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9. Thorpe, P. E., Ross, W. C., Brown. A. N., Myers, C. D., Cumber, A. J., Foxwell, B. M. & Forrester, J. A. (1984) Eur. J. Biochem. 140,63-71 10. Wawrzynczak, E. J., Drake, A. F., Watson, G. J., Thorpe, P. E. & Vitetta, E. S. (1988) Biochim. Biophys. Acta 971,55-62 11. Vitetta, E. S.(1986)J. Immunol. 136, 1880-1887 12. Lambert, J. M., McIntyre, G., Gauthier, M. N., Zullo, D., Rao, V., Steeves, R. M., Goldmacher, V. S. & Blattler, W. A. (1991) Biochemistry 30, 3234-3247 13. Youle, R. J., Murray, G. J. & Neville, D. M., Jr. (1981) Cell 23,55 1-559 Jr. ( 1 982) J. Biol. Chem. 14. Youle, R. J. & Neville, D. M., 257, 1598-1601 15. Youle, R. J. & Colombath, M. (1987) J. Biol. Chem. 262,4676-4682 16. Rutenber, E., Ready, M. & Robertus, J. D. (1987) Nature 326,624-626 17. Rutenber, E. & Robertus, J. D. (1991) Proteins Struct. Funct. Genet. 10.260-269 18. Wales, R., Richardson, P. T., Roberts, I,. M.. Woodland, H. R. & Lord, J. M. (1991) J. Biol. Chem. 266,19 172- 19179 19. Lamb, F. I., Roberts, I,. M. & Lord, J. M. (1985) Eur. J. Biochem. 148,265-270 20. Richardson, P. T.,Gilmartin, P., Colman, A., Roberts, 1,. M. & Lord, J. M.(1988) Riotechnology 6, 565-570 21. OHare. M.,Roberts, I,. M., Thorpe, P. E., Watson, G. J., Prior, €3. & Lord, J. M. (1987) FERS Lett. 216, 73-78
22. Newton, D. I,. Wales, R., Richardson, P. T.. Walbridge, S., Saxena, S. K.. Ackerman. E. J., Roberts, I,. M., Lord, J. M. & Youle, R. J. (1992) J. Biol. Chem. 267,119 17- 1 1922 23. Simmons, B. M.,Stahl, P. D. & Russell, J. H. (1986) J. Biol. Chern. 261,7912-7920 24. van Deurs, B., Tonnessen, T. I., Petersen, 0. W., Sandvig, K. & Olsnes, S. (1986) J. Cell Biol. 102, 37-47 25. van Deurs, B.,Sandvig, K., Petersen, 0. W., Olsnes. S., Simons, K. & Griffiths, G. (1988) J. Cell Hiol. 106, 253-267 26. Yoshida, T., Chen, C., Zhang, M. & Wu, H. C. (1991) Exp. Cell Res. 192,389-395 27. Hudson, T. H. & Grillo, F.G. (1991) J. Riol. Chem. 266,18586-18592 28. Sandvig, K., Prydz, K., Hansen, S. H. & van Deurs, €3. (1991)J. Cell Biol. 115,971-981 29. Seetharam, S., Chandhary, V., FitzGerald, L). & Pastan, I. (199 1)J. Biol. Chem. 266, 17376- 17381 30. Pelham, H.R. €3. (1991) Cell 67,449-451 31. Pelharn, H. R. B., Roberts, I,. M. & Lord, J. M. (1992) Trends Cell Biol. 2, 183-185 32. Peter, F.,Nguyen Van, P. & Solung, H.-D. (1992) J. Riol. Chem. 267,10631-10637 33. Van, P. N., Peter, F. & Soling, H.41. (1989) J. Biol. Chem. 264,17494-17501.
Received 10 July 1992
Structural factors influencing the pharmacokinetics and stability of immunotoxins Edward J. Wawrzynczak, Alan J. Cumber, Raymond V. Henry, Geoffrey D. Parnell and John H.Westwood Drug Targeting Laboratory, Institute of Cancer Research, Sutton, Surrey SM2 SNG, U.K.
Introduction Immunotoxins (ITS) are hybrid proteins, consisting of a monoclonal antibody (Mab) and a cytotoxin, which can exert selective systemic cytotoxic effects in cancer and autoimmune disease [l, 21. The antigen combining specificity of the Mab component dictates the selectivity with which the toxin component is targeted. The Mab component also functions as a carrier of the toxin in the circulation. The toxins are complex proteins that bind to human cells and deliver to the cytosol an enzymic component capable of irreversibly inactivating cellular protein synthesis. The catalytic portion alone of the toxin can also be linked to the Mab. a Abbreviations used: IT, immunotoxin; Mab, monoclonal antibody; SPDP, N-succinimidyl-3-(2-pyridyldithio)proprionate; SPDB, N-succinimidyl-3-(2-pyridyldithio)butyrate; SIAB, N-succinimidyl-4-(iodoacetylamino)benzoate.
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manoeuvre which has proven most satisfactory in the case of ricin A chain, the ribosome-inactivating subunit of ricin toxin. Ricin A chain contains a single reactive sulphydryl group and has conventionally been linked to Mabs by means of a disulphide bond which is essential for the expression of maximum cytotoxic activity [ 3 ] . In practice, the Mab is first modified with a heterobifunctional crosslinking agent introducing an activated disulphide bond. The covalent conjugate is then formed by disulphide exchange with the reduced A chain. The final product of these chemical procedures is heterogeneous with respect to the number of A chain molecules linked to each Mab molecule and their sites of attachment [4, 51. This review focuses on pharmacokinetic and stability studies with a unique series of immunoconjugates prepared with the mouse Mab Fib75 by standard methods of conjugate synthesis [6,7].
Endocytosis, Toxins, lmmunotoxins and Viruses
Pharmacokinetics of ricin A chain immunotoxins The first studies of ricin A chain IT pharmacokinetics in rabbits revealed that the ITS were cleared considerably more rapidly from the circulation than unmodified Mabs, and with biphasic kinetics over a 24 h period [8, 91. In one study, a disulphide-linked IT was eliminated twice as quickly as an IT prepared with a non-reducible thioether linkage, following an initial rapid phase of clearance [9], providing early evidence of instability of the disulphide linkage in vivo. A detailed study of the pharmacokinetic properties of Fib7.5-ricin A chain ITS prepared with the crosslinking agent N-succinimidyl-3-(2-pyridyldithi0)propionate (SPDP) in normal Wistar rats confirmed the original demonstrations of biphasic kinetics and accelerated clearance relative to Mabs [ 101. In addition, this study showed that the disappearance of ”‘I-labelled protein from the bloodstream with time was more rapid when the IT was selectively labelled in the A chain than in the Mab. This difference could be most readily explained by breakdown of the IT in the circulation to release either labelled A chain, which is known to be rapidly cleared by renal filtration because of its relatively small molecular size, or labelled Fib75, which by contrast possesses a long blood half-life [lo]. The behaviour of the differentially labelled Fib75 ITS was indistinguishable up to 6 h after intravenous administration of IT. This suggested that the first phase of clearance (the a-phase) could not be explained by IT breakdown alone whereas the second phase of clearance (the P-phase) was strongly influenced by IT stability. Analysis of serum samples from rats receiving radiolabelled Fib75-ricin A chain by SDS-PAGE under nonreducing conditions provided further evidence of the release of Mab from ITS in the circulation, a phenomenon confirmed by similar experiments with other ricin A chain ITS [3, 11, 121.
efficacy [ 141. In the case of Fib75-ricin A chain, the presumed mechanism was confirmed by the demonstration that the uptake of IT by the nonparenchymal hepatic cell fraction in tissue culture was inhibitable by mannose [ 131. Ricin A chain purified from natural sources consists of a mixture of two glycosylated forms: the A, chain carries a single fucosylated complex-type N-linked oligosaccharide side chain and the A? chain carries an additional N-linked oligomannose side chain. Analysis of serum samples by SDSPAGE revealed that Fib75 IT molecules containing the A, chain were preferentially and completely cleared within 1 h of intravenous administration [ 161. This very rapid loss of IT from the circulation could be avoided by preparing an analogous Fib75 IT with purified ricin A, chain [ 161. Another ricin A, chain IT has since been shown to have an improved serum half-life, reduced liver uptake and enhanced target tissue localization in vivo compared with its native A chain counterpart [17]. The pharmacokinetics of Fib75 ITS made with native ricin A chain, ricin A, chain and an aglycosyl recombinant ricin A chain produced from Escherichzh coli were compared in the Wistar rat using an enzyme immunoassay to measure the concentration of intact IT molecules in serum samples [18, 191. This comparative study revealed that the recombinant ricin A chain IT persisted in the bloodstream at a higher level than Fib75-ricin A, chain (Fig. 1) suggesting that the remaining oligosaccharide side chain on the A, chain might also have contributed to the clearance process. The improvement in IT half-life gained by substituting native ricin A chain with recombinant ricin A chain was comparable to that demonstrated for a different
Fig. I
Pharmokinetics of Fib75 ITS in the normal Wistar rat measured by enzyme immunoassay of serum samples I00
Role of A chain carbohydrate in the a-phase of IT clearance Fib75-[”’I]ricin A chain was shown to accumulate in the liver of rats soon after intravenous administration by a process that could be inhibited by coadministration of the mannosylated protein ovalbumin, but not by the galactosylated protein asialofetuin, suggesting uptake by a mannose receptor-mediated mechanism [ 131. The enhancement of rich A chain I T half-life by mannosyl agents has been demonstrated in other experimental systems [ 14, 151 and found to enhance IT
nw iA t\ \
A
Recombinant ricin A Ricin A ,
€8 2v)
I
0
24 Time after injection (h)
40
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series of ITS made with a ricin A chain preparation which had been chemically treated to destroy all mannose and fucose residues [ 11, 201. D'irect comparisons of ITS made with a single type of Mab and either recombinant ricin A chain or chemically deglycosylated ricin A chain have not demonstrated any significant differences in pharmacokinetic behaviour between the two types of IT [21,22]. In all pharmacokinetic studies, including those with the aglycosyl versions of ricin A chain, clearance of IT was biphasic indicating that the a-phase could not be explained by elimination processes mediated solely by recognition of the carbohydrate residues of the toxin component.
Fig. 2
Rates of IT breakdown by glutathione in vitro measured by enzyme immunoassay SIAB-ricin A
I SPDB-ricin A SPDP-abrin A
e
e s
Y
.c
0
E L=
Role of crosslinking agent structure in the /?-phaseof IT clearance Evidence that Fib75-ricin A chain prepared with the SPDP crosslinking agent could break down in vivo to release free Mab suggested that increasing the chemical stability of the disulphide linkage to chemical reduction would enhance IT stability in the circulation. An Fib75 IT constructed with a novel crosslinking agent based on SPDP, Nsuccinimidyl-3-(2-pyridyldithio)butyrate (SPDB), introducing a partially hindered disulphide bond sterically protected by substitution with a single methyl group on the adjacent carbon atom, had a significantly longer /?-phase half-life than the standard IT with the non-hindered linkage [23]. Comparable results have been obtained in studies of IT analogues prepared with a different hindered disulphide crosslinking agent [2 1, 241 which, moreover, demonstrated that the inclusion of a hindered disulphide linkage reduced the rate of release of free Mab and improved the therapeutic index of the IT [ZO]. In all cases, the effect of introducing a hindered disulphide linkage on half-life was confined to the /?-phase confirming previous conclusions that IT splitting was not the most important mechanism of IT loss in the a-phase. In the case of the Fib75-ricin A chain ITS, the /?-phase half-life of the SPDB-linked IT was still significantly shorter than that of an IT prepared with the crosslinking agent N-succinimidyl-4-(iodoacetylamino)benzoate (SIAB), introducing a thioether linkage stable to chemical reduction [23]. This suggested that a further increase in the resistance of the disulphide bond to reduction might further extend IT half-life. In recent studies, we have compared the stability of various Fib75-ricin A chain ITS in vitro to breakdown in the presence of glutathione, the principal reducing agent in the circulation. Using an enzyme immunoassay detecting intact IT molecules
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0
i
Ik
i4
Time of incubation (h)
[19], we found that the rate of breakdown of Fib75SPDB-ricin A chain was significantly slower than that of Fib75-SPDP-ricin A chain (Fig. 2). The measured rates of breakdown in the presence of a vast molar excess of glutathione were biphasic suggesting that each IT preparation may have contained a proportion of conjugate molecules with a higher susceptibility to reduction than the bulk of the preparation. Under comparable experimental conditions, the analogous SIAB-linked IT was completely stable to glutathione. In further experiments, the release of ricin A chain from Mab as a result of disulphide bond cleavage, monitored directly by means of a quantitative h.p.1.c. procedure, was shown to occur less extensively in the case of the SPDB-linked IT [25].
Influence of A chain polypeptide structure on IT stability The role of the A chain in IT stability was examined by studying the pharmacokinetics of a panel of SPDP-linked Fib75 ITS made with ricin A chain, with the A chain from the plant toxin abrin, and with the single chain ribosome-inactivating proteins gelonin, momordin and a-sarcin [26,27]. The most notable and unexpected finding was that Fib75SPDP-abrin A chain displayed a significantly longer /?-phase half-life than the other Fib75 ITS (Fig. 1). Abrin A chain is unglycosylated and the Fib75 abrin A chain IT was not subject to significant hepatic uptake either in vitro or in vivo [28]. Thus, the longer /?-phase half-life of the abrin A chain analogue most likely reflected a decreased suscepti-
Endocytosis, Toxins, lmmunotoxins and Viruses
bility to splitting of the disulphide bond. This supposition was confirmed by directly comparing the rates of breakdown of ricin A chain and abrin A chain Fib75 ITS by glutathione in vitro using enzyme immunoassays (Fig. 2) and gel permeation h.p.1.c. [26]. In another study, the half-life of an abrin A chain IT in the mouse was not prolonged compared with that of its ricin A chain counterpart [29]. The discrepancy between the results of the two studies may reflect the substantial differences between the methods employed. The finding that Fib75 ITS made by identical procedures and differing only in the nature of the toxin component had markedly different stabilities suggested that the structure of the A chain influenced the susceptibility of the disulphide linkage to glutathione reduction. W e have recently demonstrated that an immunoconjugate prepared in analogous fashion to an IT, but substituting ricin A chain with the C-terminal peptide WRCAPPPSSQF (ricin A25“2h7)homologous with the region of A chain structure involved in disulphide bond formation, was significantly more susceptible to glutathione reduction than Fib75-ricin A chain itself [25]. This result suggests that the intact structure of the A chain in the vicinity of the disulphide bond is necessary to exert a protective effect. In further experiments, ricin A chain or the Cterminal peptide were modified by reaction with 5,5’-dithiobis(2-nitrobenzoic acid) to introduce an activated disulphide bond. The release of the introduced 2-nitrothiobenzoate anion was more rapid from ricin A25h-267 than from the intact A chain [25]. Using identical experimental protocols, we have found that release of 2-nitrothiobenzoate from abrin A chain derivatized in similar fashion was considerably slower than from the corresponding Cterminal peptide LFVCNPPN (abrin A24”250),and significantly slower than from derivatized ricin A chain (Fig. 3). Thus, the superior protective effect of the abrin A chain appeared to be an intrinsic property of the intact protein structure. This effect further contributed to the overall stability of an abrin A chain Fib75 IT prepared with the SPDB crosslinking agent introducing a disulphide bond that is only partially hindered [30].
Influence of Mab structure on IT stability and clearance The possible contribution of the structure of the Mab component to IT stability has not previously been addressed formally. W e recently prepared a novel Fib75 I T in which the SPDP crosslinking agent was separated from the Mab by means of an
Fig. 3
Rates of 2-nitrothiobenzoate release from substituted ricin and abrin A chains and their C-terminal peptides I00
74 I
r
P) Y
Ricin A
0
Abrin A
0
10
20
30
Incubation time (min)
interspersed flexible peptide linker AAPAAAPAPA in order to distance the disulphide linkage to the A chain from the surface of the Mab and relax the constraint on the positional freedom of the A chain imposed by the use of a short chemical crosslinking agent [25]. This structural variation had no effect on the extent to which the IT broke down to its constituent parts in the presence of glutathione in vitro suggesting that the Mab had no influence on the stability of the disulphide bond in the conventional type of Fib75 IT. A comparative study of the in vivo rates of splitting of a deglycosylated ricin A chain IT and an analogue prepared with the antibody Fab’ fragment by direct attachment of the A chain to a hinge region cysteine residue, suggested that the Fab’ IT was more stable [31]. One interpretation of this result is that the structure of the Fab’ fragment also contributed to disulphide bond stability because the antibody and toxin components were more closely attached than in a standard IT employing a chemical crosslinking agent.
Conclusion Experimental studies have elucidated many of the structural features that determine the stability and pharmacokinetics of A chain ITS. Complete absence of carbohydrate from the toxin component is essential to avoid rapid and extensive uptake by the reticuloendothelial system. Protection of the disulphide linkage against cleavage by reducing agents is likewise necessary to maximize half-life. Although we now have evidence for a possible role for the toxin component in IT stability, the influence
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of the Mab component is less well defined. Importantly, we still do not fully understand the processes that occur in the a-phase of IT clearance and lead to the rapid elimination of a substantial proportion of intravenous IT from the bloodstream. Some IT molecules may be eliminated more quickly from the bloodstream than unmodified Mabs because the substitution of the Mab with the crosslinking agent or with the toxin component compromises the normal ability of the Mab to circulate. Modification of the Mab structure could promote a direct interaction with some normal tissue that leads to rapid clearance. Alternatively, modification could lead to rapid IT catabolism by interference with the function of the Mab C,2 domains which are known to be involved in the control of IgG catabolism in rodents and humans. The precise nature or extent of possible structural alterations induced by the process of chemical conjugation has not proven amenable to simple analysis. The principal difficulty impeding the further understanding of the relationship between IT structure and stability is the undefined heterogeneity introduced by chemical coupling procedures. In future, remaining questions will be resolved by examining the properties of ITS of defined stoichiometry prepared by site-specific attachment of the toxin molecule to engineered recombinant Mabs. It may indeed prove feasible to attach the toxin directly to a site on the Mab deliberately engineered to yield a stable IT without the need for a chemical crosslinking agent.
The work of the Drug Targeting Laboratory at the Institute of Cancer Research has been generously supported by the Cancer Research Campaign. We wish to thank all our past and present colleagues at the Institute and our many collaborators outside the Institute for their contribution to the work described in this review.
1. Blakey, D. C., Wawrzynczak, E. J., Wallace, P. M. & Thorpe, P. E. (1988) in Monoclonal Antibody Therapy (Waldmann, H., ed.), pp. 50-90, Karger, Basel 2. Wawrzynczak, E. J. (1991) Br. J. Cancer 64,624-630 3. Wawrzynczak, E. J. (1988) in Immunotoxins (Frankel,A. E., ed.),pp. 239-25 1, Kluwer, Boston 4. Wawrzynczak, E. J. & Thorpe, P. E. (1987) in Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (Vogel, C.-W., ed.), pp. 28-55, Oxford University Press, New York 5. Cumber, A. J. & Wawrzynczak, E. J. (1992) in Methods in Molecular Biology, Vol. 10, Immuno-
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chemical I’rotocols (Manson, M.. ed.), pp. 283-293, Humana. New Jersey 6. Cumber, A. J., Forrester, J. A., Foxwell, H. M. J.. Koss, W. C. J. & Thorpe, 1’. E:. (1985) Methods Bnzymol. 112,207-225 7. Forrester, J. A., Mclntosh, L). l’., Cumber, A. J., Parnell, G. D. & Ross, W. C. J. (1984) Cancer Drug Deliv. 1,283-293 8. Raso, V. & Basala, M. (1984) in Keceptor-Mediated Targeting of Drugs (Gregoriadis, G., I’oste, G., Senior, J. & Trouet, A., eds.), pp. 119-138, Plenum, New York 9. Jansen, F. K., Blythman, H. E., Hourrie, B., Carriere, D.. Casellas, P., Dussossoy, D., Gros, 0.. Laurent, J. C., Liance, M. C., I’oncelet, I’.$ Kicher, G. & Vidal, H. (1984) in Receptor-Mediated Targeting of Drugs (Gregoriadis, G., Poste, G., Senior, J. & Trouet, A., eds.),pp. 147-178, Plenum, New York 10. Worrell, N. R., Cumber, A. J., I’arnell, G. 11.. Koss, W. C. J. & Forrester, J. A. (1986) Biochem. I’harmacol. 3 5 4 17-423 11. Blakey, D. C., Watson, G. J., Knowles, 1’. 1’. & Thorpe, P. E. (1 987) Cancer Kes. 47,947-952 12. Greenfield, I,. & Dovey, H. F. (1992) Antibody Immunoconj. Radiopharm. 5,37-59 13. Worrell, N. R., Skilleter, D. N.. Cumber, A. J. & Price, K. J. (1986) Hiochem. Hiophys. Kes. Commun. 137, 892-896 14. Bourrie, H. J. P., Casellas. P.. Hlythman, H. E. & Jansen, F. K. (1986) Eur. J. Biochem. 155, 1-10 15. Byers. V. S., Pimm, M. V.. Pawluczyk, 1. Z.A., Lee, H. M., Scannon, P. J. & Baldwin, R. W. (1 987) Cancer Res. 47,5277-5283 16. Cumber, A. J., Parnell, G. D., Henry, R. V., Forrester, J. A. & Wawrzynczak. E. J. (1989) Biochem. SOC. Trans. 17,137-138 17. Trown, P. W., Reardan, D. T., Carroll, S. F., Stoudemire, J. B. & Kawahata, R. T. (1991) Cancer Res. 51,4219-4225 18. Wawrzynczak, E. J.? Cumber, A. J., Henry, R. V. & Parnell, G. D. (1991) Intl. J. Cancer 47, 130-135 19. Wawrzynczak, E. J. & Cumber, A. J. (1992) in Methods in Molecular Biology, Vol. 10, Imrnunochemical Protocols (Manson, M., ed.), pp. 295-305, Humana, New Jersey 20. Thorpe, P. E., Wallace, P. M., Knowles, P. P., Relf, M. G., Brown, A. N. F..Watson, G. J , Blakey, D. C. & Newell, D. R. (1988) Cancer Res. 48,6396-6403 21. Shire, D., Bourrie, B. J. P., Carillon, C., Derocq, J.-M., Dousset, P., Dumont, X., Jansen, F. K., Kaghad, M., Legoux, R., Lelong, P., Pessegue, B. & Vidal, H. (1990) Gene 93, 183-188 22. Blakey, D. C., Wright, A. F., Hewett, P. W. & Rose, M. S. (1992) in Monoclonal Antibodies: Applications in Clinical Oncology (Epenetos, A. A., ed.), Chapman and Hall, London, in the press 23. Worrell, N. R., Cumber, A. J., Parnell, G. D., Mirza, A.. Forrester, J. A. & Ross, W. C. J. (1986) AntiCancer Drug Design 1, 179- 188
Endocytosis, Toxins, lmmunotoxins and Viruses
24. Thorpe, P. E., Wallace. P. M., Knowles. P. I’.. Relf, M. G., Hrown, A. N. F.,Watson, G. J., Knyba, K. E., Wawrzynczak, E. J. & Blakey, D. C. (1987) Cancer Kes. 47.5924-593 1 25. Cumber, A. J., Westwood, J. H., Henry, R. V., Parnell, G. 11.. Coles, H. F. & Wawrzynczak. E. J. (1992) Hioconj. Chem. 3, in the press 26. Wawrzynczak, E. Cumber, A. J., Henry, K. V., May. J., Newell, D. K..Parnell, G. D., Worrell, N.R. & Forrester, J. A. (1990) Cancer Kes. 50,75 19-7526 27. Wawrzynczak, E. J., Henry, K. V.. Cumber, A. J., I’arnell, G. 11.. Derbyshire, E. J. & Ulbrich, N. (1991) Eur. J. Hiochem. 196,203-209 J.?
28. Skilleter, D. N..Price. K. J.. I’arnell, G. 1). & Cumber, A. J. (1 989) Cancer Letters 46, 161- 1Oh 29. Thorpe, P. I.:.. Hlakey, D. C., Hrown. A. N. F.. Knowles, P. P.?Knyba, K. E., Wallace, 1’. M., Watson, G. J. & Wawrzynczak, E. J. (1987) J. Natl. Canc. Inst. 79,1101-1112 30. Cumber, A. J. & Wawrzynczak, E. J. (1992) Hiochem. SOC.Trans. 20,3 12.5 31. Fulton. K.J.. Tucker, T. F.. Vitetta. E. S. & Uhr, J. W. (1988) Cancer Kes. 48,2618-26)25
Keceived 10 July 1092
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9. Thorpe, P. E., Ross, W. C., Brown. A. N., Myers, C. D., Cumber, A. J., Foxwell, B. M. & Forrester, J. A. (1984) Eur. J. Biochem. 140,63-71 10. Wawrzynczak, E. J., Drake, A. F., Watson, G. J., Thorpe, P. E. & Vitetta, E. S. (1988) Biochim. Biophys. Acta 971,55-62 11. Vitetta, E. S.(1986)J. Immunol. 136, 1880-1887 12. Lambert, J. M., McIntyre, G., Gauthier, M. N., Zullo, D., Rao, V., Steeves, R. M., Goldmacher, V. S. & Blattler, W. A. (1991) Biochemistry 30, 3234-3247 13. Youle, R. J., Murray, G. J. & Neville, D. M., Jr. (1981) Cell 23,55 1-559 Jr. ( 1 982) J. Biol. Chem. 14. Youle, R. J. & Neville, D. M., 257, 1598-1601 15. Youle, R. J. & Colombath, M. (1987) J. Biol. Chem. 262,4676-4682 16. Rutenber, E., Ready, M. & Robertus, J. D. (1987) Nature 326,624-626 17. Rutenber, E. & Robertus, J. D. (1991) Proteins Struct. Funct. Genet. 10.260-269 18. Wales, R., Richardson, P. T., Roberts, I,. M.. Woodland, H. R. & Lord, J. M. (1991) J. Biol. Chem. 266,19 172- 19179 19. Lamb, F. I., Roberts, I,. M. & Lord, J. M. (1985) Eur. J. Biochem. 148,265-270 20. Richardson, P. T.,Gilmartin, P., Colman, A., Roberts, 1,. M. & Lord, J. M.(1988) Riotechnology 6, 565-570 21. OHare. M.,Roberts, I,. M., Thorpe, P. E., Watson, G. J., Prior, €3. & Lord, J. M. (1987) FERS Lett. 216, 73-78
22. Newton, D. I,. Wales, R., Richardson, P. T.. Walbridge, S., Saxena, S. K.. Ackerman. E. J., Roberts, I,. M., Lord, J. M. & Youle, R. J. (1992) J. Biol. Chem. 267,119 17- 1 1922 23. Simmons, B. M.,Stahl, P. D. & Russell, J. H. (1986) J. Biol. Chern. 261,7912-7920 24. van Deurs, B., Tonnessen, T. I., Petersen, 0. W., Sandvig, K. & Olsnes, S. (1986) J. Cell Biol. 102, 37-47 25. van Deurs, B.,Sandvig, K., Petersen, 0. W., Olsnes. S., Simons, K. & Griffiths, G. (1988) J. Cell Hiol. 106, 253-267 26. Yoshida, T., Chen, C., Zhang, M. & Wu, H. C. (1991) Exp. Cell Res. 192,389-395 27. Hudson, T. H. & Grillo, F.G. (1991) J. Riol. Chem. 266,18586-18592 28. Sandvig, K., Prydz, K., Hansen, S. H. & van Deurs, €3. (1991)J. Cell Biol. 115,971-981 29. Seetharam, S., Chandhary, V., FitzGerald, L). & Pastan, I. (199 1)J. Biol. Chem. 266, 17376- 17381 30. Pelham, H.R. €3. (1991) Cell 67,449-451 31. Pelharn, H. R. B., Roberts, I,. M. & Lord, J. M. (1992) Trends Cell Biol. 2, 183-185 32. Peter, F.,Nguyen Van, P. & Solung, H.-D. (1992) J. Riol. Chem. 267,10631-10637 33. Van, P. N., Peter, F. & Soling, H.41. (1989) J. Biol. Chem. 264,17494-17501.
Received 10 July 1992
Structural factors influencing the pharmacokinetics and stability of immunotoxins Edward J. Wawrzynczak, Alan J. Cumber, Raymond V. Henry, Geoffrey D. Parnell and John H.Westwood Drug Targeting Laboratory, Institute of Cancer Research, Sutton, Surrey SM2 SNG, U.K.
Introduction Immunotoxins (ITS) are hybrid proteins, consisting of a monoclonal antibody (Mab) and a cytotoxin, which can exert selective systemic cytotoxic effects in cancer and autoimmune disease [l, 21. The antigen combining specificity of the Mab component dictates the selectivity with which the toxin component is targeted. The Mab component also functions as a carrier of the toxin in the circulation. The toxins are complex proteins that bind to human cells and deliver to the cytosol an enzymic component capable of irreversibly inactivating cellular protein synthesis. The catalytic portion alone of the toxin can also be linked to the Mab. a Abbreviations used: IT, immunotoxin; Mab, monoclonal antibody; SPDP, N-succinimidyl-3-(2-pyridyldithio)proprionate; SPDB, N-succinimidyl-3-(2-pyridyldithio)butyrate; SIAB, N-succinimidyl-4-(iodoacetylamino)benzoate.
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manoeuvre which has proven most satisfactory in the case of ricin A chain, the ribosome-inactivating subunit of ricin toxin. Ricin A chain contains a single reactive sulphydryl group and has conventionally been linked to Mabs by means of a disulphide bond which is essential for the expression of maximum cytotoxic activity [ 3 ] . In practice, the Mab is first modified with a heterobifunctional crosslinking agent introducing an activated disulphide bond. The covalent conjugate is then formed by disulphide exchange with the reduced A chain. The final product of these chemical procedures is heterogeneous with respect to the number of A chain molecules linked to each Mab molecule and their sites of attachment [4, 51. This review focuses on pharmacokinetic and stability studies with a unique series of immunoconjugates prepared with the mouse Mab Fib75 by standard methods of conjugate synthesis [6,7].
Endocytosis, Toxins, lmmunotoxins and Viruses
Pharmacokinetics of ricin A chain immunotoxins The first studies of ricin A chain IT pharmacokinetics in rabbits revealed that the ITS were cleared considerably more rapidly from the circulation than unmodified Mabs, and with biphasic kinetics over a 24 h period [8, 91. In one study, a disulphide-linked IT was eliminated twice as quickly as an IT prepared with a non-reducible thioether linkage, following an initial rapid phase of clearance [9], providing early evidence of instability of the disulphide linkage in vivo. A detailed study of the pharmacokinetic properties of Fib7.5-ricin A chain ITS prepared with the crosslinking agent N-succinimidyl-3-(2-pyridyldithi0)propionate (SPDP) in normal Wistar rats confirmed the original demonstrations of biphasic kinetics and accelerated clearance relative to Mabs [ 101. In addition, this study showed that the disappearance of ”‘I-labelled protein from the bloodstream with time was more rapid when the IT was selectively labelled in the A chain than in the Mab. This difference could be most readily explained by breakdown of the IT in the circulation to release either labelled A chain, which is known to be rapidly cleared by renal filtration because of its relatively small molecular size, or labelled Fib75, which by contrast possesses a long blood half-life [lo]. The behaviour of the differentially labelled Fib75 ITS was indistinguishable up to 6 h after intravenous administration of IT. This suggested that the first phase of clearance (the a-phase) could not be explained by IT breakdown alone whereas the second phase of clearance (the P-phase) was strongly influenced by IT stability. Analysis of serum samples from rats receiving radiolabelled Fib75-ricin A chain by SDS-PAGE under nonreducing conditions provided further evidence of the release of Mab from ITS in the circulation, a phenomenon confirmed by similar experiments with other ricin A chain ITS [3, 11, 121.
efficacy [ 141. In the case of Fib75-ricin A chain, the presumed mechanism was confirmed by the demonstration that the uptake of IT by the nonparenchymal hepatic cell fraction in tissue culture was inhibitable by mannose [ 131. Ricin A chain purified from natural sources consists of a mixture of two glycosylated forms: the A, chain carries a single fucosylated complex-type N-linked oligosaccharide side chain and the A? chain carries an additional N-linked oligomannose side chain. Analysis of serum samples by SDSPAGE revealed that Fib75 IT molecules containing the A, chain were preferentially and completely cleared within 1 h of intravenous administration [ 161. This very rapid loss of IT from the circulation could be avoided by preparing an analogous Fib75 IT with purified ricin A, chain [ 161. Another ricin A, chain IT has since been shown to have an improved serum half-life, reduced liver uptake and enhanced target tissue localization in vivo compared with its native A chain counterpart [17]. The pharmacokinetics of Fib75 ITS made with native ricin A chain, ricin A, chain and an aglycosyl recombinant ricin A chain produced from Escherichzh coli were compared in the Wistar rat using an enzyme immunoassay to measure the concentration of intact IT molecules in serum samples [18, 191. This comparative study revealed that the recombinant ricin A chain IT persisted in the bloodstream at a higher level than Fib75-ricin A, chain (Fig. 1) suggesting that the remaining oligosaccharide side chain on the A, chain might also have contributed to the clearance process. The improvement in IT half-life gained by substituting native ricin A chain with recombinant ricin A chain was comparable to that demonstrated for a different
Fig. I
Pharmokinetics of Fib75 ITS in the normal Wistar rat measured by enzyme immunoassay of serum samples I00
Role of A chain carbohydrate in the a-phase of IT clearance Fib75-[”’I]ricin A chain was shown to accumulate in the liver of rats soon after intravenous administration by a process that could be inhibited by coadministration of the mannosylated protein ovalbumin, but not by the galactosylated protein asialofetuin, suggesting uptake by a mannose receptor-mediated mechanism [ 131. The enhancement of rich A chain I T half-life by mannosyl agents has been demonstrated in other experimental systems [ 14, 151 and found to enhance IT
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Recombinant ricin A Ricin A ,
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series of ITS made with a ricin A chain preparation which had been chemically treated to destroy all mannose and fucose residues [ 11, 201. D'irect comparisons of ITS made with a single type of Mab and either recombinant ricin A chain or chemically deglycosylated ricin A chain have not demonstrated any significant differences in pharmacokinetic behaviour between the two types of IT [21,22]. In all pharmacokinetic studies, including those with the aglycosyl versions of ricin A chain, clearance of IT was biphasic indicating that the a-phase could not be explained by elimination processes mediated solely by recognition of the carbohydrate residues of the toxin component.
Fig. 2
Rates of IT breakdown by glutathione in vitro measured by enzyme immunoassay SIAB-ricin A
I SPDB-ricin A SPDP-abrin A
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Role of crosslinking agent structure in the /?-phaseof IT clearance Evidence that Fib75-ricin A chain prepared with the SPDP crosslinking agent could break down in vivo to release free Mab suggested that increasing the chemical stability of the disulphide linkage to chemical reduction would enhance IT stability in the circulation. An Fib75 IT constructed with a novel crosslinking agent based on SPDP, Nsuccinimidyl-3-(2-pyridyldithio)butyrate (SPDB), introducing a partially hindered disulphide bond sterically protected by substitution with a single methyl group on the adjacent carbon atom, had a significantly longer /?-phase half-life than the standard IT with the non-hindered linkage [23]. Comparable results have been obtained in studies of IT analogues prepared with a different hindered disulphide crosslinking agent [2 1, 241 which, moreover, demonstrated that the inclusion of a hindered disulphide linkage reduced the rate of release of free Mab and improved the therapeutic index of the IT [ZO]. In all cases, the effect of introducing a hindered disulphide linkage on half-life was confined to the /?-phase confirming previous conclusions that IT splitting was not the most important mechanism of IT loss in the a-phase. In the case of the Fib75-ricin A chain ITS, the /?-phase half-life of the SPDB-linked IT was still significantly shorter than that of an IT prepared with the crosslinking agent N-succinimidyl-4-(iodoacetylamino)benzoate (SIAB), introducing a thioether linkage stable to chemical reduction [23]. This suggested that a further increase in the resistance of the disulphide bond to reduction might further extend IT half-life. In recent studies, we have compared the stability of various Fib75-ricin A chain ITS in vitro to breakdown in the presence of glutathione, the principal reducing agent in the circulation. Using an enzyme immunoassay detecting intact IT molecules
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i
Ik
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Time of incubation (h)
[19], we found that the rate of breakdown of Fib75SPDB-ricin A chain was significantly slower than that of Fib75-SPDP-ricin A chain (Fig. 2). The measured rates of breakdown in the presence of a vast molar excess of glutathione were biphasic suggesting that each IT preparation may have contained a proportion of conjugate molecules with a higher susceptibility to reduction than the bulk of the preparation. Under comparable experimental conditions, the analogous SIAB-linked IT was completely stable to glutathione. In further experiments, the release of ricin A chain from Mab as a result of disulphide bond cleavage, monitored directly by means of a quantitative h.p.1.c. procedure, was shown to occur less extensively in the case of the SPDB-linked IT [25].
Influence of A chain polypeptide structure on IT stability The role of the A chain in IT stability was examined by studying the pharmacokinetics of a panel of SPDP-linked Fib75 ITS made with ricin A chain, with the A chain from the plant toxin abrin, and with the single chain ribosome-inactivating proteins gelonin, momordin and a-sarcin [26,27]. The most notable and unexpected finding was that Fib75SPDP-abrin A chain displayed a significantly longer /?-phase half-life than the other Fib75 ITS (Fig. 1). Abrin A chain is unglycosylated and the Fib75 abrin A chain IT was not subject to significant hepatic uptake either in vitro or in vivo [28]. Thus, the longer /?-phase half-life of the abrin A chain analogue most likely reflected a decreased suscepti-
Endocytosis, Toxins, lmmunotoxins and Viruses
bility to splitting of the disulphide bond. This supposition was confirmed by directly comparing the rates of breakdown of ricin A chain and abrin A chain Fib75 ITS by glutathione in vitro using enzyme immunoassays (Fig. 2) and gel permeation h.p.1.c. [26]. In another study, the half-life of an abrin A chain IT in the mouse was not prolonged compared with that of its ricin A chain counterpart [29]. The discrepancy between the results of the two studies may reflect the substantial differences between the methods employed. The finding that Fib75 ITS made by identical procedures and differing only in the nature of the toxin component had markedly different stabilities suggested that the structure of the A chain influenced the susceptibility of the disulphide linkage to glutathione reduction. W e have recently demonstrated that an immunoconjugate prepared in analogous fashion to an IT, but substituting ricin A chain with the C-terminal peptide WRCAPPPSSQF (ricin A25“2h7)homologous with the region of A chain structure involved in disulphide bond formation, was significantly more susceptible to glutathione reduction than Fib75-ricin A chain itself [25]. This result suggests that the intact structure of the A chain in the vicinity of the disulphide bond is necessary to exert a protective effect. In further experiments, ricin A chain or the Cterminal peptide were modified by reaction with 5,5’-dithiobis(2-nitrobenzoic acid) to introduce an activated disulphide bond. The release of the introduced 2-nitrothiobenzoate anion was more rapid from ricin A25h-267 than from the intact A chain [25]. Using identical experimental protocols, we have found that release of 2-nitrothiobenzoate from abrin A chain derivatized in similar fashion was considerably slower than from the corresponding Cterminal peptide LFVCNPPN (abrin A24”250),and significantly slower than from derivatized ricin A chain (Fig. 3). Thus, the superior protective effect of the abrin A chain appeared to be an intrinsic property of the intact protein structure. This effect further contributed to the overall stability of an abrin A chain Fib75 IT prepared with the SPDB crosslinking agent introducing a disulphide bond that is only partially hindered [30].
Influence of Mab structure on IT stability and clearance The possible contribution of the structure of the Mab component to IT stability has not previously been addressed formally. W e recently prepared a novel Fib75 I T in which the SPDP crosslinking agent was separated from the Mab by means of an
Fig. 3
Rates of 2-nitrothiobenzoate release from substituted ricin and abrin A chains and their C-terminal peptides I00
74 I
r
P) Y
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interspersed flexible peptide linker AAPAAAPAPA in order to distance the disulphide linkage to the A chain from the surface of the Mab and relax the constraint on the positional freedom of the A chain imposed by the use of a short chemical crosslinking agent [25]. This structural variation had no effect on the extent to which the IT broke down to its constituent parts in the presence of glutathione in vitro suggesting that the Mab had no influence on the stability of the disulphide bond in the conventional type of Fib75 IT. A comparative study of the in vivo rates of splitting of a deglycosylated ricin A chain IT and an analogue prepared with the antibody Fab’ fragment by direct attachment of the A chain to a hinge region cysteine residue, suggested that the Fab’ IT was more stable [31]. One interpretation of this result is that the structure of the Fab’ fragment also contributed to disulphide bond stability because the antibody and toxin components were more closely attached than in a standard IT employing a chemical crosslinking agent.
Conclusion Experimental studies have elucidated many of the structural features that determine the stability and pharmacokinetics of A chain ITS. Complete absence of carbohydrate from the toxin component is essential to avoid rapid and extensive uptake by the reticuloendothelial system. Protection of the disulphide linkage against cleavage by reducing agents is likewise necessary to maximize half-life. Although we now have evidence for a possible role for the toxin component in IT stability, the influence
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of the Mab component is less well defined. Importantly, we still do not fully understand the processes that occur in the a-phase of IT clearance and lead to the rapid elimination of a substantial proportion of intravenous IT from the bloodstream. Some IT molecules may be eliminated more quickly from the bloodstream than unmodified Mabs because the substitution of the Mab with the crosslinking agent or with the toxin component compromises the normal ability of the Mab to circulate. Modification of the Mab structure could promote a direct interaction with some normal tissue that leads to rapid clearance. Alternatively, modification could lead to rapid IT catabolism by interference with the function of the Mab C,2 domains which are known to be involved in the control of IgG catabolism in rodents and humans. The precise nature or extent of possible structural alterations induced by the process of chemical conjugation has not proven amenable to simple analysis. The principal difficulty impeding the further understanding of the relationship between IT structure and stability is the undefined heterogeneity introduced by chemical coupling procedures. In future, remaining questions will be resolved by examining the properties of ITS of defined stoichiometry prepared by site-specific attachment of the toxin molecule to engineered recombinant Mabs. It may indeed prove feasible to attach the toxin directly to a site on the Mab deliberately engineered to yield a stable IT without the need for a chemical crosslinking agent.
The work of the Drug Targeting Laboratory at the Institute of Cancer Research has been generously supported by the Cancer Research Campaign. We wish to thank all our past and present colleagues at the Institute and our many collaborators outside the Institute for their contribution to the work described in this review.
1. Blakey, D. C., Wawrzynczak, E. J., Wallace, P. M. & Thorpe, P. E. (1988) in Monoclonal Antibody Therapy (Waldmann, H., ed.), pp. 50-90, Karger, Basel 2. Wawrzynczak, E. J. (1991) Br. J. Cancer 64,624-630 3. Wawrzynczak, E. J. (1988) in Immunotoxins (Frankel,A. E., ed.),pp. 239-25 1, Kluwer, Boston 4. Wawrzynczak, E. J. & Thorpe, P. E. (1987) in Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer (Vogel, C.-W., ed.), pp. 28-55, Oxford University Press, New York 5. Cumber, A. J. & Wawrzynczak, E. J. (1992) in Methods in Molecular Biology, Vol. 10, Immuno-
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chemical I’rotocols (Manson, M.. ed.), pp. 283-293, Humana. New Jersey 6. Cumber, A. J., Forrester, J. A., Foxwell, H. M. J.. Koss, W. C. J. & Thorpe, 1’. E:. (1985) Methods Bnzymol. 112,207-225 7. Forrester, J. A., Mclntosh, L). l’., Cumber, A. J., Parnell, G. D. & Ross, W. C. J. (1984) Cancer Drug Deliv. 1,283-293 8. Raso, V. & Basala, M. (1984) in Keceptor-Mediated Targeting of Drugs (Gregoriadis, G., I’oste, G., Senior, J. & Trouet, A., eds.), pp. 119-138, Plenum, New York 9. Jansen, F. K., Blythman, H. E., Hourrie, B., Carriere, D.. Casellas, P., Dussossoy, D., Gros, 0.. Laurent, J. C., Liance, M. C., I’oncelet, I’.$ Kicher, G. & Vidal, H. (1984) in Receptor-Mediated Targeting of Drugs (Gregoriadis, G., Poste, G., Senior, J. & Trouet, A., eds.),pp. 147-178, Plenum, New York 10. Worrell, N. R., Cumber, A. J., I’arnell, G. 11.. Koss, W. C. J. & Forrester, J. A. (1986) Biochem. I’harmacol. 3 5 4 17-423 11. Blakey, D. C., Watson, G. J., Knowles, 1’. 1’. & Thorpe, P. E. (1 987) Cancer Kes. 47,947-952 12. Greenfield, I,. & Dovey, H. F. (1992) Antibody Immunoconj. Radiopharm. 5,37-59 13. Worrell, N. R., Skilleter, D. N.. Cumber, A. J. & Price, K. J. (1986) Hiochem. Hiophys. Kes. Commun. 137, 892-896 14. Bourrie, H. J. P., Casellas. P.. Hlythman, H. E. & Jansen, F. K. (1986) Eur. J. Biochem. 155, 1-10 15. Byers. V. S., Pimm, M. V.. Pawluczyk, 1. Z.A., Lee, H. M., Scannon, P. J. & Baldwin, R. W. (1 987) Cancer Res. 47,5277-5283 16. Cumber, A. J., Parnell, G. D., Henry, R. V., Forrester, J. A. & Wawrzynczak. E. J. (1989) Biochem. SOC. Trans. 17,137-138 17. Trown, P. W., Reardan, D. T., Carroll, S. F., Stoudemire, J. B. & Kawahata, R. T. (1991) Cancer Res. 51,4219-4225 18. Wawrzynczak, E. J.? Cumber, A. J., Henry, R. V. & Parnell, G. D. (1991) Intl. J. Cancer 47, 130-135 19. Wawrzynczak, E. J. & Cumber, A. J. (1992) in Methods in Molecular Biology, Vol. 10, Imrnunochemical Protocols (Manson, M., ed.), pp. 295-305, Humana, New Jersey 20. Thorpe, P. E., Wallace, P. M., Knowles, P. P., Relf, M. G., Brown, A. N. F..Watson, G. J , Blakey, D. C. & Newell, D. R. (1988) Cancer Res. 48,6396-6403 21. Shire, D., Bourrie, B. J. P., Carillon, C., Derocq, J.-M., Dousset, P., Dumont, X., Jansen, F. K., Kaghad, M., Legoux, R., Lelong, P., Pessegue, B. & Vidal, H. (1990) Gene 93, 183-188 22. Blakey, D. C., Wright, A. F., Hewett, P. W. & Rose, M. S. (1992) in Monoclonal Antibodies: Applications in Clinical Oncology (Epenetos, A. A., ed.), Chapman and Hall, London, in the press 23. Worrell, N. R., Cumber, A. J., Parnell, G. D., Mirza, A.. Forrester, J. A. & Ross, W. C. J. (1986) AntiCancer Drug Design 1, 179- 188
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24. Thorpe, P. E., Wallace. P. M., Knowles. P. I’.. Relf, M. G., Hrown, A. N. F.,Watson, G. J., Knyba, K. E., Wawrzynczak, E. J. & Blakey, D. C. (1987) Cancer Kes. 47.5924-593 1 25. Cumber, A. J., Westwood, J. H., Henry, R. V., Parnell, G. 11.. Coles, H. F. & Wawrzynczak. E. J. (1992) Hioconj. Chem. 3, in the press 26. Wawrzynczak, E. Cumber, A. J., Henry, K. V., May. J., Newell, D. K..Parnell, G. D., Worrell, N.R. & Forrester, J. A. (1990) Cancer Kes. 50,75 19-7526 27. Wawrzynczak, E. J., Henry, K. V.. Cumber, A. J., I’arnell, G. 11.. Derbyshire, E. J. & Ulbrich, N. (1991) Eur. J. Hiochem. 196,203-209 J.?
28. Skilleter, D. N..Price. K. J.. I’arnell, G. 1). & Cumber, A. J. (1 989) Cancer Letters 46, 161- 1Oh 29. Thorpe, P. I.:.. Hlakey, D. C., Hrown. A. N. F.. Knowles, P. P.?Knyba, K. E., Wallace, 1’. M., Watson, G. J. & Wawrzynczak, E. J. (1987) J. Natl. Canc. Inst. 79,1101-1112 30. Cumber, A. J. & Wawrzynczak, E. J. (1992) Hiochem. SOC.Trans. 20,3 12.5 31. Fulton. K.J.. Tucker, T. F.. Vitetta. E. S. & Uhr, J. W. (1988) Cancer Kes. 48,2618-26)25
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