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(1986) ]. Bacterio]. 167, 291-298 35 Kimura, S., Makino, K., Shinagawa, H., Amemura, M. and Nakata, A. (1989) Mol. Gen. Genet. 215,374-380 36 Yamada, M., Makino, K., Amemura, M., Shinagawa, H. and Nakat, A. (1989) J. Bacterio]. 171, 5601-5606 37 Ohnuki, T., Imanaka, T. and Aiba, S. (1985) J. BacterioL 164, 85-94 38 Distler, J., Ebert, A., Mansouri, K., Pissowotzki, K., Stockmann, M. and
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Piepesberg, W. (1987) Nucleic Acids Res. 15, 8041-8056 39 Murakami, T., Holt, T. G. and Thompson, C. ]. (1989) J. Bacterio]. 171, 1459-1466 40 P6rez-Martin, J., Del Solar, G. H., de la Campa, A. G. and Espinosa, M. (1988) Nucleic Acids Res. 16, 9113-9126 41 Plaskon, R. R. and Wartell, R. M. (1987) Nucleic Acids Res. 15,
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Immunotoxins: status and prospects Robert A. Spooner and J. Michael Lord Immunotoxins, which are conjugates of cell-binding antibodies and toxins, show considerable promise in the treatment of certain cancers. Genetic engineering is increasingly being used to refine and modify these conjugates, and it is now possible to design, express and purify completely recombinant therapeutic molecules. Most of the therapeutic drugs available for treatment of cancer rely on uptake by rapidly dividing cells, a poor basis for selectivity since it will still result in significant damage to normal cells. Tumour cells, though, differ not only in freedom from normal growth constraints, but also in an altered spectrum of gene expression. Where this results in expression of tumour-specific or tumour-associated antigens at the cell surface, antibodies recognizing these may be considered for use as therapeutic agents. Such antibodies may be directly cytotoxic, stimulating complement fixation that results in cell lysis or marshalling a variety of white cells to a tumour. However, despite these characteristics, there is no convincing evidence of any cancer being cured exclusively by administration of antibodies, although some remissions have been observed. Nevertheless, the impressive ability of antibodies to differentiate be-
R. A. Spooner is at the Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar EN6 3LD, UK and I. M. Lord is at the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. ~) 1990, Elsevier Science Publishers Ltd (UK)
tween closely related antigens makes them very attractive targeting devices, particularly since the required specificity can often be elicited by immunizing appropriate animals with the target tumour tissue. The animal's B lymphocytes, dedicated to the production of antibodies, can be fused with related plasmacytoma cells to produce hybridomas, which are capable of making antibodies in cell culture 1. Each individual hybridoma provides a virtually unlimited source of a monoclonal antibody that can be highly purified and screened for reactivity against the initiating tumour immunogen. If a tumour-specific monoclonal antibody is chemically linked to suitable drugs, toxins or radionuclides then cytotoxic agents with a high degree of selectivity are formed. Such molecules fit the description of 'Zauberkugeln' or 'magic bullets' proposed at the turn of this century by Paul Ehrlich, so the idea is by no means new. In this article we concentrate on the production and uses of one particular subset of magic bullets, immunotoxins, comprising a monoclonal antibody coupled to a naturally occurring plant or bacterial toxin.
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785-796 42 Struhl, K. (1985) Nature 317, 822-824 43 Reitzer, L. J. and Magasanik, B. (1986) Cell 45, 785-792 44 Buttner, M. J. (1989) Mol. Microbiol. 3, 1653-1659 45 Buttner, M. ]., Smith, A. M. and Bibb, M. J. (1988) Cell 52,599-609 46 Westpheling, J. and Brawner, M. (1989) J. Bacteriol. 171, 1355-1360
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Toxins A wide range of organisms, including bacteria, fungi and higher plants, produce protein toxins. Such toxins presumably have either a defensive role against predators or an offensive role against host or 'prey' cells. The majority of enzyme toxins produced are directed against protein translation, either against translation factors or ribosome function. In principle, any of quite a wide variety of toxins capable of acting against the target cell may be used, but bacterial toxins such as diphtheria toxin and plant toxins such as ricin have been used most frequently in the design of immunotoxins. Toxins fall into two broad categories: those with both an A (or active) chain(s) that has enzymatic activity and a B (or binding) chain(s) that binds to cell surfaces and may be involved in uptake, and those which have only A chains - for use in immunotoxins the latter type must be delivered to the cell by other means for them to have a cytotoxic effect. Bacterial toxins
Diphtheria toxin is secreted by the bacterium Corynebacterium d i p h theriae lysogenic for the fito×+ bacteriophage. It is produced as a preproprotein that is trimmed to a mature form consisting of a cytotoxic A chain disulphide-linked to a cellbinding B chain. In spite of considerable effort, the identity of its receptor on sensitive cells remains uncertain. After binding, its receptors become clustered in coated pits that are endocytotically invaginated. The endosome becomes increasingly acidified and at pH 5.3 the toxic A chain is released from the receptorbound B chain, and is translocated
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across the endosomal membrane into the cytoplasm 2. This process is almost certainly aided by a conformational change in the B chain, exposing a previously hidden hydrophobic domain. Having gained access to the cytoplasm, the A chain catalytically modifies its target, transferring the ADP-ribose moiety of NAD + to a modified histidine residue (called diphthamide) of mammalian elongation factor 2 (EF-2). Modified EF2 can no longer translocate peptidyl tRNA from the ribosome's A site to the P site, resulting in a cessation of protein manufacture and cell death. Pseudomonas exotoxin A is a single protein, with three clearly defined domains. One is responsible for cell binding, one catalytically inactivates EF2 in a similar manner to diphtheria toxin and the third is responsible for translocating the entire polypeptide, across a membrane, into the cytoplasm. A third class of bacterial proteins is represented by Shigel]a toxin and Shiga-like-toxins (SLTs). These consist of a toxic A chain linked to five smaller B chains. The A chains of these proteins show extensive similarities with the A chains of plant toxins.
Plant toxins The most commonly used plant toxin is ricin, purified from the seeds of the castor bean, Ricinus communis 3. Ricin consists of a cytotoxic A chain disulphide-linked to a cellbinding B chain. The cellular receptors are a variety of glycoproteins and glycolipids that terminate in galactose residues. In contrast to bacterial toxins, entry of plant toxins into the cell is not well understood. They are endocytosed primarily, though probably not exclusively, in coated pits and vesicles. There is no evidence that low pH is required for membrane transport; the A chains probably enter the cytoplasm from trans-Golgi cisternae rather than from acidic endosomes 4. There may be some contribution by the B chain in this process, but there is no obvious B chain hydrophobic domain. The A chains have COOH terminal hydrophobic domains and may be capable of membrane insertion if brought sufficiently close to a membrane by the B chain. Once in the cytoplasm, the A
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--Fig.
1
pit pit
t ..... ~
coated vesicle .,~lli ...~~1
|Ill,
--_
Lysosome
/
~
.,4
~
Endosor e ~/ I z
, •
| Trans-Golgi ,,~etwork
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0
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Entry of diphtheria toxin ( ~ ), ricin ( O-e ), and ricin A chain immunotoxins ( /~0 ), into a cell (A chains are shown by open squares or circles and B chains by solid squares or circles.) After binding to receptors or cell surface antigens, toxins and immunotoxins are delivered to endosomes via coated pits and coated vesicles. Ricin may also enter via smooth pits. A certain proportion may be recycled back to the cell surface or delivered to lysosomes, where it is presumably degraded. Diphtheria toxin A chain is translocated across the endosomal membrane. Ricin and immunotoxins are probably routed further, to the trans-Golgi network where release of the A chain into the cytosol occurs, and soluble translation factors or the ribosome are attacked.
chain acts as a N-glycosidase, catalytically removing one specific, highly conserved, adenine residue in a GAGA sequence, borne on a stem-loop structure in 28S rRNA 5. Ribosomes so modified are deficient in initiation and elongation functions and, as a consequence, protein manufacture ceases. The routes by which bacterial and plant toxins enter target cells is illustrated in Figure 1. A second class of similar proteins are also synthesized by plants. These are the RIPS, or ribosomeinactivating proteins, and consist of single chain molecules very similar to, and probably evolutionary related to, the A chains of plant toxins 6. Examples are pokeweed antiviral protein from the seeds and leaves of Phytolacca americana, gelonin from the seeds of Gelonium multiflavum, saporin from the seeds of Saponaria officinalis and trichosanthin from the Chinese cucumber.
Since RIPs lack a cell-binding B chain, they are relatively non-toxic. However, at least for trichosanthin, it appears that macrophages, particularly those infected with HIV, are sensitive, so these proteins may have uses in AIDS treatment 7. Immunotoxins
Immunotoxins are conjugates of ce]l-binding antibodies and intact toxins, their toxic fragments or RIPs. Since the number of antibodies that reach a tumour, particularly in solid tissue, may be small, the potency of the delivered toxin must be high. Immunotoxins prepared with intact toxins are often exceedingly toxic, but the ability of the B chain to bind cells opportunistically may override the specificity endowed by the antibody, resulting in fatal intoxication of non-target ceils. In at least one ricin-based immunotoxin, it is thought that the antibody component sterically hinders the B chain,
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producing a conjugate with acceptable specificity8. For other ricinbased immunotoxins, a blockade can be provided in vitro by provision of excess free galactose or lactose, but the rapid metabolic excretion of these sugars precludes this approach in vivo. An alternative approach that has provided some very effective immunotoxins uses isolated A chains or RIPs. These are usually constructed by introduction of an activated disulphide into an antibody, which is then reacted with the free sulphydryl of the A chain, or a free thiol that has been introduced into a RIP (Ref. 9). Since they lack B chains, these immunotoxins show good specificity but their cytotoxicity varies from powerful to very weak. At least part of the reason for this variability lies in the nature of the target antigen: an efficient binding site needs a high antigen density, high antibody affinity, must promote efficient endocytosis when bound by immunotoxin and must direct a sufficient portion of internalized immunotoxins away from the lysosomes, probably to the trans-Golgi network. For example, immunotoxins made with some antibodies to CD2 work poorly since they are directed to, and degraded within, lysosomes ~°.
Enhancing agents Reagents that reduce lysosomal degradation or alter intracellular routing should enhance the potency of immunotoxins; this may explain the effect of administration of monensin, chloroquine or ammonium chloride to culture cells treated with immunotoxin 11. Enhancement has also been noted using inhibitors of protein synthesis and glycosylation12. All the above examples have no obvious in vivo application. An alternative means of achieving in vivo enhancement might be the provision of ricin B chain, either free or as a B chain immunotoxin 13 chemically modified to reduce its cell binding activity, but still able to potentiate A chain toxicity. A further approach to immunotoxin construction depends on the use of bispecific antibodies. One arm of the antibody is specific for a cell surface antigen, the other for an RIP such as saporin. The two binding
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specificities are joined by a nonreducible thioether linkage and will bind to and deliver saporin to target cells 15. Evaluation of immunotoxins and clinical trials Guinea pigs and normal and immunodeficient mice bearing tumour cell implants have been treated with a range of immunotoxins, allowing systematic appraisal of immunotoxin efficacy. Those based on abrin A chain, saporin and Pseudomonas exotoxin A can cause significant liver damage. In the case of ricin A chain-based immunotoxins, chemical pretreatment of the toxin A chain to destroy its sugar residues resolves this problem 16. Animal models have also been used for the testing of novel heterobifunctional cross-linking agents. Overall, these models have allowed refinement of immunotoxins, and anti-tumour effects ranging from the not so dramatic (prolonged survival, inhibition • of
tumour growth), to the very dramatic (complete remissions), have been reported. These results have been sufficiently encouraging to warrant clinical trials in humans. Where cells are easily accessible to antibodies, clinical results from use of ricin A chain based immunotoxins are highly encouraging. They have been used to purge samples of bone marrow of malignant B cells prior to reinfusion into patients whose own marrow has been destroyed by radio- or chemotherapy; to remove T cells from allogeneic bone marrow grafts (marrow from matched siblings) thereby reducing the incidence of 'graft versus host disease', and also in treatment of autoimmune illnesses. In contrast, immunotoxins fail to perform well for other tumours, such as some leukaemias, breast cancer, melanoma and colon cancer, although encouraging signs such as tumour regression are seen in some patients. A full description of animal
\,,;
,7
" N o w this is a m o r e t y p i c a l a n t i - m o u s e r e s p o n s e !"
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models and clinical results can be found in Ref. 17.
Limitations ofimmunotoxins The failure of immunotoxins, so far, to fulfil their potential may be attributed to a number of causes, such as: • failure of the large conjugates (Mr /> 180000) to permeate solid turnouts; • neutralization of the therapeutic molecules by patients who are not severely immunosuppressed; • circulation of shed antigen, competing for the immunotoxin; • escapes of antigen-negative turnout variants; • the nature and density of the target antigen; • non-specific binding of the constant region of the antibody to nontarget cells. Perhaps the major limitation is the appearance of side effects, not predicted by animal models, that severely restrict the effective dose. Principal side effects include fluid retention and muscular pain 17. The answers to some of these problems lie in design of novel linkers, treatment with mixtures of different immunotoxins, alternating regimes of immunotoxin administration, and a return to the study of model systems. Some of the problems can be addressed directly through genetic engineering, and although these studies are at a preliminary stage, they do show extraordinary promise. A good example is the use of genetic "engineering to produce 'humanized' antibodies (see section below). The use of murine antibodies in human patients provokes a h u m a n anti-mouse antibody response. Because of continuing difficulties in producing appropriate h u m a n monoclonal antibodies, it will obviously be necessary to reduce the immunogenicity of murine antibodies before therapeutic use.
The role of genetic engineering Soluble, enzymically active recombinant ricin A chain has been expressed in, and purified from, Escherichia coli 18. Since it is nonglycosylated, it is eminently suitable
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for immunotoxin construction. Similarly, diphtheria toxin A chain and P s e u d o m o n a s exotoxin A have been expressed in active form in E. coli TM. These proteins (and corresponding genes) are now very well characterized, and their structures are being elucidated by X-ray crystallography. This knowledge provides a sound basis for mutagenesis: can toxicity be increased, and can non-specific cellbinding and immunogenicity be decreased? Antibodies themselves are well suited to protein engineering techniques, and enormous progress has been made in this field. Variable regions of mouse antibodies have been fused to h u m a n constant regions to give chimaeric molecules designed to reduce the immunogenic rodent component 2° ('humanizing' the antibody). The most sophisticated extension of this idea involved the painstaking replacement of just the complementarity-determining regions of a human antibody with murine counterparts, resulting in molecules that are almost completely human, but with murine specificity. Such antibodies are expected to elicit very much reduced human anti-mouse antibody responses in patients 21, and some remarkable remissions have been observed using a humanized antibody 22. The effector functions of an antibody are not strictly required for immunotoxin construction. Since the constant domains can be replaced by foreign proteins such as bacterial nucleases 23, a logical extension of this idea is the replacement of the carboxy-terminal end of an antibody heavy chain with a molecule such as ricin A chain. In practice, this may be successful at the level of protein engineering, but the extreme toxicity of ricin precludes the expression of these molecules in the eukaryotic systems that have been used for producing intact antibodies. Antibodies are normally secreted into the endoplasmic reticulum during protein translation, but just one molecule of an active antibody-ricin fusion, if it leaked into the cytoplasm, could kill the cell that made it. Such procedures, which do not result in cell death, are likely to select for protein with mutant or deleted ricin A chains. A different approach is therefore
required. One that is particularly intriguing is the genetic fusion of ricin A chain and protein A (Ref. 24). Protein A is a bacterial protein from Staphylococcus aureus that binds immunoglobulins, particularly IgG antibodies. It therefore substitutes for the chemical' linkage between antibody and toxin. Unfortunately, such immunotoxins are not toxic, probably since the ricin A chain is not capable of translocating across a membrane when synthesized as part of a fusion protein. Introduction of a trypsin-sensitive site between the ricin A chain and protein A halves of the fusion results in immunotoxins with acceptable potency (M. O'Hare, L. M. Roberts, P. E. Thorpe and J. M. Lord, unpublished). This type of strategy is very attractive since it is generally applicable to a wide range of antibodies. Is it possible to extend this idea and make a wholly recombinant immunotoxin? Until recently, the answer has been no, but the expression of antigen-binding domains of antibodies in E. cob makes this approach feasible. Antigen-binding fragments (Fvs) (Ref. 25), singlechain linked Fvs (Ref. 26), and disulphide-linked antibody arms (Fablike molecules) have been expressed in E. coB. Since these are very much smaller than whole antibodies, they are expected to diffuse into solid tumours, and are therefore good candidates for immunotoxin construction. Indeed, a single-chain immunotoxin based on an Fv recognizing the interleukin-2 receptor fused to a truncated form of Pseudom o n a s exotoxin A has been described 27. Exotoxin A is an ideal molecule for this work, since it has its own translocation domain, and it retains translocation and toxic activities even when its cell-binding domain is deleted or it is fused to other molecules such as growth factors and CD4 (Ref. 19). Animal models suggest t h a t the use of ricin A chain immunotoxins may be preferable to others, since if the A chain is deglycosylated, there is little liver damage. However, since ricin A chain cannot translocate as part of a fusion protein, single-chain ricin A immunotoxins will probably require specific engineering to allow release of the toxic component near a cell membrane. A whole range of Fv- and ricin-based recombinant im-
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m u n o t o x i n s is likely to be developed. T h e recent discovery that recomb i n a n t single variable d o m a i n s of antibodies can retain good antigenb i n d i n g properties 28 m a y in the future be extrapolated to i m m u n o toxins in order to design, express and purify v e r y small molecules, consisting p e r h a p s of o n l y two domains. Perhaps this idea c o u l d be taken even further: in one case, a p e p t i d e c o r r e s p o n d i n g to a single c o m p l e m e n t a r i t y determining region (CDR) r e p r o d u c e s the binding ability of a w h o l e a n t i b o d y 29. An i m m u n o toxin based on this w o u l d be very small indeed. If a m o l e c u l e of this size p r o v e d to be sufficient to target a toxin, this w o u l d raise an intriguing possibility - can peptides derived from growth factors, h o r m o n e s and other ligands be used as targeting devices?
Future prospects In s u m m a r y , i m m u n o t o x i n s have s h o w n e n o r m o u s , as yet unrealized, potential. W h e r e cells are accessible to antibodies and bear suitable antigens, they p r o v i d e very good targets. For other diseases, side effects not predictable from animal m o d e l s are severely limiting, and p e n e t r a t i o n of solid t u m o u r s remains a formidable problem. Protein engineering m a y p r o v i d e m o l e c u l e s more suited to these tasks. Progress is also likely to be m a d e in design of optimum chemical linkers, identification of suitable antigenic targets, and treatment with cocktails of i m m u n o t o x i n s . The field of i n f l u e n c e will almost certainly grow, and i m m u n o t o x i n s will be u s e d for t r e a t m e n t of infectious diseases (e.g. HIV), a u t o i m m u n e diseases, allergies, fungal and parasitic infections, and depletion of rejectioninitiating dendritic cells from organs prior to transplantation. W h e t h e r t h e y will p r o v e to be suitable for c a n c e r therapy, apart from m i n i m a l disease and easily accessible tumours, r e m a i n s an o p e n question, but progress is such that in the next few years it m a y be possible to create w h o l e n e w families of i m m u n o toxins capable of killing target cells w i t h great p o t e n c y whilst causing m i n i m a l h a r m to normal tissues.
Acknowledgements We wish to t h a n k D. Allen, K. Rendall and P. E. T h o r p e for their critical appraisals of this article.
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References 1 Kohler, G. and Milstein, C. (1975) Nature 256,495-497 2 0 l s n e s , S., Moskaug, J., Stenmark, H. and Sandvig, K. (1988) Trends Biochem. Sci. 13,348-351 3 0 l s n e s , S. and Pihl, A. (1982) in Mol-
16 Blakey, D. C., Watson, E. J., Knowles, P. P. and Thorpe, P. E. (1987) Cancer Res. 4 7 , 9 4 7 - 9 5 2
17 Vitetta, E. S. and Thorpe, P. E. in Biologic Therapy of Cancer: Principles and Practice (De Vita, V.,
ecular Action of Toxins and Viruses
4
5 6 7
8
9 10 11 12 13
14 15
(Cohen, P. and Van Heyningen, S., eds), pp. 51-105, Elsevier Biomedical Press Van Deurs, B., Tonnessen, T. I., Petersen, D. W., Sandvig, K. and Olsnes, S. (1986) J. Cell. Biol. 102, 37-47 Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130 Barbieri, L. and Stirpe, F. (1982) Cancer Surveys 1,489-520 McGrath, M. S., Hwang, K. M., Caldwell, S. E., Gaston, I., Luk, K. C., Wu, P., Ng, V. L., Crowe, S., Daniels, J., Marsh, J., Deinhart, T., Lekas, P. V., Vennari, J. C., Yeung, H-W. and Lifson, J. D. (1989) Proc. Natl Acad. Sci. USA 86, 2844-2848 Thorpe, P. E., Ross, W. C. J., Brown, A. N. F., Meyers, C., Cumber, A. J., Foxwell, B. M. J. and Forrester, J. A. (1984) Ear. J. Biochem. 140, 63-71 Thorpe, P. E. and Ross, W. C. J. (1982) Immunol. Rev. 62,119-158 Press, O. W., Vitetta, E. S., Farr, A. G., Hansen, T. A. and Martin, P. J. (1986) Cell. Immunol. 102, 10-20 Blakey, D. C., Wawrzynczak, E. J., Wallace, P. M. and Thorpe, P. E. (1988) Progress in Allergy 45, 50-90 Sandvig, K., Tonnessen, T. I. and Olsnes, S. (1986) Cancer Res. 46, 6418-6422 McIntosh, D. P., Edwards, D. C., Cumber, A. J., Parnell, G. D., Dean, C. J., Ross, W. C. J. and Forrester, J. A. (1983) FEBS Lett. 164, 17-20 Youle, R. J. and Neville, D. M. (1982) J. Biol. Chem. 257, 1598-1601 Glennie, M. J., McBride, H. M., Worth, A. T. and Stevenson, G. T. 1987) J. Immunol. 139, 2367-2375
18
19 20
21 22
23 24 25 26
27
28 29
Hillman, S. and Rosenberg, S., eds), J. B. Lippincott (in press) O'Hare, M., Roberts, L. M., Thorpe, P. E., Watson, G. J., Prior, B. and Lord, J. M. (1987) FEBS Lett. 216, 73-78 Pastan, I. and Fitzgerald, D. (1989) J. Biol. Chem. 264, 15157-15160 Morrison, S. L., Johnson, M. J., Herzenberg, L. A. and Oi, V. T. (1984) Proc. Natl Acad. Sci. USA 81, 6851-6855 Reichmann, L., Clark, M., Waldmann, H. and Winter, G. (1988) Nature 332,323-327 Hale, G., Clark, M. R., Marcus, R., Winter, G., Dyer, M. J. S., Phillips, J. M., Reichmann, L. and Waldmann, H. (1988) Lancet i, 1394-1399 Neuberger, M. S., Williams, G. T. and Fox, R. O. (1984) Nature 312, 604-608 Kim, J. H. and Weaver, R. F. (1988) Gene 72,315-321 Skerra, A. and Pliickthun, A. (1988) Science 240, 1038-1041 Huston, J. S., Levinson, D., MudgettHunter, M., Tae, M-S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R. and Opperman, H. (1988) Proc. Natl Acad. Sci. USA 85, 5879-5883 Chaudhury, V. K., Queen, C., Junghams, R. P., Waldmann, T. A., Fitzgerald, D. J. and Pastan, I. (1989) Nature 339, 394-396 Ward, E. S., Giissow, D., Griffiths, A. D., Jones, P. T. and Winter, G. (1989) Nature 341, 544-546 Williams, W. V., Moss, D. A., KeiberEmmons, T., Cohen, J. A., Myers, J. N., Weiner, D. B. and Greene, M. I. (1989) Proc. Natl Acad. Sci. USA 86, 5537-5541
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(1986) ]. Bacterio]. 167, 291-298 35 Kimura, S., Makino, K., Shinagawa, H., Amemura, M. and Nakata, A. (1989) Mol. Gen. Genet. 215,374-380 36 Yamada, M., Makino, K., Amemura, M., Shinagawa, H. and Nakat, A. (1989) J. Bacterio]. 171, 5601-5606 37 Ohnuki, T., Imanaka, T. and Aiba, S. (1985) J. BacterioL 164, 85-94 38 Distler, J., Ebert, A., Mansouri, K., Pissowotzki, K., Stockmann, M. and
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Piepesberg, W. (1987) Nucleic Acids Res. 15, 8041-8056 39 Murakami, T., Holt, T. G. and Thompson, C. ]. (1989) J. Bacterio]. 171, 1459-1466 40 P6rez-Martin, J., Del Solar, G. H., de la Campa, A. G. and Espinosa, M. (1988) Nucleic Acids Res. 16, 9113-9126 41 Plaskon, R. R. and Wartell, R. M. (1987) Nucleic Acids Res. 15,
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Immunotoxins: status and prospects Robert A. Spooner and J. Michael Lord Immunotoxins, which are conjugates of cell-binding antibodies and toxins, show considerable promise in the treatment of certain cancers. Genetic engineering is increasingly being used to refine and modify these conjugates, and it is now possible to design, express and purify completely recombinant therapeutic molecules. Most of the therapeutic drugs available for treatment of cancer rely on uptake by rapidly dividing cells, a poor basis for selectivity since it will still result in significant damage to normal cells. Tumour cells, though, differ not only in freedom from normal growth constraints, but also in an altered spectrum of gene expression. Where this results in expression of tumour-specific or tumour-associated antigens at the cell surface, antibodies recognizing these may be considered for use as therapeutic agents. Such antibodies may be directly cytotoxic, stimulating complement fixation that results in cell lysis or marshalling a variety of white cells to a tumour. However, despite these characteristics, there is no convincing evidence of any cancer being cured exclusively by administration of antibodies, although some remissions have been observed. Nevertheless, the impressive ability of antibodies to differentiate be-
R. A. Spooner is at the Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Potters Bar EN6 3LD, UK and I. M. Lord is at the Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK. ~) 1990, Elsevier Science Publishers Ltd (UK)
tween closely related antigens makes them very attractive targeting devices, particularly since the required specificity can often be elicited by immunizing appropriate animals with the target tumour tissue. The animal's B lymphocytes, dedicated to the production of antibodies, can be fused with related plasmacytoma cells to produce hybridomas, which are capable of making antibodies in cell culture 1. Each individual hybridoma provides a virtually unlimited source of a monoclonal antibody that can be highly purified and screened for reactivity against the initiating tumour immunogen. If a tumour-specific monoclonal antibody is chemically linked to suitable drugs, toxins or radionuclides then cytotoxic agents with a high degree of selectivity are formed. Such molecules fit the description of 'Zauberkugeln' or 'magic bullets' proposed at the turn of this century by Paul Ehrlich, so the idea is by no means new. In this article we concentrate on the production and uses of one particular subset of magic bullets, immunotoxins, comprising a monoclonal antibody coupled to a naturally occurring plant or bacterial toxin.
0167 - 9430/90/$2.00
785-796 42 Struhl, K. (1985) Nature 317, 822-824 43 Reitzer, L. J. and Magasanik, B. (1986) Cell 45, 785-792 44 Buttner, M. J. (1989) Mol. Microbiol. 3, 1653-1659 45 Buttner, M. ]., Smith, A. M. and Bibb, M. J. (1988) Cell 52,599-609 46 Westpheling, J. and Brawner, M. (1989) J. Bacteriol. 171, 1355-1360
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Toxins A wide range of organisms, including bacteria, fungi and higher plants, produce protein toxins. Such toxins presumably have either a defensive role against predators or an offensive role against host or 'prey' cells. The majority of enzyme toxins produced are directed against protein translation, either against translation factors or ribosome function. In principle, any of quite a wide variety of toxins capable of acting against the target cell may be used, but bacterial toxins such as diphtheria toxin and plant toxins such as ricin have been used most frequently in the design of immunotoxins. Toxins fall into two broad categories: those with both an A (or active) chain(s) that has enzymatic activity and a B (or binding) chain(s) that binds to cell surfaces and may be involved in uptake, and those which have only A chains - for use in immunotoxins the latter type must be delivered to the cell by other means for them to have a cytotoxic effect. Bacterial toxins
Diphtheria toxin is secreted by the bacterium Corynebacterium d i p h theriae lysogenic for the fito×+ bacteriophage. It is produced as a preproprotein that is trimmed to a mature form consisting of a cytotoxic A chain disulphide-linked to a cellbinding B chain. In spite of considerable effort, the identity of its receptor on sensitive cells remains uncertain. After binding, its receptors become clustered in coated pits that are endocytotically invaginated. The endosome becomes increasingly acidified and at pH 5.3 the toxic A chain is released from the receptorbound B chain, and is translocated
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across the endosomal membrane into the cytoplasm 2. This process is almost certainly aided by a conformational change in the B chain, exposing a previously hidden hydrophobic domain. Having gained access to the cytoplasm, the A chain catalytically modifies its target, transferring the ADP-ribose moiety of NAD + to a modified histidine residue (called diphthamide) of mammalian elongation factor 2 (EF-2). Modified EF2 can no longer translocate peptidyl tRNA from the ribosome's A site to the P site, resulting in a cessation of protein manufacture and cell death. Pseudomonas exotoxin A is a single protein, with three clearly defined domains. One is responsible for cell binding, one catalytically inactivates EF2 in a similar manner to diphtheria toxin and the third is responsible for translocating the entire polypeptide, across a membrane, into the cytoplasm. A third class of bacterial proteins is represented by Shigel]a toxin and Shiga-like-toxins (SLTs). These consist of a toxic A chain linked to five smaller B chains. The A chains of these proteins show extensive similarities with the A chains of plant toxins.
Plant toxins The most commonly used plant toxin is ricin, purified from the seeds of the castor bean, Ricinus communis 3. Ricin consists of a cytotoxic A chain disulphide-linked to a cellbinding B chain. The cellular receptors are a variety of glycoproteins and glycolipids that terminate in galactose residues. In contrast to bacterial toxins, entry of plant toxins into the cell is not well understood. They are endocytosed primarily, though probably not exclusively, in coated pits and vesicles. There is no evidence that low pH is required for membrane transport; the A chains probably enter the cytoplasm from trans-Golgi cisternae rather than from acidic endosomes 4. There may be some contribution by the B chain in this process, but there is no obvious B chain hydrophobic domain. The A chains have COOH terminal hydrophobic domains and may be capable of membrane insertion if brought sufficiently close to a membrane by the B chain. Once in the cytoplasm, the A
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--Fig.
1
pit pit
t ..... ~
coated vesicle .,~lli ...~~1
|Ill,
--_
Lysosome
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.,4
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Entry of diphtheria toxin ( ~ ), ricin ( O-e ), and ricin A chain immunotoxins ( /~0 ), into a cell (A chains are shown by open squares or circles and B chains by solid squares or circles.) After binding to receptors or cell surface antigens, toxins and immunotoxins are delivered to endosomes via coated pits and coated vesicles. Ricin may also enter via smooth pits. A certain proportion may be recycled back to the cell surface or delivered to lysosomes, where it is presumably degraded. Diphtheria toxin A chain is translocated across the endosomal membrane. Ricin and immunotoxins are probably routed further, to the trans-Golgi network where release of the A chain into the cytosol occurs, and soluble translation factors or the ribosome are attacked.
chain acts as a N-glycosidase, catalytically removing one specific, highly conserved, adenine residue in a GAGA sequence, borne on a stem-loop structure in 28S rRNA 5. Ribosomes so modified are deficient in initiation and elongation functions and, as a consequence, protein manufacture ceases. The routes by which bacterial and plant toxins enter target cells is illustrated in Figure 1. A second class of similar proteins are also synthesized by plants. These are the RIPS, or ribosomeinactivating proteins, and consist of single chain molecules very similar to, and probably evolutionary related to, the A chains of plant toxins 6. Examples are pokeweed antiviral protein from the seeds and leaves of Phytolacca americana, gelonin from the seeds of Gelonium multiflavum, saporin from the seeds of Saponaria officinalis and trichosanthin from the Chinese cucumber.
Since RIPs lack a cell-binding B chain, they are relatively non-toxic. However, at least for trichosanthin, it appears that macrophages, particularly those infected with HIV, are sensitive, so these proteins may have uses in AIDS treatment 7. Immunotoxins
Immunotoxins are conjugates of ce]l-binding antibodies and intact toxins, their toxic fragments or RIPs. Since the number of antibodies that reach a tumour, particularly in solid tissue, may be small, the potency of the delivered toxin must be high. Immunotoxins prepared with intact toxins are often exceedingly toxic, but the ability of the B chain to bind cells opportunistically may override the specificity endowed by the antibody, resulting in fatal intoxication of non-target ceils. In at least one ricin-based immunotoxin, it is thought that the antibody component sterically hinders the B chain,
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producing a conjugate with acceptable specificity8. For other ricinbased immunotoxins, a blockade can be provided in vitro by provision of excess free galactose or lactose, but the rapid metabolic excretion of these sugars precludes this approach in vivo. An alternative approach that has provided some very effective immunotoxins uses isolated A chains or RIPs. These are usually constructed by introduction of an activated disulphide into an antibody, which is then reacted with the free sulphydryl of the A chain, or a free thiol that has been introduced into a RIP (Ref. 9). Since they lack B chains, these immunotoxins show good specificity but their cytotoxicity varies from powerful to very weak. At least part of the reason for this variability lies in the nature of the target antigen: an efficient binding site needs a high antigen density, high antibody affinity, must promote efficient endocytosis when bound by immunotoxin and must direct a sufficient portion of internalized immunotoxins away from the lysosomes, probably to the trans-Golgi network. For example, immunotoxins made with some antibodies to CD2 work poorly since they are directed to, and degraded within, lysosomes ~°.
Enhancing agents Reagents that reduce lysosomal degradation or alter intracellular routing should enhance the potency of immunotoxins; this may explain the effect of administration of monensin, chloroquine or ammonium chloride to culture cells treated with immunotoxin 11. Enhancement has also been noted using inhibitors of protein synthesis and glycosylation12. All the above examples have no obvious in vivo application. An alternative means of achieving in vivo enhancement might be the provision of ricin B chain, either free or as a B chain immunotoxin 13 chemically modified to reduce its cell binding activity, but still able to potentiate A chain toxicity. A further approach to immunotoxin construction depends on the use of bispecific antibodies. One arm of the antibody is specific for a cell surface antigen, the other for an RIP such as saporin. The two binding
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specificities are joined by a nonreducible thioether linkage and will bind to and deliver saporin to target cells 15. Evaluation of immunotoxins and clinical trials Guinea pigs and normal and immunodeficient mice bearing tumour cell implants have been treated with a range of immunotoxins, allowing systematic appraisal of immunotoxin efficacy. Those based on abrin A chain, saporin and Pseudomonas exotoxin A can cause significant liver damage. In the case of ricin A chain-based immunotoxins, chemical pretreatment of the toxin A chain to destroy its sugar residues resolves this problem 16. Animal models have also been used for the testing of novel heterobifunctional cross-linking agents. Overall, these models have allowed refinement of immunotoxins, and anti-tumour effects ranging from the not so dramatic (prolonged survival, inhibition • of
tumour growth), to the very dramatic (complete remissions), have been reported. These results have been sufficiently encouraging to warrant clinical trials in humans. Where cells are easily accessible to antibodies, clinical results from use of ricin A chain based immunotoxins are highly encouraging. They have been used to purge samples of bone marrow of malignant B cells prior to reinfusion into patients whose own marrow has been destroyed by radio- or chemotherapy; to remove T cells from allogeneic bone marrow grafts (marrow from matched siblings) thereby reducing the incidence of 'graft versus host disease', and also in treatment of autoimmune illnesses. In contrast, immunotoxins fail to perform well for other tumours, such as some leukaemias, breast cancer, melanoma and colon cancer, although encouraging signs such as tumour regression are seen in some patients. A full description of animal
\,,;
,7
" N o w this is a m o r e t y p i c a l a n t i - m o u s e r e s p o n s e !"
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models and clinical results can be found in Ref. 17.
Limitations ofimmunotoxins The failure of immunotoxins, so far, to fulfil their potential may be attributed to a number of causes, such as: • failure of the large conjugates (Mr /> 180000) to permeate solid turnouts; • neutralization of the therapeutic molecules by patients who are not severely immunosuppressed; • circulation of shed antigen, competing for the immunotoxin; • escapes of antigen-negative turnout variants; • the nature and density of the target antigen; • non-specific binding of the constant region of the antibody to nontarget cells. Perhaps the major limitation is the appearance of side effects, not predicted by animal models, that severely restrict the effective dose. Principal side effects include fluid retention and muscular pain 17. The answers to some of these problems lie in design of novel linkers, treatment with mixtures of different immunotoxins, alternating regimes of immunotoxin administration, and a return to the study of model systems. Some of the problems can be addressed directly through genetic engineering, and although these studies are at a preliminary stage, they do show extraordinary promise. A good example is the use of genetic "engineering to produce 'humanized' antibodies (see section below). The use of murine antibodies in human patients provokes a h u m a n anti-mouse antibody response. Because of continuing difficulties in producing appropriate h u m a n monoclonal antibodies, it will obviously be necessary to reduce the immunogenicity of murine antibodies before therapeutic use.
The role of genetic engineering Soluble, enzymically active recombinant ricin A chain has been expressed in, and purified from, Escherichia coli 18. Since it is nonglycosylated, it is eminently suitable
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for immunotoxin construction. Similarly, diphtheria toxin A chain and P s e u d o m o n a s exotoxin A have been expressed in active form in E. coli TM. These proteins (and corresponding genes) are now very well characterized, and their structures are being elucidated by X-ray crystallography. This knowledge provides a sound basis for mutagenesis: can toxicity be increased, and can non-specific cellbinding and immunogenicity be decreased? Antibodies themselves are well suited to protein engineering techniques, and enormous progress has been made in this field. Variable regions of mouse antibodies have been fused to h u m a n constant regions to give chimaeric molecules designed to reduce the immunogenic rodent component 2° ('humanizing' the antibody). The most sophisticated extension of this idea involved the painstaking replacement of just the complementarity-determining regions of a human antibody with murine counterparts, resulting in molecules that are almost completely human, but with murine specificity. Such antibodies are expected to elicit very much reduced human anti-mouse antibody responses in patients 21, and some remarkable remissions have been observed using a humanized antibody 22. The effector functions of an antibody are not strictly required for immunotoxin construction. Since the constant domains can be replaced by foreign proteins such as bacterial nucleases 23, a logical extension of this idea is the replacement of the carboxy-terminal end of an antibody heavy chain with a molecule such as ricin A chain. In practice, this may be successful at the level of protein engineering, but the extreme toxicity of ricin precludes the expression of these molecules in the eukaryotic systems that have been used for producing intact antibodies. Antibodies are normally secreted into the endoplasmic reticulum during protein translation, but just one molecule of an active antibody-ricin fusion, if it leaked into the cytoplasm, could kill the cell that made it. Such procedures, which do not result in cell death, are likely to select for protein with mutant or deleted ricin A chains. A different approach is therefore
required. One that is particularly intriguing is the genetic fusion of ricin A chain and protein A (Ref. 24). Protein A is a bacterial protein from Staphylococcus aureus that binds immunoglobulins, particularly IgG antibodies. It therefore substitutes for the chemical' linkage between antibody and toxin. Unfortunately, such immunotoxins are not toxic, probably since the ricin A chain is not capable of translocating across a membrane when synthesized as part of a fusion protein. Introduction of a trypsin-sensitive site between the ricin A chain and protein A halves of the fusion results in immunotoxins with acceptable potency (M. O'Hare, L. M. Roberts, P. E. Thorpe and J. M. Lord, unpublished). This type of strategy is very attractive since it is generally applicable to a wide range of antibodies. Is it possible to extend this idea and make a wholly recombinant immunotoxin? Until recently, the answer has been no, but the expression of antigen-binding domains of antibodies in E. cob makes this approach feasible. Antigen-binding fragments (Fvs) (Ref. 25), singlechain linked Fvs (Ref. 26), and disulphide-linked antibody arms (Fablike molecules) have been expressed in E. coB. Since these are very much smaller than whole antibodies, they are expected to diffuse into solid tumours, and are therefore good candidates for immunotoxin construction. Indeed, a single-chain immunotoxin based on an Fv recognizing the interleukin-2 receptor fused to a truncated form of Pseudom o n a s exotoxin A has been described 27. Exotoxin A is an ideal molecule for this work, since it has its own translocation domain, and it retains translocation and toxic activities even when its cell-binding domain is deleted or it is fused to other molecules such as growth factors and CD4 (Ref. 19). Animal models suggest t h a t the use of ricin A chain immunotoxins may be preferable to others, since if the A chain is deglycosylated, there is little liver damage. However, since ricin A chain cannot translocate as part of a fusion protein, single-chain ricin A immunotoxins will probably require specific engineering to allow release of the toxic component near a cell membrane. A whole range of Fv- and ricin-based recombinant im-
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m u n o t o x i n s is likely to be developed. T h e recent discovery that recomb i n a n t single variable d o m a i n s of antibodies can retain good antigenb i n d i n g properties 28 m a y in the future be extrapolated to i m m u n o toxins in order to design, express and purify v e r y small molecules, consisting p e r h a p s of o n l y two domains. Perhaps this idea c o u l d be taken even further: in one case, a p e p t i d e c o r r e s p o n d i n g to a single c o m p l e m e n t a r i t y determining region (CDR) r e p r o d u c e s the binding ability of a w h o l e a n t i b o d y 29. An i m m u n o toxin based on this w o u l d be very small indeed. If a m o l e c u l e of this size p r o v e d to be sufficient to target a toxin, this w o u l d raise an intriguing possibility - can peptides derived from growth factors, h o r m o n e s and other ligands be used as targeting devices?
Future prospects In s u m m a r y , i m m u n o t o x i n s have s h o w n e n o r m o u s , as yet unrealized, potential. W h e r e cells are accessible to antibodies and bear suitable antigens, they p r o v i d e very good targets. For other diseases, side effects not predictable from animal m o d e l s are severely limiting, and p e n e t r a t i o n of solid t u m o u r s remains a formidable problem. Protein engineering m a y p r o v i d e m o l e c u l e s more suited to these tasks. Progress is also likely to be m a d e in design of optimum chemical linkers, identification of suitable antigenic targets, and treatment with cocktails of i m m u n o t o x i n s . The field of i n f l u e n c e will almost certainly grow, and i m m u n o t o x i n s will be u s e d for t r e a t m e n t of infectious diseases (e.g. HIV), a u t o i m m u n e diseases, allergies, fungal and parasitic infections, and depletion of rejectioninitiating dendritic cells from organs prior to transplantation. W h e t h e r t h e y will p r o v e to be suitable for c a n c e r therapy, apart from m i n i m a l disease and easily accessible tumours, r e m a i n s an o p e n question, but progress is such that in the next few years it m a y be possible to create w h o l e n e w families of i m m u n o toxins capable of killing target cells w i t h great p o t e n c y whilst causing m i n i m a l h a r m to normal tissues.
Acknowledgements We wish to t h a n k D. Allen, K. Rendall and P. E. T h o r p e for their critical appraisals of this article.
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References 1 Kohler, G. and Milstein, C. (1975) Nature 256,495-497 2 0 l s n e s , S., Moskaug, J., Stenmark, H. and Sandvig, K. (1988) Trends Biochem. Sci. 13,348-351 3 0 l s n e s , S. and Pihl, A. (1982) in Mol-
16 Blakey, D. C., Watson, E. J., Knowles, P. P. and Thorpe, P. E. (1987) Cancer Res. 4 7 , 9 4 7 - 9 5 2
17 Vitetta, E. S. and Thorpe, P. E. in Biologic Therapy of Cancer: Principles and Practice (De Vita, V.,
ecular Action of Toxins and Viruses
4
5 6 7
8
9 10 11 12 13
14 15
(Cohen, P. and Van Heyningen, S., eds), pp. 51-105, Elsevier Biomedical Press Van Deurs, B., Tonnessen, T. I., Petersen, D. W., Sandvig, K. and Olsnes, S. (1986) J. Cell. Biol. 102, 37-47 Endo, Y. and Tsurugi, K. (1987) J. Biol. Chem. 262, 8128-8130 Barbieri, L. and Stirpe, F. (1982) Cancer Surveys 1,489-520 McGrath, M. S., Hwang, K. M., Caldwell, S. E., Gaston, I., Luk, K. C., Wu, P., Ng, V. L., Crowe, S., Daniels, J., Marsh, J., Deinhart, T., Lekas, P. V., Vennari, J. C., Yeung, H-W. and Lifson, J. D. (1989) Proc. Natl Acad. Sci. USA 86, 2844-2848 Thorpe, P. E., Ross, W. C. J., Brown, A. N. F., Meyers, C., Cumber, A. J., Foxwell, B. M. J. and Forrester, J. A. (1984) Ear. J. Biochem. 140, 63-71 Thorpe, P. E. and Ross, W. C. J. (1982) Immunol. Rev. 62,119-158 Press, O. W., Vitetta, E. S., Farr, A. G., Hansen, T. A. and Martin, P. J. (1986) Cell. Immunol. 102, 10-20 Blakey, D. C., Wawrzynczak, E. J., Wallace, P. M. and Thorpe, P. E. (1988) Progress in Allergy 45, 50-90 Sandvig, K., Tonnessen, T. I. and Olsnes, S. (1986) Cancer Res. 46, 6418-6422 McIntosh, D. P., Edwards, D. C., Cumber, A. J., Parnell, G. D., Dean, C. J., Ross, W. C. J. and Forrester, J. A. (1983) FEBS Lett. 164, 17-20 Youle, R. J. and Neville, D. M. (1982) J. Biol. Chem. 257, 1598-1601 Glennie, M. J., McBride, H. M., Worth, A. T. and Stevenson, G. T. 1987) J. Immunol. 139, 2367-2375
18
19 20
21 22
23 24 25 26
27
28 29
Hillman, S. and Rosenberg, S., eds), J. B. Lippincott (in press) O'Hare, M., Roberts, L. M., Thorpe, P. E., Watson, G. J., Prior, B. and Lord, J. M. (1987) FEBS Lett. 216, 73-78 Pastan, I. and Fitzgerald, D. (1989) J. Biol. Chem. 264, 15157-15160 Morrison, S. L., Johnson, M. J., Herzenberg, L. A. and Oi, V. T. (1984) Proc. Natl Acad. Sci. USA 81, 6851-6855 Reichmann, L., Clark, M., Waldmann, H. and Winter, G. (1988) Nature 332,323-327 Hale, G., Clark, M. R., Marcus, R., Winter, G., Dyer, M. J. S., Phillips, J. M., Reichmann, L. and Waldmann, H. (1988) Lancet i, 1394-1399 Neuberger, M. S., Williams, G. T. and Fox, R. O. (1984) Nature 312, 604-608 Kim, J. H. and Weaver, R. F. (1988) Gene 72,315-321 Skerra, A. and Pliickthun, A. (1988) Science 240, 1038-1041 Huston, J. S., Levinson, D., MudgettHunter, M., Tae, M-S., Novotny, J., Margolies, M. N., Ridge, R. J., Bruccoleri, R. E., Haber, E., Crea, R. and Opperman, H. (1988) Proc. Natl Acad. Sci. USA 85, 5879-5883 Chaudhury, V. K., Queen, C., Junghams, R. P., Waldmann, T. A., Fitzgerald, D. J. and Pastan, I. (1989) Nature 339, 394-396 Ward, E. S., Giissow, D., Griffiths, A. D., Jones, P. T. and Winter, G. (1989) Nature 341, 544-546 Williams, W. V., Moss, D. A., KeiberEmmons, T., Cohen, J. A., Myers, J. N., Weiner, D. B. and Greene, M. I. (1989) Proc. Natl Acad. Sci. USA 86, 5537-5541
