f surface a n t i g e n s w h i c h are l a r g e l y absent f r o m n o r m a l cells. M o n o c l o n a l a n t i b o d i e s against these surface a n t i g e n s can be c o u p l e d to p o t e n t toxins to f o r m conjugates ( i m m u n o t o x i n s ) w h i c h s e l e c t i v e l y kill the a n t i g e n - b e a r i n g cells. T h i s article describes the c o m p o n e n t s o f i m m u n o t o x i n s , their m o d e s o f action and their c l i n i c a l a p p l i c a t i o n s . One o f the major achievements o f medical research has been the development of anti-bacterial agents (antibiotics) which selectively kill a wide variety of pathogens. Antibiotics are either analogues o f normal intermediates of bacterial metabolism or inhibitors of metabolic pathways unique to bacteria. Since the eukaryotic host cells use different J. M. Lord and L. M. Roberts are at the Department of Biological Sciences, University of Warwick, Coventry, CV4 7AL, UK; P. E. Thorpe is at the Drug Targeting Laboratory, Imperial Cancer Research Fund Laboratories, Lincoln's Inn Fields, London, WC2A 3PX, UK, and E. S. Vitetta is at the Department of Microbiology, University of Texas Health Center, Dallas, TX 75325, USA.

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D. B. L. (1984) Trends Biotechnol. 2, 70 77 Jolly, M. F., Wang, C. H. J., Ekenberg, S. J., Zueike, M. S. and Ketso, D. M. (1984) J. Immunol. Methods 67, 21-35 Jarvis, A. P. and Gridina, T. A. (1983) Biotechniques 1, 22-27 Ku, K., Kuo, M. J., Delente, J., Wilde, B. S. and Feder, J. (1981) Biotechnol. Bioeng. 23, 79-95 Lydersen, B. K., Pugh, G. C., Paris, M. S., Sharma, B. P. and Noll, L. A. (1985) Bio/Technol. 3, 63-67 Tolbert, W. R. and Feder, J. (1983) in Annual Reports of Fermentation Processes

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(Tasco, G., ed.), pp. 35-74, Academic Press Cabilly, S., Riggs, A. D., Pande, H., Shively, J. E., Holmes, W. E., Rey, M., Perry, L. J., Wetzel, R. and Heyneher, H. L. (1984)Proc. Natl Acad. Sei. USA 81, 3273-3277 Jenske, D. J., Jarvis, J., Milstein, C. and Capra, J. D. (1984)J. Immunol. 133, 1090-1092 Milstein, C. and Cuello, A. C. (1984) Immunol. Today 5, 299-304 Sherman, D. M., Acres, S. D., Sadowski, P. L., Springer, J. A., Bray, B., Raybould, T. J. and Muscoplat, C. C. (1983) Infect. Immun. 42, 653-658 Weil, G. J., Malane, M. S., Powers, K. G. and Blair, L. S. (1985)J. Immunol. 134, 1185-1191

minor subpopulations o f normal cells which are not life-sustaining. Antibodies against these molecules can be coupled to potent drugs or toxins to form reagents which are powerfully and selectively toxic towards the normal and malignant cells that express the target antigen.

Monoclonal antibodies Until 1975, the generation of tumour-reactive antibodies for use as drug or toxin carriers was complicated metabolic pathways, they are by the fact that heterologous antiunaffected by such agents. In the case bodies raised against tumour cells had of tumour cells, however, it has been to be absorbed with normal tissues to difficult to identify specific metabolic remove antibodies recognizing strucdifferences between the normal cells of tures common to both normal and neothe host and their neoplastic counter- plastic cells. This was technically parts. M o s t anti-cancer drugs elimi- difficult and different batches of nate rapidly dividing cells and so can absorbed antibody showed large variaalso cause significant damage to tion in their reactivity. With the development of the hybridoma techdividing cells in normal tissues. T h e r e is, however, another way to nology 1, however, it became possible distinguish normal from malignant to produce monoclonal antibodies ceils, which could provide a basis for which would recognize individual therapy. M a n y types of tumour cell determinants on tumour cells. Large express high levels of certain dif- quantities o f standardized antibodies ferentiation antigens, receptors for could then be produced in tissue culgrowth factors or viral antigens on t u r e or in mice. In principle, the their surface. These molecules are delivery o f drugs or toxins to tumour often expressed in lower densities on cells could also be accomplished by normal tissue or are expressed only on hormones, lectins, growth factors, or © 1985,ElsevierSciencePublishersB.V.,Amsterdam0166 9430/85l$02.00

176 other molecules with innate ability to bind to tumour cells. However, monoclonal antibodies are generally more versatile as delivery systems, because antibodies of the desired specificity can be elicited by immunizing appropriate animals with the target tissue itself. It should be recognized that specificity is only relative and that even monoclonal antibodies cross-react to a greater or lesser extent with other molecules on normal cells. Nevertheless, the idea of selective targeting of cytotoxic agents with cell-reactive antibodies is rapidly materializing into a new modality for tumour therapy. Toxins Several toxins produced by bacteria and plants are potent cytotoxic agents. These toxins are proteins which in their active forms consist of a cellbinding B-chain and a toxic A-chain joined by a disulphide bond. The most widely used of these toxins are ricin and abrin from the seeds of the plants Ricinus communis and Abrus prec a t o r i J , respectively, and diphtheria toxin, the exotoxin secreted by Corynebacterium diphtheriae carrying the D N A phage/3 tox + (Ref. 3). Many plants also synthesize A-chain-like proteins (e.g. gelonin, saporin, pokeweed anti-viral protein, etc. 4) that inh~bit protein synthesis by eukaryotic ribosomes apparently in the same manner as the A-chains of the plant toxins. These ribosome-inactivating proteins (RIPs) are not themselves highly toxic because they lack an equivalent of the B-chain by which to bind to and enter cells. M o d e of action of toxins The toxins bind by means of the B-chain to receptors that are on the surface of virtually all cell types in sensitive species of animal. The receptors for abrin and ricin are galactose-containing glycoproteins and glycolipids whereas the receptor for diphtheria toxin is probably a 150 kDa glycoprotein 5. The receptors become clustered in specialized areas of the cell surface known as coated pits 6. An invagination step then produces a vesicle (or endosome) inside the cytoplasm which contains the receptor and its attached toxin. A complex series of events ensues. The endosome moves deeper into the cell and becomes acidified. With natural ligands, such as growth

Trends in Biotechnology, VoL 3, No. 7, 1985 factors, this acidification would cause the ligand-receptor complex to dissociate: the endosome would then fragment into two vesicles, one containing the receptors which would usually recycle to the plasma membrane, and the other containing the ligand which would be delivered to the lysosomes for degradation7. However, the toxins probably do not dissociate from their receptors at acid pH and so some are recycled to the plasma membrane and the rest are eventually degraded in the lysosomes. For diphtheria toxin, there is compelling evidence that the B-chain of the molecule spontaneously inserts into the membrane of an acidic cellular compartment (probably the endosome) and that the A-chain then crosses the membrane and is released into the cytoplasm8. For abrin and ricin, however, there is no evidence that low p H is required for A-chain entry and it is unclear which of the endocytic events is important for toxicity2. The final step in the cytotoxic process is that the A-chain of the toxin, having gained access to the soluble phase of the cytoplasm, inactivates the cell's machinery for protein synthesis. This it does with great efficiency: it is thought that just one molecule of Achain can kill the cell9. This extraordinary potency is attributed to the enzymatic activity of the A-chain. Diphtheria toxin A-chain catalytically transfers the ADPR moeity of NAD+ to a novel amino acid called 'diphthamide' in the elongation factor, EF2: NAD + + EF2~ADP-ribose-EF2 + nicotinomide. The ADP-ribose-EF2 can still bind to ribosomes in the presence of G T P but can no longer perform its normal role, namely the translocation of peptidyl t R N A from the A-site to the P-site on the ribosome. How the plant toxins and RIPs inactivate protein synthesis is less well understood. They directly inactivate the 60S subunit of the ribosomes but the enzymatic reaction involved has not been elucidated2. Immunotoxins Immunotoxins are defined as conjugates of cell-binding antibodies and either intact toxin, A-chains or RIPs. Two main strategies for making immunotoxins have been adopted. The first is to link the intact toxin

molecule to the antibody: the second is to link just the toxin A-chain or an RIP to the antibody. Immunotoxins made with intact toxins have consistently proved to be outstandingly powerful as cytotoxic agents for cells with appropriate antigens, often matching or surpassing the native toxin in potency. Predictably, however, they suffer from a lack of complete specificity because they can also bind by the B-chain moiety to non-target cells. A simple and very effective way of blocking the nonspecific binding properties of intact abrin or ricin conjugates in vitro is by competition with high concentrations of free galactose or lactose ~°. For therapy in animals, however, a permanent or semi-permanent blockade is needed because lactose co-administered with abrin or ricin conjugates is excreted too rapidly for protective levels to be maintained. One such method of blockade was recently described in which intact ricin immunotoxins were prepared with the galactose-binding site of the toxin sterically hindered by the antibody moiety itself. Monoclonal anti-Thy 1.1 antibody linked in this way to ricin was about 10 000 times more toxic to Thy 1.1-expressing AKR-A lymphoma cells than it was to EL 4 lymphoma cells which express the alternative Thy 1.2 allele11. Most workers have preferred to eliminate non-specific toxicity by linking isolated toxin A-chains or RIPs to antibodies. The coupling is usually achieved by introducing an activated disulphide (e.g. dithiopyridine) into the antibody component which reacts with the free sulphydryl group of the A-chain to form a conjugate in which the linkage is a disulphide bond. The RIPs lack a free thiol group and so one is introduced chemically before conjugation. Immunotoxins prepared by methods which do not introduce a disulphide bridge between the two protein components are usually much less active. This is probably because the A-chain moiety of the conjugate has, at some stage in the cytotoxic process, to be released by reduction from the carrier in order to diffuse to the ribosomes. Many immunotoxins have now been made by linking Achains to polyclonal- and monoclonal-antibodies against tumourassociated antigens, immunoglobulin determinants, T- and B-lymphocyte

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antigens, and haptens ~2. The toxicity of the intact toxin immunoimmunotoxins are almost wholly toxins. The B-chain of the toxin, in selective in their toxic effect upon addition to its cell-binding property, cultured cells bearing the corres- also appears to facilitate the entry of ponding antigen but their potency is the A-chain portion of the conjugate variable and often unpredictable. into the cytoplasm ~3.The way in which Some A-chain type immunotoxins are the B-chain assists A-chain entry is powerfully toxic to target cells while unknown. It could be that the B-chain others are weakly- or non-cytotoxic. simply protects the A-chain from A number of factors appear to deter- proteolysis. Alternatively, the B-chain mine the effectiveness of an A-chain- could insert into the membrane of the type immunotoxin. These factors endosome or lysosome and, either include the density of the surface alone or in conjunction with its cellantigen, the affinity of the antibody surface receptor molecule, form a used to prepare the immunotoxin, the channel through which the A-chain route of entry of the antigen- immuno- can pass to the cytoplasm. toxin complex into the cell, and the nature of the target antigen itself. Enhancing immunotoxin potency The variable cytotoxicity of the ATwo ways of enhancing the potency chain immunotoxins contrasts with of A-chain immunotoxins have been the regular and generally superior described. Firstly, the immunotoxins

BINDINGTO SURFACE RECEPTOR

often show a moderate to dramatic increase in potency in the presence of monensin ~4, ammonium chloride ~5 or chloroquine ~6.This is possibly because these reagents elevate endosomal and lysosomal pH, thereby inhibiting proteolytic digestion of the immunotoxin. Such agents are useful in vitro but may be too toxic to use in vivo. Secondly, ricin A-chain immunotoxins usually cooperate with free ricin Bchain to give a markedly enhanced and accelerated cytotoxic effect ~7. Similarly, the delivery of A- and B-chains to the target cell via two independent antibodies is usually much more effective in killing cells than the delivery of the A-chain immunotoxin alone ~3. The synergy afforded by Bchain-immunotoxins is obtained even when the B-chain has been chemically

ENDOCYTIC VESICLE

A-CHAIN IN CYTOPLASM

(D LYSOSOME

ENDOSOME

INHIBITION OF PROTEIN SYNTHESIS

ENZYMICALLY-ACTIVE A-CHAIN SEQUENCES~ ~ S HUMANCONSTANT REGION DOMAINS

I

B-CHAIN SEQUENCES LACKINGTHEGALACTOSE*--BINDING DOMAINS

'S-S iS 2

~KED-TOXlN CONJUGATES

MOUSE VARIABLE REGION DOMAINS 3

Fig. I. A model for the cytotoxic action of heterodimeric toxins such as ricin or diphtheria toxin. Fig. 2. Antibody-toxin conjugates for use in vivo. Single-chain toxins such as gelonin or saporin have been used in place of cytatoxin A chain. The B-chain cell binding site can be blocked by chemical or genetic modification and by steric obstruction by the antibody molecule. Fig. 3. Antibody-toxin hybrid molecules produced by genetic engineering.

L78 modified to attenuate its capacity to bind to cells, suggesting that this approach is probably feasible in vivo.

Anti-tumour effects in experimental animals Using the IgD-expressing murine leukaemia BCL1 as a model for chronic lymphocytic leukaemia in man, Krolick and his colleagues18 induced prolonged remissions in mice with advanced BCL1 tumours by a sequence of total lymphoid irradiation, splenectomy and intravenous administration o f an immunotoxin; the immunotoxin was constructed by linking ricin A-chain to antibodies against immunoglobulin 6-chain. Mice that received cytoreductive therapy and unconjugated antibody suffered early leukaemic relapse. Less dramatic, but nevertheless convincing, anti-tumour effects have been obtained by several other groups by administering ricin A-chain immunotoxins intratumourally to m i c e m-21 . Two reports of impressive antitumour effects of immunotoxins in guinea pigs have been published. Bernhard et aL = and Hwang et alfl 3 showed that the intravenous administration of anti-L10 hepatoma immunotoxins, made with diphtheria toxin A-chain or abrin A-chain, delayed or abolished the growth of established intradermal L 10 tumours. The abrin A-chain immunotoxin was also able to delay or inhibit tumour metastases to the lymph nodes: 20-40% of the treated animals had long-term, complete remissions. Recently, Thorpe and coworkers 24 linked saporin (an RIP from Saponaria officinalis) to monoclonal anti-Thy 1.1 antibody. A single intravenous injection of the immunotoxin into mice bearing a Thy l.l-expressing lymphoma allograft prolonged their survival time by an extent corresponding to that expected if 99.999% of the tumour cells had been eradicated. Interestingly, ricin A-chain coupled to the same antibody was 100-1000 times less effective than the saporin immunotoxin as an anti-tumour agent in vivo, even though the two immunotoxins were equally cytotoxic to the lymphoma cells in tissue culture. This difference suggests that the saporin immunotoxins are either more stable in vivo than those made with ricin A-chain, or that they do not interact as extensively with cellular or non-cellular components of the host (in ways

Trends in Biotechnology, Vol. 3, No. 7, 1985

that would promote their clearance or inhibit their diffusion throughout the tissues of the animal). Another unexpected finding with the saporin immunotoxin was that it caused gross necrosis in the liver of the recipients, an effect that was not seen with the ricin A-chain immunotoxin. Clearly, a better understanding of the interactions of immunotoxins with host tissues is needed before their activity in vivo can be optimized and before they can be regarded as safe for administration to patients.

Clinical applications of immunotoxins Bone marrow transplantation

Immunotoxins have found their first clinical application in depleting T-cells or tumour cells from bone marrow before allogeneic or autologous bone marrow transplantation, respectively. Bone marrow transplantation is used in conjunction with radiotherapy and chemotherapy in vivo to treat a variety of neoplastic diseases, particularly leukaemia. T h e doses of anti-tumour drugs or radiotherapy which must be used to eliminate tumour cells from the host are frequently toxic to the bone marrow, resulting in an aplastic state. In such cases, higher doses of the conditioning regimens can be used if the patient is reinfused with the bone marrow after supralethal chemotherapy or radiotherapy. The infused bone marrow reconstitutes the haematopoietic system and reverses the aplastic state. In autologous bone marrow transplantation, the marrow is removed from the patient during remission. When the patient relapses, he or she is given intensive radiochemotherapy and is then reinfused with his or her own bone marrow. Since the bone marrow is autologous, it engrafts and does not induce graft-versus-host disease (GVHD). Unfortunately, the marrow frequently contains residual tumour cells which are reinfused with the marrow stem cells. These tumour cells may cause a relapse later on. Therefore, in autologous marrow transplantation, it is desirable to purge the marrow oftumour cells before reinfusion. In animal models, this has been accomplished using immunotoxins containing ricin or ricin A-chain2% In humans, results are difficult to evaluate because relapses can be due either to the failure to eradicate the

primary disease in the patient, or to reinfusion of residual tumour cells in the bone marrow. In allogeneic transplantation, the patient is given intensive radiochemotherapy and is then infused with bone marrow ceils from a matched sibling. In such cases, G V H D can occur. It has been shown in animal models 26,and more recently in humans, that the use of anti-T-cell immunotoxins to kill the T-cells in the donor bone marrow can greatly reduce the incidence of GVHD. It remains to be determined whether reinfused natural killer (NK) cells will cause late graft rejection as they do in animal models27: if this is the case, immunotoxins directed against both T-cells and NKcells could be used. Tumour cell elimination in vivo

The other major application of immunotoxins is in the elimination of tumour cells in vivo. Judging from results in animal experiments, the most fruitful strategy will probably be to use cytoreductive therapy to eliminate the vast majority of tumour cells non-specifically and immunotoxin to kill any residual cells specifically. Before this approach can be tested in humans, a number of problems remain. Firstly, immunotoxins can damage the reticuloendothelial system (RES). The cells of the RES have receptors that bind the mannoseterminating oligosaccharides present on ricin and other toxins, necessitating deglycosylation of the toxins before their linkage to the antibody 2a. Similarly, the cells of the RES have receptors for the Fc portion of immunoglobulin and so may be damaged by immunotoxins prepared from intact antibody. Secondly, the stability of the linkage formed by the disulphide cross-linking agents currently available may not be sufficient for optimal effects in vivo. Thirdly, there is the problem oftumour cell heterogeneity. Antigen-negative tumour variants are likely to emerge unless cocktails of tumour-reactive immunotoxins are used. Similarly, toxin-resistant tumour variants may emerge if use is not made of cocktails ofimmunotoxins prepared from toxins whose mode of entry and toxicity do not overlap. Fourthly, neutralizing antibodies are certain to be produced by patients who are not severely immunosuppressed, making it imperative to keep changing

Trends in Biotechnology, VoL 3, No. 7, 1985

the toxin and antibody components to ones antigenically unfamiliar to the patient. Finally, the most promising results in experimental animals have been with leukaemia models in which the cells are freely available to systemically administered immunotoxin. The problems related to the penetration o f solid tumours with immunotoxins are formidable. Fragments of antibody may have to be used to make immunotoxins and agents employed to facilitate escape from the vascular system supplying the solid tumour.

Genetically engineered toxins and immunotoxins D N A sequences representing the entire structural genes of diphtheria toxin and the ricin precursor, preproricin, have been cloned 29,3°. Genes encoding ricin and diphtheria toxin A-chains were expressed in E.coli and the expressed proteins found to be enzymatically active. Several groups are now attempting to identify the amino acids in the B-chain that are responsible for cell binding activity. This knowledge should enable the nucleotide sequences which specify the cell binding sites to be deleted or altered by site-specific mutagenesis. The modified gene sequences, incorporated into appropriate vectors, could then be introduced into prokaryotic or eukaryotic cells and the modified toxin produced in large quantity. These approaches should enable the manufacture of modified toxins whose B-chains retain the ability to insert into membranes and transfer the A-chain to the cytosol but which no longer bind non-specifically to cells. It may also be possible using similar approaches to identify the most immunogenic stretches o f the A- and B-chains and to delete the D N A encoding these stretches from the gene. Another goal is to produce immunotoxins entirely by recombinant D N A technology. Transfection of myeloma cells with immunoglobulin genes ligated to toxin genes could facilitate the production of large quantities of immunotoxins provided that variants of the cells can be selected which are insusceptible to the toxic effects o f the A-chain. T h e feasibility of this approach has beefi elegantly

TIB- 7

179 demonstrated by Neuberger and his (1984) Microbiol. Rev. 48, 199-221 9 Uchida, T. (1982) In Molecular Action of colleagues for monoclonal anti-hapten Toxins and Viruses (Cohen, P. and Van antibodies ligated to staphylococcal Heyningen, S., eds), pp 1 31, Elsevier nuclease 31. The linkage o f the A-chain Biomedical Press to an antibody via conventional D N A 10 Youle, R. J. and Neville, D. M. (1980) splicing methods might prove probProc. Natl Acad. Sci. USA 77, 5483-5486 lematic since the A-chain and the 11 Thorpe, P. E., Ross, W. C. J., Brown, A. N. F., Meyers, C., Cumber, A. J., Foxwell, antibody must be cleaved in the B. M. J. and Forrester, J. A. (1984) Eur..7. endosome. However, it is conceivable Biochem. 140, 63-71 that these genes could be ligated with 12 Thorpe, P. E. and Ross, W. C. J. (1982) an intervening sequence encoding a Immunol. Rev. 62, 119-158 polypeptide that can be split by pro- 13 Youle,R. J. and Neville, D. M. (1982).,7. Biol. Chem. 257, 1598-1601 teolytic enzymes inside the target cell 14 Casellas, P., Bourrie, B. J. P., Gros, P. and or associated with its surface. Jansen, F. K. (1984) J. Biol. Chem. 259, 9359-9364 Future prospects 15 Casellas, P., Brown, J. P., Gros, P., Hellstrom, I., Jansen, F. K., Poncelet, P., In summary, immunotoxins have Roncucci, R., Vidal, H. and Hellstrom, K. enormous potential in bone marrow E. (1982)Int. J. Cancer 30, 437-443 transplantation and in the treatment of 16 Ramakrishman, S. and Houston, L. L. cancer in vivo. T h e y may also be valu(1984) Science 223, 58-61 able in the treatment o f infectious and 17 McIntosh, D. P., Edwards, D. C., Cumber, A. J., Parnell, G. D., Dean, C. J., autoimmune diseases. In such cases, Ross, W. C. J. and Forrester, J. A. (1983) the elimination of helper or suppressor FEBS Lett. 164, 17-20 T-cells may alter the disease state. The 18 Krolick, K. A., Uhr, J. W., Slavin, S. and construction of the ideal reagents will Vitetta, E. S. (1982)ft. Exp. Med. 155, require more information concerning 1797-1809 the generation of stable linkers, the 19 Blythman, H. E., Casellas, P., Gros, D., Jansen, F. K., Pashucci, F., Pan, B. and properties of immunotoxins conVidal, H. (1981) Nature 290, 145-146 structed with antibody fragments, the 20 Kishida, K., Mashuo, Y., Saito,M., Hara, pathways of entry of immunotoxins T. and Fuji, H. (1983) Cancer Immunol. into cells, and the reasons why some Immunother. 16, 93-97 immunotoxins are effective while 21 Seto, M., Umemoto, N., Saito, M., Masuno, Y., Hara, T. and Takahashi, T. others are not. Within the next decade, (1982) Cancer Res. 42, 5209-5215 it may be possible to generate a new 22 Bernhard, M. I., Foon, K. A., Oehmann, family ofimmunotoxins which will kill T. N., Key, M. E., Hwang, K. M., Clarke, target cells with great potency in vivo G. C., Christensen, W. L., Hoyer, L. C., Hanna, M. G. and Oldham, R. K. (1983) while causing little or no harm to norCancer Res. 43, 4420-4428 mal host tissue. 23 Hwang, K. M., Foon, K. A., Cheung, P. H., Pearson, J. W. and Oldham, R. K. Acknowledgement (1984) CancerRes. 44, 4578 4586 T h e authors wish to thank Dr Ellen 24 Thorpe, P. E., Brown, A. N. F., Bremner, J. A. G., Foxwell, B. M. J. and Stirpe, F. S. Vitetta for her valuable comments J. Natl Cancer Inst. (in press) and suggestions during the prepara25 Thorpe, P. E,, Mason, D. W., Brown, tion of the manuscript. A. N. F., Simmonds, S. J., Ross, W. C. J., Cumber, A, J. and Forrester, J. A. (1982) Nature 297, 594-596 References 26 Vallera, D. A., Youle, R. J., Neville, 1 Kohler, G. andMiistein, C. (1975)Nature D. M. and Kersey, J. H. (1982)J. Exp. 256, 495-497 Med. 155, 949-954 2 Olsnes,S. and Pihl, A. (1982) InMolecular 27 Warner, J. F. and Dennert, G. (1985)J. Action of Toxins and Viruses (Cohen, P. and Exp. Med. 161, 563-576 Van Heyningen, S., eds), pp 51 105,Else- 28 Thorpe, P. E., Detre, S. I., Foxwell, vier Biomedical Press B. M. J., Brown, A. N. F., Skilleter, 3 Pappenheimer, A. M. (1977) Annu. Rev. D. N., Wilson, G., Forrester, J. A. and Biochem. 256, 69-94 Stirpe, F. (1985) Eur. J. Biochem. 147, 4 Barbieri, L. and Stirpe, F. (1982) Cancer 197-206 Surveys 1,489-520 29 Greenfield, L., Bjorn, M. J., Horn, G., Fong, D., Buck, G. A., Collier, R. J. and 5 Eidels, L., Proia, R. L. and Hart, D. A. Kaplan, D. A. (1983) Proc. NatlAcad. Sci. (1983) Microbiol. Rev. 47, 596-620 USA 80, 6853-6857 6 Goldstein, J. L., Anderson, R. G. W. and Brown, M. S. (1979)Nature 279, 679 685 30 Lamb, F. I., Roberts, L. M. and Lord, M. J. (1985) Eur. ,,7. Biochem. 148, 265-270 7 Dautry-Varsat, A. and Lodish, H. F. (1983) Sci. Am. 249, 48-54 31 Neuberger, M. S., Williams, G. T. and Fox, R. O. (1984) Nature 312, 604-608 8 Middlebrook, J. L. and Dorland, R. B.